The 4th International Rice Blast Conference


ABSTRACTS


Changsha, China


October 9-14, 2007


Oral Presentations


Current Status and Future Prospects of Researchon Blast Disease in Rice (Oryza sativa)


Gurdev S. Khush and K. K. Jena


University of California, Davis CA 95616-7008, USA

Email:gurdev@khush.org


Rice is the most important food security crop and staple of half the world population. Major increases in rice production occurred during last four decades of last century as a result of wide scale adoption of green revolution technology. Demand for rice continues to increase as a result of population increase and improvement in living standards particularly in Africa and Latin America. However, rate of increase of rice production has slowed down. It is estimated we will have to produce 30% more rice in 2030. For this purpose we need rice varieties with high yield potential and greater yield stability. Breakdown of blast resistance is the major cause of yield instability in several rice growing areas. Efforts are underway to develop rice varieties with durable blast resistance.

More than 40 major genes as well as QTL for resistance to blast have been identified. Monogenic resistance is less stable but varieties with pyramided monogenes or QTL are durably resistant. Rice research should focus on identifying more durably resistant genes, tagging of these genes with molecular markers and pyramiding these genes or QTL through molecular marker aided selection. Candidate gene identification through rice functional genomics has great potential for developing more durably resistant varieties.



Progress in Breeding of Super Hybrid Rice


Long-Ping Yuan


China National Hybrid Rice R&D Center, Changsha, Hunan, 410128 China


In order to meet food requirement in the 21st century, a super rice breeding program was set up by China Ministry of Agriculture in 1996. The yield targets for single season crop of rice hybrids are as follows.

Phase Ⅰ (1996~2000): 10.5 t/ha.

Phase Ⅱ (2001~2005): 12 t/ha.

Several pioneer super hybrid rice varieties had been developed by 2000, which met the yield target of the Phase Ⅰ. More than 20 demonstration locations with 6.7 or 67ha each where the average yield was over 10.5 t/ha in 2000. The yield of these pioneer super rice hybrids in large-scale commercial production (1.2-2.0 million ha) is 8.5 t/ha in recent years.

Good progress had been made on developing Phase Ⅱ super rice hybrids. A promising two-line indica/japonica combination, P88s/0293, yielded more than 12 t/ha at five demonstration locations with around 7 ha each in 2003 and at twelve locations in 2004. This second generation super hybrid rice was released for commercial production in 2006. Based on the above achievements, the phase Ⅲ super hybrid rice breeding program is proposed, in which the yield target is 13.5 t/ha and can be fulfilled by 2010. The technological approaches for breeding super hybrid rice mainly are: ①Morphological improvement, ② Using inter-subspecific heterosis and ③ Utilization of biotechnology. The details of these technical issues are discussed in the full paper.




An “omics” Interrogation of Pathogenesis

by the Rice Blast Fungus


Ralph Dean


Center for Integrated Fungal Research, Dept. Plant Pathology, North Carolina State University, Raleigh, NC 27606, USA


Magnaporthe grisea is the causal agent of rice blast, the most devastating disease of rice worldwide and is a seminal model to elucidate the basis of pathogen–host interactions. Following the completion of the genome sequence of both the fungus and its host, rice, research is focused on functional and comparative approaches to uncover the molecular and evolutionary foundation of fungal pathogenesis. Whole genome microarray analysis of appressorium initiation, development and maturation induced by hydrophobic surfaces and by cAMP revealed a core set of 357 genes that were differentially expressed when compared to conidial germination under non-inductive conditions. In addition to genes involved in lipid, carbohydrate and secondary metabolism, numerous genes involved in protein turn over and amino acid catabolism were significantly induced. The critical requirement for protein catabolism, including endo-proteases and key enzymes involved in shuttling carbon back into the Kreb cycle, was demonstrated by gene knockout. To examine the evolution of pathogenesis, a semi-automated high throughput computational platform to facilitate large-scale comparative analyses was developed. Comparison of gene sets from 11 fully sequenced fungal pathogens and related non-pathogens revealed evidence for duplication of genes associated with several functional categories including signaling and hydrolytic activities. A database, FEGA (Fungal Evolutionary Genomic Analyses), was created and provides information about gene copy number in a genome, gene family constituency among genomes, and functional descriptions of gene families, including Gene Ontology (GO)-based functional annotation. Indices of selection (Ka/Ks ratios) and divergence (Ks, synonymous nucleotide substitution) are also included. Other efforts are currently focused on examination of novel non-coding transcripts, transcriptional networks and protein-protein interactions to define the circuitry regulating rice-rice blast interactions.



Genome-wide Identification of Genes Controlling Hyphal Growth of Magnaporthe Oryzae


You-Liang Peng


The State Key Laboratory for Agrobiotechnology and Department of Plant Pathology, China Agricultural University, Beijing 100094


Magnaporth oryzae infects host plants by a process involving conidiation, appressorium formation, penetration and invasive growth of infection hyphae. Understanding of molecular mechanisms regulating the infection process will contribute to design of novel approaches for the disease control. We suppose that many of genes required for in vitro colony growth are also necessary for invasive growth of infection hyphae in M. oryzae, and thus started genome-wide identification of genes that control colony growth. In order to identify genes that control colony growth of M. oryzae, an insertion mutagenesis library was generated with a field isolate P131. The library contained about 68000 independent transformants. Through screening the library, over 600 mutants were identified, showing slow colony growth. Genetic co-segregation analysis was carried out on about 400 of the mutants, which showed that the phenotype change in about 40% of the mutants was co-segregated with the selection marker of hygromycin resistance, suggesting that not all the mutations were caused by the insertion. TAIL PCR was used to isolate the sequences flanking the insertion sites. So far, the sequence in about 60 co-segregation mutants was determined, and through gene complementation and targeted gene disruption, more than ten genes were proven to control the colony growth. A detailed report will be given on the genetic network controlling colony growth of M. oryzae.



Systems Biology Initiatives for Magnaporthe oryzae


Yong-Hwan Lee


Department of Agricultural Biotechnology and Center for Fungal Genetic Resources, Seoul National University, Seoul 151-921, Korea


Magnaporthe grisea is a causal agent of rice blast and considered as a model pathogen for studying plant-microbe interactions. This is due to not only economic significance of rice blast disease worldwide but genetic and molecular tractability of this fungal pathogen. Currently whole genome sequence of strain 70-15 is available in the public database. To decipher fungal pathogenicity factors at genome-wide level in this fungus, we initiated a large-scale insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation (ATMT). This project includes (1) construction of transformants library (2) development of high throughput phenotype screening and DNA extraction systems, and (3) rescuing flanking sequences of T-DNA insertion from selected transformants. We generated 21,070 transformants and screened for 7 phenotypes as a high throughput manner; conidiation, conidial morphology, conidial germination, appressorium formation, mycelial growth, pigmentation, and pathogenicity. Over 1,000 loss of virulence and development-defective mutants were obtained from ATMT mutant library. The T-DNA tagged sequences from the selected transformants are being rescued by TAIL-PCR and sequencing and 741 unique loci are identified thus far. To verify phenotype changes by T-DNA insertion, crossing with a wild type and targeted gene knock-out are being applied. Distribution and integration patterns of T-DNA on fungal chromosomes are also analyzed. Furthermore, we developed the ATMT data management system to handle all phenomics and genomics data of these transformants.




Characterization of T-DNA Insertion Patterns and Identification of Pathogenicity-deficient Mutants

in Rice Blast Fungus Magnaporthe Oryzae


Guihua Li, Zhuangzhi Zhou, Chunhua Lin and Chaozu He*


State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101

* Corresponding author: E-mail: hecz@im.ac.cn


Agrobacterium tumefaciens-mediated transformation (ATMT) has been proven to be a powerful strategy for gene disruption in plants and fungi. Using ATMT, a T-DNA tagged population consisting of 6179 transformants of Magnaporthe oryzae was constructed. With thermal asymmetric interlaced-PCR (TAIL-PCR), 623 right border (RB) flanking sequences and 124 left border (LB) flanking sequences were generated. Analysis of these flanking sequences indicated a significant integration bias toward non-coding sequences, suggesting distribution of T-DNAs was not random. Comparing to T-DNA RB, LB was nicked inaccurately and truncated frequently during integration. Chromosomal rearrangements, such as deletion, inversion and translocation, were associated with T-DNA integration in some transformants. Our data suggest that, comparing with plant cells, T-DNA integrates into this filamentous fungus with more precise and simpler patterns. After innoculation of these transformants against a compatible rice cultivar Nipponbare, some pathogenicity-deficient mutants were identified. Of them, one mutant was identified that the insertion of T-DNA dramatically reduced the expression of MoUROD, a gene encoding uroporphyrinogen decarboxylase (UROD). UROD is an enzyme of heme biosynthesis. Mutation of MoUROD attenuated virulence of M. oryzae to compatible rice but not affected in induction of hypersensitive response in incompatible rice. Our results suggest that MoUROD plays an important role in tolerance of oxidative stress during expansion of invasive hyphae in rice cell.



Characterization on Transcriptional Circuitry Regulating Pathogenicity Using a Proteomics Approach


Thomas Mitchell1,, Jin-Rong Xu2, Cornelia Koten2, Heng Zhu3, Heesool Rho3, Yeon Yee Oh4, Malali Gowda4, and Ralph Dean4


1 The Ohio State University, Department of Plant Pathology, Columbus OH

2 Purdue University, Department of Botany and Plant Pathology, West Lafayette, IN

3 Johns Hopkins University, School of Medicine, Baltimore, MD

4 North Carolina State University, Center for Integrated Fungal Research, Raleigh, NC


A key component to deciphering the molecular underpinnings of pathogenicity for Magnaporthe grisea is the modeling of signaling networks the fungus uses to coordinate gene expression. These signaling networks continue to be fertile ground for generating insights into fungal biology and virulence, however full characterization of the downstream transcription factors they directly regulate is lacking. Through a combined automated and manual annotation process, we have identified over 500 putative transcription factors in the M. grisea genome and confirmed the expression of > 90% using expression data from EST, RL-SAGE and MPSS studies. We cloned >80% of these transcription factors and expressed each in yeast in order to print a M. grisea transcription factor protein microarray. Protein arrays were used to assay kinase phosphorylation specificity and activity. Gene disruption and over-expression strategies are now being used to map transcription factor binding motifs and identify genes regulated by selected transcription factors using a ChIP-chip hybridization strategy. We will present results from annotation, cloning, and phosphorylation studies as well as describe ChIP-chip studies.



The MGOS (M. grisea O. sativa) Interaction Community Database


Carol Soderlund


University of Arizona Bio5 Institute, 1657 E. Helen Street, University of Arizona, Tucson AZ 85721 USA

E-mail: cari@agcol.arizona.edu


The MGOS (Magnaporthe grisea Oryza sativa, www.mgosdb.org) database was

developed to contain genomic, gene expression and mutant data fromexperiments on the interaction between Oryza sativa and Magnaporthe grisea(M. oryzae) (1,2). The experiments were planned by a consortium of fungal and rice geneticists, to dissect early stages of the interaction betweenhost and pathogen. The data include ESTs and RL-SAGE information from infection in both resistant and susceptible interactions; phenotypic analysis of >50,000 M. grisea mutants; and results from a dual O. sativa-M. grisea microarray. MGOS also contains the genomic sequence from both rice and M. grisea along with automated annotation. A recent grant was awarded (3) to make MGOS a community database, which includes community annotation, enhanced microarray submission capabilities, and literature submissions. The community annotation functions have been implemented, and work on microarray submission is near completion. The annotation information includes the gene name and description, transcript structure and location, gene ontology terms, fungal anatomy terms, publication information, and the name of the person making the annotation submission.We request the communities' active participation to make this a model community database. We want the MGOS database to greatly enhance Magnaporthe research for everyone in the community.Towards this end, your feedback and requests will be greatly appreciated.


1. NSF-PGRP #0115642, PIs: R.Dean, D.Ebbole, M.Farman, M.Orbach, C.Soderlund, G.Wang, R.Wing, J.Xu.

2. C.Soderlund, K.Haller, V.Pampanwar, D.Ebbole, M.Farman, M.Orbach,G.Wang, R.Wing, J.Xu, D.Brown, T.Mitchell, R.Dean (2006) MGOS: a resource for studying Magnaporthe grisea and Oryza Sativa interactions.Mol Plant Microbe Interact 19: 1055-1061.

3. NSF-MGS #0627159, PIs: C.Soderlund, M.Orbach, B.Valent.




Investigating the Biology of Appressorium-mediated Plant Infection by the Rice Blast Fungus Magnaporthe grisea


Nicholas J Talbot*, Richard A. Wilson, Michael J. Kershaw, Darren M. Soanes, Diane O. C. Saunders, Elise Lambeth, Thomas A. Richards, Martin J. Egan, Han-Min Wong, Zaira Caracuel-Rios, Romain Huguet, Ana-Lilia Martinez-Rocha


School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter, EX4 4QD, United Kingdom

*Corresponding author:E-mail: N.J.Talbot@exeter.ac.uk


During plant infection, the rice blast fungus elaborates a specialised infection structure known as an appressorium. This unicellular, dome-shaped structure generates turgor that is translated into mechanical force to allow rupture of the rice cuticle and entry into plant tissue. We set out to explore whether the development of a functional appressorium was linked to the control of cell division. This was based on the observation that following germination of a conidium on the rice leaf surface, a single round of mitosis always occurs during germ tube elongation, prior to the formation of an appressorium. We found that blocking completion of mitosis by generation of a temperature-sensitive MgnimA mutant prevented appressorium morphogenesis. Furthermore, we found that following mitosis, conidia always undergo cell collapse and cell death, which appears to be a programmed, autophagic process. Deletion of MgATG8 prevented autophagy in M. grisea and rendered the fungus non-pathogenic. Taken together, our results indicate that appressorium morphogenesis requires genetic control by completion of mitosis and autophagic cell death of the conidium. We have also recently demonstrated the appressorium morphogenesis is accompanied by a burst of reactive oxygen species. Deletion of NOX1 or NOX2 which encode NADPH oxidases is sufficient to prevent plant infection by interfering with appressorium function. A third NOX-encoding gene NOX3 plays a role in hyphal morphogenesis. Once the M. grisea appressorium has formed, cellular turgor is generated by accumulation of osmotically-compatible solutes, notably glycerol. We have used genetic, biochemical, proteomic and, most recently, metabolomic analysis to investigate how turgor is generated and to define the key genetic determinants of appressorium function. Of particular interest is the central role of trehalose metabolim to the genetic control of fungal virulence and the interplay between sugar signaling and nitrogen source utilization and the role of peroxisomal fatty acid beta-oxidation in appressorium physiology. The appressorium brings about plant infection by elaborating a penetration hypha that differentiates further into invasive hyphae which grow rapidly within the host plant cells. One of the key challenges in understanding rice blast disease is to determine how fungal proteins are delivered to the host during plant infection and to define the mechanisms by which the fungus proliferates biotrophically within the rice leaf. Progress towards determining the secretory processes necessary for M. grisea plant tissue colonization will also be presented.




Fungal Secondary Metabolism is an Essential Component of the Complex Interplay between Rice and Magnaporthe


Marc Henri Lebrun, Jerome Collemare, Isabelle Fudal, Heidi Bohnert


UMR5240 CNRS/UCB/INSA/BCS, Functional Genomics of Plant Pathogenic Fungi, Bayer Cropscience, 14-20 rue P Baizet, 69263 Lyon Cedex 09, France


Functional analyses of fungal genomes are expanding our view of the metabolic pathways involved in the production of secondary metabolites. These genomes contains a significant number of genes encoding biosynthetic enzymes such as PKS and hybrid PKS-NRPS involved in the production of polyketides, NRPS involved in the production of peptides and TS involved in the production of terpenes. Magnaporthe grisea has a high number of such key enzymes (22 PKS, 8 NRPS, 10 PKS-NRPS, 5 TS), suggesting that this fungal species produce a large number of diverse secondary metabolites. In particular, it has the highest number of PKS-NRPS in fungi. Among them, 4 are expressed during infection (ACE1, SYN2, SYN6, SYN8). Targeted gene replacement of SYN6, expressed in mycelium, spores and infected leaves, does not impair infection. ACE1, SYN2 and SYN8 share the same pattern specific of the early stages of infection (penetration). Ace1 was shown to be expressed only in appressoria during the penetration of the fungus into host plant suggesting that the corresponding metabolite is delivered to the first colonized cells. Targeted gene replacement of ACE1 and SYN2 does not impair infection of susceptible rice cultivars suggesting a possible functional redundancy between these pathways. However, only ACE1 null mutants are able to infect resistant rice cultivars carrying Pi33 blast resistance gene and recognition of M. grisea by Pi33 resistant rice cultivars was shown to require ACE1 that behaves as a classical avirulence gene. However, fungal AVR genes are known to encode small peptides secreted into host tissues during infection. ACE1 from M. grisea differs from these fungal AVR genes as it likely controls the production of a secondary metabolite recognized by resistant rice cultivars carrying Pi33. Arguments toward this hypothesis involves the fact that the protein Ace1 is only detected in the cytoplasm of appressoria and not in infectious hyphae differentiated inside infected epidermal cells. Furthermore, Ace1-ks0, a non-functional ACE1 allele obtained by site-directed mutagenesis of an amino acid from polyketide synthase KS domain essential for its enzymatic activity, is unable to confer avirulence. According to this hypothesis, resistant plants would have evolved mechanisms to recognize microbial pathogens through the perception of the secondary metabolites they produced during infection. In order to characterize the metabolite produce by ACE1, this enzyme is currently constitutively expressed in M. grisea and under the control of an inducible promoter in Fusarium venenatum.




Regulation of Infectious Growth by the TBL1 Complex in Magnaporthe grisea


Shengli Ding and Jin-Rong Xu


Dept. of Botany and Plant Pathology, Purdue University, West Lafayette, IN47907


Magnaporthe grisea is pathogenic to economically important crops such as rice and wheat. Although appressorium formation and penetration are well characterized, there are only limited studies on infectious hyphal growth in M. grisea. We have generated gene replacement mutants for the transducin beta-like gene TBL1, which was first identified as a novel fungal pathogenicity factor in Fusarium graminearum. Similar to the F. graminearum mutant, the M. grisea tbl1 deletion mutant was defective in conidiogenesis and non-pathogenic. Appressoria formed by the tbl1 mutant were able to penetrate but failed to form secondary infectious hyphae. TBL1 is homologous to the Tbl1 nuclear receptor corepressor. In transformants expressing a TBL1-GFP fusion construct, nuclear localization of the GFP fusion protein was observed. Deletion analysis revealed that the N-terminal LisH domain that is normally involved in protein-protein interactions is essential for Tbl1 function. Homologs of TBL1 in yeast and mammalian cells are part of a conserved HDAC transcription co-repressor complex. Several components of the yeast Set3C complex, including the Set3 and Snt3 homologs, also are highly conserved in M. grisea. Mutants deleted of the MgSET3 gene had the same defects in conidiation and plant infection as the tbl1 mutant. In transformants expressing a TBL1-3Flag construct, we were able to pull down MgSnt3. These data indicate that TBL1 is a component of this well-conserved regulatory complex, which has been evolved to regulate the development and growth of infectious hyphae in M. grisea.




Surface Sensing and Signaling during Initiation

of Rice-blast Disease


Naweed Naqvi*, Ravikrishna Ramanujam, Angayarkanni Suresh, Liu Hao


Fungal Patho-Biology Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604

*Corresponding author:E-mail: naweed@TLL.org.sg


Conidial germ tubes of rice-blast fungus Magnaporthe grisea must differentiate into an infection structure called the appressorium in order to penetrate its host. Apart from hydrophobicity, the other host-surface characteristics responsible for appressorium initiation are poorly understood. In a forward genetics approach, we screened for Magnaporthe mutants defective in early surface signaling events during infection. This lead us to the identification of TMT1390 mutant (disruption in the Regulator of G-protein Signaling/RGS1 locus) which was capable of forming appressoria on non-inductive surfaces both hydrophobic and hydrophilic but couldn’t form appressoria on soft surfaces. Further characterisation, molecular identification and analysis of cells lacking RGS1 function helped us identify and define a thigmotropic response as being essential for initiation of pathogenesis. Involvement of a G-protein signaling network was identified as a downstream effector module of such early surface-hardness dependent signaling. Chemical genetic studies and global transcriptome analyses related to surface hardness indicated that such thigmo-morphogenesis is initiated within two hours after conidia germination and uses calcium signaling mediated by ion channels. Biophysical analyses allowed us to estimate the critical hardness necessary for efficient initiation of infection in Magnaporthe. Our preliminary results suggest a possible role for stretch-activated ion channels and a non-canonical GPCR in hardness sensing and host infection. These will be discussed along with a function for G proteins in elaborating the extra-cellular matrix during the pathogenic development in Magnaporthe.




Organ-specificity and Pathogenesis in the Rice Blast

Fungus Magnaporthe oryzae


Sara Tucker1, Maria Besi1, Stephan Goetz1, Yueh-Mei Zhou1, Anne Osbourn2 and Ane Sesma1,*


1 Dept. of Disease and Stress Biology, John Innes Centre NR4 7UH Norwich, UK

2 Dept. of Metabolic Biology, John Innes Centre NR4 7UH Norwich, UK

*Corresponding author: E-mail: ane.sesma@bbsrc.ac.uk


Compared to foliar fungal pathogens, very little is known about the requirements of soil-borne fungal pathogens for successful colonisation of root tissues, despite the fact that root-infecting fungi are extremely important as disease-causing agents. Recently, fungi regarded as foliar pathogens have been found to infect plant roots under field conditions, e.g. Leptosphaeria maculans and Cercospora beticola. The rice blast fungus Magnaporthe oryzae is regarded as a foliar pathogen. It belongs to the Magnaportaceae family, which contains root-infecting pathogens such as Magnaporthe poae, the turfgrass pathogen, and Gaeumannomyces graminis, the causal agent of “take-all” disease of cereals. We have previously shown that M. oryzae can also infect roots under laboratory conditions. Instead of the appressoria formed during leaf infection, simple hyphopodia structures allow M. oryzae to penetrate roots. Now we have found that M. oryzae can penetrate through the root hairs and natural openings present on the root surface. Preliminary investigations using characterised M. oryzae mutants from other laboratories have allowed us to classify mutants with leaf-specific, root-specific and general defects in the ability to cause lesion formation on leaves and/or roots. We have undertaken plant infection tests of ~1,000 T-DNA insertional transformants generated in our lab. This has enabled us to identify several M. oryzae mutants deficient in the ability to infect leaves and/or roots. Among them, we have started to functionally characterise two mutants. One of the mutants is tagged in a gene encoding for a putative RNA-binding protein which may have a role in the maturation and transport of mRNA precursors that are required for pathogenesis. The second mutant has undergone insertional inactivation in a gene encoding a predicted protein with DNA-binding functions. Both of them are defective in infection of rice roots.

At present, there is a lack of understanding of root diseases in part, due to the difficulty of studying such processes below ground, and also because of the genetic intractability of many root-infecting organisms. Therefore, understanding the root infection-related developmental process in M. oryzae will greatly enhance our understanding of fungal pathogenesis.




Telomere Instability in Magnaporthe oryzae Caused by Highly Active Telomere-targeted Retrotransposons


John H. Starnes, Cathryn J. Rehmeyer, Shouan Zhang, David Thornbury and Mark L. Farman*


Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA

*Corresponding author:E-mail: farman@email.uky.edu


Magnaporthe oryzae isolates that infect perennial ryegrass (prg) have unusually unstable telomeres that undergo continual rearrangements in culture and in planta. By comparison, telomeres in other host-specific forms of this fungus are quite stable. Sequencing revealed that the chromosome ends of the ryegrass pathogens are organized very differently to those in a strain with stable telomeres. Specifically, the subtelomeres consist of tandem arrays of three types of repetitive elements, with canonical telomere repeats at the ends of the arrays. The elements are found only in subtelomeric locations, so we have named them M. oryzae telomere-exclusive repeats (MoTERs). MoTER1 is 4.6 kb in length and codes for a reverse transcriptase. It lacks terminal repeats and, therefore, appears to be a non-LTR retrotransposon. MoTER2 is only 1.7 kb long and potentially codes for a 204 aa protein of unknown function, while MoTER3 (4.95 kb) is a recombinant of MoTER1 and MoTER2. We have identified new insertions of MoTER1, thereby demonstrating that it is an active transposon. We are currently trying to identify new insertions of MoTER2. The structures of MoTER1 and MoTER2, and the way they are organized at the chromosome ends, are reminiscent of the retrotransposons TART and HeT-A, which are responsible for maintaining the Drosophila telomeres. Therefore, we hypothesize that M. oryzae strains from ryegrass have dual systems for telomere maintenance - one based on telomerase, and another which uses terminally-targeted retrotransposons to repair degraded ends that arise during telomere crisis. Telomere instability was observed in all of the progeny from genetic crosses between a prg pathogen and an isolate with stable telomeres, although it was largely restricted to telomeres that were inherited from the prg parent. This suggests that the presence of the MoTER elements causes the telomeres to be unstable in the first place. Currently, we are testing effects of MoTER activity on the expression of neighboring genes. Results from these studies will be discussed.




Studying the Role of Heat Shock Proteins in Pathogenicity

of Magnaporthe oryzae


Nicole M. Donofrio


152 Townsend Hall, 531 S. College Ave, Newark, DE, 19716, USA

E-mail: ndonof@udel.edu


Heat shock proteins (HSPs), or chaperones, range in cellular function from folding of newly synthesized proteins, to repair of misfolded proteins during stress. Currently, there is a paucity of data describing whether HSPs are involved in fungal virulence on plants. We hypothesize that plant pathogens must be able to cope with stressful conditions upon gaining ingress to plant hosts, and one such mechanism may employ stress-related genes, including heat shock. Our main goal is to more fully characterize and understand the suite of HSPs in the rice blast pathogen, Magnaporthe oryzae, and ultimately determine their role in pathogenicity. Towards achieving this goal, we first performed inhibition experiments using the antibiotic, geldanamycin. This chemical is known to bind the active site of the major chaperone, HSP90, rendering it function-less. We wished to determine whether GDA had effects on the disease cycle of this pathogen, including germination, appressorial development, hyphal growth, and lesion production. Our results revealed that while GDA, at varying concentrations, appeared to have no effect on germination and appressorial development, this chemical did inhibit both hyphal growth in vitro as well as lesion development in planta by up to 75% at the highest concentrations used. Controls determined that lesion inhibition was most likely a result of GDA’s effect on the fungus, rather than the plant reacting to presence of the chemical. Second, we wished to determine whether GDA had any effect on the expression of putative heat shock genes, which RT-PCR tests revealed to be the case for several genes. We then performed a microarray experiment on GDA-treated versus untreated fungal hyphae, and results will be presented. Third, we wished to genetically determine whether several chaperones had any impact on pathogenicity. To this end, we generated knock-out mutants in two genes known to be molecular co-chaperones for HSP90 in other organisms, including yeast. Results of these knock-out experiments will also be presented.




Effector Function and Secretion during Biotrophic

Invasion by the Rice Blast Fungus


Barbara Valent1, Gloria Mosquera1, Chang-Hyun Khang1,2, Romain Berruyer1,4, Prasanna Kankanala1, Martha Giraldo1, Kirk Czymmek3, Sook-Young Park and Seogchan Kang2


1 Department of Plant Pathology, Kansas State University, Manhattan, KS 66503, USA

2 Pennsylvania State University, University Park, PA, USA

3 University of Delaware, Newark, DE, USA

4 Current Address: Université d'Angers, 49045 Angers, France


Magnaporthe oryzae is a hemibiotropic fungus that sequentially invades living plant cells using intracellular invasive hyphae (IH) that grow from cell to cell. Using live-cell imaging of a highly compatible interaction, we reported that IH are tightly wrapped in plant-derived extra-invasive hyphal membrane (EIHM). IH appear to seek out plasmodesmata for moving into the next cell, and filamentous IH that first grow in these cells have distinctive membrane caps at their tips (Kankanala et al, The Plant Cell, February 2007). To begin to identify and characterize effector proteins secreted by IH into live plant cells to control host cellular processes, we developed a reproducible procedure for obtaining infected sheath tissue with 20% IH RNA at 36 hpi, when most IH were still growing in the first-invaded rice cell. These samples were used for hybridization of both the whole genome M. oryzae microarray and a rice microarray to identify the interaction transcriptome. Fungal genes that were induced >50-fold in IH (compared to mycelium in culture) were enriched in putative secreted proteins with unknown functions, and rice genes that were induced >50-fold in infected tissue (compared to mock-inoculated tissue) were enriched in MAP kinase kinase kinase genes and transcription factors. These results are consistent with the hypothesis that IH secrete novel effector proteins into rice cells to reprogram their gene expression. Our initial gene replacement experiments have not shown major phenotypes associated with putative effectors. To study effector secretion in planta, we fused known and putative blast effectors to green fluorescent protein. Predicted effector signal peptides directed secretion of GFP from the fungus, and fusion proteins accumulated at predictable locations inside the EIHM. Fluorescence concentrated in the EIHM caps and in previously unrecognized structures, Blast Interfacial Complexes (BICs). Fusion proteins accumulated in BICs as long as IH grew in the cell. Correlative light and electron microscopy suggest that BICs are complex membrane-rich and vesicle-rich structures between the fungal wall and EIHM. We will discuss a potential role for BICs in blast effector secretion.




Toward the Understanding of Magnaporthe-rice Interactions: a Multi-faceted Genomics Approach


Ryohei Terauchi1,*, Joe Win2, Sophien Kamoun2, Hideo Matsumura1, Saitoh1, Hiroyuki Kanzaki1, Kentaro Yoshida1, Matt Shenton1, Thomas Berberich1, Shizuko Fujisawa1, Akiko Ito1, Yoshitaka Takano3, Yukio Tosa4


1 Iwate Biotechnology Research Center, Kitakami, Japan

2 Department of Plant Pathology, Ohio State University, OARDC, Wooster Ohio, USA

3 Laboratory of Plant Pathology, Kyoto University, Kyoto, Japan

4 Laboratory of Plant Pathology, Kobe University, Kobe, Japan

*Corresponding author: E-mail: terauchi@ibrc.or.jp


Since whole genome sequences are available for both Magnaporthe and rice, this pathogen-host system provides a unique opportunity to address microbe-plant interactions from genomics perspectives. To select candidate genes directly involved in the interactions, we are employing (1) bioinformatics approach through identification of 1,884 Magnaporthe putative secreted protein genes, (2) simultaneous gene expression analysis of Magnaporthe and rice by SuperSAGE with comparison of expression profiles between compatible and incompatible interactions, (3) population genetics analysis of the polymorphism of Magnaporthe secreted protein genes by EcoTILLING. Screens (1) – (2) allowed us to select 452 Magnaporthe effector candidate genes and 150 rice genes presumably involved in resistance. Screen (3) identified a gene under strong diversifying selection, possibly involved in direct pathogen-host interaction. Function of these genes is tested by Agrobacterium-mediated transient overexpression in Nicotiana benthamiana. Through this assay, we have so far identified several Magnaporthe cell death inducing proteins including Nep1-like protein, and several rice cell death inducing proteins encoding a novel transcription factor. These are further functionally tested in Magnaporthe and rice by gene knockout and overexpression.




Expression of Secreted Proteins from Magnaporthe grisea and Analysis of Elicitor Activity toward Rice


Hanno Wolf1, Guodong Lu2, Kiran Bhattarai1, Cristina Filippi1, Yue Shang1, Dan Li1, Daniel J. Ebbole1,*


1 Department of Plant Pathology & Microbiology, Texas A & M University, College Station, USA 77843

2 College of Plant Protection, Fujian Agricultural and Forestry University, Fuzhou, China 350002

* Corresponding author:E-mail: d-ebbole@tamu.edu


Extracellular proteins of fungal pathogens are candidate effector molecules. We have cloned a large number (~300) of putative secreted proteins from Magnaporthe oryzae. The proteins were fused to a His(x6) tag to allow for affinity purification of the proteins. The genes were transformed into M. oryzae to test for expression. Approximately one-third of the genes could be expressed and secreted to sufficient levels to allow detection by western blot analysis of culture filtrates. In almost all cases, constitutive expression of the proteins in the transformed strains did not markedly alter their interaction with rice. Proteins were purified from culture filtrates of M. oryzae and symptoms were observed on rice leaves in only very few cases. A directed approach to examination of candidate factors is being pursued with emphasis on unique gene families and genes implicated in virulence in other systems.




Large-scale Isolation and Functional Analysis of Putative Effectors from M. oryzae Using Integrated

Genomics Approaches

Songbiao Chen1, Pattavipha Songkumarn1, Malali Gowda1, Venu Reddyvari Channarayappa1, Chan Ho Park1, Maria Bellizzi1, Daniel Ebbole2, Guo Liang Wang1,*

1 Department of Plant Pathology, The Ohio State University, USA
2 Department of Plant Pathology and Microbiology, Texas A&M University, USA
* Corresponding author E-mail:
wang.620@osu.edu

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a leading constraint to rice production and is a serious threat to food security worldwide. To elucidate the function of effector proteins from M. oryzae in pathogenesis and interaction with the host, we have performed RL-SAGE and MPSS approaches to study the gene expression profiles of M. oryzae during the interaction. One RL-SAGE library from leaf tissues after 96-h compatible blast infection, 5 MPSS libraries from leaf tissues after 3-h, 6-h, 12-h, 24-h, 48-h incompatible blast infection, and 6 MPSS libraries from leaf tissues after 3-h, 6-h, 12-h, 24-h, 48-h, and 96-h compatible blast infection have been made and analyzed. The RL-SAGE and MPSS analyses identified 3,441 and 3,004 annotated M. oryzae genes, respectively. Among them, 217 in-planta expressed putative secreted protein genes of M. oryzae were identified. We have then applied integrated functional genomic approaches to analyze the function of the identified putative effectors from M. oryzae. These approaches include a high throughput vector system for cloning and expression of M. oryzae genes in rice cells, a highly efficient rice protoplast transient expression system for cell death and defense response assays after expression of the fungal genes and a yeast secretion trap system for confirming the secretion function of signal peptide of M. oryzae secreted proteins. Through these integrated approaches, ~30 in-planta expressed effector proteins have been isolated and characterized. Among them, several cell-death inducing proteins in rice protoplasts have been identified. Further characterization of these genes and identification of their host targets will provide new insights into the interaction between rice and M. oryzae.

Acknowledgements: This project is supported by the NSF-Plant Genome Research Program (#0605017).




The MGOS (M. grisea O. sativa) Interaction Community Database Workshop


K.A. Greer and C. Soderlund


Bio5 Institute, 1657 E. Helen Street, University of Arizona, Tucson AZ 85721 USA


The MGOS (Magnaporthe grisea Oryza sativa, www.mgosdb.org) database was originally developed to contain genomic, gene expression and mutant data fromexperiments on the interaction between Oryza sativa and Magnaporthe grisea (M. oryzae) (1,2). A grant was awarded in December 2006 (3) to extend MGOS for community annotation and microarray submission, thereby, making it acommunity database. 

This workshop will provide a detailed demonstration of the functionality added to the MGOS database as part of NSF grant #0627159 (3), and will cover:

  1. Adding a community annotation for a gene, including the gene name, symbol, and description of function, synonyms, transcript information (orientation, start, stop, exon boundaries, coding sequence start and stop), citations, and GO and fungal anatomy annotations.

  2. Adding a citation for a journal article.

  3. Entering, and viewing the results of, a microarray experiment.

  4. Using BLAST and BLAT to align a nucleotide or amino acid sequence to the genomic or transcript sequences in MGOS.

  5. Using the forum to communicate with the Magnaporthe grisea community.

An essential part of this workshop will be obtaining input from participants regarding improvements that they would like made to the MGOS database. The current grant will last through 2008, providing an incredible opportunity for the Magnaporthe grisea community to develop a world-class bioinformatics tool to aid in scientific research.


1. NSF-PGRP #0115642, PIs: R.Dean, D.Ebbole, M.Farman, M.Orbach, C.Soderlund, G.Wang, R.Wing, J.Xu.

2. C.Soderlund, K.Haller, V.Pampanwar, D.Ebbole, M.Farman, M.Orbach, G.Wang, R.Wing, J.Xu, D.Brown, T.Mitchell, R.Dean (2006) MGOS: a resource for studying Magnaporthe grisea and Oryza Sativa interactions. Mol Plant Microbe Interact 19: 1055-1061.

3. NSF-MGS #0627159, PIs: C.Soderlund, M.Orbach, B.Valent.




Rac GTPase-mediated Blast Resistance in Rice


Ko Shimamoto1,*, Letian Chen1, Nguyen Phuong Thao1, Ayako Nakashima1,Yoji Kawano1, Keiko Imai1, Hann Ling Wong1, Kenji Umemura2, Tsutomu Kawasaki1


1 Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma 630-0101, Japan

2 Meiji Seika Ltd., 5-3-1 Chiyoda, Saitama 350-0289, Japan

* Corresponding author :E-mail: simamoto@bs.naist.jp


We have been studying the role of Rac GTPase in innate immunity of rice and found that it is a key molecular switch for defense response in rice. To identify components of the Rac GTPase-mediated innate immunity in rice we have taken a number of approaches including proteomics and reverse genetics and found components associated with OsRac1. They include known components such as RAR1, SGT1, and Hsp90 and novel components such as RWD and Sti1/Hop and they interact with OsRac1. Various studies on protein-protein interactions among these components indicate that at least 10 proteins form a network in the Rac GTPase-mediated innate immunity. Since most of those components are involved in both PAMP-mediated and R protein-mediated resistance in rice we propose a model in which early signaling events involved in these two types of resistance occur in essentially the same protein complex we call “defensome” at the plasma membrane. Details of individual components in the protein complex will be discussed.




Insights into the Rice Defense Response


Pamela Ronald


Department of Plant Pathology University of California, Davis, 95616 USA


The plant innate immune response includes perception of pathogen molecules as well as perception of signals released by the plants in response to pathogens. The molecular events of and connections between these processes are as yet ill defined, especially in the agriculturally paramount monocot clade. Our work focuses on three participants in rice (Oryza sativa) innate immunity, the pathogen recognition receptor, Xa21, and the proteins NH1 (NPR1 homolog1) and NRR (negative regulator of resistance) that respond to elevated levels of SA produced by the plant in response to pathogen attack. Xa21 allows the plant to perceive and defend against most strains of Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight. NH1 and NRR play key roles in perception of salicylic acid, a signal that is elevated during the plant defense response.

Using yeast two-hybrid screening, we have identified 51 putative components of the rice innate immunity protein-protein interaction network that includes the Xa21 intracellular domains, NH1, and NRR. In addition, we have gathered microarray data comparing Xa21, NH1 over-expressing (ox), and NRR-ox plants before and after inoculation. We are using these data to support and prioritize further analyses of the members of the interaction map. We have found that another pathogen recognition receptor that confers resistance to the fungal pathogen Magnaportha grisea, Pi-d2, also interacts with at least three of the members of this network including Xa21 binding protein 15 (Xb15), a protein phosphatase 2C (PP2C). Functionaly analysis of Xb15 indicate that Xb15 is a PP2C phosphatase and may act as a negative regulator in XA21-mediated resistance.




Towards Understanding of Signal Perception and Transduction in Rice Blast Resistance


Yinong Yang


Department of Plant Pathology and Huck Institutes of Life Sciences, 405C Life Sciences Bldg., Pennsylvania State University, University Park, PA 16802, USA

E-mail: yuy3@psu.edu


A combination of molecular, biochemical, genomic and proteomic approaches have been taken to understand the early signal perception during the rice-Magnaporthe grisea interaction as well as the downstream signaling pathways leading to blast resistance and susceptibility. Using transgenic rice lines defective in salicylic acid (SA), jasmonic acid (JA), ethylene (ET) or abscisic acid (ABA) signaling, we have shown that (i) SA is not an effective signal molecule in rice but acts as a constitutive antioxidant to protect rice plants from the pathogen-induced oxidative damage; (ii) JA signal pathway is important for mediating rice defense gene expression and blast resistance; (iii) ET signal pathway appears to be very critical for rice blast resistance; and (iv) ABA interacts antagonistically with ET signaling in rice and significantly increases blast susceptibility. We also identified 17 members of rice mitogen-activated protein kinase gene (OsMPK) family and found that a half of them were induced by M. grisea infection. Transgenic analysis demonstrated that pathogen-induced OsMPKs were capable of mediating JA, ET and/or ABA pathway interactions and modulating rice blast resistance. Our preliminary data revealed that pathogen-responsive OsMPKs may interact with upstream NBS-LRR proteins and phosphorylate key components of downstream defense pathways. It is hypothesized that NBS-LRR proteins may detect the interaction between M. grisea effectors and host cell targets, and relay the early signal through the OsMPK cascades, leading to the activation of defense pathways and subsequent blast resistance or susceptibility.




Functional Dissection of a Rice Mitogen-activated Protein Kinase Kinase Kinase OsACDR1


Jung A Kim and Nam-Sooc Jwa*


Department of Molecular biology, Sejong University, Seoul 143-747, Korea

* Corresponding author: E-mail: nsjwa@sejong.ac.kr


The OsACDR1 (OsEDR1) gene of rice has been previously characterized to encode a Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK), showed multistress responsive regulations. The OsACDR1 protein, as a MAPKKK, displayed autophosphorylation and kinase activity. OsACDR1 positively regulated HR-like leaf spots and its overexpression induced typical lesion mimic phenotype. Defense-related molecules including phenolic compounds and phytoalexins were accumulated around individual lesions. Pathogens-related (PR) genes were also up-regulated associated with lesion development. Lesion development in OsACDR1-OX transgenic lines increases resistance to both fungal (M. grisea) and bacterial (Xanthomonas oryzae pv. oryzae) pathogens through activation of basal resistance. Over-expression of OsACDR1 enabled transgenic lines to inhibit appressoria penetration of M. grisea on the leaf surface. Deletion mutant of OsACDR1 showed susceptible phenotype to rice blast fungus. Those results suggest that OsACDR1 is the first identified rice MAPKKK, positively regulating lesion development as well as basal resistance.




Functional Analysis of Rice WRKY89 in Rice Defense Response and UV-B Stress


Zejian Guo, Haihua Wang, Junjie Hao, Xujun Chen


Department of Plant Pathology, China Agricultural University, Beijing 100094, China


WRKY proteins are a large family of transcriptional regulators involved in a variety of biological processes in plants. In this study, we characterized a rice WRKY gene, OsWRKY89. RNA gel blot analysis indicated that OsWRKY89 was induced strongly by treatments of methyl jasmonate and UV-B radiation. Transient expression analysis using an OsWRKY89-eGFP fusion gene in onion epidermal cells revealed that the OsWRKY89 protein was targeted to nuclei. Protein fusion analysis of OsWRKY89 and its mutants with a GAL4 DNA binding domain indicated that the 67 C-terminal amino acids were required for transcriptional activation and that the leucine zipper region at the N-terminus enhanced transcriptional activity. Overexpression of OsWRKY89 led to retarded growth at the early stage and reduced internode length. Scanning electron microscopy revealed an increase in wax deposition on leaf surfaces of the overexpressing lines and a decrease in wax loading in the RNAi lines. Moreover, extractable and cell-wall-bound phenolics were decreased in the overexpressor lines, whereas the SA levels increased. Staining experiments demonstrated an increase in lignification in culms. Interestingly, overexpression of the OsWRKY89 gene enhanced resistance to rice blast fungi and white-backed plant hoppers and tolerance to UV-B tolerance. These results suggest that OsWRKY89 is involved in response to biotic and abiotic stresses.




Functional and Evolutionary Analysis of the Pi9 Resistance Gene Cluster in Rice


Bo Zhou1, 2, Liangying Dai3, Xionglun Liu3, Xunbo Li, Jun Wu3, Yajun Hu3, Jinling Liu3, Shaohong Qu1, Guifu Liu1,, Bellizzi Maria1,, Hajime Sakai4, Bin Han2, and Guo-Liang Wang1,3


1 Department of Plant Pathology, the Ohio State University, Columbus OH 43210 USA,

2 National Center for Gene Research, Chinese Academy of Sciences, Shanghai 200233, China

3 Rice Genomics Laboratory, Hunan Agricultural University, Changsha, Hunan 410128, China

4 DuPont Crop Genetics, Experimental Station, Wilmington, DE 19880, USA,


The complex Pi9 locus contains at least six resistance (R) alleles, i.e., Pi2, Pi9, Piz-t, Piz, Pigm(t) and Pi40(t), and each of them confers broad-spectrum resistance to diverse strains of Magnaporthe oryzae. To understand the molecular mechanism underlying the broad spectrum resistance mediated by these R genes, we successfully cloned the Pi2, Pi9, and Piz-t genes via map-based cloning and PCR homology cloning strategies. They all encode highly homologous proteins with a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) domain and belong to a multiple NBS-LRR gene family in each donor line. The three genes share over 96% identity in amino acid sequence to each other. Sequence analysis indicated that the LRR region plays a critical role in the determination of their resistance specificities. Only eight amino acid changes distinguish the Pi2 from the Piz-t, which are exclusively confined within three consecutive LRRs. The Pi2/Pi9 chimeric gene, which is derived from the replacement of the Pi2’s LRR region with the Pi9’s, was found to confer the Pi9 resistance specificity, further suggesting that the LRR region is the major determinant of the resistance specificity. Comparative analysis of the Pi9 locus in five different rice cultivars revealed contrasting genomic dynamics at the intra- and inter-haplotype levels. To further elucidate the evolutionary mechanism of the Pi9 cluster in wild rice species, we sequenced five BAC clones spanning the Pi9 locus in AA, BB, CC and BBCC genomes. Preliminary sequence analysis indicated that the Pi9 locus within each wild rice genome have a similar genomic dynamics to that in the cultivated species, in which the NBS-LRR paralogues have the same phase and position of their first intron but show significant sequence variation with each other. In contrast, a clear orthologous relationship of the NBS-LRR genes was not observed among the five genomes. Furthermore, each genome comprises different copy numbers of NBS-LRR genes, of which the AA genome has the least number of the NBS-LRR genes. A sequence rearrangement among different NBS-LRR genes was found in the five genomes, suggesting that the Pi9 locus has undergone intergenic unequal recombination during its evolution. We also amplified 119 Pi9 homologous DNA fragments containing both NBS and LRR domains from 60 cultivated and wild rice lines. These comparative analyses of the Pi9 cluster will provide valuable insights into the genomic dynamics and evolutionary mechanism of the Pi9 NBS-LRR resistance gene complex in the Genera Oryzae.




Understanding the Coevolution of Rice Blast Resistance Hene Pi-ta and Magnaporthe oryzae

Avirulence Gene AVR-Pita


Yulin Jia


USDA-ARS Dale Bumpers National Rice Research Center Stuttgart, AR 72160

E-mail: yjia@spa.ars.usda.gov


Rice blast disease caused by the filamentous ascomycetous fungus Magnaporthe oryzae remains to be one of the most serious threats for food security globally. Using resistance (R) genes in integrated cultural practices has been the most powerful practice for rice crop protection. Genetic analysis suggests that resistance mediated by the R gene Pi-ta in rice is effective at preventing infection of fungal races containing the corresponding avirulence gene AVR-Pita. To develop effective strategies to control rice blast disease, a comprehensive study of the co-evolution of Pi-ta with AVR-Pita has been undertaken at USDA-ARS Dale Bumpers National Rice Research Center in cooperation with scientists from US, China and Colombia. A survey in the USDA rice collection of 52 Oryza species identified a total of 14 haplotypes of the Pi-ta allele. Translation of these Pi-ta haplotypes revealed 10 highly similar Pi-ta proteins. Bootstrapping and neighbor joining analysis suggest that these Pi-ta haplotypes belong to two major clades. Tijma’s D value suggests that the Pi-ta allele existed before the divergence of Oryza species, and the Pi-ta allele is under neutral mutation.

In contrast, the AVR-Pita allele was present in most fungal isolates examined. The virulent isolates, with altered AVR-Pita alleles, were detected from US, China and Colombia. Deletions and transposon insertions in the coding regions were found to be responsible for the structure variation of the AVR-Pita gene in virulent isolates. More point mutations leading to amino acid substitutions (nonsynonymous substitution, Ka) were observed than point mutation leading to silenced mutation (synonymous substitutions, Ks) in avirulent isolates suggesting that AVR-Pita is under diversified selection. These data indicate that the ratios of Ka/Ks is less than 1 for Pi-ta and greater than 1 for AVR-Pita. We suggest that Pi-ta and AVR-Pita evolve through trench warfare and will present the implications for crop protection.




The Relationship between the Pseudogenization of NBS-LRR Genes and the Functional Loss of Rice Blast Resistance Genes in Asian Cultivated Rice (Oryza sativa L.)


Jun Shang1, Yong Tao1, Cailin Lei2, Xuewei Chen1, Yan Zou1, Jing Wang1,

Meijun Zhang3, Z Jun hi Ke Lu3, Lihuang Zhu1


1 State Key Laboratory of Plant Genomics & National Plant Gene Research Centre (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

2 Institute of Crop Research, Chinese Academy of Agricultural Sciences, Beijing 100081, China

3 Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101300, China



Rice blast, caused by Magnaporthe grisea, is one of the most devastating diseases. The two major subspecies of Asian cultivated rice (Oryza sativa L.), indica and japonica, have been shown obvious difference in rice blast resistance. We performed a genome-wide comparison of the NBS-LRR genes between the two sequenced rice genomes, 93-11 (indica) and Nipponbare (japonica). The pseudogenization of the NBS-LRR genes were found to some degree associated with the loss of the blast resistance. Using the NBS-LRR pseudogene sequences as genetic landing markers, we identified a novel blast R gene, Pid3. The allelic loci of Pid<?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />3 in most of the tested japonica varieties were found as pseudogenes, suggesting that the pseudogenization of Pid3 in japonica is a gene loss event occurred in the evolution of cultivated rice in agreement with the “birth-and-death” model.




Development of Targeted Evolution System of Resistance Genes LRR (Leucine Rich Repeat) to Recognize

Blast Surface Protein


Shinji Kawasaki, Ken'Ichi Ikeda, Ko Hirano

NIAS (Natl. Inst. Agrobiological Sciences) Kannon-dai, Tsukuba, Ibaraki, JAPAN 305-8602


It seems that in most cases the specificity of the plant disease resistance genes may be held in their product’s LRR structure, probably often with the help of the other factors. Still, there are a few cases known that the resistance gene products interact directly with the avirulence gene products, such as that of Pita-AvrPita pair. Really, LRR is a state of art ligand recognizing system with stacked LRR units each with specific inner side amino acids, with much simpler genomic structure than that of immunoglobulins. Only the deficit of the plant disease resistance genes is that they can not keep in pace with the mutations of the ligand-coding avirulence genes of the parasites, at least in the artificial monoculture system. If an LRR of a resistance gene can be made to recognize the Achilles tendon of the parasite; the indispensable structure or domain of the vital enzyme or structural protein, this will be a very powerful and durable resistance gene. Or several kinds of such LRRs may be used as material of multi-line strategy, with no afraid of using up the resistance genes resource.

Therefore, we have developed a targeted evolution system of LRR. As a model case, we have preferred the blast surface protein MPG1 of the rice blast fungus Maganporthe grisea as the target, and the material LRR was supplied from those of Pib and FLS2. After constructing a randomly shuffled library of the LRR repeating units of these two genes in the bacterial two hybrid system, with E coli harboring the bait (target) plasmid of MPG1 gene, the best surviving clones were chosen in the selective condition. The resultant LRR showed significant affinity to the MPG1, comparable to those of Pita/AvirPita or Galacturonase/Inhibitor pairs. Although the affinity was weaker than that of the positive control of this hybrid system (bait: Gal4-LGF2/target: Gal11P), after fine tuning with the Error-prone PCR system, finally the refined LRR was obtained, with the affinity comparable to the above control. We are now trying to confer the signal transducing ability to this LRR by swapping it to the Xa21 LRR, to induce the defense reactions in rice on infection.




Molecular Cloning and Characterization of the Rice Blast Resistance Gene Pi37 in the Well-known Cultivar St. No. 1


Fei Lin, Shen Chen, Zhiqun Que, Qinzhong Yang, Chunzhai, Lixia Hua, Xinqiong Liu, Ling Wang, and Qinghua Pan*


Laboratory of Plant Resistance and Genetics, College of Natural Resources & Environment, South China Agricultural University, Guangzhou 510642, China

* Corresponding author :E-mail: panqh@scau.edu.cn


The resistance (R) gene Pi37, present in the rice cultivar St. No. 1, was isolated by an in silico map-based cloning procedure. The equivalent genetic region in Nipponbare contains four NBS-LRR type loci. These four candidates for Pi37 (Pi37-1, -2, -3 and -4) were amplified separately from St. No. 1 via long-range PCR, and cloned into a binary vector. Each construct was individually transformed into the highly blast susceptible cultivar Q1063. The subsequent complementation analysis revealed Pi37-3 to be the functional gene, while -1, -2 and -4 are probably pseudogenes. Pi37 encodes a 1290 peptide NBS-LRR product, and the presence of substitutions at two sites in the NBS region (V239A and I247M) is associated with the resistance phenotype. Semi-quantitative expression analysis showed that in St. No. 1, Pi37 was constitutively expressed and only slightly induced by blast infection. Transient expression experiments indicated that the Pi37 product is restricted to the cytoplasm. Pi37-3 is thought to have evolved recently from -2, which in turn was derived from an ancestral -1 sequence. Pi37-4 is likely the most recently evolved member of the cluster, and probably represents a duplication of -3. The four Pi37 paralogs are more closely related to maize rp1 than to any of the currently isolated rice blast R genes Pita, Pib, Pi9, Pi2, Piz-t and Pi36.




Functional and Evolutionary Analysis of the Broad Spectrum Resistance Locus Pigm(t) to

Rice Blast Magnorpathe oryzae


Zuhua He1, Yiwen Deng1, Jing Xu1, Hongqi Chen2, Xudong Zhu2


1 National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

2 China National Rice Research Institute, Hangzhou 31006, China


Rice blast, caused by the fungal pathogen Magnorpathe oryzae, is one of the most destructive diseases of rice worldwide. The identification and utilization of broad-spectrum or durable resistance genes has been proven the most effective and economical approach to control the disease. A native Chinese variety, GM4, was identified with broad-spectrum and durable resistance and has been used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to different races in GM4 is controlled by the same dominant locus designated as Pigm(t). The allelism test showed that Pi-gm(t) was allelic to Pi2 and Pi9, two known blast resistance genes. The map-based cloning strategy was employed with a large mapping population consisting of 1556 recessive individuals. Pigm(t) was finally mapped on chromosome 6 region between two markers c5483 and c0428. A BAC contig covering the Pigm(t) region was constructed and completely sequenced. An NBS-LRR gene cluster encompassing 10 NBS-LRR candidate resistance genes was identified in the 120-kb sequenced region, which contains 6 resistance genes in the Pi2/Pi9 gene cluster. Sequence comparison of the orthologous and paralogous genes in the Pigm(t) locus in both resistant and susceptible backgrounds showed that the Pigm(t) loci had undergone duplication during the evolution of the resistance cluster, suggesting that broad-spectrum disease resistance of GM4 might be conferred by a pair of duplicated R genes with sequence variation. Our results of resistance spectrum analysis showed that Pigm(t) confers a broader resistance to blast isolates from different cultivated regions than Pi9/Pi2/Pizt/Piz, making Pigm(t) a good genetic resource for hybrid rice breeding for durable blast resistance with markers-assisted selection. Extensive genetic complement analysis of Pigm(t) is underway to dissect the Pigm(t)-mediated blast resistance.




Molecular Cloning and Gene Pyramiding of QTLs Controlling Field Resistance to Blast in Rice


Shuichi Fukuoka1,*, Norikuni Saka3, Hironori Koga4, Takehiko Shimizu2, Kaworu Ebana1, Akira Takahasi1, Hirohiko Hirochika1, Masahiro Yano1, Kazutoshi Okuno5


1 National Institute of Agrobiological Sciences, Kannondai 2-1-2 Tsukuba, Ibaraki 305-8602, Japan

2 Institute of the Society for Techno-Innovation of Agriculture, Forestry and Fisheries, Ippaizuka, Tsukuba, Ibaraki 305-0854, Japan

3 Mountainous Region Agricultural Research Institute, Aichi Agricultural Research Center, Inahasi, Toyota 441-2513, Aichi, Japan

4 Bioproduction Sciences, Ishikawa Prefectural University, Suematsu 1-308, Nonoichi-machi, Ishikawa 921-8836, Japan

5 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305–8572, Japan

* Corresponding author: E-mail: fukusan@affrc.go.jp


Field resistance in the Japanese upland rice cultivar Owarihatamochi is controlled by quantitative trait loci (QTLs) that confer durable resistance to diverse races of blast. We previously identified three QTLs and determined the map position of the QTL with the largest effect, the recessive resistance locus pi21 on chromosome 4. To characterize field resistance, we cloned the pi21 gene by a map-based strategy and used marker-assisted selection to develop experimental lines that contained multiple resistant QTLs.

A large-scale linkage analysis at the pi21 locus identified the sequence variations associated with the resistant/susceptible phenotypic difference. These variations cause two deletions at the amino acid level in a protein of unknown function encoded by the resistance allele. Transgenic plants with genomic fragments containing the Pi21 gene from a susceptible cultivar showed increased susceptibility to blast, whereas those with fragments containing the gene from a resistant cultivar showed no change in phenotype.

Backcrossed progeny lines, each carrying one of four resistant QTL (including one newly identified one) were developed in the genetic background of the susceptible cultivar, and two to five resistant QTLs were combined by marker-assisted selection. The disease severity of lines with multiple QTLs was lower than that of lines with a single QTL. In the pi21 resistant lines into which other resistance QTLs had been combined, the level of resistance was comparable to that of the original upland rice cultivar Owarihatamochi.

The results suggest that pi21 is a major QTL that belongs to a novel type of gene for plant defense. The combined effect of pi21 with some other resistance alleles may be the basis for the high level of field resistance in Japanese upland rice.




Identification, Mapping and Positional Cloning Rice Blast Resistance Gene Pi-kh from Rice Line Tetep


T.R. Sharma*, S. P. Kumar, N. Gautam, A.K. Rai, M.S. Madhav, H.C. Upreti and N. K. Singh


*Genoinformatics Laboratory National Research Centre on Plant Biotechnology

IARI, New Delhi –110012, India

* Corresponding author: E-mail: trsharma@nrcpb.org


Biotic stresses like Rice blast, Bacterial leaf blight, Sheath blight and Stem borer limiting rice productivity where ever rice is grown. Of these stresses, rice blast caused by Magnaporthe grisea (hebert) Barr is a serious constraint in rice production at global level. None of the rice cultivars possesses durable blast resistance because of the highly variable nature of the pathogen in the North-Western Himalayan region of India. Although chemical control of rice blast disease is feasible yet it remains environmentally unsafe. Developing blast resistance cultivars is important for sustainable management of the disease. Therefore, We have identified and mapped a blast resistance gene Pi-kh in rice line Tetep by using SSR markers at on long arm of rice chromosome 11. Once a skeleton genetic map was constructed using a mapping population of HP2216 (blast susceptible) and Tetep (blast resistant) rice lines, STMS markers linked to Pi-kh genes were mapped on the rice genome sequence of japonica type using sequence homology approach and a physical map having 1MB region was constructed. Since, known DNA markers were not available in the I MB region of constructed physical map, we identified simple sequence repeat (SSR) markers and designed 40 new SSR markers. Of these, only two were found flanking to the Pi-kh at 0.5 and 0.7 cM distance. The physical distance between these two markers was 142 kb. In this 142 kb genome sequence we predicted only one candidate gene for disease resistance belonging to the NBS and LRR categories of R-genes. PCR primers were designed from the Nipponbare sequence and used for amplification of the gene from blast resistant indica rice line Tetep. The Pi-kh gene was finally cloned and characterized at molecular level. Our experiment showed pathogen inducible nature of the gene. For functional validation of the gene we successfully produced transgenic Taipei 309 lines containing Pi-kh gene. Molecular analysis of these transgenics has shown stable integration of Pi-kh gene in the Taipei genome. Functional analysis of this gene using complementation test with M. grisea inoculation revealed that Pi-kh gene is expressing in the transgenic lines and imparting resistance to the pathogen. A complete story of the identification, mapping, cloning and functional validation of rice blast resistance gene Pi-kh will be presented.




Microarray-assisted Identification of Genes Associated with Blast Resistance in Rice


Yan Liu1, Xiaoyuan Zhu2, Shaohong Zhang2, Menchu Bernardo1, Jeremy Edward3, David Galbraith3, Jan Leach4, Hei Leung1, Bin Liu2


1 Plant Breeding, Genetics and Biotechnology Division, IRRI, Los Banos, Philippines

2 Guangdong Academy of Agricultural Sciences, Guangdong, China

3 University of Arizona, Tucson, Arizona, USA

4 Colorado State University, Fort Collins, Colorado, USA

E-mail: lbgz_2006@yahoo.com


Previous work showed that SHZ-2, an indica cultivar grown in China, has broad spectrum resistance to multiple races of the blast pathogen. A number of QTL have been mapped using recombinant inbred lines derived from SHZ-2. We crossed SHZ-2 to TXZ-13, a blast susceptible but high-quality variety to produce two BC3 lines, BC10 and BC116. These two lines showed strong to moderate blast resistance over eight cropping seasons in the field. In particular, BC10 has been successfully used in the hybrid rice program in Guangdong Province to produce high-yielding and blast resistance hybrids.

To further dissect the QTL responsible for durable blast resistance, 451 BC4F3 lines were developed by backcrossing BC10 and BC116 to TXZ-13. The BC4F3 population consisted of 244 lines from BC10 and 207 lines from BC116. The two BC4 populations were evaluated for blast resistance in the greenhouse and the blast nursery at IRRI, Philippines. Chromosomal introgressions from SHZ-2 were identified by using a genome-wide, genotyping microarray produced at University of Arizona. This array consists of 880 oligos that detect single feature polymorphisms (insertions and deletions) in unique genes evenly spaced along the chromosomes (medium spacing about 250 kb). Hybridization was done by pairing Cy3/Cy5 labeled DNA of BC10 and BC116 with that of the recurrent parent TXZ-13 and graphical genotypes of BC10 and BC116 generated using the software of Graphical Genotyping (GGT) version 2.0. Based on data from four replicated hybridization experiments, 39 and 22 regions with SHZ-2 introgression segments were detected in BC10 and BC116, respectively. Some of the regions of SHZ-2 introgression shown by microarray data were checked by SSR markers located near the introgression regions. Of 42 SSR markers tested, 19 were consistent with the microarray data; the other SSR markers were monomorphic and hence not informative.

To determine the relationship between introgression regions and blast resistance, extreme resistant and susceptible BC4F3 lines were selected for analysis. We monitored the introgression of SHZ-2 alleles in BC4F3 lines using SSR markers and SNPs in defense genes assayed by TILLING (a procedure that detects SNP between two target sequences). Through a combination of genome-wide genotyping, SSR marker genotyping and detection of specific alleles, we found four regions were on chromosome 2, one on chromosome 6 (about 250 kb) and one on chromosome 9 (about 400 kb) associated with blast resistance in the advanced backcross lines. Within the narrow region on chromosome 6 and chromosome 9, several candidate defense genes including NB-LRR genes were found. We are testing whether coordination expression of these genes are responsible for the quantitative resistance observed in advanced backcross families.




Genomic Approaches for Development of Broad-spectrum Rice Variety for Leaf and Neck Blast


Chatchawan Jantasuriyarat1, Pattama Sirithunya 2, Tanee Sreewongchai1, Saengchai

Sriprokhon 1, Chanakarn Wongsaprom1, Apichart Vanavichit 1,3,* ,Theerayut Toojinda1,

and Didier Tharreau 4


1 Rice Gene Discovery, National Center for Genetic Engineering and Biotechnology

(BIOTEC), Kasetsart University, Kamphangsaeng, Nakornpathom, 73140, Thailand

2 Rajamangala University of Technology Lanna, Science and Agricultural Technology

faculty, Chiengmai, 50000, Thailand

3 Agronomy Department, Kasetsart University, Kamphangsaeng Campus,

Nakornpathom, 73140, Thailand

4 UMR BGPI, INRA-ENSAM-CIRAD, TA73/09, 34398 Montpellier Cedex 05, France.

* Corresponding auther: E-mail: vanavichit@gmail.com


Rice blast, caused by the fungus, Magnaporthe grisea, is one of the most important diseases in rice production worldwide. Host resistance is the most effective way to control the disease. Genomic approaches including a survey of rice germplasm for new sources of resistance, a study of genetic structure of rice blast fungus, mapping of resistant and avirulence genes, and using of marker assisted selection (MAS) for gene pyramiding were used to develop broad spectrum blast resistant rice varieties. For the result, new source of rice leaf and neck blast resistant genes from Thai rice variety “Jao Hom Nin” (JHN), which shows broad-spectrum resistance to all but one group of rice blast isolates in Thailand, was mapped on rice chromosome 1 and 11. Pyramiding these QTLs for rice blast resistance from JHN with QTLs for rice blast resistance from IR64, which located on chromosome 2 and 12, result a broad-spectrum rice varieties which is resistant to all Thai blast isolates. Currently this rice variety is being used in rice breeding program in Thailand. A QTL for avirulence gene of rice blast fungus corresponding to leaf and neck blast resistant QTLs in JHN was mapped to the same region on blast chromosome 2 which can explain approximately 20 percent of phenotypic variance. Fine mapping of both resistance genes and avirulence genes are underway and positional cloning and identifying of their functions are our ultimate goal.




Regulation of Reactive Oxygen Production by the Binding of Rac GTPase to the N-terminal Extension of NADPH Oxidase of Rice


Hann Ling Wong1, Reinhard Pinontoan1, Kokoro Hayashi2, Ryo Tabata2, Takashi Yaeno3, Kana Hasegawa1, Chojiro Kojima2, Hirofumi Yoshioka4, Koh Iba3, Tsutomu Kawasaki1, and Ko Shimamoto1


1 Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology (NAIST), Ikoma, Japan

2 Laboratory of Biophysics, Nara Institute of Science and Technology (NAIST), Ikoma, Japan

3 Department of Biology, Kyushu University, Fukuoka, Japan. 4Laboratory of Defense in Plant-Pathogen Interactions, Nagoya University, Nagoya, Japan


Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities including defense against pathogens by plants. In contrast to the large multi-protein NADPH oxidase complex of phagocytes, in plants only the homologues of the catalytic subunit gp91phox and the cytosolic regulator small GTPase Rac are found. Plant homologues of the gp91phox subunit are known as Rboh (respiratory burst oxidase homologue). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+-binding EF-hand motifs. The involvement of Ca2+ in plant immune response has been implicated in many studies, but its exact role in ROS signaling remains unclear. Previously, we showed that a small GTPase Rac (OsRac1) enhanced elicitor-induced ROS production and resistance to pathogens in rice. In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac-Rboh interaction was confirmed in further studies using in vitro pull-down assay, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET microscopy and in vitro NADPH oxidase assay also suggest that the direct Rac-Rboh interaction and the activation of NADPH oxidase activity are modulated by cytosolic Ca2+ concentration. Therefore, our study provides evidence that Ca2+ and Rac GTPase coordinately regulate ROS production in plant immune response.




Host Active Defense Responses Occur within 24 Hours after Pathogen Inoculation in the Rice Blast System



Zhonghua Wang1, Yulin Jia2, Hui Lin3, Barbara Valent4, J. Neil Rutger5

1 Institute of Biotechnology, Zhejiang Wanli University, Ningbo 315100, P. R. China

2 USDA-ARS Dale Bumpers National Rice Research Center, P. O. Box 1090, Stuttgart, AR 72160, USA

3 Adair Intern, Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA

4 Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Science Center, Manhattan, KS 66506-5502, USA

5 USDA-ARS Dale Bumpers National Rice Research Center, P. O. Box 1090, Stuttgart, AR 72160, USA


Phenotypical, cytological and molecular responses of rice to the fungus M. grisea were studied using rice cultivars and lesion mimic plants. Cultivar Katy was susceptible to several virulent Magnaporthe grisea isolates, and a Sekiguchi like-lesion mimic mutant of Katy (LmmKaty) was shown enhanced resistance to these isolates. Lesion mimic phenotype of LmmKaty was rapidly induced by virulent M. grisea isolates or by avirulent isolates only at high levels of inoculum. Autofluorescence (a sign of an active defense response) was visible under ultraviolet light 24 h after localized inoculation in the incompatible interaction whereas, autofluorescence was not evident in the compatible interaction. Autofluorescence was also observed in LmmKaty 20 h after pathogen inoculation, thus indicating that rapid cell death is a mechanism of LmmKaty to restrict pathogen invasion. Rapid accumulation of defense related (DR) gene transcripts, phenylalanine ammonia lyase and ß-glucanase, was observed beginning at 6 h and was obvious at 16 h and 24 h in an incompatible interaction. Rapid transcript accumulation of PR-1 and chitinase had occurred by 24 h after inoculation in an incompatible interaction. Accumulation of these transcripts was delayed in a compatible interaction. These results indicate that host active defense responses occur 24 h after pathogen inoculation and that LmmKaty exhibits enhanced resistance to M. grisea. We suggest that the autofluorescence and expression of the DR genes after heavy inoculation be important cytological and molecular markers respectively for early determination of the host response to M. grisea in the rice blast system.




RacGEFs Function as Activators of Small GTPase OsRac1 in Innate Immunity of Rice


Tsutomu Kawasaki, Keiko Imai, Hann Ling Wong, Yoji Kawano, Keita Nishide, Jun Okuda and Ko Shimamoto


Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma, Japan.

E-mail: kawasaki@bs.naist.jp


We have demonstrated that the small GTPase OsRac1 is a key regulator for induction of immune responses in rice. Activation of OsRac1 induces NADPH oxidase-mediated ROS production, PR gene expression, production of antimicrobial compounds, and lignification. Transgenic rice plants expressing constitutively active mutant of OsRac1 showed enhanced resistance of virulent races of Magnaporthe grisea and Xanthomonas oryzae, whereas hypersensitive response induced by avirulent M. grisea is suppressed by dominant-negative form of OsRac1. These results indicate that OsRac1 plays important roles in R gene-mediated resistance and basal resistance. In addition, OsRac1 was found to interact with important immune components including NBS-LRR, RAR1 and HSP90. However, how OsRac1 is activated during immune responses remains to be identified.

Recently, a new type of GDP-GTP exchange factor (GEF) for Rac/Rop has been found in plants, termed PRONE-type GEF. We found twelve PRONE-type RacGEFs in rice by data base search. All of them are constitutively expressed in most of rice tissues, suggesting that these RacGEFs may be activated at the post-translational level. To identify the RacGEFs that activate OsRac1, we purified all of the recombinant RacGEF proteins and examined the in vitro GEF activities against OsRac1. Four of them exhibited strong GEF activities for OsRac1, indicating that these RacGEF genes are possible candidates that regulate activation of OsRac1 in immune responses. Recently, the PRONE-type GEF was found to interact with LRR-type protein kinase similar to PAMPs receptors such as FLS2 and EFR. Therefore, the RacGEFs may regulate both PAMPs- and R gene-mediated disease resistances through activation of OsRac1 in rice.




Identification and Application of a Novel Bi-directional Promoter that Drives High Level Expression

of Genes in Monocot and Dicot Plants


Wensheng Zhao, Junhua Liu, Kezhen Yang, De Ye and You-Liang Peng


The State Key Laboratory for Agrobiotechnology, The MOA Key Laboratory for Molecular Plant Pathology and Department of Plant Pathology,

China Agricultural University.


In order to isolate a pathogen-inducible bi-directional promoter for engineering plant disease resistance, the authors firstly carried out large-scale identification of genes that were induced in rice leaves by infection of Magnapothe oryzae. A cDNA library was constructed using mRNA from the pathogen-infected leaves. Out of about 30000 recombinants from the library, about 2000 clones were identified as the induced cDNA and are subjected to sequencing. Sequence analysis showed that the cDNA clones were originated from 1075 rice genes and that two genes, named OsSCI2 and OsSCI3 occupying 21 and 12 copies of the cDNA clones were linked head by head in the genome. Northern blot analysis confirmed that these two genes are highly induced in rice leaves during blast infection. The above results suggest that the intergenic region between OsSCI2 and OsSCI3 is putative a bi-directional promoter.

To test the hypothesis, a DNA segment with 1152 bp length upstream of the ATG codon of the two genes was fused with GUS gene in opposite directions respectively. The fusion constructs were introduced into rice plants by Agrobaterium-mediated transformation. Analyses on T1 transgenic plants showed that the DNA segment drove the expression of a GUS gene in both directions, but the expressions were constitutive in roots and induced in leaves. The induced expressions were very high, 3 times and 1.2 times, and 28 and 12 times as high as that of CaMV 35S or rice ACTIN1 promoter, respectively. Transgenic rice lines were also generated containing a construct in which the GUS and LUC genes were fused to each of the ends respectively. Northern blot analysis showed that all tested T1 transgenic plants expressed the GUS and LUC genes at a high level in the leaves in response to blast infection. These results proved that the intergenic region between OsSCI2 and OsSCI3 could drive simultaneous expression of two genes.

In addition, transgenic lines of Arabidopsis thaliana with above constructs were generated. Histochemical staining, GUS enzyme activity assays and RT-PCR analysis showed that the promoter drove constitutive expression of transgenes in both orientation in roots and leaves of Arabidopsis. The expression was as similar levels as that driven by the CaMV 35S. This demonstrated that the bi-directional promoter is applicable for engineering dicotyledonous plants.

A detailed report will be also given regarding the motif identification in the promoter.




The Rice Defensome Database: Centralizing and Unifying R-genes, Resistance QTL Mapping and Defense Gene Expression for Rice Blast


Elsa Ballini1, Emilie Vergne1, Gaétan Droc2, Brigitte Courtois2, Adam Price3, Jean-Loup Notteghem1, Didier Tharreau1, Jean-Benoît Morel1,*


1 BGPI, CIRAD-INRA-SupAgro, Campus International de Baillarguet, TA 41/K

2 DAP, CIRAD-INRA-UMII, Campus de Lavalette, TA 40/0334398 Montpellier Cedex 5, FRANCE

3 School of Biological Sciences, University of Aberdeen, Aberdeen, AB24 3UU, UK

*Corresponding author:E-mail: jbmorel@cirad.fr


Along years, the rice community has produced an incredible amount of information on the rice blast system: R-genes and resistance QTLs were mapped while there has been a recent increase in the information relative to gene expression upon infection. There is a need for integrating all these informations. Since 2002, the availability of the rice genome provides a new frame for integrating genetic and molecular information into a unified physical map. Some efforts were made in the past to anchor genetic information to the rice physical map (Wisser et al, 2005). We have extensively reviewed more than 60 publications on R-gene mapping, more than 20 published and unpublished studies on QTL mapping and more than 60 publications on gene expression in infected rice plants. This allowed the positioning of 85 R-genes, 343 QTLs and more than 5000 defence-related genes. The density of defence genes along chromosomes varies greatly, highlighting chromosome regions rich in defence genes. A metaQTL analysis was done to reduce the number of regions of the genome potentially useful for breeding blast resistance, leading to the identification of 160 blast resistance metaQTLs. Examination of physical distances indicated that more than 16 R-genes are found in intervals of less than 500 kb and that many QTLs represent very small intervals in which defence gene expression is found. This database was inserted into the OrygenesDB web site (Droc et al, 2007) and all associated tools. This database offers new opportunities for developing markers useful for marker-assisted selection and pinpoints some regions of the genome that could be targeted using candidate gene strategies.




Analysis of Calcium Signaling Proteins in the Genome of the Rice Blast Fungus Magnaporthe oryzae Using

an RNA Silencing Approach


Nguyen Bao Quoc, Naoki Kadotani, Yukio Tosa, Shigeyuki Mayama, Hitoshi Nakayashiki*


Laboratory of Plant Pathology, Kobe University, 657-8501 Kobe, Japan

*Corresponding author:E-mail: hnakaya@kobe-u.ac.jp


We developed an RNA silencing vector, pSilent-Dual1 (pSD1), with a convergent dual pol II promoter system. pSD1 has an advantage over hairpin RNA-expressing vectors as it shortens the cloning steps. Independent transcription from each promoter produces a pool of sense and anti-sense RNAs that combine together into long dsRNAs, resulting in siRNA generation by the action of Dicer in vivo. A study on the model gene, green fluorescent protein (GFP), indicated that pSD1-based vectors induced GFP silencing with a little lower efficiency than did hairpin GFP RNA-expressing vectors. To demonstrate the applicability of pSD1 for elucidating gene function in the rice blast fungus Magnaporthe oryzae, 37 calcium signaling-related genes in the genome were targeted for gene silencing by the vector. The 37 genes included 11 different protein classes; phospholipase C, calreticulin, calnexin, calpactin heavy chain, calmodulin (CAM), calcineurin (regulatory), calcineurin (catalytic), Ca2+ transporter, Ca2+ permeable channel, Ca2+/CAM-binding protein, and Ca2+ pump. The target gene in pSD1 was designed to be transcribed as a chimeric RNA with eGFP RNA so that we could screen the resulting transformants using GFP fluorescence as an indicator of gene silencing when the silencing vector was introduced into a GFP expressing parent strain. Northern analysis revealed that the level of gene silencing differed among screened transformants. Nevertheless, phenotypic analyses of the silenced transformants identified many calcium signaling proteins involved in growth, pigmentation, sporulation, appressorium formation, conidial morphology, and pathogenicity. In particular, 35 of the 37 genes analyzed were involved in sporulation, indicating that calcium signaling was a key regulator in the sporulation pathway of the fungus. The phenotype of the silencing mutants was mostly similar to one reported for mutants of the paralogous gene in yeasts or other fungi. This suggested the functional conservation of calcium signaling proteins across a wide range of fungi and yeasts.




Functional Analysis of a Sublitase Gene in

Magnaporthe Grisea


Yicong Wei, Guo-dong Lu *, Zonghua Wang


Fuctional genomic center, Fujain Agriculture & Forestry University

*Corresponding author: E-mail: guodonglu@yahoo.com


Secreted proteins, by virtue of their being presented on the outside of the fungus, are the most likely candidates for being elicitors or virulence factors. A high-throughput approach was established in our lab to identify and functionally analyze the secreted proteins in Magnaporthe grisea. MGG_07965.5 is a M. grisea annotation protein, which predicted to be a secreted protein by the computer algorithms. The protein is a serine proteinase like protein, which belongs to sublitase family. Twenty-four putative sublitases were found in the genome of M. grisea. The MGG_07965.5 was selected for functional analysis. The gene encodes the protein, names as MgSP3, was isolated by PCR amplification. The expression of MgSP3 was detected in the stage of mycelium, spore and in the infected rice tissues. The MgSP3 was over-expressed with C-terminal his-tag in the M. grisea under the RP27 promotor. The expressed fusion protein was purified from culture filtrate using NiNTA agarose. The proteins activity assay showed that the purified protein gave necrotic symptom in rice-detached leaves.

MgSP3 over-expressed mutants and knock-out mutants were constructed for further analysis. Comparing to wild type strain, the knock-out mutants showed a great decrease in aerial mycelium and sporulation, but with no difference in spores germination and appressorium formation. The over-expressed mutants showed obvious increase in aerial mycelium and sporulation but also with no difference in spores germination and appressorium formation. The pathogenicity tests showed that the over-expressed mutants decreased greatly and the knock-out mutants decreased lightly in rice infection. It is suggested that MGG_07965.5 is involved in aerial mycelium development, sporulation, and as well as pathogenicity.




Autophagy in Magnaporthe oryzae


Xiaohong Liu1, Jianping Lu2, Tongbao Liu2, Bo Dong1, Fu-Cheng Lin1,*

1 Biotechnology Institute, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, China

2 College of life Sciences, Zhejiang University, Yuhangtang Road 388, Hangzhou 310058, China

*Corresponding author: E-mail: fuchenglin@zju.edu.cn;


In a model fungus, Magnaporthe orzyae, well-known as the causal agent of rice blast, typically rupures the rice cuticle by using of a infection structure, an appressorium. Recently, we isolated MgATG1 gene (the orthologue of Saccharomyces cerevisiae ATG1), encoding a serine/threonine protein kinase, an autophagic gene, which is highly conserved among other eukaryotes, including humans and plants. Disruption of MgATG1 gene influenced the capability of surviving starvation, conidiation, conidial germination, lipid turnover, and appressorium turgor generation. As a result, the ∆mgatg1 mutant loses its penetration and pathogenicity into the host plants. Besides, MgATG2 (the orthologue of S. cerevisiae ATG2) encoding 2077 amino acids was isolated as well. MgATG2 restored the corresponding defects in the starvation-sensitive phenotype of Δatg2 mutant of S. cerevisiae. We got a MgATG2-deleted mutant in M. oryzae. The null mutant has similar phenotypes with Δmgatg1 including the abilities of conidiation, appressorium turgor, pathogenicity and sex reproductive.

Based on PCR, MgATG5 gene was also isolated. MgATG5 (GenBank No. EF486490) is encoding 314 amino acids. MgAtg5 has a conserved domain with Apg5 and is essential to autophagosome formation in M. oryzae. The MgATG5 and ATG5s from other organisms show very high homology. MgATG5 restored the corresponding defects in autophagy of Δatg5 mutant of S. cerevisiae. The MgATG5-deleted mutant was got by using targeted gene disruption strategy. The Δmgatg5 mutant shows defects in conidiation, conidia germination, appressorium formation, appressorium turgor, sex reproductive ability and pathogenicity. By reintroducing the MgATG5 to the Δmgatg5 mutant, all the defects were restored.

Similarly, MgATG18 was isolated based on PCR. MgATG18 gene encodes 469 amino acids. MgATG18 is the orthologue of S. cerevisiae ATG18. MgATG18 is phosphatidylinositol 3,5-bisphosphate-binding protein of the vacuolar membrane. By using targeted gene disruption strategy, a MgATG18 gene deleted mutant was obtained. The pathogenicity of Δmgatg18 didn’t lost completely. It is different to the null mutants of MgATG1, MgATG2 and MgATG5. Moreover, we also have isolated MgATG4 and MgATG9 and the gene functional analyses are undergoing now.

Taken together, clarification of the functions and network of these autophagic genes will lead to well understanding of the role of autophagy in this fungal pathogenesis.




Genetic Diversity for Rice Blast Management


Youyong Zhu, Yunyue Wang, Xiahong He, Chunming Lu, Shusheng Zhu,

Chengyun Li


Key Laboratory for Agricultural Biodiversity and Pest Management of China Education Ministry, Yunnan Agricultural University, Hei Longtan, Kunming,650201,China


Previous studies have showed that rice genetic heterogeneity provides blast disease suppression significantly. To understand the mechanism of that, 668 field experiments in 202 counties of 13 provinces in China from 1997 to 2006, resistance of rice cultivars, fungal lineages, microclimate of rice field were investigated by comparison between monoplanting and interplanting. The results suggested that dilution of host inoculum and microclimate variation for the susceptible type substantial contributor to blast management in imterplanting. At the same time, 3.6 million ha was extended in Yunnan, Sichuan and other provinces during 2000-2006. Averaged over years, interplanting of susceptible and resistant cultivars reduced the incidence and severity of panicle blast on susceptible cultivars by >86%, and on resistant cultivar by 30-50%. Interplanting increased the rice yield by 7 to 10%, and farmers’ income was increased as well.


This work is supported by National Basic Research Program of China (2006CB100203), Scientific Foundation of Education Ministry of China (307025).




World Population Structure and Migration of the Rice

Blast Fungus, Magnaporthe Oryzae


Didier Tharreau1,*, Isabelle Fudal2, Dodelys Andriantsimialona3, Santoso4, Dwinita Utami5, Elisabeth Fournier1, Marc-Henri Lebrun6, and Jean-Loup Notteghem1


1 UMR BGPI, CIRAD-INRA-SupAgro.M, Montpellier, France.

2 UMR BIOGER, INA.PG-INRA, Versailles, France

3 FOFIFA, URP SCRID, BP230, Antsirabe, Madagascar

4 Research Institute for Rice, Subang, Indonesia

5 Biology Molecular Division, RIFCB, Bogor, Indonesia

6 UMR5240, CNRS-UCB-INSA-Bayer CropScience, Lyon, France

*Corresponding author: E-mail: tharreau@cirad.fr


The diversity and structure of Magnaporthe oryzae populations was described in many countries during the last 20 years. The expected clonal structure of the populations has been illustrated in various studies. The relationship between the genetic structure, deduced from neutral molecular markers, and the pathotypic structure, showed a range of situations varying from a one lineage-one race to almost a one genotype-one race correspondence. These studies have helped in choosing appropriate isolates for genetic characterization and to propose strategies to breed for durable resistance to blast disease. This background information is the basis to understand rice blast population evolution. But, to date, our understanding of how new virulent races appear and spread is limited. For example, the relative importance of short and long distance migration in the spreading of new virulent races is unknown. However, this information is needed to determine at which scale the deployment of a resistance strategy is likely to be effective and durable.

We recently developed and used a set of 18 microsatellite markers for population studies (Adreit et al. 2007). Studies on populations from the Central part of Madagascar and from France show that populations can be differentiated at a local scale (10-20 km). These results suggest limited migration.

We also studied the distribution of the genotypes of the cloned avirulence gene ACE1 at the worldwide scale. We determined the ACE1 genotype of more than 800 isolates. Avirulent isolates were the most frequent, were sampled all over the world, and shared the same ACE1 allele. Two major virulent genotypes were identified. Their frequencies vary with geographic origin. These genotypes appeared by a complex duplication/deletion event of ACE1. These two genotypes are widely distributed over different continents. Altogether, these results suggest a unique selection event followed by long distance migration(s).

Our apparently contradictory results from studies at two different geographic scales are explained by two distinct modes of dissemination. Structuration at a local scale is consistent with short distance spore dispersal (1-5 m) observed during natural epidemics. Long distance migrations, including intercontinental, are possible through the transport of infected materials (probably seeds).




Examination of the Rice Blast Pathogen Population Diversity between 1990 and 2006 – Stable or Unstable?


Jim C. Correll*, E. J. Boza, E. Seyran, R. D. Cartwright, Y. Jia, and F. N. Lee


University of Arkansas, Fayetteville, Arkansas, 72701, USA and Dale Bumpers National Rice Research Center, Stuttgart, AR USA

*Corresponding author: E-mail: jcorrell@uark.edu


Over the past 17 years, isolates of Pyricularia grisea (= P. oryzae) have been recovered from commercial rice fields in Arkansas. Annual samples have typically included 200-500 isolates recovered from 5-10 cultivars from 10 different counties with the majority of the isolates being recovered from neck blast samples. All isolates were subsequently single-spored and stored desiccated on filter paper. Isolates of P. grisea were characterized using a number of tests including DNA fingerprinting with MGR586, mitochondrial DNA RFLPs, mating type, vegetative compatibility, and virluence. Although up to eight different MGR586 DNA fingerprint groups (A-H) have been identified among contemporary and archived isolates, only 4 MGR586 DNA fingerprint groups (groups A, B, C, and D) have been identified since we have been monitoring populations beginning in 1990. These four DNA fingerprint groups correspond with four distinct genetic groups, or vegetative compatibility groups (VCGs). In addition, some yearly samples have shown that a single haplotype often makes up the majority of the isolates within a given fingerprint group. For example, over 60% of the isolates recovered in a given season belonged to 1 of 4 distinct clones. Thus, it is evident that the rice blast pathogen population in Arkansas has remained stable over the past 17 years with regard to these 4 MGR586 DNA fingerprint groups. Although all 4 MGR586 groups can typically be found in the annual samples of the contemporary population, there appears to be a strong bias for group A isolates in recent samplings. Over 90% of the isolates recovered between 2000 and 2006 were in MGR586 group A, belonged to VCG US001, had a single mtDNA RFLP haplotype, and were a single mating type (mat1-1). The data indicate that the population is strongly influenced by host genotype. Evaluation of virulence indicates that isolates within a group are clearly more similar within a group than between groups; however, there is some virulence diversity within each of the genetic groups identified. A distinct shift in virulence among field isolates to overcome the Pi-ta resistance gene among MGR586 group B isolates is associated with changes in AVR-Pita.




Genetic Diversity of Rice Blast Pathogen, Pyricularia grisea in Karnataka State, India


Meena.B.S, S.K. Prashanthi.S.K*, Srikant Kulkarni, Y.R.Hegde and S.D.Sagar


Department of Plant Pathology University of Agricultural Sciences, Dharwad -580007. Karnataka, India

*Corresponding author:E-mail: prasamhi@rediffmail.com

Rice is the staple food of seventy per cent of the Indian population and being cultivated under varying agroclimatic conditions in India. Blast disease is the major threat to rice production under both rainfed and transplanted situations of Karnataka state in India. Understanding the organization and distribution of the blast pathogen population in specific geographic location helps us to employ the R-gene/s specific to that region.

In the present study genetic polymorphism of Pyricularia grisea isolates on different rice varieties grown in various agro regions of Karnataka state was analysed during 2005-06 using random Amplified polymorphic DNA marker (RAPD). OPB and OPF series primers were used to determine the genetic diversity and to construct the dendrogram. OPB2, OPB4, OPB7, OPB9, OPB10, OPB12, OPB14 and OPF3 primers showed cent per cent polymorphism. The isolates exhibited overall polymorphism of about 87.8 per cent. Total 135 bands were obtained of which 122 were polymorphic and 13 were monomorphic. The genetic similarity coefficient values (SM) for the isolates ranged from 0.34 to 0.86. UPGMA analysis of total data set of RAPD marker classified the twelve isolates into five DNA finger print groups. The first group consisted of only two isolates with 57 per cent genetic similarity. The second group comprised five isolates collected from same as well as different agro regions. This indicates the existence of local and geographical polymorphism in P. grisea. The varieties with diversified genetic background exert enormous selection pressure on the pathogen’s variability. Molecular variability observed in our study may be attributed to distinct geographic regions and host genetic diversity.




Studies on the Complementary Identification Varieties and Differentiation of Local Physiologic Races of

Magnaporthe grisea in Sichuan Province


Hongli Ji1, Yunjia Xiang1, Zhenyu Zhang1, Huamin Liao2, Linmin Luo2, Zhen Qin2, Yunliang Peng1


1 Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Jingjusilu Road 20, Chengdu 610066, China

2 Plant Protection Station under Sichuan Provincial Ministry of Agriculture, Wuhouci Street 4, Chengdu 610041, China


Since the popularization of hybrid rice in Sichuan Province in the late 1970s, it has witnessed three surges of rice blast disease causing severe yield losses due to the braking down of the resistance in the prevalent hybrid rice combinations respectively during 1983-1985, 1989-1995 and 2002-2005. But the changes in the composition of physiologic races differentiated by Chinese national identification varieties failed to provide clues for the boo-bust history of rice blast disease in the province. So Lijiang Xintuan Heigu (LTH), IR24, Minhui 63 (MH63), Duohui No 1 (DH1), Neihui 99-14 (99-14), Chenghui 448 (CH448) and RHR-1 were used as complementary identification varieties to differentiate 243 isolates of Magnaporthe grisea from Sichuan Province into 41vilulent types in 2005-2006. The annual occuring frequencies of 18 virulent types were higher than 2.0% and wre respectively named as local physiologic races CB0 and CB1-CB17. The prevaling Chinese national identification varieties differentiated 128 isolates into 19 physiologic races in ZA ZBZCZE groups, which was only half of the the number virulent types by the local complementary identification varieties. There were no distinct links between the races differentiated by two sets of identifiaction varieties. Base on the the links of virulence between different local races and the boomand-bust of hybrid rice in Sichuan Province, the evolution of virulence of M. griesea population in Sichuan were brifely decribed: evolved from CB0, the other local races acquired the virulence to IR24, MH63, DH1, CH448 or (/and) 99-14 which were successively as the major resistant donor of hybrid rice prevalent in Sichuan Province. RHR-1, a resistant source variety used now in breeding programs could be infected by several local races including CB17 which was virulent to all of the seven complementary identification varieties. While a part of the local race qcured virulence to MH63 bypassing that to IR24 the the races to 4 other lately introduced reistant lines were all evolved from strains pathogenic to MH63. Similar to IR24, the virulence to DH1, CH448, 99-14 could be bypassed during virulent evolution.CB2 and CB3 were respectively the most frequently detected local races in 2005 and 2006.The total frequencies of CB5, CB14, CB16 and CB17which were respectivley virulent to 90, 89, 80 and 87 out of the 101 tested hybrid rice combination prevailing in Sichuan Province were was 20.3% in 2006, indicating possible turn for the worse of the blast disease in Sichuan when weather condition is suitable for the epidemic. The vurluent frequencies of each local race to Lu You 502 is lower than 33.3%.




What It Takes to Achieve Durable Resistance to Blast?


Hei Leung


Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Manila, Philippines

E-mail: h.leung@cgiar.org


Achieving stable resistance to blast is perhaps the most important goal in managing blast disease. Over the past two decades, a shift from an over-reliance on major R-genes to selection for polygenic quantitative resistance has shown success in some breeding programs. In recent years, however, we have seen an increased severity of blast in Indonesia, Vietnam, and the Philippines. This suggests either an erosion of resistance due to pathogen evolution or a lessening of screening efforts in breeding programs, or both. There is an urgency to maintain the stability of blast resistance in these production systems, and to intensify efforts to understand the genetic mechanisms underpinning durable resistance. Here, I wish to briefly assess the situation of applying quantitative and qualitative resistance in breeding, and then discuss our attempts to define the genetic basis of quantitative resistance through analysis of germplasm and gain/loss-of-resistance mutants.

Germplasm analysis: We have analyzed two varieties--Shan-Huang-Zhan No.2 from south China and Moroborekan from Africa with reputation for exhibiting durable?blast resistance. QTL analysis using advanced backcross populations suggested that relatively few chromosomal regions (5-10) can confer adequate resistance (Wu et al. 2004; Liu et al. 2004; Y. Liu et al., unpublished data), indicating that it is feasible to reconstitute the level of resistance as seen in the donor parents. Some candidate defense genes, notably members of the gene families of oxalate oxidase (OsOXO) and oxalate oxidase-like proteins (OsOXLP) are associated with blast resistance. Contribution to blast resistance can be demonstrated by gene silencing of selected defense genes (R. Davidson and J. Leach, unpublished). Resistance transcriptome analysis suggests that aggregated and correlated expression patterns within defined chromosomal regions may contribute to resistance (R. Mauleon, K. Satoh, S. Kikuchi et al., unpublished). This raises the interesting possibility that coordinated expression of groups of adjacent genes may function as complex QTL?

Mutational analysis: IR64 is a popular variety with a good level of blast resistance, presumably due to the presence of multiple R-genes and quantitative resistance. To identify the genomic regions with effects on blast resistance, we screened for loss-of-resistance in 10,000 IR64 mutants under field conditions. So far, over 30 susceptible mutants (0.3%) have been recovered. Genomic deletions in some of these mutants were detected through hybridization with whole-genome oligoarrays (M. Bruce, G. Diaz, J. Leach et al., unpublished data). Systematically analysis of such deletion lines may reveal all chromosomal regions important for blast resistance in IR64.

So what it takes to achieve durable resistance? First, we need to know what genes/chromosomal regions have phenotypic effects in a range of germplasm with records of good blast resistance. This will tell us the consensus regions important for blast resistance. Combining QTL analysis of durable resistance?germplasm with mutagenesis can help pinpoint regions or genes responsible for quantitative resistance. Second, we need multiple screening sites to validate the effectiveness of the reconstituted resistance? Third, we need integration of mapping/mutant datasets and collaboration among breeders, pathologists and geneticists across institutions. This would be a worthy objective for implementation by an international working group.




Breeding Rice Cultivars with Durable Blast

Resistance in Colombia


Fernando Correa Victoria* and Cesar Martinez


Centro Internacional de Agricultura Tropical, CIAT AA 6713 Cali, Colombia

* Corresponding author E-mail: f.correa@cgiar.org


Rice blast disease (Pyricularia grisea), is the most important rice production constraint in Latin America. One strategy to improve the durability of blast resistance is to pyramid resistance genes. To do this we have, conducted extensive studies on the genetic structure of blast pathogen populations in Colombia and Latin America; determined composition, distribution and frequency of the avirulences that underlie race variation; identified and incorporated resistance gene combinations into commercial rice cultivars using genetic markers; and continuously evaluated and selected breeding lines under high disease pressure and pathogen diversity. These studies have allowed the grouping of the pathogen in just a few genetic families, each conserving specific avirulence genes despite the great pathogenic variability observed. Rice differentials with known blast resistance genes have been used to study avirulence gene composition and frequency in the blast pathogen and to identify relevant resistance genes. The combination of blast resistance genes (Pi-1, Pi-2, Pi-33) interacting with conserved avirulence genes present in the most predominant genetic lineages of the pathogen has proven to confer stable blast resistance after several years of testing under high blast pressure in the field and greenhouse inoculations. Near-isogenic lines segregating for these three blast resistance genes were used to demonstrate the simple inheritance of the genes and to identify candidate microsatellite markers linked to them from public database sources. Several of these markers were found to cosegregate with the blast resistance genes Pi-1, Pi-2, and Pi-33 on rice chromosomes 11, 6, and 8, respectively. These markers are polymorphic among different commercial rice cultivars from Latin America, which are blast susceptible, appearing to be ideally suited for marker assisted selection for improving their blast resistance. Additional pathogen characterization of spontaneous mutations of the blast pathogen using rice blast differentials allowed the identification of the blast resistance genes Pi-b (chromosome 2), Pi-9 (chromosome 6), and Pi-ta2 (chromosome 12), which will be needed for protecting rice cultivars from potential future changes in the avirulence/virulence genes in the blast pathogen population. Microsatellite markers highly linked to these additional blast resistance genes were also found from the public database. These markers are facilitating the introgression and pyramiding of each of these six blast resistance genes into Latin American rice cultivars and elite lines. Our studies will be discussed considering the potential benefits of pyramiding the blast resistance genes found, in rice breeding programs aiming at developing rice cultivars with durable blast resistance.




Defense Response Genes Contributing to Race Non-specific Resistance to Blast in Rainfed Upland Rice


Mukund Variar 1, C M Vera Cruz2, G Carrillo2, J C Bhatt3 and R B S Sangar4

1 Central Rainfed Upland Rice Research Station; Hazaribag 825 301;

2 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines;

3 VPKAS, Almora, Uttaranchal, India;4IGAU, Raipur, Chattisgarh, India


Resistance gene deployment for major biotic stresses is a primary objective in rainfed upland rice grown in Eastern India where endemic diseases like blast and brown spot are often enhanced by drought stress. Quantitative, race-nonspecific resistance against blast is desirable but it is difficult to accumulate quantitative resistance without the knowledge of the underlying genetic control and corresponding mechanisms involved in resistance expression. Recent work has shown that selection for candidate defense response genes (CGs) is effective in accumulating a high level of quantitative resistance. The level of resistance was proportional to the number of candidate gene alleles accumulated in advanced breeding lines and the resulting resistance was race non-specific and effective in multiple environments. We evaluated a population of rice lines containing none to six CGs (thaumatin, oxalate oxidase, oxalate oxidase-like proteins, chitinase, peroxidise, HSP90) in three blast endemic locations during 2004-2006 and compared their performance with the level of resistance in monogenic lines. The population was obtained by intermating advanced backcross derived lines of Vandana/Moroberekan (V4M-14-1-B with V4M -5-3-B, V4M -6-1-B and V4M -82-2-B). Disease progress curves in lines carrying five and six CGs were comparable to the monogenic lines carrying R genes Piz and Pi9 effective at all three locations. While the monogenic lines generally exhibited an ‘all or nothing effect’ with high or low disease, the introgressed population had a range of disease intensities that declined progressively with the addition of each CG. Some defense response (DR) genes individually conferred a higher level of resistance compared to others and hence resistance was not proportional to the number of CGs present in all cases. Nevertheless, significant reduction in leaf blast intensity with increasing CGs in the introgressed lines at different locations and years suggested that accumulation of CGs conferring different mechanisms of resistance may contribute to non-specific resistance effective in multiple environments.

Leaf and neck blast intensity was well correlated in the monogenic differentials at different locations and at Almora for the intermated population. The number of defense response genes and panicle blast severity was not well correlated. When exposed to brown spot, these lines exhibited varying degrees of disease irrespective of the number of CGs present in them indicating that DR genes could also be pathogen specific. Agronomic evaluation of lines with 4-6 CGs, however, led to selection of highly productive lines with resistance to leaf/neck blast and brown spot suitable for Eastern Indian uplands.




Characterization of Monogenic –lines to Korean Rice

Blast Fungus for Durable Resistance


Jae-Hwan Roh2, Byung-Ryun Kim2, Young-Chan Cho2, Dong- Soo Ra1,

Seong-Sook Han1*


1 National Institute of Agricultural Science and Technology, RDA, Suwon, 441-707, Korea

2 National Institute of Crop Science, RDA, Suwon, 441-857, Korea

* Corresponding author: E-mail : sshan@rda.go.kr; Tel: +82-31-290-0411


Rice blast, caused by Magnaporthe grisea, is one of the most economically important diseases of rice in various ecosystems such as in upland, lowland fields and irrigated ecosystems in temperate regions. Since use of the host resistance has been regarded as the most efficient way to control the disease, a systemic breeding program and improved techniques of screening methods are still needed. Present study was conducted to identify durable blast resistance in Korean rice cultivars by a simple greenhouse assay enforced with sequential planting. First planting of monogenic rice lines, harboring different major resistance gene for blast developed at International Rice Research Institute (from Dr. Fukuda of IRRI), Pi-a, Pik-s, Pk-h, Pi-ta, Pi-b, Pi-sh, Pi-7(t), Pi-z, Pi-i, Pi-3, Pi-5(t), and Pi-9(t), in a seedling box (57x30cm) was inoculated by sprayer with mixture of 27 blast isolates consisted of genetically different lineages (23 races).

There was a difference in time and quantity of susceptible response among cultivars. IRBL9-W harboring Pi-9 produced very small and few lesions in the 1st inoculation. The size and number of susceptible lesions increased as planting set increased from 2nd to 7th. At the 3rd plant set, the IRBL9-W already showed abundant big size susceptible lesions and disease severity became severe at the 5th planting time. The isogenic line harboring Pi-ta and Pi-b etc showed higher than 40% of DLA at the 1st planting time and continuously showed high incidence of disease. Some commercial cultivars or isogenic lines including Palgongbyeo, IRBL5-M(Pi-5(t)) and IRBL3-M(Pi-3) showed less than 40% DLA during the whole time until 7th planting. The sequential planting method in this experiment used enough number of compatible isolates under favorable condition for disease development, suggesting that this method might be useful to evaluate durability of blast resistance




Identification of Resistance Genes and Their Effect to Blast in Korean Rice Varieties (Oryza sativa L.)

Using DNA Markers


Yeon-Gyu Kim1*, Young-Chan Cho1,*, Soon-Wook Kwon1, Jung-Pil Suh2, Im-Soo Choi1, Sang-Kyu Lee3, Jong-Seong Jeon3, Jae-Hwan Roh1, Myung-Kyu Oh1, In-Bae Choi1, Sae-Jun Yang1, Young-Tae Lee1


1 National Institute of Crop Science, RDA, Korea;

2 IRRI-Korea Office, NICS, RDA, Korea;

3 Kyung-Hee Univ., Yongin, Korea

*Corresponding authors:Yeon-Gyu Kim: E-mail: ygkim55@rda.go.kr; Tel: +82-31-290-6650Young-Chan Cho: E-mail: yccho@rda.go.kr; Tel: +82-31-290-6666

The 11 major blast resistance (R) genes against Magnaporthe grisea were screened in a number of Korean rice varieties using DNA markers. Of the 98 rice varieties tested, 28 were found to contain the Pia gene originating from Japanese japonica rice genotypes, whereas 49 contained the Pib gene. We also found that 17 of the japonica varieties contain the Pii gene inherited from a Korean japonica Nongbaeg, and Japanese japonicas Hitomebore, Inabawase and Todorokiwase. The Pi5 gene, which clusters with Pii on chromosome 9, was identified only in Taebaeg. Thirty four varieties were found to contain alleles of resistance gene Pita inherited from the Japanese japonica genotypes, Fuji280, Sadominori and Shimokita. Seventeen japonica and one Tongil-type varieties contained Piz gene, which in the japonica varieties originates from Fukuhikari and 54BC-68. Piz-t gene contained in three Tongil-type varieties was derived from IRRI-bred indica rice varieties. Pi9(t) gene locus that is present in Korean japonica and Tongil-type varieties was not inherited from the original Pi9 gene from wild rice O. minuta. Pik-multiple alleles Pik, Pik-m and Pik-p were identified in 24 of 98 varieties tested. Pit gene inherited from the indica rice K59 line was not found in any of the Korean japonica or Tongil-type varieties tested. In haplotype analysis, Piz and Pita genes were stable effective to Korean blast isolates yearly across the regions tested




DNA Markers for Durable Resistance to Blast

Disease in Rice (Oryza Sativa )

Nguyen thi Lang, and Bui Chi Buu


CuuLong Delta Rice Research Institute Omon, CanTho, VietNam


Blast, caused by Pyricularia grisea Cav., is one of the major fungal diseases infected rice (Oryza sativa L.) in VietNam .This disease occurs world wide which cause the yield loss of up to 20% particularly in a year with long wet season. Local varieties have been considered as genetic sources of disease resistance among crops. The breeding program was aimed at improvement blast resistance in improve varieties. The five crosses OM 2514/ IR 64, IR 24/ OM 2514, C 53/ IR 64, C53/ OM 2514 and IR 36/ C53 plants were obtained. SSR marker with RM 483 was used to detect 100 local varieties to find gene Pi-33(t) in chromosome 6 and SSR marker with RM21 to find genes Pi-5(t) and Pi-3(t) in chromosome 4. Phenotypic selection was used to compare with genotype to check how accurate the methods were. Polymorphisms in varieties show that MAS reached an accuracy of 100% in SSR marker with RM 483. These methods are enough accurate to apply in practice to select varieties that have blast resistance genes for breeding rice. Nang Huong, Trang Phieu, Hai Hoanh, Than Nong Lun, Neo Vo Dua, Trang Tep, Nep Do, Mong Chim vang Nghe and Nep Chuot che resistance with race P(OM 1).Phenotypic selection was used to compare with genotype to check how accurate the methods were. Polymorphisms in varieties show that MAS reached an accuracy of 100% in STS marker with RG64 and 99.49% in SSR marker with RM21. These methods are enough accurate to apply in practice to select varieties that have blast resistance genes for breeding rice. Nang Huong, Lem Bui, Soi Da, Nang Tra, Nang Tra Ran Doc, Soc Nau, and Te Tep having 3 blast resistance genes Pi-2(t), Pi-5(t), and Pi-3(t), .These are considered as valuable material for pyramiding resistance genes to created durably resistant varieties.




Genetic Analysis of Bacterial Leaf Blight and Blast Disease Resistance in a Japanese Rice Cultivar, Asominori


Takashi Endo1,*, T. Nakamura1,, J. Yonemaru1, G. Ishikawa1, M. Yamaguchi1,

T. Kataoka1, K. Nakagomi1, and N. Yokogami2


1 National agricultural research center for Tohoku region

2 National agricultural research center for Hokkaido region Yotsuya, Daisen, Akita, JAPAN, 014-0102

*Corresponding author:E-mail: t_endo@affrc.go.jp


The Japanese rice cultivar Asominori shows a high resistance to bacterial leaf blight(BLB). The resistance locus is closely linked with the Ph gene, which is located on chromosome 4 and induces a brown color reaction on the grain surface when grain is soaked in phenol solution. Japanese rice breeders have noticed empirically that lines showing resistance not only to BLB but also to rice leaf blast (LB) segregated from descendants of crosses with Asominori. However, the cause of this co-segregation was unclear. The purpose of this study is to clarify the relationship between the two types of resistance and to develop a DNA marker tightly linked to the LB resistance gene.

In 2004, we obtained 5 backcrossed inbred lines of Asominori, Ukei851, Ukei852, Ukei854, Ukei856, and Ukei857, which all possessed a high resistance to both BLB and LB. First, to test whether BLB resistance (BLBR) and LB resistance (LBR) are controlled by independent loci, segregation of BLBR and LBR were monitored using 118 F3 lines obtained from a cross between Ukei854 and Hitomebore, a cultivar that is a susceptible cultivar to both BLB and LB. Most of the resulting F3 lines showing BLBR also carried LBR but several lines showed only BLBR or BLR. This indicated that BLBR and LBR were controlled by very closely linked loci. Secondly, we used Asominori and the 5 backcrossed inbred lines to conduct a genotype analysis of the long arm of chromosome 4, where BLBR and ph genes are located, using 20 PCR-based DNA markers. We found that all 5 inbred lines carried a common chromosomal segment derived from Asominori. From the positions of the markers used, the segment size was estimated at 1388 kbp. In addition, we surveyed the genotypes of 118 F3 lines using the marker, RM5473-2, which is located on the common segment. Most of the F3 lines with a high LBR showed the same genotype as Asominori and the 5 inbred lines.

Consequently, in this study, it was clarified that the LBR locus detected in Asominori was tightly linked to the BLBR locus on the long arm of chromosome 4, and the LBR gene was expected to be located near the position of the marker RM5473-2. In further studies, we will identify the position of the LBR gene and develop a practical perfect marker to select LBR lines carrying the LBR gene.




Flood-Induced Resistance Expressed when the Pi-ta R-gene is Compromised by Magnaporthe grisea


Fleet N. Lee


Rice Research and Extension Center, University of Arkansas
2900 Hwy 130 E Stuttgart, AR 72160 USA


Research data will be presented to show the efficacy of flood-induced resistance to rice blast when the Pi-ta R-gene is compromised by Magnaporthe grisea Cav. The Pi-ta R-gene conferred complete resistance to US blast races when first introduced during 1989 in the Arkansas rice cultivar Katy.

Previously unidentified Pi-ta virulent races IB-33 and IE-1k were subsequently detected in laboratory tests. During the years 1989 through 2003, only race IE-1k was detected in a few random plants from assays of commercial production fields and test plots. In 2004 and again in 2005, however, race IE-1k severely damaged small drought stressed areas of the newly released cultivar Banks which contained Pi-ta. In 2006 and 2007, race IE-1k incidence and severity increased significantly and limited rough rice yield in drought-stressed Banks fields.

In replicated greenhouse tests, plants grown in either continuous upland (dry) or flood conditions were inoculated with a blast spore suspension and visually evaluated using a zero to nine blast severity index. Test isolates used were a Pi-ta-avirulent race IB-49, Pi-ta-virulent races IE-1k and IB-33 or Pi-ta-virulent isolate MSG-19. When inoculated with race IB-49 or IE-1k, continuously flooded plants of susceptible cultivars Cypress, Mars, Wells, Newbonnet, LaGrue and M-201 had a lower blast index than comparable plants grown in upland conditions. Similar responses were observed when the Arkansas cultivars Katy, Kaybonnet, Drew, and Ahrent which contain Pi-ta were inoculated with Pi-ta-virulent isolates IE-1k, IB-33 or MGS-19 and when international cultivars Tetep (donor parent for Arkansas cultivars containing Pi-ta), CICA 9, and Tadukan which contain Pi-ta were inoculated with race IB-33 or isolate MSG-19.

Results define flood-induced field resistance to be an effective control measure when the blast fungus has compromised R-gene resistance. Although inherent susceptibility is important, to date all cultivars inoculated with a virulent blast race have exhibited some degree of flood-induced resistance.

Research and field observations show flood-induced blast resistance to be cumulative with duration and depth of flood and, in certain cultivars, comparable to R-genes. Currently, Arkansas growers use flood-induced resistance as a primary blast control measure to produce record rough rice yields in high-yield blast-susceptible cultivars lacking R-genes.




Development of Mass Screening Method for Rice Panicle Blast in the Greenhouse


Jae-Hwan Roh1, In-Seok Oh1, Seong-Sook Han2, and Byung-Ryun Kim3


1National Institute of Crop Science, Suwon, 441-857, Korea

2 National Institute of Agricutural Sci. & Tech., Suwon, 441-857, Korea

3 Chungnam Agricultural Research and Extension Services, Yesan, Chungnam 340-861, Korea


Rice blast disease caused by the fungal pathogen Margnaporthe grisea is one of the most serious and widespread disease of rice worldwide. Through the screening the resistant of leaf blast in rice breeding lines, most of rice cultivars were bred as a resistant rice cultivar against rice blast. But panicle blast is more dangerous than leaf blast in rice production. Up to now, nursery test, seedling test, greenhouse test, and polycyclic test were only used for screening the resistant against rice leaf blast but field screening was only used for screening the resistant both of leaf and panicle blast. Screening for panicle blast in the greenhouse is not established using different ecotype rice cultivars. In this study, a mass screening method for panicle blast in greenhouse condition was developed. Optimum inoculation time and sowing time in different ecotype rice cultivars to synchronize the heading time were established. Through this screening method, resistant characteristics of panicle blast in rice cultivars were classified into resistance or susceptible effectively and it will be seriously aimed to develop resistant rice cultivars to rice blast diseases.




Studies on Broad-spectrum Durable Resistance of a New Blast Resistance Gene by Sequential Planting Method


Jung-Pil Suh1,2,*, Ji-Ung Jeung2, Jae-Hwan Roh2, Young-Chan Cho2, Kshirod K. Jena 1,2


1 Plant Breeding Genetics and Biotechnology Division, International Rice Research Institute, Los Banos, Laguna, Philippines

2 National Institute of Crop Science, RDA, Suwon 441-857, Republic of Korea

* Corresponding author:E-mail : suhjp@rda.go.kr


Rice blast caused by the fungus, Magnaporthe grisea is a serious disease of japonica rice. Durable resistance to multiple rice blast pathogen population is an important goal for japonica rice breeding program. A new source of blast resistance to blast isolates of Korea was identified in an indica introgression line, IR65482-4-136-2-2 that has inherited the resistance gene from an EE genome wild Oryza species, O. australiensis (Acc. 100882). The gene has been fine mapped in the 70kb chromosomal regions flanking the resistance gene and has shown resistance to a number of virulent blast isolates of Korea as well as Philippines suggesting broad-spectrum of resistance. This major dominant gene designated as Pi40(t) has been located on the short arm of chromosome 6. PCR marker, 9871.T7E2b was completely linked with the blast resistance gene, Pi40(t) and used as a selective DNA marker in the backcrossing. Four introgression lines (BC3F5) carrying the Pi40(t) gene were selected to study durability of blast resistance in the gene, Pi40 along with other known blast resistance genes, Pita2, Pizt, Pi9, Piz5, Piz, Pi5, Pii, Pit, and Pi1. Using a sequential planting method (SPM), we characterized blast resistance of the four introgression lines expressing strong resistance to Korean blast isolates. Our results suggest broad-spectrum resistance of the Pi40 gene to a diverse blast pathogen population of Korea. However, the resistance genes, Piz5, Pi9, Pi5 expressed different degrees of resistance and susceptibility to Korean blast isolates. The four introgression lines with Pi40(t) gene showed resistance reaction in SPM as well as against most isolates in Korea and Philippines. Our results from the SPM study were significantly similar to the farmer’s field data and suggest that the Pi40(t) gene may have a broad-spectrum durable blast resistance. Information of the backcrossed lines carrying the Pi40(t) gene on bioassay for blast resistance and gene-marker association is useful for pyramiding effective gene combinations for durable blast resistance in rice.




Bacterial Determinants and Plant Defense Mechanisms Underpinning Rhizobacteria-mediated Systemic

Resistance in Rice


David De Vleesschauwer* and Monica Höfte


Lab of Phytopathology, Faculty of Bioscience Engineering, Ghent University,

Coupure Links 653, 9000 Ghent, Belgium

*Corresponding author: E-mail: david.devleesschauwer@ugent.be


Selected strains of benign plant growth-promoting rhizobacteria (PGPR) are capable of mounting a type of induced resistance that is commonly denoted as Induced Systemic Resistance (ISR). Compared to the relative wealth of information available in dicotyledoneous plant species, little is known about ISR in economically important cereal crops such as rice. In this work, we assessed several PGPR strains, which have previously been shown to trigger ISR in dicots, for eliciting resistance against the rice blast fungus Magnaporthe oryzae.

Whereas Pseudomonas aeruginosa 7NSK2 elicits ISR in dicots through a synergistic interaction of the siderophore pyochelin and the phenazine antibiotic pyocyanin, only mutations interfering with pyocyanin production led to a significant reduction in ISR to M. oryzae. In trans complementation for pyocyanin production restored the ability to mount ISR. Furthermore, application of purified pyocyanin mimicked 7NSK2-triggered ISR, indicating that redox-active pyocyanin plays a pivotal role in 7NSK2-induced ISR. Low-level systemic generation of pyocyanin-induced reiterative H2O2-microbursts, leading to expression of hypersensitive response-like cell death upon pathogen challenge, most likely constitutes the in situ mechanism of 7NSK2-mediated ISR because the resistance could be counteracted by exogenously added H2O2-quenching ascorbate.

Extensive bacterial mutant analysis identified the iron-chelating fluorescent siderophore pseudobactin (also called pyoverdine) as a crucial determinant of Pseudomonas fluorescens WCS374r-elicited ISR. Root application of pure pseudobactin was shown to trigger a pronounced multifaceted cellular defense response comprising a localized oxidative burst, accumulation of phenolics and accelerated deposition of callose at appressorial interaction sites, cell wall fortifications, protein cross-linking and the expression of infection peg-embedding tubules. Intriguingly, pseudobactin from P. aeruginosa 7NSK2 did not trigger ISR. Several lines of evidence suggest that the ISR-triggering potential of pseudobactin from P. fluorescens WCS374r is due to its capacity to deprive rice seedlings from iron. Currently, we are investigating the signaling pathways underlying ISR using several transgenic and mutant lines, which are impaired in various structural components of known signal transduction cascades.




Study on Genetic Architechture of Selected Rice Cultivars for Blast Resistance and its Componenets


Ali Moumeni


Rice Research Institute of Iran, Km5 Tehran Rd.,Rasht,Guilan 41996-13475 I.R.Iran

E-mail: alimoumeni@yahoo.com


Characterizing of genetic architechture of different traits inluding inheritance, gene action and partitioning of phenotypic variance to it’s components are one of the major issues in plant breeding. Hence, to study genetic architecture of resistance of rice to blast disease and other important quality and agronomic traits, five diverse rice genotypes viz. Neda, Dasht, Domsiah, SHZ2 and Ahlami-tarom has been selected and all cross combinations has been made among them (a half-diallel). All genotypes, 10 F1 together with their parents, totally 15, have been evaluated for components of resistance to blast againt 4 different blast races, quality characters, some agronomic traits, yields and its component in 3 different experiments with 3 replication for each in Rice Research Institute of Iran in Rasht and Amol during 2004-2006. Analysis of variance showed that there was significantly difference among all rice cultivars and their cross combinations for all traits. Combining Ability analysis based on Method II and Model Mixed B (Grffing 1956) revealed that mean of squares for GCA and SCA were significantly difference, hence both additive and non-additive components of genetic variance were important in controlling of traits. For most of traits additive components was predominant but for yield, number of tiller and gel temperature non-additive genetic variance was predominant. Cultivars Neda, Dasht and SHZ2 were good combiner for increasing resistance to blast disease, yield and its componens. Most of combinations showed high significant sca for components of resistance. Cross combinations including Domsiah/Neda, Neda/Ahlami-tarom and Dasht/Ahlami-tarom showed the best sca for yield and some of its component. Genetic analysis through Hayman (1954) also proved the results of Griffing’s method.


Key words: blast disease, grain quality, combining ability, gene action, rice




An Unprecedented Outbreak of Rice Blast on a Newly Released Cultivar BRS Colosso in Brazil


A S Prabhu *, Filippi M.C, Silva G.B, Silva-Lobo V.L.& Morais O P. Embrapa


Rice and Beans, Caixa Postal 179, CEP 75375-000, Santo Antonio de Goi.s, GO, Brazil, Fax (0xx62) 533-2172

*Corresponding author: E-mail: prabhu@cnpaf.embrapa.br


Rice blast occurred in epidemic proportions on a newly released upland rice cultivar BRS Colosso, in the rice growing season 2004/2005. Of 20 isolates collected from the affected panicles of cv BRS Colosso from three different States, two pathotypes IB-1 and IB-17 were identified. They were classified into thirteen Brazilian pathotypes based on reaction on eight upland rice cultivars, utilized as local differentials. Differences in aggressiveness of the isolates on cv BRS Colosso were evident. Ten highly aggressive isolates were used to determine the partial resistance in the cvs BRS Colosso and cv BRS Bonan. There was no significant isolate x cultivar interaction for partial resistance. The mean leaf blast severity was significantly higher in cv BRS Colosso than in cv BRS Bonan. Inoculation of culms with the same 10 isolates showed cultivar x isolate interaction. Some isolates were more aggressive resulting in severe panicle blast. There was no correlation among the aggressiveness of the isolates on leaf and panicle. The disease outbreak in cv BRS Colosso?could be attributed to the absence of adequate degree of partial resistance, the preexistence of compatible pathotypes IB-1 and IB-17 and their dissemination through seed.




Effect of Magnaporthe grisea on Seed Germination,

Yield and Quality of Wheat


Urashima A.S*, Grosso C.R.F, Stabili A, Freitas E.G, Silva C.P, NettoD.C.S, Franco I, M ola Bottan, J.H


Universidade Federal Sao Carlos,Via Anhanguera, km 174,Araras,Sao Paulo 13600-000,Brazil

*Corresponding author:E-mail: alfredo@cca.ufscar.br


The effect of wheat blast, caused by Magnaporthe grisea (Pyricularia grisea), on seed germination, yield loss and grain quality were evaluated in wheat cultivars BRS208 and CD104 under field conditions of Sao Paulo State, Brazil during the growing season of 2005. Each field was divided in an imaginary diagonal line crossing the center and then five areas of 1 m2 each separated 10 m were selected. Cultivars differed significantly in their reaction, the BRS208 proved to be more susceptible with incidence of 76.2%, higher severity and yield loss of 662.2 kg/ha or 32.2% whereas CD104 showed lower incidence (32%), severity and yield loss (399.1 kg/ha or 13.9%). Although a clear difference was detected between weight of 100 seeds from healthy-looking and blast diseased heads there was no difference in seed infection which ranged from 68.6 to 83.1% in these two cultivars. Furthermore there was no correlation between seed infection and germination in any cultivar. Seeds from blast diseased heads had higher proteins contents whereas lipids remained unaffected. Finally it is worth stressing that incidence of disease, yield loss and other parameters evaluated in this work were carried out in a commercial field with two applications of fungicides. The yield loss shown here proved a poor efficacy of chemicals and the importance of blast disease on wheat in Brazil.


Key words: Pyricularia grisea, blast, yield loss, incidence, germination, centesimal composition




NATIVO, A Broad Spectrum Rice Blasticide

Gilbert Labourdette and Jingquan Guo


Bayer CropScience AG, Agronomic Development Fungicide Lyon La Dargoire,France


Nativo WG75% - An innovative tool to control Pyricularia oryzae and other important rice disease pathogens in Asia.

Among the cultivated cereal species, Rice brings a large contribution to world’s food requirement. According to FAO, the world production 2006-2007 could slightly decrease by 0.2% (631Mt of Paddy rice against 632Mt in 2005-2006) caused by climatic problems but also insects and diseases. Production in Asia accounts for the 90% of the world total. In areas where this crop is grown intensively it is necessary to use appropriate technologies such as adapted varieties and improved crop management. The control of pathogens causing important yield losses, such as blast disease (Pyricularia oryzae), Sheath blight (Rhizoctonia oryzae) and panicle infection (a fungal complex including grain spot symptoms following Helminthosporium oryzae infection ) or destruction by false smut (Ustilaginoides virens), is an important tool to ensure yield quality and quantity. Bayer CropScience propose to the farmers a new product, Nativo 75WG applied in a range of 120 to 150g active substance/Ha, corresponding to 0,16Kg -0,2Kg commercial product/Ha, to control in an effective way, most of the rice diseases. This new fungicide is a balanced mixture of two active ingredients with different mode of action: the triazole Tebuconazole, an ergosterol synthesis inhibitor and Trifloxystrobin, a strobilurin of the new generation inhibiting electron transport across the mithochondrial membrane. The different behaviour of the two actives in the plant, combining penetration and redistribution, gives to Nativo 75WG the potential to control a broad spectrum of diseases at different stages of the evolution and an excellent flexibility of use from booting to maturation stage. In different trials carried out in asian countries between 2004 and 2006, Nativo 75WG, with 2 applications at panicle formation and panicle emergence gave an excellent protection, similar or superior to narrow and broad spectrum fungicides targeting Pyricularia oryzae and other diseases affecting yield quantity and harvest quality. An increased healthy greening effect contributes to keep a longer flag leaf efficiency without delaying maturation. In the development phase, a yield increase from 10% to 50% according to the main pathogen infection, was measured in a significant number of trials across asian countries. A better yield quality was also able to provide higher marketable harvest. The broad and balanced spectrum of activity of Tebuconazole and Trifloxystrobin against rice diseases combined with a good flexibility of use and physiological effect on rice plant, contributed to prevent yield losses and to improve the quality of harvest for a better income to the farmer.




Pathogenicity-related Compounds from Pyricularia grisea


Tetsu Tsurushima*1, Hitoshi Nakayashiki2, Yukio Tosa2, Shigeyuki Mayama2


1 Faculty of Business, Hannan University, Matsubara 580-8502, Japan

2 Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan

* Corresponding author: E-mail: tsuru@hannan-u.ac.jp


Several phytotoxic metabolites have been isolated from cultures of Pyricularia grisea. They are pyriculol-related compounds [pyriculol, epipyriculol, pyriculariol, pyricuol, 3-(1',3'-pentadienyl)-3,4- dihydro-1H-2-benzopyran-4,8-diol, and 4-(1'-hydroxy-2'-butenyl)-1,4-dihydro-2,3-benzodioxocin-10-ol], tenuazonic acid and pyrichalasin H. However, these toxins have not been compared their effects on their host plants. We undertook to reinvestigate whether they could be detected in cultures of Pyricularia isolates from various plants and discussed about their roles on pathogenicity of P. grisea. We searched the necrosis-inducing factor in cultures of P. grisea, because blast fungi produce necrotic lesions on their host. A Triticum isolate, Br48, was inoculated on petri dishes containing oat-meal agar plates cultures. After 10 days, their cultures were dipped in acetone. An acetone extract was evaporated to obtain an aqueous fraction, which was extracted with ethyl acetate. The extract was chromatographed on a silica gel column by stepwise gradient elution (chloroform-MeOH). The active fraction was analyzed with HPLC and fractions of two peaks showed the necrosis-inducing activity. They were identified as pyriculol and epipyriculol. We also examined their toxins in extracts from a Triticum isolate, an Avena isolate, two Setaria isolates, two Elusine isolates, two Digitaria isolates and two Panicum isolates. Their toxins were detected in extracts of all isolates. From this result, we discussed that they might be mainly necrosis-inducing principles by P. grisea. Recently, we found a novel toxin from cultures of an Avena isolate, Br 58. This toxin showed chlorosis-inducing activity on oat leaf segments in the light but not on segments in the dark. This was identified as 8-oxo-octadeca-9E, 12Z-dienoic acid, which was named Mag-toxin. Mag-toxin also induced ROS generation and cell death in oat cells. Interestingly, their inductions were light-independence.

We have also examined pyrichalasin H in cultures filtrates of 72 isolates of Pyricularia from 16 gramineous plants. Pyrichalasin H was only detected in culture filtrates of isolates to infect Digitaria plants. There is a correlation between pyrichalasin H production and the ability of Pyricularia isolates to infect Digitaria. Pretreatment of leaf sheaths of crabgrass with pyrichalasin H led to penetration and colonization by nonhost isolates. Thus, we propose that pyrichalasin H may be responsible for the specific pathogenicity of Pyricularia isolates on Digitaria genus.




A Blast Research Network for Stable Rice Production


Yoshimichi Fukuta1, Nobuya Kobayashi2, Casiana M. Vera Cruz2


1 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1, Ohwashi, Tsukuba, Ibaraki 305-8686, Japan

2 International Rice Research Institute (IRRI). DAPO Box 7777, Metro Manila, Philippines

E-mail: Fukuta: zen@jircas.affrc.go.jp; Kobayashi: n.kobayashi@cgiar.org; Vera Cruz: c.veracruz@cgiar.org


The Japan International Research Center for Agricultural Sciences (JIRCAS) and the International Rice Research Institute (IRRI) are organizing a collaborative study, begun in 2006, focusing on rice blast resistance. The aim is to develop and apply a universal differential system, designed to strengthen the sustainability of rice production systems against disease. A differential system is a basic tool for understanding host-pathogen interactions that consist of rice varieties, each ideally carrying a single gene for blast resistance, and blast isolates differing in their corresponding avirulence/virulence genes. They are classified based on the specificity of the reaction between the particular differential variety and differential blast isolates, and can be used to identify resistance gene(s) in the varieties and avirulence/virulence genes in the blast isolates. Seven institutes have so far joined this research project: the China National Rice Research Institute (CNRRI) and Institute of Crop Sciences (ICS) in China, the Cuu Long Delta Rice Research Institute (CLRRI) in Vietnam, the Philippines Rice Research Institute (PhiliRice) in the Philippines, the Indonesian Center for Rice Research (ICRR) and the National Nuclear Energy Agency, Center for Research and Development of Isotopes and Radiation Technology (CRDIRT) in Indonesia, and the National Institute of Agrobiological Sciences (NIAS) in Japan. The National Institute of Crop Science (NICS) in Korea is attending as an observer. Based on collaborations between pathologists and rice breeders, the following projects are planned: (1) diversity research into blast pathogens, (2) development of differential systems for blast resistance, and (3) diversity research and identification of novel resistance genes in each region and globally as part of a collaborative effort with all participating scientists. This networked research configuration should enable the discovery of the relationship and differentiation between blast races and resistance genes and lead to the development of a universal differential system. The characterization of novel resistance genes and differential isolates, development of a new designation system of blast race, and conservation of blast isolates are anticipated. Although this network research project as yet has few members and is targeting only East and Southeast Asian countries, it is an open collaboration. It is the first attempt to develop a universal differential system that can act as a common tool and language that links breeders, geneticists, and pathologists.




Situation of Blast Research in 14 Countries:

a Summary of Survey


Casiana M. Vera Cruz1, N. Kobayashi1, and Y. Fukuta2


1 International Rice Research Institute (IRRI), DAPO Box 777, Metro Manila, Philippin

2 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, 305-8686, Japan

E-mail: Vera Cruz: c.veracruz@cgiar.org; Kobayashi: n.kobayashi@cgiar.org; Fukuta: zen@jircas.affr.go.jp


A survey of blast research situation was conducted in 2006 in 14 countries to obtain a global view of blast damage in rice production environment and approaches used to solve the problem, the differential system used in many countries, and strategies proposed to attain durable resistance to blast. Blast was considered a major constraint in irrigated and rainfed lowlands, and upland ecosystem with yield losses ranging from10-70%. A few resistant varieties released in many countries were effective in 1-3 years only, thus farmers still rely on fungicides to control the disease. There is, however, limited information on the resistance of local varieties and effective resistance genes against contemporary pathogen population due to (1) lack of standard differential varieties useful in distinguishing the diverse pathogen population, thus each country use different varieties as differentials, (2) differential lines with similar resistance genes have inconsistent reactions, and (3) target resistance genes differ from one country to another depending on the local pathogen population. Sixty percent of the institutions have developed the differential system in their own country. Despite this, different countries have used a range of differential varieties/lines in their study. Several differential varieties for these resistance genes, Pi33, Piar, Pi3, Pi5, Pia, Pib, Pik-h (Tetep), Pi1 (C101 LAC), Pi2 (C101A51), Pita (C101 PKT), Pi9 (DM360), Pi20 (IR64), were evaluated in some institutions with well-selected blast isolates (n = 5-1,500). To attain durable resistance, researchers employ several approaches: (1) pyramiding resistance genes in advanced lines/susceptible commercial cultivars using markers associated with the most effective genes, (2) application of partial resistance, (3) identification of new genes, and (4) a combination of these approaches. This is complemented by studies on (1) genetic diversity and virulence/avirulence gene frequencies of blast populations, (2) development of NILs using popular upland and irrigated varieties, (3) identification of effective resistance genes against known avirulence genes, phenotyping elite lines against well-characterized isolates and conducting selections in multi-environment trials under high blast pressure. The lack of common differentials and a diverse blast isolate collection in some countries may slow down development of durable resistance to blast. Making these genetic resources freely available, and combined with advances in knowledge and tools in M. oryzae-rice pathosystem, 60% of the researchers/scientists acknowledged that a collective effort through collaboration among institutions is a means to address problems aimed at attaining broad-spectrum, durable resistance to blast.




Genetic Characterization of Universal Differential Varieties’ sets Developed Under the IRRI-Japan Collaborative Research Project


Donghe Xu1, Nobuya Kobayashi2, Mary Jeanie T. Yanoria2, Aris Hairmansis3, Yoshimichi Fukuta1

1 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1, Ohwashi, Tsukuba, Ibaraki 305-8686, Japan

2 International Rice Research Institute (IRRI). DAPO Box 7777, Metro Manila, Philippines

3 Indonesian Center for Rice Research. JL. Raya Muara No.25A Ciapus Bogor, West Java, Indonesia

E-mail: Fukuta: zen@jircas.affr.go.jp; Kobayashi: n.kobayashi@cgiar.org; Yanoria: m.yanoria@cgiar.org; Hairmansis: a.hairmansis@yahoo.com

The International Rice Research Institute (IRRI) and the Japan International Research Center for Agricultural Sciences (JIRCAS) have developed four kinds of differential sets of varieties: monogenic lines (MLs) with a Japonica-type variety Lijianxintuanheigu (LTH) genetic background; near isogenic lines (NILs) with three different genetic backgrounds, (LTH, Indica type variety CO39; and a universal susceptible line US-2) under the IRRI-Japan Collaborative Research Project. These target 24 kinds of resistance genes: Pia, Pib, Pii, Pik, Pik-h, Pik-m, Pik-p, Pik-s, Pish, Pita, Pita-2, Pit, Piz, Piz-5(Pi2), Piz-t, Pi1, Pi3, Pi5, Pi7, Pi9, Pi11(t), Pi12, Pi19 and Pi20, and the MLs have been distributed to more than 15 countries. To characterize these chromosome components and confirm the introgression of chromosome segments harboring resistance genes, the graphical genotypes were investigated using 162 simple sequence repeat (SSR) markers distributed on whole rice genome chromosomes. These chromosome components of the three sets’ NILs were more uniform than those of MLs when compared to the corresponding recurrent parent. Several introgression segments, which corresponded to the locations of blast resistance genes, were also confirmed. Almost all the MLs were developed by backcrossing one or two times, while some lines were backcrossed three to five times with the recurrent parent, LTH. The restoration of genomic chromosomes of 31 MLs to LTH ranged from 50 to 90.0%, averaging 77.3%. All LTH, CO39, and US-2 NILs were developed by backcrossing six times with each recurrent parent. The frequencies of genome restoration to the parent in 34 LTH NILs ranged from 75.6% to 96.9%, averaging 90.6%, lower than theoretical value of 99%. The 31 CO39 NILs showed greater than 90% genome restoration to the recurrent parent, averaging 97.3%. The 16 US-2 NILs were closely similar to those of the recurrent parent: these genome restorations ranged from 88.9% to 98.8%, averaging 94.6%. Four resistance genes, Pik, Pik-h, Pi1, and Pi7 in the LTH NILs confirmed introgression by co-segregation analysis with DNA markers of F3 family lines derived from the crosses between the LTH NILs and the recurrent parent. Genetic characterizations of four kinds of universal differential sets of varieties were carried out using DNA markers, and the introgression of several resistance genes was confirmed by co-segregation analyses using DNA markers. This information on DNA markers linked with resistance genes is potentially very useful for marker-assisted selection (MAS) in the breeding program, since the differential varieties can be applied as gene sources.




Identification of Blast Resistance Genes in IRRI-bred Rice Varieties by Segregation Analysis Based on a

Differential System


Nobuya Kobayashi1, Leodegario A. Ebron 1, Daisuke Fujita1, and Yoshimichi Fukuta2

1 International Rice Research Institute (IRRI), Los Baños, Philippines

2 Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Japan

E-mail: Kobayash: n.kobayashi@cgiar.org; Ebron: l.ebron@cgiar.org;

Fujita: d.fujita@cgiar.org; Fukuta: zen@jircas.affr.go.jp


A differential system for rice blast disease consists of standard blast isolates (Pyricularia grisea Sacc.) and differential rice varieties (Oryza sativa L.) with known resistance genes. Monogenic lines for blast resistance have been newly developed at the International Rice Research Institute (IRRI) as international differential varieties and differential blast isolates from the Philippines were selected. Using a differential system based on the gene-for-gene theory, blast resistance genes were estimated in the IRRI-bred elite rice varieties. At least seven kinds of resistance genes -- Pi20, Pita, Pik* (one of the Pik allele genes except Pik-s), Pik-s, Pib, Piz-t, and Pii or Pi3 -- were estimated in 42 rice varieties on the basis of reaction patterns to 14 standard blast isolates. These were compared with those of the blast monogenic lines. To confirm this gene estimation, genetic analysis was done using segregating populations derived from crosses between the IRRI varieties and a susceptible Indica-type variety, CO 39. The BC1F2 populations (with CO 39 as recurrent parent) were segregated for reaction to the specific standard isolates. Furthermore, the estimated genes were confirmed by allelism test against the blast resistance monogenic lines. As a result of the segregation analysis of 10 of 42 IRRI-bred varieties, seven genes -- Pi20, Pita, Pik*, Pia, Pib, Pik-s -- and Piz-t, were identified. Genes Pia, Pib, Pik-s, and Piz-t were not estimated by reaction patterns to the blast isolates but identified by genetic analysis in some varieties. The effectiveness of the differential system, which is based on conventional methods and which does not require advanced facilities, is discussed as a fundamental tool to provide essential information to develop breeding programs for blast resistance.




DNA Marker Analysis of Blast Resistance Genes Pib and Pita in IRRI-bred Rice Varieties Comparing with Gene Estimation by a Differential System


Daisuke Fujita1, Leodegario A. Ebron1, Nobuya Kobayashi1 and Yoshimichi Fukuta2


1 International Rice Research Institute (IRRI), Los Baños, Laguna, Philippines

2 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1, Ohwashi, Tsukuba, Ibaraki 305-8686, Japan

E-mail: Fujita: d.fujita@cgiar.org; Ebron: l.ebron@cgiar.org; Kobayashi: n.kobayashi@cgiar.org; Fukuta: zen@jircas.affrc.go.jp


The rice blast resistance genes are important in improvement programs of rice (Oryza sativa L.). The DNA markers linked to resistance genes are a powerful tool to detect the presence of genes and are widely used to select breeding materials through marker-assisted selection. This study was conducted to evaluate the detection ability of DNA markers for rice blast resistance genes, Pib and Pita, in IRRI-bred rice varieties. Forty-two Indica-type varieties, which have been previously analyzed for the presence of Pib and Pita by conventional genetic analysis using a differential system involving standard blast isolates (Pyricularia grisea Sacc) from the Philippines, were tested. To estimate the presence of Pib and Pita, PCR-based dominant markers previously reported by Wang et al. (1999) and Jia et al. (2002) were used. The DNA markers, Sub3-5 (Pib), YL153/YL154, YL155/YL87 and YL100/YL102 (Pita), have been developed on the basis of sequence information of Pib and Pita. The target DNA fragments of Pib using Sub3-5 were amplified in 40 varieties but not in 2 varieties. Also, the target DNA fragments of Pita using YL153/YL154 and YL155/YL87 were amplified in 28 varieties but not in 14 varieties and those using YL100/YL102 were amplified in 26 varieties but not in 16 varieties. The results of DNA marker analysis of 42 IRRI-bred rice varieties were compared with previous gene estimation of Pib and Pita by the differential system. In 6 varieties, detection of DNA fragments for Pib by marker analysis did not correlate with estimation by the differential system. On the other hand, detection of DNA fragments for Pita corresponded well with estimation by the differential system excluding 2 varieties. The use of DNA markers enabled the detection of Pita in most IRRI-bred rice varieties. These suggest that the efficiency of detecting blast resistance genes through use of DNA markers depend on the rice variety and the DNA markers. The proper markers for the Pita gene provide a basis for stacking other blast resistance genes into high-yielding and good-quality advanced breeding rice lines.




Proposal for a New International System of Differentiating Races of Pyricularia Oryzae Cavara by Using LTH Monogenic Lines


Nagao Hayashi1 and Yoshimichi Fukuta2


1 National Institute of Agrobiological Sciences (NIAS), 2-1-2 Kannondai, Tsukuba, 305-8602, Japan

2 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, 305-8686, Japan

E-mail: Hayashi: nhayash@affrc.go.jp; Fukuta: zen@jircas.affrc.go.jp


Each country tends to use its own system for differentiating rice blast fungus races. The races differentiated by each system cannot be compared with each other, because there are differences in the genetic backgrounds of the rice cultivars used for differentiation in each country. Recently, 29 LTH(Lijiangxintuanheigu) monogenic lines, each containing one of 23 resistance genes, were developed at IRRI. The development of these monogenic lines has allowed us to prepare a new international differential system (IDS). We examined the components of the lines and designated an order for their adoption by the IDS. This IDS based on monogenic lines is expected to promote the utilization of rice blast resistance genes. First, the response of each LTH monogenic line to more than 50 fungal isolates (including standard strains of blast and isolates from a different ecosystem in Japan) was recorded according to infection type (generally, 0–2 resistant; 3–5 susceptible). The monogenic lines that had the same reaction to all blast fungus were eliminated except one, as a result we chose 20 representative lines. Lines with the multiallelic loci Pii, Pik, Piz, and Pita were handled as a group. To delineate clearly the relationship between race code number and resistance gene, we divided the selected lines into groups with 3 lines, given the code numbers 1, 2, and 4 in accordance with Gilmour’s method. Each race code number has 9 digits divided by commas (i.e. NN,NNN,NNNN). In the IDS, the names of the LTH monogenic lines are ordered as follows in the race codes. The 1st digit of the race code number is composed of LTH, IRBLa-A, and IRBLsh-S. The 2nd digit is IRBLb-B and IRBLt-K59; the 3rd digit is IRBLi-F5, IRBL3-CP4, and IRBL5-M, which are multiallelic or closely linked; the 4th and 5th digits are IRBLks-S, IRBLk-Ka, IRBLkp-K60, IRBL7-M, IRBLkm-Ts or IRBL1-CL, and IRBLkh-K3, which are multiallelic or closely linked; the 6th and 7th digits are IRBLz-Fu, IRBLz5-CA, IRBLzt-T, and IRBL9-W, which are multiallelic or closely linked, and the 8th and 9th digits are IRBLta-K1, IRBLta2-Pi, IRBL20-IR24, and IRBL19, which are also multiallelic or closely linked. We plan to use more fungal isolates to verify this system.




Monitoring of Blast Races and Stable Use of Blast Resistance in Rice


Shinzo Koizumi


Lowland Crop Rotation Research Team, National Agricultural Research Center for Tohoku Region, Yotsuya, Daisen, Akira, 014-0102 Japan

E-mail: skoizumi@affrc.go.jp


The breakdown of complete resistance to blast in rice cultivars in the 1960s led to the conduct of gene analyses of blast resistance in Japan, and currently complete resistance genotypes and levels of partial resistance of most of Japanese rice cultivars are already known. Differential systems of blast races have also been established, and monitoring of blast races had been done all over the country in 1976, 1980, 1994 and 2001. The results of monitoring blast races had been used for analyses of virulence genes in the pathogen population (Kiyosawa, 1980; Kiyosawa, 1986), and proportions of components in three Japanese released multilines for rice blast control, which consist of three to four near-isogenic lines with different complete resistance genes, have been also based on the monitoring results. Moreover, the results accelerated development of rice cultivars with high levels of partial resistance to blast because of its stability. Monitoring of blast races in areas where complete resistance genotypes of rice cultivars planted are not sufficiently known, is also useful in the selection of effective complete resistance genes for blast control, and inoculations with the isolated races reveal promising rice cultivars for blast control and levels of partial resistance in some of them.

Although monitoring of blast races has been conducted for rice blast control in several countries, sufficient quantitative analyses of blast race epidemics, which are necessary for stable use of blast resistance, have not been carried out. This is due to the fact that monitoring of blast races is very laborious and reliable epidemiological data on population interaction between host and pathogen is lacking. For stable use of blast resistance in rice, it is therefore necessary to develop easy means of monitoring blast races like the use of DNA markers tightly linked to avirulence genes in the fungus; accumulating epidemiological data on population interaction between host and pathogen; and construction of reliable epidemiological models, which can simulate increase of blast races in host populations with different resistance.




Development of Differential System for Blast

Resistance in China


Cailin Lei1, Jianli Wu2, Zhongzhuan Ling1, Jiulin Wang1, Jieyun Zhuang2,

Jianmin Wan1


1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (ICS-CAAS), No. 12 Zhongguancun Nandajie Street, Beijing 100081

2 China National Rice Research Institute (CNRRI), No.359 Tiyuchang Road, Hangzhou 310006, China


To better determine pathotypes of blast pathogen and clarify the relationship between resistance genes in rice and avirulence genes in blast pathogen in China, we have been trying to develop universal differential varieties since late 1980s. Ling et al (1995) developed six near-isogenic lines (NILs) with the background of a universally susceptible variety Lijiangxintuanheigu (LTH). By inoculating the 6 NILs and 24 monogenic lines (MLs) with LTH background (kindly provided by Dr. Fukuta in 2002) using 290 isolates from 8 provinces, we selected out 12 lines with higher differential ability, i.e., IRBLi-F5, IRBLk-Ka, IRBLkp-K60, IRBLta-K1, IRBLb-B, IRBL3-CP4, IRBL7-M, IRBL12-M, F-124-1(Pita), F-128-1(Pita-2), F-129-1(Pik-p) and F-145-2 (Pib), as a new set of differentials (Ling et al., unpublished data). In order to improve the differentials’ set and construct more ideal one, we continue the pathotyping of Chinese blast isolates using the 6 NILs and 24 /31 MLs in the past several years, and have performed the pathotyping for nearly 800 blast isolates during the past several years. Based on their resistance reaction on 24 MLs, 738 isolates could be classified into 609 pathotypes, among which 296 indica-derived and 422 japonica-derived isolates belonged to 263 and 360 pathotypes, respectively. Based on their resistance reaction on 31 MLs, 233 japonica-derived isolates from Heilongjiang and Ningxia could be classified into 217 pathotypes. According to the unique reaction patterns and differential ability to the resistance genes, dozens of isolates were picked up as the candidates of representative ones in China, and their sporulating ability and stable reactions are under testing. The JIRCAS research project “Blast Research Network for Stable Rice Production” has officially initialized recently. Following the Contract Research Agreement between ICS-CAAS /CNRRI and IJIRCAS, this year we will focus on collection of blast isolates, characterization of the pathogenicities, and collection of land and improved rice lines for characterization of blast resistance. Our work plan from July 2007 to March, 2008 are as follows: (i) to collect about 200 blast isolates from 5 regions (northeast China, East China, South China and southwest China, about 50 each) this autumn; (ii) to characterize about 100 isolates using differential varieties /MLs, (iii) to collect 60-100 land and improved core lines for seed increase and use some candidate representative differential isolates for characterization of blast resistance later.




Pathogenicity of Rice Blast Isolate from some Irrigated Areas of Southern Vietnam


Pham Van Du, Le Cam Loan


Cuu Long Rice Research Institute (CLRRI), Co Do, Can Tho, Vietnam

E-mail: Du: phamvandu@hcm.vnn.vn; Loan: lecamloan1@yahoo.com


To develop differential system and to have good achievement in rice blast breeding program in Vietnam; collection, isolation, reservation and pathogenicity evaluation of blast isolates have been carried out. Two hundred twenty blast samples including 30 (from Long An province), 25 (Tien Giang), 15 (Ben Tre), 20 (Vinh Long), 30 (Dong Thap), 15 (Tra Vinh), 15 (Soc Trang), 5 (Can Tho),15 (Hau Giang), 7 (An Giang), 30 (Kien Giang), and 13 (Bac Lieu) were collected in irrigated areas of Southern Vietnam and 31 monogenic lines carrying 24 single blast resistance genes were used in virulence analysis. Preliminary results showed that IRBL7, IRBL25, IRBL18 and IRBL21 having the resistance genes Pik-p, Pik-m, Pi1 and Pi7t, respectively were resistant to most of tested isolates. Compatible reactions were determined on the monogenic lines IRBL1, IRBL2, IRBL4, IRBL11, IRBL12, IRBL13, IRBL14, IRBL15, IRBL23, IRBL24, IRBL27, IRBL28 and IRBL29 carrying the resistance genes Pia, Pia, Pik-s, Piz-t, Pita, Pita, Pib, Pit, Pi12t, Pi19t, Pita-2, Pita-2, and Pita, respectively. The monogenic lines were higher susceptible than their donors. The additional blast samples will be collected in upland areas in Vietnam and identification of blast resistance genes in Vietnamese local rice varieties will be conducted. This has been carrying out under a Japan International Research center for Agricultural Sciences (JIRCAS) research project, Blast Research Network for Stable Rice Production.




Distribution and Occurrence of Rice Blast Fungus, Pyricularia grisea in the Philippines


Fe dela Pena, Mary Ann Manalo, Loida Perez1, Edna Ardales2, Yoshimichi Fukuta3, Analiza Tagle4, Isabelita Ona4, Nobuya Kobayashi4, Casiana Vera Cruz4


1 Philippine Rice Research Institute (PhilRice), Maligaya, Mu oz 3119, Nueva Ecija, Philippines
2 University of the Philippines at Los Ba os (UPLB), College, Laguna, Philippines
3 Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki, Japan
4 International Rice Research Institute (IRRI), Los Ba os, Laguna, Philippines
E-mail: Fe:
delapenafe@yahoo.com; Moreno-Perez: lmperez@philrice.gov.ph; Fukuta: zen@jircas.affrc.go.jp; Kobayashi: n.kobayashi@cgiar.org; Vera Cruz: c.veracruz@cgiar.org
 
The Philippines has a total of 4,159,930ha palay area harvested in 2006 (BAS, 2006) which are also beset with disease problems. Blast caused by Pyricularia grisea, which is a destructive disease of rice worldwide is one of the major obstacles in rice farming in the Philippines particularly in water-stressed and in cool-elevated areas.  However, in recent years, it has also become popular in most irrigated areas planted with recently released varieties like IR64, PSBRc14, PSBRc82, NSICRc112 and NSICRc122 whose resistance has broken down due to increase in new blast races. With this growing problem on breakdown of resistance of varieties due to increase in new blast races, PhilRice is doing pathogenicity research on blast isolates collected from different rice growing areas in the Philippines and the reaction of IRRI monogenic lines to the different blast isolates is being determined. Analyses of 196 monoconidial isolates showed that there were 33 Pot2 and 27 URP5 haplotypes defined. Based on Nei’s genetic diversity index, there was high genetic diversity estimates of 0.94 (Pot2) and 0.89 (URP5) obtained for the entire population. Furthermore, the phylogenetic relationship among the isolates was determined by cluster analysis by the Unweighted Pair Group Method with Arithmetic Means (UPGMA) using the NTSYS statistical package. Using Pot2, 7 clusters were formed at 75% similarity level while using URP5 revealed that there were 10 major clusters at 76% similarity level. Majority of the isolates having the same haplotype was detected from more than one location and in most cases; isolates exhibiting particular haplotypes that clustered together were derived from different geographical locations indicating no correlation between haplotype and geographical location among the isolates. Moreover, the reaction of monogenic lines to the different rice blast isolates revealed significant differences in terms of virulence. Future activities will be on the collection and characterization of rice varieties for blast resistance, which will include DNA polymorphism of 100 varieties including landraces and improved rice cultivars using a panel of 50 SSRs selected for diversity analyses by SR McCouch et al. Furthermore, study on resistance genes through reaction to blast differential isolates will also be done. Hybrid populations will be developed to detect novel resistance genes that can be used in the improvement of blast resistance in Philippine rice breeding programs.
 



Development of Differential System for Rice Blast Pathogen and Identification of Novel Resistance Gene in Indonesia


Suwarno1, Sobrizal2, Santoso1, Anggiani1, Aris Hairmansis1 and Erwina Lubis1


1 Indonesian Center for Rice Research, Jalan Raya Sukamandi 9, Subang, West Java, 41256. Email: balitpamuara@telkom.net

2 Center for Research and Development of Isotopes and Radiation Technology, National Nuclear Energy Agency, Jalan Cinere Pasar Jumat PO BOX 7002, JKSKL, Jakarta 12070. Email: sobrizal@lycos.com


Collaborative research project between Indonesia, Japan International Research Center for Agricutural Sciences (JIRCAS), and International Rice Research Institute (IRRI) under “Blast Research Network for Stable Rice Production” have been started since 2006. The objectives of this project were to develop differential system for rice blast pathogen and to identify novel resistance genes from Indonesian germplasm. The project consisted of four major activities include: 1) collection of blast isolate from different ecosystem, 2) characterization of collected blast isolate, 3) characterization of resistance of rice varieties against Indonesian blast races, and 4) identification of novel resistance genes. This paper will describe progress results of the project. A total of 85 blast isolates have been collected from three different ecosystems comprised lowland, upland and swampy areas. The blast isolates from upland area were collected in Lampung, Indramayu, and Sukabumi. For lowland area, the isolates were collected in Lampung, Sukabumi, and Kuningan; while for swampy area in Kayu Agung and Karang Agung. Collections of blast isolates from other areas are being conducted. Twenty out of collected isolates have been characterized based on their reaction patterns to the monogenic lines of blast resistance and differential blast varieties of Indonesia. Characterization of blast resistance of rice varieties were comprised Indonesian germplasm and monogenic lines for blast resistance. A total of 22 rice varieties that used as blast resistant gene source in Indonesian breeding program were selected, and their reaction pattern to four Indonesian blast races have been characterized. Characterizations of other rice varieties are in progress. Blast resistances of monogenic lines also have been evaluated in the blast endemic area, Sukabumi. Ten monogenic lines, IRBLk-Ka, IRBLkp-K60, IRBL7-M, IRBLkm-Ts, IRBLkh-K3, IRBLz-Fu, IRBLz5-CA, IRBLz5-CA(R), IRBL1-CL and IRBL9-W, showed resistant reaction to blast disease in this area. To study the genetic of blast resistance, seven rice varieties with different blast resistance i.e. Padi Banten, Asahan, Genjah Arak, Mutu, Laka, IR54 and Nippon; have been crossed with US-2 line. The F1 populations of these crosses have been produced and segregation populations for genetic study are being developed. Genetic analysis is also being conducted on Laka/Kencana Bali cross. Reaction pattern of Laka and Kencana Bali to 12 blast races have been obtained. The seeds of 126 recombinant inbred lines (RIL’s) derived from Laka/Kencana Bali cross are being multiplied


Poster Presentations


1


Pathogen Molecular Biology, Signaling and Interaction with Host




Cell Biology of Biotrophic Invasion by the Rice

Blast Fungus Magnaporthe oryzae


Prasanna Kankanala 1, Kirk Czymmek 2, and Barbara Valent 1


1 Department of Plant Pathology, 4024 Throckmorton Plant Sciences Center, Kansas State University, Manhattan, KS 66506, USA;

2 Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA


Magnaporthe oryzae, the causal agent of rice blast disease, produces intracellular Invasive Hyphae (IH) that grow from cell to cell to colonize rice tissue. Many studies have focused on understanding the mechanisms by which the fungus penetrates the outer plant surface using its specialized appressorium. However, cellular and molecular details of biotrophic blast colonization strategies inside the plant remain largely unknown. We applied live-cell imaging to characterize spatial and temporal development of IH and plant responses inside successively-invaded rice cells (1). Early loading experiments with the endocytotic tracker dye, FM4-64, showed dynamic plant membranes around IH. The IH exhibited pseudohyphal growth and were sealed in plant membrane, termed the Extra-Invasive Hyphal Membrane (EIHM). The fungus spent up to 12 hours in the first-invaded cell, often tightly packing it with IH. IH that moved into neighboring cells were biotrophic, although they were initially thinner and grew more rapidly. IH in neighboring cells were wrapped in EIHM with distinct membrane caps at the hyphal tips. IH showed remarkable constrictions as they crossed the plant cell wall. Time-lapse imaging showed IH scanning plant cell walls before crossing them, and transmission electron microscopy showed crossing occurring at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Our studies provided insights into a novel hemibiotrophic strategy employed by the blast fungus. To further understand the molecular basis of this infection strategy we employed laser microdissection technology and identified several hundreds of infection-specific IH genes. This data will contribute to identification of fungal effectors that are secreted inside host cells to control normal plant cellular processes.


  1. Kankanala, P., Czymmek, K., and Valent, B. (2007). Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus. The Plant Cell 19: 706-724.




Separation of Virulent Proteins from Infiltration of Magnaporthe grisea by Using Rotofor


Yuan Su, Chengyun Li, Chuisi Kong, Jing Zhou, Jing Yang, Lin Liu, Jinbin Li,

Yunyue Wang, Youyong Zhu


Key Laboratory for Agricultural Biodiversity and Pest Management of China Education Ministry,Yunnan Agricultural University, Hei Longtan, Kunming, 650201,China . Email: jzsmkm@126.com


The Rotofor (Bio-Rad product) preparative isoelectric focusing apparatus was used for separation of secreted proteins in phytopathogenic fungus, Magnaporthe grisea. The total secreted proteins infiltrated from nitrogen starvation liquid medium from isolates Y99-63 and Y98-16 of M. grisea were lyophilized. After dialysis, 3% Bio-lytepH 3 - pH10was added. The infiltrated proteins were separated to 20 fractions using by Rotofor. All fractions were tested the virulence by wounded inoculation on Lijiangxintunheigu (LTH) leaves, a susceptible rice cultivar.

The results showed that there were 4 fractions in acidic proteins and 4 ones in basic proteins which could induced the necrosis on rice leaves from isolate Y 99-63. The acidic proteins had stronger virulence than basic proteins and induced similar lesion caused by total proteins. The most virulent fractions in acidic proteins were 7, 8, 9 and 10 (pH 5.8 - pH 6.9); The most virulent fractions in basic proteins were14, 15, 16 and 17 (pH 8.1 - pH 8.5).

Furthermore, there were 5 fractions in acidic proteins and 5 ones in basic proteins could induced the necrosis on rice leaves from isolate Y98-16. In contrast, The basic proteins had stronger virulence than acidic proteins in infiltration of Y98-16. The most virulent fractions in acidic proteins were 1, 3 and 9, 10, 12 (pH 2.0-pH 2.6 and pH 4.7- pH 6.0 respectively). The most virulent fractions in basic proteins were 15, 16, 17, 18 and 20 (pH 7.0 - pH 8.5; pH 9.0 respectively). Our results showed that Rotofor is a useful tool for separation and identification novel proteins primarily, and can provides the key parameters for the further purification procedure, such as ionic chromatography.


This work is supported by National Basic Research Program of China (2006CB100202), Scientific Foundation of Education Ministry of China (307025), and Doctorial Foundation of Education Ministry of China (20050676001).




Genitic Analysis on a Temperature Sensitive Locus Associated with Penetration Peg Formation in the

Rice Blast Fungus, Magnaporthe grisea


Bo Dong, Hong-Kai Wang, Fu-Cheng Lin, De-Bao Li


Biotechnology Institute, Zhejiang University, Hangzhou, China 310029


Magnaporthe grisea, the rice bllast fungus, can differentiate a penetration peg from deep of appressorium to penetrate the epidermis cells into host tissue,this structure play an essential role during the pathogenesis process. A temperature sensitive (TS) mutant mutagenesis by UV rediationin Magnaporthe grisea, named T-154, is defaulted in penetration peg formation at non-permissive temperature, 33℃. Genetic analysis using 60 progenies showed that the defective of penetration peg formation in T-154 is controlled by a single locus. RAPD analysis and SSR analysis were performed and a RAPD marker and a SSR marker were obtained which closely linked to the temperature sensitive locus. Sequence analysis results of RAPD marker showed that this locus was located at supercontig 196, Contig662, linkage group Ⅰin genome of Magnaporthe grisea. These results are useful for further studies on the TS gene related with penetration peg formation.temperature sensitive




Screening for Rice Receptors to Magnaporthe grisea Secreted Proteins Using Yeast Two-hybrid Approach


Li-hua Liu 1, 2, Shi-hua Wang 1,2*, Guo-dong Lu 1, Zong-hua Wang 1


1 The Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002

2 The School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002

*Corresponding author: wshyyl@sina.com


The interaction between the fungal pathogen Magnaporthe grisea and its host, rice, is a well-described gene for gene system. Several resistance genes of rice and matching fungal avirulence (Avr) genes have been characterized. So far, a lot of studies showed that most of elicitors and virulence factors are secreted proteins. Secreted proteins are the most likely candidates for being elicitors that are potentially recognized by resistance gene products in rice and other host plants. We characterized a secreted protein, a putative extracellular chitinase from M.grisea. The putative extracellular chitinase isolated from M.grisea, gave an elicitor reaction on detached leaf of rice plants in vitro test, suggesting its possible key role in initiation of rice hypersensitive response. To screen for the possible receptors of rice, the cDNA of extracellular chitinase encoding gene was used as a bait in a yeast two-hybrid assay, and 180 interacting proteins from rice were found. Further confirmation and characterization of these candidates were under way.


Keywords: yeast two-hybrid, extracellular chitinase, receptor, Magnaporteh grisea


National Nature Foundation of China (Project No.30471132, 30500325)




Secretion Property and Its Gene Expression Pattern of a Putative Feruloyl Esterase in Magnaporthe grisea


Xiang-zi Zheng2, Jie Zhou1,2, Chen-zeng Lin 2 , Xiong-jie Lin1, Lan Lan2, Zong-hua Wang 1,2* ,Guo-dong Lu 1*


1 The Key Laboratory of Biopesticide and Chemistry Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China

2 The School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China


The plant cell wall is a pivotal battleground between microbial pathogens and their plant hosts. Microbial pathogens secrete an array of cell wall-degrading enzymes enable to depolymerize the noncellulose polysaccharides of primary cell walls. Besides, some cell-wall degrading enzymes may involve in the pathogenicity.

The predicted protein encoded by MGG_01403.5 from Magnaporthe grisea has a high degree of sequence identity with the ferulic acid esterase A (so to be designated as MgFaeA) from Penicillium funiculosum. Tested by the SignalP, TMHMM and Protcomp programs, the hypothetic protein, consisting of 284 amino acids, contained a cleavable signal peptide at the N terminus without transmembrane structure, and located mainly in extracellular matrix.

In the work presented here, MgFaeA, with a His6 tag at its C terminus, was introduced into M. grisea for overexpression, which resulted in the purification of a fusion protein from the culture filtrate. Therefore, it confirmed the secretion property of MgFaeA. To further investigate the role of MgFaeA on rice infection process of the fungus, the gene expression was analyzed by RT-PCR during five different stages of infected rice seedlings. The results indicated that MgFaeA transcripts were detectable as early as 72 hpi when inoculated with M.grisea conidium suspension, and peaked at 7 dpi. It suggested that the MgFaeA gene might be involved in the fungal pathogenesis.


Key words: Magnaporthe grisea; Ferulic acid esterase; Secreted protein




Possible Roles of the Extracellular Matrix (ECM) from Magnaporthe oryzae During the Fungus-host Adhesion


Kanako Inoue, Ken-ichi Ikeda and Pyoyun Park


Laboratory of Stress Cytology, Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan


Spores and infection structures such as germ tubes and appressoria of Magnaporthe oryzae are always accompanied with the mass production of an extracellular matrix (ECM) in the fungal morphogenesis on plant surface. The ECM, appeared to act as cementing substance to plant surfaces, has been regarded as a one of fungal pathogenesity factors. Until now, however, the roles of the ECM had not been determined yet. To understand the roles, the chemical components and function of the ECM were studied on artificial membranes and plant surfaces by two immunological techniques or the adhesion inhibiting test of infection structures with various enzymes and inhibitors. The ECM was characterized by fibrous and amorphous materials which were located at the spaces between fungal cell walls and plant cuticles. Immunohistochemical and immunoelectron microscopy suggested that the ECM includes components positively reacted with antibodies of four animal cell adhesion factors (collagen VI, vitronectin, fibronectin and laminin) and an animal integrin α3. Ultrastructural immunohistochemical studies showed that the ECM was much more produced from the appressoria than from the spores and germ tubes. Spore solution with tunicamycin (protein glycosylation inhibitor) or colchicine (glycoprotein secretion inhibitor) developed appressoria but failed to adhere to the surface, suggesting that the ECM was secretory glycoprotein. The infection structures after 6 h of spore germination, when matured appressoria had formed, were effectively detached by treatment with glycoprotein-degrading enzymes such as pronase E and matrix metalloproteinases (MMPs; collagenase and gelatinase), while glucanase, lipase and proteinase was not. The lesion formation on wheat leaves, inoculated with compatible strain Br48 and treated with collagenase, was restricted. The results suggested that ECM was compatible with the complex of glycoproteins, similar to animal cell adhesion factors, and are contributed to a powerful adhesion force for infection structures to penetration into plant cuticle.


Contact Address

Name: Kanako INOUE

Affiliation: Faculty of agriculture, Kobe University, Nada, Kobe 657-8501, Japan

Tel&Fax: +81-78-803-6487

E-mail: 049d810n@stu.kobe-u.ac.jp




Ca2+ Signal Pathway is Required for the Conidial Germination, Appressorial Formation and

Pathogenicity of Magnaporthe oryzae


Yi Gu1, Jian-Ping Lu2, Fu-Cheng Lin1*


1 Biotechnology Institute, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, China

2 College of life Sciences, Zhejiang University, Yuhangtang Road 388, Hangzhou 310058, China

*Corresponding Author: E-mail: fuchenglin@zju.edu.cn, Tel (+86) 571 86971185; Fax (+86) 571 86971516

The process of conidial germination, appressorial differentiation and infection to plant in the rice blast fungus, Magnaporthe oryzae (M. grisea), is precisely and complicatedly controlled by a serial of genes. Several of these genes are clustered as three known signal pathway, namely cAMP signaling pathway, MAP kinase pathway, Ca2+ signaling pathway. cAMP pathway and MAP kinase pathway is necessary to appressorial differentiation. Some cues also showed several calcium modulators and calmodulin antagonists, such as EGTA, Neomycin, Verapamil, U-73122 and KN-93, inhibited appressorial formation in M. oryzae, while conidial germination remained affected less.

In this study, we have isolated three Ca2+ signaling pathway-related genes, CMK1, PLA2 and PLC, from the rice blast fungus, and deleted it in the fungus strain Guy-11. CMK1 gene (MG09912.5) encodes a CaM dependent protein kinase; PLA2 gene (MGG14014.5) encodes a Ca2+-activated phospholipase A2; and PLC gene (MG02444.5) encodes a phosphoinositide-specific phospholipase C. ΔCMK1 null mutants showed defective phenotypes, such as sparse hyphae for mycelium growing on CM medium plates, decreased conidiation, delayed conidial germination and appressorial formation, and highly mild pathogenicity to rice and barley. The mating ability of ΔCMK mutants crossing with strain 2539 was also deadly impaired. ΔPLA2 null mutants and ΔPLC null mutants also showed defects in colonial morphology, conidiation, conidial germination and appressorial formation; the pathogenicity of these null mutants on rice and barley was weakened too. However, different from ΔCMK1, the mating ability of ΔPLA2 mutants crossing with strain 2539 was not impaired.

These results revealed by ΔCMK, ΔPLA2 and ΔPLC mutants suggested that Ca2+ signal pathway is required for the conidial germination, appressorial formation and pathogenicity of Magnaporthe oryzae.




Functional Analysis of Two Laccase Genes in

Magnaporthe grisea


Xin Chen2, Wende Liu1, Chuanzhi Zhao2, Shuji Liu2, Minno Razee, Guodong Lu, Zonghua Wang


1 The Key Laboratory of Biopesticide and Chemistry Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China

2 The School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China


Laccase is found to be involved in pathogenicity of Cryphonectria parasitica and Cryptococcus neoformans. In this report we demonstrate that laccase is not necessary for pathogenicity in Magnaporthe grisea, which might due to functional redundancy in some or all of laccase genes. The major laccase activity in M. grisea is not encoded by the gene MGG_00551.5 and MGG_02876.5, which show only slightly decrease in laccase activity compared to wild-type strains. Targeted deletion of gene MGG_00551.5 and MGG_02876.5 generated mutants that have the same growth rate, conidiation and pathogenicity as wild-type strains. Taken togethor, our findings provide evidence that gene MGG_00551.5 and MGG_02876.5 are not essential for the differetiation and development of M. grisea.




High Throughput Protoplast System for Protein-Protein Interactions at the Host-Pathogen Interface


Abdelaty Saleh, Thomas Mitchell, and Ralph Dean


Center for Integrated Fungal Research (CIFR), Plant Pathology Department, North Carolina State University, Raleigh, NC 27695, USA

Email: asaleh@ncsu.edu, Thomas_Mitchell@ncsu.edu, radean2@ncsu.edu


Recent genome based research has revealed many insights into the nature of host-pathogen interactions. For the rice blast disease, both host and pathogen genomes have been sequenced, which aid us to understand the host-pathogen interactions at the protein level. In our research project, we are developing a rice protoplast system with bimolecular fluorescence complementation (BiFC) technique to visualize protein-protein interactions directly in living cells. The BiFC relies on the formation of a fluorescent protein complex by two split fragments of the yellow/green fluorescent protein (YFP/GFP) brought together by interacting proteins. We are currently testing this system using rice proteins that are known to interact and subsequently, test the applicability of this system to interrogate the interaction of rice with the rice blast fungus, Magnaporthe grisea. Initially, we will test a known fungal avirulence gene (AVR-Pita) product with its cognate plant resistance gene receptor (Pita). Then, we will scale up protoplast system and BiFC strategy using fluorescence activated cell sorting (FACS) to screen for unknown fungal and plant proteins that interact with encoded products of recently identified resistance genes. To facilitate the mobilization of cDNA libraries and genes of interest into the appropriate expression vectors, the gateway cloning system will be used. The high throughput protoplast system will be adapted to screen for unknown fungal elicitors. Some of these newly identified elicitors will be used to identify plant interacting proteins.




MGLIG4, a Neurospora Crassa MUS-53 homolog, is Involved in Non-homologous Endjoining

Events in Magnaporthe grisea


Hideki kito, Takashi Fujikawa and Marie Nishimura


National Institute of Agrobiological Sciences (NIAS), Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.

Email: marie@affrc.go.jp


In eukaryote, two major pathways are present to deal with DNA double strand breaks (DSB); homologous recombination (HR) and non-homologous end-joining (NHEJ) systems. In many organisms, including filamentous fungi, the NHEJ system seems to be used to repair the DSB that is induced by a number of exogenous and endogenous agents. Ku70/80 heterodimer, DNA-PKcs and Xrcc4-Lig4 complex are known to be involved in the NHEJ system. In filamentous fungi, NHEJ components including KU70, KU80 and LIG4 homologues were identified from Neurospora crassa and Aspergillus species. In N. crassa, Ku70/80 heterodimer and Lig4 is important for NHEJ. To investigate functions of Ku70/80 complex and Lig4 homologues in M. grisea, we replaced MGKU70 and MGLIG4, homologues of MUS-51 and MUS-53 respectively, with hygromycin phosphotransferase gene. No defect was observed in vegetative growth, pathogenic developments and fertility in the mgku70 and mglig4 deletion mutants. Although the mgku70 deletion mutant exhibited no change in HR frequency and tolerance to DSB mutagens as compared to those of wild type, the mglig4 deletion mutant presented dramatically increased HR frequency and showed sensitivity to UV, 4-nitroqunoline 1-oxide and camptothectin. The complementation test of mglig4 restored the NHEJ activity. Our results demonstrated that Mglig4 was important for the NHEJ system whereas Mgku70 was not.  


Presenter address

Name: Hideki Kito

Affiliation: National Agricultural Research Center for Tohoku region (NARCT), Yotsuya, Daisen, Akita, 014-0102, Japan. E-mail: hkito@affrc.go.jp ;

Tel/ Fax: +81-187-66-2772 (Tel), +81-187-66-2639 (Fax)




Mapping of Avirulence QTLs of Magnaporthe grisea Corresponding to Leaf and Neck Blast Resistant

QTLs in Jao Hom Nin

Tanee Sreewongchai 1, Saengchai Sriprokhon 2, Chanakarn Wongsaprom 2, Apichart Vanavichit 1,2, Theerayut Toojinda 2, Didier Tharreau 3, and Pattama Sirithunya 4


1 Rice Gene Discovery, National Center for Genetic Engineering and Biotechnology (BIOTEC), Kasetsart University, Kamphangsaeng, Nakornpathom, 73140, Thailand.

2 Agronomy Department, Kasetsart University, Kamphangsaeng Campus, Nakornpathom, 73140, Thailand.

3 UMR BGPI, INRA-ENSAM-CIRAD, TA73/09, 34398 Montpellier Cedex 05, France.

4 Rajamangala University of Technology Lanna, Science and Agricultural Technology Faculty , Chiengmai, 50000, Thailand.

Email: taneesree@yahoo.com


Magnaporthe grisea (Hebert) Barr (anamorph: Pyricularia grisea Sacc.), a filamentous heterothallic ascomycetous fungus, is the causal organism of rice blast disease. Recently, the cultivar specificity of blast fungus has now confirmed gene-for-gene interactions which elicitors are produced from avirulence genes in pathogen through direct or indirect interactions and reacted with receptors produced from resistance genes in rice to activate resistance reaction. Joa Hom Nin, JNH, a rice variety that shows broad spectrum resistance against Thailand blast pathogens has been used as resistant source in many rice breeding programs. In this study, QTL approach was applied to locate avirulence gene(s) location on blast genome and QTL corresponding to blast resistance in JHN. Crossing between the pathogen isolate B1-2 and TH16 was performed in order to produce 140 progenies for mapping population. Linkage was created with 44 microsatellite markers which distributed throughout the blast genome and map was spanned 7 linkage groups with average distance 11.9 cM per marker. According to the results, distribution of pathogenic and non-pathogenic isolates in mapping population inoculated onto JHN was found to be 1:1 for both leaf and neck blast. QTL analysis for avirulence gene(s) corresponding to two traits was located at the same region on chromosome 2 between markers Pyms305/306 to Pyms435/436 while LOD score and percentage of phenotypic variance explain on these traits were 5.01/16.69 and 6.73/20.26, respectively. This discovery is useful for positional cloning and identifying any function of avirulence genes linking to leaf and neck blast resistance.




Funtional Analysis of MER1 Required for Spore Morphogenesis and Pathogenicity in Magnaporthe grisea


Jaeduk Goh, Sook-Young Park, Myung-Hwan Chi, Sung-Yong Yoo, Junhyun Jeon, Hee-sool Rho, Soonok Kim and Yong-Hwan Lee

Email: fri28@snu.ac.kr


Magnaporthe grisea, a causal fungus of rice blast, forms a specialized infection structure, an appressorium, that is essential for successful infection on plants. To identify novel genes involved in fungal pathogenicity, 21,070 transformants generated by Agrobacterium tumefaciens-mediated transformation were screened by high-throughput screening system. One of transformants, ATMT0659D4, was identified and further characterized as a pathogenicity-defective transformant. T-DNA was inserted on MG02423.4 locus, a yeast homologue of ERD2, and names as MER1 (Magnaporthe ER protein retaining Receptor 1). Both ATMT0659D4 and ∆mer1, generated by targeted gene disruption, form small and round-shaped conidia and are defective in mycelial growth and sporulation. Although frequencies of appressorium formation were not affected, appressoria formed by both transformants were unable to penetrate plant surface. However, they incited blast lesions when wound-inoculated. These data indicate that MER1 is required for conidial morphogenesis and appressorial function in M. grisea.




Functional Analysis of bZIP Transcription Factors in the Rice Blast Fungus, Magnaporthe oryzae


Sunghyung Kong, Jongsun Park, Hee-Sool Rho, Soonok Kim, and Yong-Hwan Lee


Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea. Email: yonglee@snu.ac.kr; liebehada@gmail.com


Pathogenic lifestyle of fungi depends largely on their versatility and adaptability in environments. This implies that the fungi have to adapt their transcriptional programs to environmental conditions such as varying nutrient supply, physical and chemical stress, and host-dependent constraints. It is therefore pivotal to elucidate the transcriptional programs operating during a host relationship. However, genome-wide understanding of transcription factors (TFs) in plant pathogenic fungi is still lacking. Magnaporthe oryzae has been a model organism due to its genetic tractability and availability of molecular tools. The completion of M. oryzae genome sequence enables identification of 819 putative TF genes accounting for 6.3% of the total 12841 annotated genes. For a large scale functional study of TF, 3406 candidate deletion mutants for 197 TFs were generated using a high-throughput gene deletion and screening pipeline. These TFs include 83 Zn (II)2Cys6 fungal binuclear cluster, 66 C2H2 Zinc finger, 16 bZIP, 12 Myb, 7 bHLH, 5 GATA type zinc finger, 5 homeobox, 3 APSES. Out of these, the 3 members of bZIP family are being characterized. Phenotypic changes of mutants were examined for seven traits: mycelial growth, pigmentation, conidiation, conidial morphology, conidial germination and appressorium formation, and pathogenicity. Remaining bZIP mutants will be subjected in-depth functional characterization. Taken together, this work would provide valuable insights on transcriptional regulation of bZIP transcription factors to fungal development and pathogenicity.




Functional Analysis of APSES Type Transcription Factors in Magnaporthe oryzae


Se-Eun Lim, Jaeduk Goh, Kyongyong Jung, Hye-Young Han, Jongsun Park, Hee-Sool Rho, Soonok Kim, and Yong-Hwan Lee


Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea.

Email: yonglee@snu.ac.kr


The APSES (Asm1, Phd1, StuA, Efg1, Sok2) transcription factors (TFs) are a conserved class of transcriptional regulator which is unique to fungi, regulating morphogenetic process and cellular differentiation in the ascomycetes including yeast. In M. oryzae, three APSES TF genes were identified: MGG00692.5 (MStuA), MGG098695 (MOA1, Magnaporthe oryzae APSES1) and MGG08463.5 (MOA2). Both △moa1 and △moa2 showed similar phenotype in cellular differentiation and development. They displayed reduced mycelial growth and lower virulence toward rice cv. Nakdong when compared with wild type strain, KJ201. Notably, they were impaired in their ability to undergo asexual reproduction. Conidiophore development was markedly delayed, and small numbers of conidia were produced (25% of wild type). Abnormally strong melanization was observed in their culture, suggesting that MOA1 and MOA2 can function as transcriptional repressors. Taken together, this work would provide valuable insights on transcriptional regulation of asexual development and pathogenicity in M .oryzae.




MoDim2, the Magnaporthe oryzae Methyltransferase Orthologous to Neurospora crassa Dim-2 is Dispensable

for the Life Cycle of the Fungus in Nature


Ken-ichi Ikeda1, Kohta Shi-na2, Naoki Kadotani2, Masaki Tanaka2, Toshiki Murata2, Izumi Chuma2, Yukio Tosa2, Pyoyun Park1, Shigeyuki Mayama2, and Hitoshi Nakayashiki2


1 Laboratory of Stress Cytology, Faculty of Agriculture, Kobe University, Kobe, 657-8501, Japan

2 Laboratory of Plant Pathology, Faculty of Agriculture, Kobe University, Kobe, 657-8501, Japan

Email: ikeken@phoenix.kobe-u.ac.jp

Cytosine methylation is a chemical modification of DNA that potentially serves as an epigenetic mark in a variety of biological processes such as embryogenesis, cell differentiation, X-chromosome inactivation, genomic imprinting, and gene silencing in various organisms. In the Magnaporthe genomes, the LTR retrotransposon MAGGY was targeted for cytosine methylation in a subset of field isolates but not in the other subset. Analysis of F1 progenies from a genetic cross between methylation proficient (Br48, a wheat pathotype) and methylation deficient (GFSI1-7-2, a foxtail millet pathotype) isolates revealed that methylation of the MAGGY elements was governed by a single dominant allele. Positional cloning was used to identify the responsible gene, MoDim-2, a M. oryzae ortholog of Neurospora crassa Dim-2 methyltransferase. Dim-2 is reported to be responsible for all known cytosine methylation in N. crassa. Sequencing revealed that MoDim-2 protein in GFSI1-7-2 was truncated due to a single base change in the methyltransferase domain. Cytosine methylation on MAGGY sequences was detected in GFSI1-7-2 when the MoDim-2 locus of Br48 was introduced in the isolate. Using the methylation proficient transformant and wild-type GFSI1-7-2, we then performed phenotypic analyses on growth rate, spore formation, fertility, and pathogenicity on the host plant. However, we failed to detect a significant difference between the transformant and wild-type in any of the phenotypes examined. In addition, cytosine methylation on MAGGY sequences in the transformant did not appear to have a negative effect on the rate transposition and the stress-induced transcriptional activation of the element. A MoDim-2 disruptant of Br48 also did not cause any apparent phenotypic changes compared to wild-type. A comparison of MoDim-2 sequences among methylation proficient and deficient Magnaporthe isolates from different host plants identified various SNPs as well as insertions and deletions. Interestingly, statistical analysis did not support a preference to synonymous substitutions over nonsynonymous substitutions in the MoDim2 genes. Overall, our results suggested that cytosine methylation could be dispensable for the life cycle of this plant parasitic fungus in nature.




Saccharomyces Cerevisiae SSD1 Orthologus of Magnaporthe grisea and Colletotrichum Lagenarium are Essential for Host Infection by Circumventing Host Defense Responses


Shigeyuki Tanaka1, Hironori Koga2, Koji Dohi3, Masashi Mori3, Nobuaki Ishihama4, Hirofumi Yoshioka4, Gento Tsuji1, Seiji Tsuge1 and Yasuyuki Kubo1


1 Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522, Japan.

2 Laboratory of Plant Protection, Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, Ishikawa 921-8836, Japan;

3 Laboratory of Plant Gene Technology, Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Ishikawa, 921-8836, Japan;

4 Laboratory of Defense in Plant-Pathogen Interactions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan.

Email: y_kubo@kpu.ac.jp


We identified MgSSD1 of M. grisea and ClaSSD1 of C. lagenarium as orthologs of Saccharomyces cerevisiae SSD1, a regulator of cell wall biogenesis. Gene disrupted mutants of mgssd1 and classd1 produced appressoria with normal structure but could not establish penetration to host epidermal cells. Cytological observation revealed that the failure of those mutants accompanied by host defense responses. In support of this, mgssd1 mutants recovered their infectivity when host defense responses were compromised by abscisic acid treatment that suppresses basal resistance. The involvement of induced defense responses in the failure of classd1 mutant infection was verified by VIGS (virus induced gene silencing) experiments of defense response related genes using Nicotiana benthamiana, a susceptible host of C. lagenarium. We generated knock-down plants of defense-related genes, MEK1, MEK2, SIPK, WIPK, NbrbohA, NbrbohB, RAR1, SGT1 and HSP90, respectively by VIGS, and the classd1 mutant was inoculated to these silenced plants. MEK2 (MAPKK) silenced plants and SIPK/WIPK (MAPKs) double-silenced plants showed typical lesions similar to non-silenced plants infected with wild-type. However, neither MEK1 silenced plants nor SIPK, WIPK single-silenced plants showed lesions. These results suggest that defense reaction under the control of MEK2-SIPK/WIPK cascade is involved in the failure of infection by the classd1 mutant and that appropriate assembly of the fungal cell wall as regulated by SSD1 orthologs allow these pathogens to establish infection by avoiding the induction of host defense responses.




Analysis of Genes Involved in Post-penetration Phase of Rice Blast Fungus


Junhyun Jeon


Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, Center for Agricultural Biomaterials, and Fungal Bioinformatics Lab, Seoul National University , Seoul 151-921,


Magnaporthe oryzae is a hemibiotrophic ascomyceteous fungus that causes the most devastating disease on cultivated rice, the rice blast. The fungus has been an important model organism for understanding the fungal pathogenicity due to its genetic tractability. As a hemibiotroph, M. oryzae invades plant cells biotrophically following penetration, and switches to necrotrophy thereafter. During these processes, the fungus has to develop bulbous invasive hyphae and secrete effector proteins inside host cells. Despite the importance of understanding post-penetration phase of fungal pathogenesis, extensive mutational analysis of the fungus during the last two decades, have been mainly focused on identification of genes that are required for appressorium formation and function. Two major developments recently made provided the ground for investigating the genetic basis of post-penetration phase. First, the method for cytological observation of fungal infection was developed and applied to dissection of invasive growth of M. oryzae, providing the checkpoints in functional analysis of mutations. The second development is that a total of 202 new pathogenicity genes were identified in M. oryzae using forward genetics approaches. Based on phenotype data of mutants, 81 out of 202 genes were selected to be involved in either penetration or post-penetration phase, with 11 genes predicted to be secreted. Penetration assay using yielding onion epidermis will be employed to screen mutants for the ones defective in post-penetration phase. The genes tagged by T-DNA in these mutants will be subjected to molecular analysis including targeted gene knockout and localization study, and function of genes concerning post-penetration phase will be concurrently pinpointed through cytological analysis of mutant phenotypes using rice sheath inoculation method. Considering that few genes that affect fungal growth inside plant cell have been identified, our attempts to unveil pivotal players of post-penetration phase of rice blast fungus would open up a new avenue in understanding disease mechanisms as well as the potential molecular mechanisms of disease resistance.




Cloning and Functional Characterization of a Methionine Sulfoxide Reductase B Gene in Magnaporthe oryzae


Hyejeong Kim1, Yoonhee Kim1, Miyeon Jeong2, Soyoung Park2, Jinsoo Kim2and Woobong Choi1,2


1 Department of Biomaterial Control, Dongeui University, Busan 614-714, Korea,

2 Department of Biotechnology and Bioengineering, Dongeui University, Busan 614-714, Korea

Email: wbchoi@deu.ac.kr


Magnaporthe oryzae, the causal agent of rice blast, is one of the most destructive fungal pathogens throughout the world. Plant pathogenic fungi may act as virulence factors if they provide protection against plant defence compounds during disease development. Methionine is among the amino acids the most susceptible to oxidation by almost all forms of ROS. This malformation can be repaired specifically by the methionine sulfoxide reductase (msr). Degenerate oligonucleotide primers were designed based on the genome sequence of the msrB gene in M. oryzae strain 70-15. A polymerase chain reaction (PCR) was carried out by using M. oryzae KJ201 genomic DNA as a template. The copy number of msrB in the genome of KJ201 was examined by Southern blot analysis, which revealed that gene exists as a single copy. Expression of msrB gene in the presence of hydrogen peroxide was examined by Northern blot analysis and RT-PCR, in which the expression of msrB gene was detected without significant correlation with hydrogen peroxide treatment. To analyze the function of important gene of the pathogen, knock-out analysis is necessary. To confirm the gene replacement, transformants were screened by Southern blot analysis indicating that the selection marker, hygromycin phosphotransferase gene cassette, was integrated into the target gene and the gene was eliminated. Analysis of knock-out mutants of msrB could elucidate the function of the gene against oxidative stress presented by host plant.




A Putative Secreted Protein MC69 is Required for Invasion, Invasive Growth and Pathogenicity of the Rice Blast Fungus Magnaporthe oryzae


Hiromasa Saitoh


Iwate Biotechnology Research Center, Narita 22-174-4, Kitakami, Iwate 024-0003, Japan.

Email: msaitoh@ibrc.or.jp


Treatment with cAMP induces appressorium formation in rice blast fungus Magnaporthe oryzae. A cAMP-induced gene MC69 was identified by SuperSAGE. The MC69 codes for an unknown putative secreted protein of 54 amino acids containing 18 amino acids of signal peptides. Targeted gene disruption of MC69 produced mutants that show reduction in penetration rate and infectious growth in rice leaf sheath cells, and a reduction in expression of blast symptoms in rice and barley. These phenotypes were complemented by re-introduction of an intact copy of MC69. MC69-green fluorescent protein fusion protein under the native MC69 promoter was expressed in conidia, germ tubes, appressoria and infectious hyphae. GFP was strongly detected in appressoria and at the tip of invaded mycelia at 24 hours after incubation on glass coverslips and on rice leaf sheath cells, respectively. Analysis of localization of MC69 protein is in progress. Function of MC69 will be discussed on the basis of phenotype of mc69 mutant, and MC69 localization.




Analysis of Two AVR-Pita Homologues in Japanese Isolates of Rice Blast Fungus.

Mami Takahashi1, Taketo Ashizawa1, Kazuyuki Hirayae1, Jouji Moriwaki1, Teruo Sone2, Ryoichi Sonoda3, Masako Tsujimoto Noguchi4


1 National Agricultural Research Center, Inada 1-2-1, Jouetsu, Niigata 943-0193, Japan

2 Research Faculty of Agriculture, Hokkaido University, Kita9 Nishi9, Kita-ku, Sapporo Hokkaido 060-8589, Japan;

3 National Institute of Vegetable and Tea Science, Kanaya 2769, Shimada, Shizuoka 428-8501, Japan;

4 National Institute for Agro-Environmental Sciences, kannondai 3-1-3 Tsukuba, Ibaraki 305-8604, Japan.

Email:mamitaka@affrc.go.jp


In order to investigate the molecular mechanisms by which Japanese rice blast fungus becomes virulent to Pita-containing rice cultivars, AVR-pita homologues were examined in Japanese isolates. We first analyzed AVR-pita homologues in an avirulent isolate, OS99-G-7a (G7a), and 14 virulent mutants obtained from paddy fields with severe blast epidemics. The mutants were trapped using Pita-containing seedlings of cv. Yashiromochi. G7a was found to contain two AVR-pita homologues, AVR-pita A (AtaA) and AVR-pita B (AtaB), with sequences that are 98.6% and 98.3% identical respectively, to that of AVR-pita reported by Orbach et al. (2000). AtaA and AtaB differed by three nucleotides. Hybridization and sequencing analysis of the 14 mutants revealed that AtaA was completely deleted from 11 mutants, while two contained base substitutions in AtaA and one contained a 31-bp duplication in AtaA. No changes in AtaB were seen in any mutants. This suggests that AtaA functions as an avirulence gene in G7a, but AtaB does not. To investigate whether AtaA commonly acts as an avirulence gene in Japanese rice blast fungus, alleles of AVR-pita homologues were then examined in 13 isolates collected from various localities. Five of 12 avrirulent isolates contained only AtaA, and seven avirulent isolates contained both AtaA and AtaB. One virulent isolate contained neither AtaA nor AtaB. These results indicate that avirulent Japanese rice blast fungus commonly carries AtaA, which may act as an avirulence gene.




Molecular Cloning of Avr-Pia, the Avirulence Gene in Magnaporthe oryzae Toward the Rice

Blast Resistance Gene Pi-a


Shinsuke Miki1, Kotaro Matsui1, Taketo Ashizawa2, Hideki Kito3, Kazuyuki Hirayae2, Fusao Tomita4, and Teruo Sone1


1 Hokkaido Univ., Sapporo Japan;

2 Nat. Agric. Res. Cen., Niigata Japan;

3 Nat. Agric. Res. Cen., Akita Japan;

4 Univ. of Air, Sapporo Japan. Kita-9 Nishi-9, Kita-ku, Sapporo city, Hokkaido, Japan. Email: sonet@chem.agr.hokudai.ac.jp; mi-ke@chem.agr.hokudai.ac.jp


Pi-a is the resistance gene found in the rice cultivar Aichi-asahi and used in blast resistant rice cultivars. It is important to understand the mechanism of the mutation of the host specificity of the pathogen because of making stabile crops. So, we aimed to clone and analyze the avirulence gene, AVR-Pia, in the Magnaporthe oryzae which confers avirulence toward rice cultivars carrying resistance gene Pi-a. First, we obtained the mutant of strain Ina168, named Ina168m95-1, which was virulent toward Aichi-asahi. RAPD primers were screened for polymorphic amplification between the mutant and the parent. One primer, named OPM-1, resulted a DNA (named PM01) fragment absent in the mutant. Three cosmid clones including PM01 flanking region were screened from genomic DNA library. One of these clones named 46F3 could complement the mutant phenotype, and thus strongly indicated that this clone contains the avirulence gene, Avr-Pia. This positive clone 46F3 contained many truncated fragments of transposable elements. Therefore, we divided 46F3 insert into six fragments, I to VI, which didn’t contain transposable elements and we introduced each of the six fragments into the mutant Ina168m95-1. The spray inoculation test revealed that the fragment V (3.5 kbp) contained the Avr-Pia. Further, 11 deleted versions of the fragment V were produced by PCR; Va to Vf were deleted from 5' end and Vg to Vk were deleted from 3' end of the fragment V. We introduced each 11 fragments into the mutant Ina168m95-1 and carried out the spray inoculation test. The test revealed that the 1.2 kb DNA region contained the Avr-Pia. This region contained various ORFs and the amino acid sequence of the longest ORF had the similarity with the cytochrome c family protein of Geobactor metallireduence GS-15. 10 field isolates with the AVR-Pia has also the 1.2 kb DNA region and have the longest ORF with 100% homology.




The Analyses of Recombinational Repair Gene Rhm54

in Rice Blast Fungus


Kudo R., Abe A., Ashizawa T., Hirayae K., and Sone T.


Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido, 060-8589, Japan.

Email: sonet@chem.agr.hokudai.ac.jp; arigato@chem.agr.hokudai.ac.jp


In rice blast fungus, one possibility of the generation system of genetic diversity, such as avirulence gene mutation, is homologous recombination. Rad52 epistasis group genes play important roles in homologous recombination. We had isolated and characterized Rhm54 (Rad54 homolog Magnaporthe), a member of the epistasis group in rice blast fungus. Rhm54 transcription was induced by oxidative stress, DNA damage and heat shock. It suggests that Rhm54 expression might increase relative to host -specific resistant reactions 1).

For quantification and visualization of the expression of Rhm54 in planta, a reporter vector was constructed by multisite gateway method. The vector had a putative region of Rhm54 promoter and a GFP gene. The vector was transformed into strain Ina168 by a PEG-mediated method and some transformants with single copy insertion were isolated. As a control experiment, they were incubated in liquid 2YEG and treated by 1h heat shock. The expression of GFP was increased by 1.9-fold by fluorometric quantification. A suspension of spores of the transformants was inoculated on a polycarbonate slide and observed under fluorescent microscope until the development of appressoria. The continuous GFP fluorescence was observed during each infection stage.

Rhm54 defective mutants in Ina86-137 were constructed using the gene disruption vector, pDESTR2). The mutants grew more slowly than wildtype on prune agar media. The growth defect of the mutants was severe on other prune plates including DNA-damaging agent, methyl methanesulfonate (MMS), or oxidative reagents, methyl viologen or hydrogen peroxide, especially on the MMS containing media. On the other hand, neither morphological change during early infection-related development nor spore production were detected between mutant and wildtype. There results suggest that Rhm54 is not essential for normal growth but functions for the successful growth or the repair of DNA damages. Moreover, the constitutive expression of Rhm54 might indicate that the fungus is continuously suffering damages to DNA during its life cycle.


1) Elegado et al., Journal of General Plant Pathology, 72, 16-19 (2006)

2) Abe et al., Current Microbiology, 52, 210-215 (2006)




Characterization of Two Transcription Factors Differentially Expressed During Appressoria Formation

in Magnaporthe grisea


Gregory C. Bernard, Thomas Mitchell, and Ralph Dean


Center for Integrated Fungal Research, Department of Plant Pathology, North Carolina State University, Raleigh, NC 27606

Email: gcbernar@ncsu.edu


Over the past two decades, significant efforts have been made to elucidate the molecular controls of rice blast. International efforts have been conducted to characterize genes involved in pathogenecity of M.grisea. Two genes, MGG_11512.5, and MGG_3196.5 were found to be expressed during appressoria formation through microarray analysis. Hypothetical proteins MGG_11512.5 and MGG_3196.5 have homologies with bromodomain containing proteins and tetratricopetide (TPR) containing proteins in other fungi, respectively. Bromodomain containing proteins may be involved in protein-protein interactions and transcriptional activation. TPR containing proteins are involved in various functions such as filamentous growth, glucose repression, and transcriptional regulation. The purpose of this research is to characterize MGG_11512.5 and MGG_3196.5 via homologous recombination gene deletion. Four transformants have been recovered for MGG_11512.5. The mutants show a decrease in appressoria formation and a coordinate decrease in virulence versus the wildtype. A complete characterization of these two genes will be presented.




Development of New SSR Markers within ABC Transporter Genes in Phytopathogenic Fungus, Magnaporthe grisea


Lin Liu, ChengYunLi, Jing, Yang Jin Bin Li, Yuan Su, Yun Yue Wang,

You Yong Zhu


Key Laboratory for Agricultural Biodiversity and Pest Management of China Education Ministry, Yunnan Agricultural University, Hei Longtan, Kunming,650201,China

Email: li.chengyun@gmail.com


To understand the variation of simple sequence repeat (SSR) sequence within protein coding sequence in the genome of rice blast fungi, Magnaporthe grisea. Bioinformatics analysis showed that SSRs nonrandomly distribute between expressed sequence tags (ESTs) and genes and non-coding region. Thirteen of forty-eight ABC-transporter genes contain SSRs, that is about 27 percent of all the ABC-transporter genes. The consequences of SSRs repeat-number’s changes are different in those regions. The distance between SSRs and the locations of function domains is different too. Based on those genes, thirteen polymorphic microsatellite markers were suitable for population genetic structure analysis and ABC transporter coding genes variation measurement were developed. Polymorphism was evaluated by using forty-six isolates collected from diverse geographical locations and rice varieties of Yunnan Province, China. Thirteen polymorphic loci produced amplicons from a majority of 46 isolates, and displayed as ten alleles. Observed heterozygosity and expected heterozygosity values of all microsatellite markers were calculated by GENEPOP32. The results showed that there were diversities in M.grisea of Yunnan province, China. These genes containing these SSR sequences are also used analysis of diversity too. And the degree of polymorphism in this set of microsatellite markers can be used to analyze the population structure and strain distribution associated with particular locations, as well as complemented for understanding functions of regulatory genes in the fungus.




Expression Pattern of Magnaporthe grisea Gene in

Infected Leaves of Rice Detected by Real-time

Flourescence Quantitative PCR


Hua Li, Jing Yang, Lin Liu, Yuan Su, Youyong Zhu, Yunyue Wang, Chengyun Li


Key Laboratory for Agricultural Biodiversity and Pest Management of China EducationMinistry, Yunnan Agricultural University, Hei Longtan, Kunming, 650201,China;

*Corresponding author, E-mail: li.chengyun@gmail.com


The expression pattern of candidate virulence genes MGNIP10, MGNIP18, MGNIP24, MGNIP34, MGNIP38, MGNIP53, MGNIP74, MGNIP97 and MgNIP04 in phytopathogenic fungus, Maganporthe grisea were detected by Real-time flourescence quantitative PCR in different isolates, different mediums and different phases of susceptible rice cultivar Lijiangxintuanheigu (LTH) infection process were analyzed in present study.The infection phases is at 24 h, 48 h, 72 h, 96 h and 168 h post inoculation (HPI), respectively. MgNIP04 was a novel characterized virulence protein by our group recently.The SYBR Green I intercalating dye was used in the study for displaying the kinetic accumulation of PCR product. And with the standard curve, copy number of a gene could be quantified. All expression level of target genes that normalized by actin housekeeping gene, quantified by both the comparative threshold method and standard curve method.

Our results showed that all target genes were significantly difference among isolates 94-64-1b Y99-63, 95-23-4a, Y98-16 and 94-64-1b on expression level. Relative expression amount of genes of Y99-63 and Y98-16 cultured in different media showed that nitrogen starvation treated for 24 h could induce some genes’ expression. Contrasted with the Y99-63 mycelium cultured in liquid culture medium, the results showed that at 24 HPI and 48 HPI, all tested genes expression level strongly up-regulated. At 48 HPI the expression level achieved the maximum. At 72 HPI the expression level became lower.The expresson pattern of these genes in rice leaves of different phases of infected leaves were similar with MgNIP04, suggested that these genes were involved in pathogenicity of M. grisea.

We also showed that comparative threshold method was sufficient for quantified the gene expression level, when we wanted to know fungal pathogenicity gene expression specificity. It could avoid constructing standard samples, and calculating the copy number. Our study confirmed that real-time flourescence quantitative PCR is an accurate, sensitive, reliable and rapid approach for quantification of target fungal pathogenicity involved gene in various treated samples, e.g. in different hosts tissues, in different phase of infection processes.

Key words: real-time flourescence quantitative PCR; Magnaporthe grisea; gene expression; amplification curve; standard curve; secreted protein


This work is supported by National Basic Research Program of China (2006CB100202), Scientific Foundation of Education Ministry of China (307025), and Doctorial Foundation of Education Ministry of China (20050676001).




A Novel Gene Involved to Lesion Formation in

Magnaporthe grisea


J. Yang1, H. Li1, L. Liu1, Y. Su1, J.B. Li1, Q. Chang1, L.J. Qu2,Y.Y. Wang1, Y.Y. Zhu1, C.Y. Li1


1 Key Laboratory for Agricultural Biodiversity and Pest Management of China Education Ministry, Yunnan Agricultural University, Heilongtan, Kunming, Yunnan Province, 650201, China;

2 National Laboratory of Protein Engineering and Genetic engineering, College of Life Sciences, Peking University, Beijing, 100871, China

Correspondence Author: C. Y. Li, E-mail: li.chengyun @gmail.com


To date, a number of genes that are expressed in the early stages of infection have been reported, while few genes that are expressed during the course of colonization, after the initiation of plant infection, have been studied. Plant inoculations, real-time PCR, gene cloning, protein expression, and bioinformatics analysis were combined to identify a novel gene, MgNIP04, in the rice blast fungus, Magnaporthe grisea. Bioinformatics analysis showed that the amino acid sequence contained a signal peptide. The wounded inoculation resulted in necrosis specks when the total expressed proteins including MBP-MgNIP04 was inoculated on the wounded rice leaves, which demonstrated the protein directly interacted with rice. The real-time PCR result of infected leaves showed that it is up-regulated during late stages of infection of rice, and the copies of the gene expression absolute quantity of the gene were 7.61×102, 1.06×103, 1.31×103, 4.13×103, and 4.00×103, at 24, 48, 72, 96 and 168 HPI respectively. The higher copies of MgNIP04 were found at 96 HPI and 168 HPI. The copies of MgNIP04 in mycelia of Y98-63C, Y99-16, 94-64-1b and 95-23-4a were significantly lower than one of infected leaves. Based on the gene expression pattern of wounded inoculation and bioinformatics analysis, we deduced the gene was the higher expressed at the later stage of rice infection, and so the gene was selected as a candidate effector for studying M. grisea genes that involved in necrosis spots formation and pathogenicity. Combining with the experiment of wound inoculation and gene expression pattern, it was suggested that the MgNIP04 was a novel pathogenicity–related protein in rice blast fungus. Although there was no direct evidence that MgNIP04 was a secretory protein yet, the signal peptide at N-terminal of the protein implied that it was a potential secretory protein. Our results imply that these signal peptide-containing proteins are candidate effectors, and need further study functionally.


Keywords: Magnaporthe grisea, lesion formation stage, expression pattern, real-time PCR,




Autophagy is Essential for Magnaporthe

Conidiogenesis and Virulence


Yizhen Deng, Marilou Ramos-Pamplona and Naweed I. Naqvi


Fungal Patho-Biology, Temasek Life Sciences Laboratory, Singapore-117604


Autophagy is a conserved process in eukaryotes that is responsible for membrane turnover, organellar homeostasis and cytoplasmic proteins degradation. Autophagy plays an important role in the cellular response to environmental stress. We decided to analyze the role of Autophagy in rice blast fungus Magnaporthe grisea. We created deletion mutants for Magnaporthe ATG8 gene and for ATG1 in order to study the loss of autophagy function. The orthologous ATG8/AUT7 is essential for the biogenesis of autophagosomes in yeast, and usually used as a marker for autophagosomes. Loss of ATG8 resulted in the failure of autophagosome formation and severely impaired asexual development in Magnaporthe, leading us to propose that autophagy plays an important role during M. grisea conidiogenesis. Interestingly, we found that the impaired conidiation in atg8 mutant could be rescued by supplying with high concentration of carbohydrates, such as sucrose, glucose, or maltose. The posttranslational modifications and the Nitrogen starvation based regulation of Atg8p were found to be conserved in M. grisea. By mass spectrometry we identified several proteins that were differentially regulated in atg8 mutant, one of which was Gph1p, a glycogen phosphorylase. Deletion of GPH1 could partially restore the conidiation in atg8, indicating that autophagy-regulated glycogen metabolism plays an important role in Magnaporthe conidiogenesis.




A Non-canonical GPCR Required for Surface Signaling

and Pathogenicity in Magnaporthe


Ravikrishna Ramanujam, Hao Liu, Angayarkanni Suresh, Naweed I. Naqvi


Fungal Patho-Biology, Temasek Life Sciences Laboratory, Singapore-117604


Among the various complex morphogenetic processes that take place during the life cycle of the rice-blast fungus Magnaporthe grisea, the germination of a conidium and the initiation of an infection structure (appressorium) requires the fungus to perceive and respond to a wide range of environmental cues. These include signals from the contact surface, such as surface hydrophobicity, surface hardness, as well as signals in the form of cell permeable solutes. The signal transduction pathways, in particular the cell surface receptor proteins involved in directing these morphological and physiological transitions in Magnaporthe, remain poorly understood.

Heterotrimeric G-protein signaling plays an important role in perceiving extracellular signals in various eukaryotic organisms, including M.grisea. We identified MGG_05871.5 a putative non-canonical GPCR that is upregulated during growth on inductive surfaces, in a transcriptome analysis (Hard surface vs. Soft surface). Deletion of MGG_05871.5 (allelic to Pth11 1) lead to a complete loss of pathogenicity. The loss of pathogenicity was due to the inability of the mutant to form mature appressoria on host plant surfaces, as well as on artificial inductive surfaces.The ability of cAMP analog 8-Br-cAMP to rescue the pth11Δ phenotype suggested that Pth11 lies upstream of thecAMP signaling cascade, and possibly acts as a cell surface receptor for the G-proteins.Based on epifluorescence microscopy, preliminary observations suggest that the Pth11-GFP fusion protein localizesto the plasma membrane of the germ tube as well as to the immature/mature appressorial membrane. In addition, co-localization with Lysotracker dyes indicate an endosomal/vacuolar destination for the Pth11 protein. We hypothesize that Pth11 plays an important and critical role in Magnaporthe response to host surface cues during the early stages of infection , as well as controlling appressorial maturation at a later stage of the infection process.


References:

1. DeZwaan T M et.al, (1999) The Plant Cell 11, 2013-2030




2


Pathogen Population Analysis




Monitoring on Pathogenicity Variations of Magnaporthe grisea and Resistance Variations of Rice Varieties


Fu Huang1, Rong Xie2, Cheng-yuan Liu2 , Yan Zheng1, Shi-qi Zhao1, Fu-ling Peng1, Xue-mei Zhang1, Cong Luo1, Hua-zhi Ye1

1 Agricultural College, Sichuan Agricultural University, Yaan 625014Sichuan, China;

2 Rice and Sorghum Research Institute, Sichuan Academy of Agricultural Sciences, Luzhou 646100, Sichuan, China


The pathogenicity variations of Magnaporthe grisea often lead to the loss of blast resistance of rice varieties. Consequently, comprehensive understanding of the composition, distribution and variation tendency of physiological races of blast fungus, as well as the resistance variations in main rice varieties, plays an important role in making breeding strategy for resistance and in arranging appropriately resistant rice varieties. The systematically monitoring results using China’s 7 standard assessing varieties showed that the races composition varies annually and the frequency of ZA group rose from 8.3-3.4in 2002 and 2003 to 30.3-36.7in 2004 and 2005, reaching the summit. The pathogenicity of ZA group is more severe than ZB and ZC, so we have to make strategies aiming at ZA group. The races distribution varies among different ecological regions such as Yaan, Xuyong and Pujiang disease nurseries; as a result it is necessary to assess the resistance of rice varieties in different ecological regions. In addition, the relationship between the variation in blast fungus races and the epidemics of this disease from the year of 1979 to 2005 shows that the higher the predominant races takes up in the frequency, the hasher the disease is in this region/season. A monitor system has been established to watch the trend of resistance variation in major rice varieties, which successfully predicted the loss of blast resistance in major hybrid rice varieties Shanyou 63, Gangyou22, etc. recently. Enlargement of growth area of hybrid rice varieties with similar parental linage greatly accelerates homogeneity and results in an enhanced adaptability and pathogenicity of rice blast fungus, and the strains infecting the major varieties gradually became predominant races or groups, causing the loss of blast resistance in Shanyou 2 in 1984, Shanyou 63 and Dyou 63 in 1993, and Gangyou 22 in 2000, which lead to the epidemics of rice blast. Further results of this study showed that the resistance of hybrid rice combination was influenced by both restoring and infertile lines simultaneously. But in practice, the resistance of hybrid rice combination is mainly determined by restoring lines, since most infertile lines are susceptible to blast. Therefore, it has been postulated that using resistance variation trend in restoring lines monitors resistance of hybrid rice combinations. For the infertile line breeding, the fertility, cross hybridization capability and grain quality etc. should be taken into consideration, consuming much more time and efforts. Consequently, the breeding strategy for blast resistance of hybrid rice is to enhance the resistance of restoring lines.




Analysis of Population Genetic Diversity of Magnaporthe grisea in Hunan by SSR marker


Ya Li 1, Er-ming Liu1*, Liang-ying Dai1, Cheng-yun Li2, Lin Liu 2, Zhi-xiang Zhao 1


1 College of Bio-Safety Science and Technology, Hunan Agricultural University, Hunan Changsha 410128;

2 Key Laboratory for Plant Pathology, Yunnan Agricultural University, Yunnan Kunming 650201


The genetic diversity of 230 monoconidial isolates of Magnaporthe grisea from 96 cultivars in Hunan was analysed using 8 pair of SSR primers. All isolates tested were classified into seven genetic lineages at 0.23 lever of the genetic dissimilarity through the clustering analysis of unweighted pair-group method with arithmetic average (UPGMA). The lineagesI, V, VI were the predominant lineages with the ratio 23.9%, 23.5%, 23.0%, respectively. Only one isolate located in the lineage IV and five isolates belonged to the lineage VII, which both them were possessed of 0.43% and 2.17% in the total isolates.


KeywordsOryzae sativa, Magnaporthe grisea, simple sequence repeat(SSR), genetic diversity




Diversity of rice blast pathotypes and Effectiveness of main resistance genes in China


Cailin Lei1, Jiulin Wang1, Ke Shi1, Juntao Ma2, Guomin Zhang2, Hua Li3, Zhongzhuan Ling1, Jianmin Wan1


1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China

2 Crop Cultivation Institute, Heilongjiang Academy of Agricultural Sciences, Haerbin, China

3 Rice Research Institute, Ningxia Academy of Agricultural Sciences, Yinchuan, China

A total of 738 blast isolates, including 296 indica-derived ones from southern China and 442 japonica-derived ones from northern China, were pathotyped by using 24 rice monogenic differentials harboring 20 resistance genes (IRBLa-A, IRBLa-C, IRBLi-F5, IRBLks-F5, IRBLks-S, IRBLk-Ka, IRBLkp-K60, IRBLkh-K3, IRBLz-Fu, IRBLz5-CA, IRBLzt-T, IRBLta-K1, IRBLta-CT2, IRBLb-B, IRBLt-K59, IRBLsh-S, IRBLsh-B, IRBL1-CL, IRBL3-CP4, IRBL5-M, IRBL7-M, IRBL9-W, IRBL12-M and IRBL19-A). As a result, the 738 isolates could be classified into 609 pathotypes, among which 296 indica-derived and 422 japonica-derived isolates belonged to 263 and 360 pathotypes, respectively. In addition, 223 japonica-derived isolates from Heilongjiang and Ningxia were classified into 217 pathotypes by using the 24 differentials and additional 7 monogenic differentials (IRBLkm-Ts, IRBL20-IR24, IRBLta2-Pi, IRBLta2-Re, IRBLta-CP1, IRBL11-Zh and IRBLz5-CA(R)). These results, though further confirmation is needed by multiple repeated inoculation, indicated that the differentials could have high differential ability to Chinese blast isolates, and in other words, the blast pathogen populations could be highly diverse in pathotypic differentiation.

Among the 20 resistance genes in the above 24 differentials, four genes, Pi5(t) (IRBL5-M), Piz (IRBLz-Fu), Piz-5 (IRBLz5-CA) and Pi9 (IRBL9-W), showed broader resistance spectrum (RS) to the 738 isolates (72%-97%); eight genes, Piz-t (IRBLzt-T), Pi5(t) (IRBL5-M), Pik (IRBLk-Ka), Pi1 (IRBL1-CL), Piz (IRBLz-Fu), Pik-h(IRBLkh-K3), Piz-5 (IRBLz5-CA) and Pi9(IRBL9-W), showed broader RS to the 296 indica-derived isolates (72%-94%), and four genes, Pi5(t) (IRBL5-M), Pi12(t) (IRBL12-M), Piz-5 (IRBLz5-CA) and Pi9(IRBL9-W) to the 422 japonica-derived isolates (73%-99%). Two genes Pi9 and Piz-5 showed the broadest RS to both indica-derived and japonica-derived isolates, reaching 93.9% and 89.5%, and 98.4% and 89.1%, respectively. However, some differentials with same resistance gene expressed different RS, meaning their monogenic status could need further confirmation.

The JIRCAS research project “Blast Research Network for Stable Rice Production” has officially initialized under the collaboration with IRRI recently. As a member, we focus our research on diversity of blast pathogens and development of differential systems for blast resistance in China for the network’s goal to discover the relationship and differentiation between blast races and resistance genes, and develop a universal differential system.




Pathogenicity of Rice Blast Isolates from Some Irrigated Areas of Southern Vietnam


Pham Van Du1 and Le Cam Loan1

1 Cuu Long Rice Research Institute (CLRRI), Co Do, Can Tho, Vietnam

E-mail: phamvandu@hcm.vnn.vn, lecamloan1@yahoo.com


To develop differential system and to have good achievement in rice blast breeding program in Vietnam; collection, isolation, reservation and pathogenicity evaluation of blast isolates have been carried out. Two hundred twenty blast samples including 30 (from Long An province), 25 (Tien Giang), 15 (Ben Tre), 20 (Vinh Long), 30 (Dong Thap), 15 (Tra Vinh), 15 (Soc Trang), 5 (Can Tho),15 (Hau Giang), 7 (An Giang), 30 (Kien Giang), and 13 (Bac Lieu) were collected in irrigated areas of Southern Vietnam and 31 monogenic lines carrying 24 single blast resistance genes were used in virulence analysis. Preliminary results showed that IRBLkp-K60, IRBLkm-Ts, IRBL1-CL and IRBL7-M having the resistance genes Pik-p, Pik-m, Pi1 and Pi7t, respectively were resistant to most of tested isolates. Compatible reactions were determined on the monogenic lines IRBLa-A, IRBLa-C, IRBLks-F5, IRBLzt-T, IRBLta-K1, IRBLta-CT2, IRBLb-B, IRBLt-K59, IRBL12-M, IRBL19-A, IRBLta2-Pi, IRBLta2-RE and IRBLta-CP1 carrying the resistance genes Pia, Pia, Pik-s, Piz-t, Pita, Pita, Pib, Pit, Pi12(t), Pi19(t), Pita-2, Pita-2, and Pita, respectively. The monogenic lines were higher susceptible than their donors. The additional blast samples will be collected in upland areas in Vietnam and identification of blast resistance genes in Vietnamese local rice varieties will be conducted. These have been carrying out under a Japan International Research Center for Agricultural Sciences (JIRCAS) research project, Blast Research Network for Stable Rice Production.




Proposal for a New International System of Differentiating Races of Pyricularia oryzae Cavara by Using

LTH Monogenic Lines


Nagao Hayashi1 and Yoshimichi Fukuta2


1 National Institute of Agrobiological Sciences (NIAS), 2-1-2 Kannondai, Tsukuba, 305-8602, Japan; Email: nhayash@affrc.go.jp

2 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, 305-8686, Japan

Email: zen@jircas.affrc.go.jp


Each country tends to use its own system for differentiating rice blast fungus races. The races differentiated by each system cannot be compared with each other, because there are differences in the genetic backgrounds of the rice cultivars used for differentiation in each country. Recently, 29 LTH(Lijiangxintuanheigu) monogenic lines, each containing one of 23 resistance genes, were developed at IRRI. The development of these monogenic lines has allowed us to prepare a new international differential system (IDS). We examined the components of the lines and designated an order for their adoption by the IDS. This IDS based on monogenic lines is expected to promote the utilization of rice blast resistance genes.

First, the response of each LTH monogenic line to more than 50 fungal isolates (including standard strains of blast and isolates from a different ecosystem in Japan) was recorded according to infection type (generally, 0–2 resistant; 3–5 susceptible). The monogenic lines that had the same reaction to all blast fungus were eliminated except one, as a result we chose 20 representative lines. Lines with the multiallelic loci Pii, Pik, Piz, and Pita were handled as a group. To delineate clearly the relationship between race code number and resistance gene, we divided the selected lines into groups with 3 lines, given the code numbers 1, 2, and 4 in accordance with Gilmour’s method. Each race code number has 9 digits divided by commas (i.e. NN,NNN,NNNN).

In the IDS, the names of the LTH monogenic lines are ordered as follows in the race codes. The 1st digit of the race code number is composed of LTH, IRBLa-A, and IRBLsh-S. The 2nd digit is IRBLb-B and IRBLt-K59; the 3rd digit is IRBLi-F5, IRBL3-CP4, and IRBL5-M, which are multiallelic or closely linked; the 4th and 5th digits are IRBLks-S, IRBLk-Ka, IRBLkp-K60, IRBL7-M, IRBLkm-Ts or IRBL1-CL, and IRBLkh-K3, which are multiallelic or closely linked; the 6th and 7th digits are IRBLz-Fu, IRBLz5-CA, IRBLzt-T, and IRBL9-W, which are multiallelic or closely linked, and the 8th and 9th digits are IRBLta-K1, IRBLta2-Pi, IRBL20-IR24, and IRBL19, which are also multiallelic or closely linked. We plan to use more fungal isolates to verify this system.




Prospect of Rice Blast in Thailand: From the Pathogen Population Structure to an Application in

Molecular Breeding


Pattama Sirithunya1, Tanee Sreewongchai2, Saengchai Sriprokhon2, Chanakarn Wongsaprom2, Apichart Vanavichit 2, 3, and Theerayut Toojinda 2


1 Rajamangala University of Technology Lanna, Science and Agricultural Technology faculty , Chiengmai, 50000, Thailand.

2 Rice Gene Discovery, National Center for Genetic Engineering and Biotechnology (BIOTEC), Kasetsart University, Kamphangsaeng, Nakornpathom, 73140, Thailand.

3 Agronomy Department, Kasetsart University, Kamphangsaeng Campus, Nakornpathom, 73140, Thailand.

Email:patttamasirithunya@yahoo.com


Rice blast, caused by Pyricularia grisea, is one of the most devastate diseases of rice. In planting season 2006, blast ruins rice-planting fields cost millions baht in Thailand. Hence, knowledge in population structure and pathogen genetic diversity are necessary for effective employment of resistant gene leading to a successful breeding programme. One thousand two hundred and eighty four isolates of P. grisea was collected from various hosts such as rice, wild relatives of rice, barley and weed throughout the country to study genetic structure using both conventional and molecular methods such as RAPD, AFLP, SSR and AVR markers. Analysis reflected sophisticated population structure of the pathogen in Thailand especially in north, north-east and central of Thailand and also suggested that gene pyramiding should be considered for mean of durable resistance. Meanwhile, two cultivars were identified to be good donor since they were able to resist infection of representative isolates of blast. Hence, these two cultivars, IR64 and Jao Hom Nin (JHN), were focused on type of resistant gene and location in order to use as resistant donors.

QTLs discovered in IR64 (chromosome 2 and 12) and JHN (chromosome 1 and 11) resulted in tremendous advantages in both line conversion and gene pyramiding. For example, integrations of QTLs from IR64 with susceptible cultivars such as RD.6 help to improve resistant qualities in short time while other good qualities were maintained. In addition, durable resistance is a desirable goal for breeders for prolongation any break in resistance by pathogenadaptation and mutation. Pyramiding is one of desirable method for durable achievement by combining more than one resistance to prolong resistance period. Therefore, each of 2 QTLs from IR64 and JHN were introgressed to target cultivar such as Kao Dok Mali 105 (KDML105) in response to increase efficiency against Thai blast. Inoculation of progenies with isolates that were able to attack KDML105, IR64 and JHN offered the results that proved broader resistance compared to parents. This is a meaningful success because it should be use as source of resistance against blast isolate in Thailand with an advantage in higher resistance and length of resistance that can be employed as resistant donor for all improved cultivars.




Pathogenic Specificity of Magnaporthe Grisea Species Complex Isolated from Several Weeds


Jaehyuk Choi1, Byung-Ryun Kim2, Jae-Hwan Roh2, In-Seok Oh2, Seong-Sook Han2, and Yong-Hwan Lee1


1 School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea

2 National Institute of Crop Science, RDA, Suwon 441-857, Korea


The blast fungus Magnaporthe grisea has been known as a species complex causing disease on a wide range of gramineous hosts, not only cultivated rice, but also other cereals and weeds; such as wheat, barley and grassy weeds. Fifty one isolates collected from ten weed species grown nationwide in Korea were screened to analyze their pathogenicity spectrum to rice using two susceptible cultivars, LTH and Nagdongbyeo. Pathogenicity assays gave an evidence of cross-infection between rice and crab grass (Digitaria sanguinalis). Sixty six percent of Digitaria isolates were found to infect on rice, while 23% of isolates from other weeds produced lesions on rice. Some Digitaria isolates maintained strong virulence for rice in repeated pathogenicity assays. The various spectrums in pathogenicity among Digitaria isolates exhibits limitation for this species complex to be divided into two discrete species, although recent study suggested that M. oryzae was distinct from M. grisea, the Digitaria isolates in point of phylogenetic view. In addition, phylogenetic analysis for our field collection indicated that some isolates had different origin from their isolation hosts as an evidence for cross-infection in fields and another lineage sharing both hosts’nucleotide change was found. Therefore the separation of this species complex would require more specific and precise measurement in pathological and geneological aspects.




Monitoring of Blast Races and Stable Use

of Blast Resistance in Rice


Shinzo Koizumi


Lowland Crop Rotation Research Team, National Agricultural Research Center for Tohoku Region, Yotsuya, Daisen, Akira, 014-0102 Japan

E-mail: skoizumi@affrc.go.jp


The breakdown of complete resistance to blast in rice cultivars in the 1960s led to the conduct of gene analyses of blast resistance in Japan, and currently complete resistance genotypes and levels of partial resistance of most of Japanese rice cultivars are already known. Differential systems of blast races have also been established, and monitoring of blast races had been done all over the country in 1976, 1980, 1994 and 2001. The results of monitoring blast races had been used for analyses of virulence genes in the pathogen population (Kiyosawa, 1980; Kiyosawa, 1986), and proportions of components in three Japanese released multilines for rice blast control, which consist of three to four near-isogenic lines with different complete resistance genes, have been also based on the monitoring results. Moreover, the results accelerated development of rice cultivars with high levels of partial resistance to blast because of its stability. Monitoring of blast races in areas where complete resistance genotypes of rice cultivars planted are not sufficiently known, is also useful in the selection of effective complete resistance genes for blast control, and inoculations with the isolated races reveal promising rice cultivars for blast control and levels of partial resistance in some of them.

Although monitoring of blast races has been conducted for rice blast control in several countries, sufficient quantitative analyses of blast race epidemics, which are necessary for stable use of blast resistance, have not been carried out. This is due to the fact that monitoring of blast races is very laborious and reliable epidemiological data on population interaction between host and pathogen is lacking. For stable use of blast resistance in rice, it is therefore necessary to develop easy means of monitoring blast races like the use of DNA markers tightly linked to avirulence genes in the fungus; accumulating epidemiological data on population interaction between host and pathogen; and construction of reliable epidemiological models, which can simulate increase of blast races in host populations with different resistance.




Pyricularia grisea, the Blast Fungus: Its Distribution and Occurrence in Philippine Ricefields


Fe A. dela Peňa* and Mary Ann S. Manalo


Crop Protection Division, Philippine Rice Research Institute (PhilRice), Maligaya, Science City of Muňoz 3119, Nueva Ecija, Philippines.

Email: delapenafe@yahoo.com, fadelapena@philrice.gov.ph



Collection was done in 2004-2006 to determine the distribution and occurrence of Pyricularia grisea, the blast fungus in Philippine ricefields. Leaves showing blast lesions or panicles with panicle/neck blast symptoms were gathered in Luzon (Nueva Ecija, Pangasinan, Isabela, Benguet, Camarines Sur, Tarlac, Bulacan, Ilocos Sur, Ilocos Norte and Oriental Mindoro), Visayas (Leyte, Iloilo, Aklan, Bohol and Biliran Province) and Mindanao (Agusan del Sur, Zamboanga del Sur, Zamboanga City and South Cotabato). Samples from the same variety within the same barangay/municipality though different ricefields were treated as one. The unknown varieties represent the collection with no available information. Due to time and financial limitations, collection was not done in all reported areas with rice blast occurrence. Distribution was mapped depending on the geographical location where the sample was collected.

A total of 146 blast samples were gathered from different varieties on which 79 were from Luzon, 31 from the Visayas and 36 from Mindanao. The most affected varieties were unknown varieties (23%) and PSB Rc82 (18%) in Luzon; NSIC Rc112 (48%) and unknown varieties (19%) in the Visayas; and NSIC Rc122 (22%), PSB Rc82 and unknown varieties (19%) and IR64 (17%) in Mindanao.

Moreover, P. grisea was collected from different rice ecosystems like irrigated lowlands, rainfed lowlands, uplands and cool-elevated areas. Of the 146 blast samples collected, 59% were from irrigated lowlands, 36% were from rainfed lowlands, 4% from uplands and the least from cool-elevated areas. This indicates that rice blast is no longer limited in occurrence in rainfed, upland and cool-elevated areas in the Philippines as it used to be. It has now become very common in irrigated areas particularly those planted with varieties known to have succumbed to the rice blast pathogen in recent years like PSB Rc14, PSB Rc82, NSIC Rc112, NSIC Rc122 and IR64 that had intermediate to resistant reaction when released as varieties. As proven, resistance to P. grisea can be broken down within several years after the variety is released due to increase in new blast races virulent to the resistance. It is therefore suggested that these varieties should not be planted in hot spot areas.




Genetic and Phenotypic Diversity of Magnaporthe grisea from Leaf and Panicles of Rice in Farmers’ Fields


G. B. Da Silva1,2,4, Anne S. Prabhu3,4, Maria da G. Trindade3, Leila G. Araújo3,4 , Marta C. Filippi3,4 and L. Zambolim1,4


1 Federal Rural University of Amazonia, Av. Pres. Tancredo Neves, 2508, 66077-530, Belém, PA,Brazil.; 2 Federal University of Viçosa; 3 Embrapa Arroz e Feijão, 4 Bolsista Cnpq.

E-mail: gisele.barata@ufra.edu.br


Genetic and phenotypic structure of Magnaporthe grisea, both among and within the populations, retrieved from leaves and panicles of two upland rice cultivars was determined. For establishing monoconidial isolates of M. grisea, leaf and panicle blast samples were collected from eight farmers’ fields, four from cv. Bonança and four from cv. Primavera, in the State of Goias, Brazil, during two consecutive rice growing seasons. The pathotypes IB-41 and IB-9 were predominant in both leaf and panicle isolates of Bonança and IF-1 in populations of Primavera. Of the 35 and 27 pathotypes identified among isolates obtained from cvs. Bonança and Primavera, 42.8% and 66.6% were common in both leaf and panicle subpopulations, respectively. A total of 15 pathotypes which were not in leaf were found in low frequency in panicles of cv. Bonança. The analysis of variance of phenotypic virulence data based on 32 genotypes showed high variability within population of each cultivars. The effect of cultivar on population structure was significant. There was no significant change in virulence pattern of isolates from leaves and panicles, independent of collection site and cultivar. The molecular characterization of isolates was done employing the rep-PCR analysis with two primer sequences from Pot2. The genetic analysis showed the presence of 103 haplotypes of cv. Bonança and 49 of cv. Primavera. The migration of pathotypes from leaves to panicles in each field was 70.8% and 36.6% for the cvs.Primavera and Bonança, respectively. Rep-PCR analysis of 538 isolates of M. grisea showed a high genotypic diversity in both leaf and panicle pathogen population. There was no correlation between virulence pattern and molecular analysis.




Evaluation of Stress Induced Genetic Variations in Magnaporthe grisea by Inter Simple Sequence

Repeats (ISSR) Technique


Sonia Chadha and T. Gopalakrishna


Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India

Email:soniachaddha@yahoo.com

Magnaporthe grisea, the causative agent of rice blast disease has been a topic of debate for its high degree of genomic variability and instability. The studies on various factors contributing to the high genetic variability are important in the blast disease management. The important question is how the pathogen alters its genome in response to selective pressures? Various multistep genetic processes are likely to be responsible for the genomic instability. In this study, stress induced genetic variations were detected using inter simple sequence repeat (ISSR) technique. For the analysis, M. grisea was exposed to copper and thermal stresses. Screening with 85 ISSR primers to obtain DNA fingerprints from control and stress treated samples resulted in the selection of 20 ISSR primers. The selected primers based on di-, tri- and tetra-nucleotide repeats were used for further analysis. In samples exposed to copper, the mutation rate was observed to increase in a dose dependent manner. The average genome template stability was found to be 92.1 % and 80.7 % for samples exposed to copper and thermal stress respectively. Bands specific to stress treatments were observed. Out of the 20 ISSR primers, 8 primers yielded large number of stress induced bands. These primers by analogy can be regarded as stress indicative primers. Results suggest that environmental stresses are one of the key factors contributing towards the genomic instability of Magnaporthe and the effects of these stresses can be easily evaluated by ISSR technique.




Population Shift in Magnaporthe oryzae at Screening

Sites in the Philippines


I. Ona1,*, S. Castro2, A. Tagle1,*, E. Ardales2, P. Goodwin3, S.S. Han4, J.H. Roh4, C.M. Vera Cruz1,*


1 Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines

2 University of the Philippines at Los Banos, College, Laguna, Philippines

3 Department of Environmental Biology, University of Guelph, Guelph, ON, Canada N1G2W1

4 Crop Environment and Biotechnology Division, National Institute of Crop Science, RDA, Suwon 441-857

* Corresponding authors:E-mail: cveracruz@cgiar.org and iona@cgiar.org


To ensure effective screening of rice germplasm against blast, virulence and genetic variation of the rice blast pathogen, Magnaporthe oryzae, was studied using 130 isolates collected in 2005 from three screening sites at the International Rice Research Institute (IRRI) and compared with previous populations collected 20 years ago at these sites. Virulence analysis was done using 23 blast monogenic lines with specific blast resistance (R) genes. Molecular characterization was done using Pot2 and URP2 primers that amplify repetitive elements. Assessment of virulence on 23 monogenic lines showed that 30 % of the isolates were virulent to more than 12 monogenic lines. Over 50% of the isolates were virulent to monogenic lines with Pia, Pita, Piz-t, Pib, Pik-s, Pi11(t), Pi19(t), Pi20(t), and Pi12(t) genes. Genetic diversity estimates was higher at IRRI experiment station (0.79 Pot2 and 0.95 URP2) than at the IRRI Blast Nursery (IRRI-BN) (0.66 Pot2 and 0.93 URP2) and Cavinti (0.74 Pot2 and 0.92 URP2) screening sites. The highest number of haplotypes (13) was found at IRRI experiment station while the IRRI-BN and Cavinti screening sites had 9 and 8 haplotypes, respectively. Cluster analysis revealed that the isolates from IRRI experiment station grouped together whereas the isolates collected from IRRI-BN and Cavinti screening sites clustered together at >80% similarity level by UPGMA. Very few haplotypes were shared among the three screening sites. Similar results were obtained from the analysis of 145 M. oryzae isolates collected from different provinces in the Philippines in 2004 ?2005 where high genetic diversity (0.89 to 0.94) and completely different virulence patterns were observed. Haplotypic analysis of M. oryzae isolates collected 20 years ago (classical isolates) and isolates collected after 2002 (new isolates, particularly those from the screening sites) showed two distinct groups suggesting a shift in the M. oryzae populations. The virulence pattern on blast monogenic lines of the new isolates was also different from the classical isolates. To assess the effectiveness of major R genes against the new and genetically diverse population of M. oryzae, we are evaluating the different monogenic lines by sequential planting (polycyclic tests) for 5-7 cycles under natural infection at IRRI-BN and artificial inoculation in the screenhouse.




3


Host Defense Signaling and Gene Expression




Sti1/Hop, a Hsp90 Cochaperone, Is a Novel Regulator in Defense Signaling in Rice


Letian Chen1, Nguyen Phuong Thao1, Ayako Nakashima1, Kenji Umemura2, Akira Takahashi3, Ken Shirasu3, Tsutomu Kawasaki1, and Ko Shimamoto1


1 Plant Molecular Genetics, NAIST , 8916-5 Takayama, Ikoma 630-0101, Japan;

2 Meiji Seika Ltd., 5-3-1 Chiyoda, Saitama 350-0289, Japan;

3 The Sainsbury Lab., John Innes Centre, Colney Lane, Norwich NR4 7UH, UK. Email:lotichen@naist.jp


Basal and R gene-mediated resistance are the most common systems for plant defense against pathogen attacks. Differences exist between the two systems, but host reactions are qualitatively similar. Rac GTPase, RAR1, SGT1 and Hsp90 are characterized as important components in plant defense signaling. Unveiling the relationship and context of important components will lead to a better understanding of resistance signal transduction. Here, we identified novel components, Sti1/Hop and RWD, by Rac1 affinity chromatography and characterized the role of Stil/Hop in disease resistance signaling. We found Sti1-knockdown plants were compromised to virulent fungus, while Sti1-overexpressing plants became more resistant. R gene Pi-a-mediated pathway was also affected by the loss of Sti1. Our data showed Sti1 interacted with Hsp90, RWD interacted with SGT1 and RAR1, while Rac1 interacted with both Sti1 and RWD.

Our results indicate Stil/Hop is a novel player in defense signaling, thus extending the existing tripartite interaction of RAR1, SGT1 and Hsp90 to a gene network including Sti1, Rac1, and RWD. Furthermore, we hypothesize these components may form a dynamic complex termed as defensome. The defensome may be required for both basal and R gene-mediated resistance and its compositions may vary depend on different triggering signal and signaling stage.




Isolation and Characterization of BWMK1-Interacting

Proteins in Rice


Liangying Dai, Xionglun Liu, Jia Gao, Yajun Hu, Sujun Pan, Jinling Liu, Jun Wu, Guoliang Wang*


Rice Genomics Laboratory, Hunan Agricultural University, Changsha, Hunan 410128, China

*Corresponding author: Email: glwang@hunau.net


Mitogen-activated protein kinases (MAPKs) play important roles in various developmental processes as well as biotic and abiotic stress responses. The first reported rice MAPK gene BWMK1 is induced by blast fungus (Magnaporthe grisea) infection and by mechanical wounding. In this study, we used the yeast two-hybrid system to identify the proteins that interact with BWMK1. Rice cDNA library was screened using BWMK1 cDNA as a bait. Nine positive clones were isolated and sequenced. DNA database searching showed that they encode novel proteins such as myo-inositol phosphate synthease, unknown protein, hypothetical protein, putative phosphate transporter, WD-40 repeat protein-like, 5-methyltetrahydropteroyltriglutamate-homocysteineS-methyltransferase and N-acetyl glutamate kinase 2. RT-PCR analysis indicated that most of the genes are induced by M. grisea infection. RNAi and overexpression constracts for these genes were generated and transformed into the japonica rice cultivar Nipponbare. Functional analysis of the BWMK1-interacting genes will provide valuable information for the function of the BWMK1 associated proteins in rice.




This work supported by National Nature Foundation of China (.30470990) and Scientific Research Fund of Hunan Provincial Education Department (04A024).



Integrated Approaches Towards Understanding

the Rice Blast System

Elsa Ballini1, Emilie Vergne1, Henri Adreit1, Véronique Chalvon1, Loic Fontaine1, Elisabeth Fournier1, Hirsch1, Thomas Kroj1, Corinne Michel1, Joelle Milazzo1, Nabila Yahiaoui2, Marc-Henry Lebrun3, Jean-Benoît Morel1, Didier Tharreau1, Jean-Loup Notteghem1

1 BGPI unit, CIRAD-INRA-SupAgro, Campus International de Baillarguet TA 41/K;

2 DAP unit, CIRAD-INRA-UMII, Campus de Lavalette, TA 40/03, 34398 Montpellier cedex 5, FRANCE;

3 CNRS Lyon, UMR 2847, CNRS-BAYERCROP SCIENCE, 14 rue Pierre Baizet BP 9163, 69263 Lyon cedex 9, FRANCE.

Our group has focused its interest on the rice blast system since several years. Both plant and pathogen biology is studied in order to establish basic knowledge on this plant-pathogen model. M. grisea populations are studied to identify virulence evolution in natural situations. Classical genetic approaches are taken to identify pathogen avirulence genes such as the unusual ACE1 gene (1) and genes required for infection (2). Our aim now is to identify pathogen proteins effectors that are injected into the plant apoplast or cell. On the plant side, different forms of resistance are studied: gene-for-gene resistance (Pi33/ACE1), partial resistance, non-host resistance and susceptibility. Major R-genes like Pi33 are studied (1). The regulation of defence response upon infection is also studied at the transcriptional level using DNA chips (4) and also at the post-transcriptional level by studying small RNAs expression. This allowed the identification of a large part of the rice “defensome”. Using RNAi or insertion mutant lines, we could demonstrate the role of several of these genes into resistance. We also proceeded to forward genetic screens in rice in order to identify rice genes that regulate disease resistance. Several genes were potentially identified, including genes that control cell death. Finally, we developed several resources: more than 2500 M. grisea isolates were collected and characterized, one Tilling and several mutant rice populations were built and a database centralizing information on R-gene, QTL and defence gene expression was inserted into the OryGenesDB web site. These different converging approaches are likely to increase our knowledge and tools to tackle this economically important disease.

(1) Bohnert HU, Fudal I, Dioh W, Tharreau D, Notteghem JL, Lebrun MH. A putative polyketide synthase/peptide synthetase from Magnaporthe grisea signals pathogen attack to resistant rice. Plant Cell. 2004 Sep;16(9):2499-513.

(2) Gourgues M, Brunet-Simon A, Lebrun MH, Levis C. The tetraspanin BcPls1 is required for appressorium-mediated penetration of Botrytis cinerea into host plant leaves. Mol Microbiol. 2004 Feb;51(3):619-29.

(3) Ballini E, Berruyer R, Morel JB, Lebrun MH, Notteghem JL, Tharreau D. Modern elite rice varieties of the 'Green Revolution' have retained a large introgression from wild rice around the Pi33 rice blast resistance locus. New Phytol. 2007;175(2):340-50.

(4) Vergne E, Ballini E, Marques S, Sidi Mammar B, Droc G, Gaillard S, Bourot S, DeRose R, Tharreau D, Notteghem JL, Lebrun MH, Morel JB. Early and specific gene expression triggered by rice resistance gene Pi33 in response to infection by ACE1 avirulent blast fungus. New Phytol. 2007;174(1):159-71.

(5) Droc G, Ruiz M, Larmande P, Pereira A, Piffanelli P, Morel JB, Dievart A, Courtois B, Guiderdoni E, Perin C. OryGenesDB: a database for rice reverse genetics. Nucleic Acids Res. 2006 Jan 1;34(Database issue):D736-40.




Constitutive Expression of Rice Defence Genes Correlates with Partial Resistance Against Magnaporthe grisea


Emilie Vergne1, Didier Tharreau1, Marc-Henry Lebrun2, Jean-Loup Notteghem1, Jean-Benoît Morel1


1 INRA et CIRAD Montpellier, UMR BGPI, CIRAD Campus International de Baillarguet TA 41/K, 34398 Montpellier cedex 5, France

2 CNRS Lyon, UMR 2847, CNRS-BAYERCROP SCIENCE, 14 rue Pierre Baizet BP 9163, 69263 Lyon cedex 9, France

Email: jbmorel@cirad.fr


Besides the development of the gene for gene resistance, one can observe a limitation of the infection by the installation of a resistance of more or less high level called partial resistance. The mechanisms of partial resistance are poorly understood despite its importance for the breeding programs. We used the rice/Magnaporthe grisea system to decipher the mechanisms involved in partial resistance. We hypothesised that some preformed defence could explain some parts of partial resistance. The expression of more than 20 regulatory and classical defence genes was measured in a set of well-characterized cultivars (japonica and indica) representative of rice diversity and partial resistance. We demonstrated that preformed defence as measured by the expression of these genes accounts for almost 50% of partial resistance, in particular in japonica type of cultivars. The indica-type of cultivars seem to use a different set of genes and regulation systems to control partial resistance. The OsMAPK6 negative regulator of defence gene expression could be a major regulator of this phenomenon. A quantitative analysis of constitutive expression of defence genes allowed the identification of eQTLs representing regions of the rice genome involved in the control of constitutive expression of several defence genes. In contrast, salicylic acid levels did not correlate with consitituve expression of defence. This work demonstrates that constitutive defence has been underscored in the past and opens new opportunities for breeding programs.




Evaluation of SNP Markers in Candidate Defense Genes for Selection of Blast Resistance in Philippines

Rice Breeding Programs


Loida M. Perez1, Haizel M. Pastor1, Teodora E. Mananghaya1 and

Noriel M. Angeles2


1 Plant Breeding and Biotechnology Division, Philippine Rice Research Institute, Mgaya, Science City of Muñoz, 3119 Nueva Ecija, Philippines

2 International Rice Research Institute, Los Banos, 4031 Laguna, Philippines

Email: lmoreno_perez@yahoo.com, lmperez@philrice.gov.ph


In wet season of 2005, severe infection of rice blast disease caused by Magnaporthe oryzae was observed in NSIC Rc112, a popular irrigated rice variety in the Philippines. Currently the disease is becoming severe in other rice growing areas particularly in upland and rainfed lowland environments. Rapid introduction of genes associated with blast resistance into breeding populations is important to avoid escalation of the disease problem in the Philippines. Previous studies showed that several defense-related genes identified in the variety Shan-Huang-Zhan-2 (SHZ-2) were associated with blast resistance. This study aimed to apply SNP markers in genes encoding oxalate oxidase (OsOXO), oxalate oxidase-like (OsOXLP) to track the introduction of quantitative resistance from SHZ-2 into local popular varieties. Using the modified agarose TILLING (Targeting Induced Local Lesions in Genomes) approach, seven SNP markers in OsOXO3, OsOXLP6, OsOXLP7, OsOXLP9, OsOXLP10, OsOXLP8-1 and OsOXLP8-2 were evaluated in 35 popular rice cultivars (recurrent parents) and SHZ-2. Results indicated that OsOXLP7 was generally polymorphic between the recurrent parents and SHZ-2, representing a potential marker for tracking the gene clusters of OsOXLP on chromosome 8. In AR32-19-3-3 (NSIC Rc142), a variety developed for bacterial blight resistance through marker assisted selection, OsOXLP8-2 showed polymorphism among the markers tested. Additional SNP markers are being developed for other candidate defense genes (e.g., heat shock protein, kinase) for testing association with blast resistance in advanced breeding populations. The potential use of SNPs in MAS for blast resistance in Philippine rice breeding programs will be further discussed.

Keywords: Rice blast, SNP, agarose TILLING, oxalate oxidase, heat shock proteins




Functional Analysis of the MOM-Like Gene Spin3 that is Involved in the Spl11-mediated

Ubiquitination Pathway in Rice


Xiao-shan Zeng 1, Liang-ying Dai 1, Li-Rong Zeng 2, Park Chan Ho2, Xiong-lun Liu 1, Yuese Ning 1, Yajun Hu 1, Jingling Liu 1, Jun Wu 1, Suhua Wang 1, Gao Jia1, Guo-liang Wang1*


1 Rice Genomics Laboratory, Hunan Agricultural University, Changsha 410128, China;

2 Department of Plant Pathology, the Ohio State University, Columbus 43210, USA


Spl11 encodes U-box E3 ligase and plays an important role in cell death and defense response in rice. A MOM-like protein named Spin3 was screened in the yeast two hybrid screens using Spl11’s ARM repeat domain as the bait. RNAi and Overexpression construction were generated to determine the function of Spin3. Two RNAi constructs were made using the pANDA vector with the sequences in the MOM-conserved region and 3’ Spin3 specific region, respectively. For the overexpression (OX) construct, the Spin3 full-length cDNA was inserted into the binary vector pubix.nc1300.ntap.gck. All constructs were delivered into the japonica rice cultivar Nipponbare by Agrobacterium-mediated transformation procedure. To investigate the function of Spin3 in defense response to rice pathogens, the RNAi and OX transgenic plants and the wild type plants were inoculated with Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae isolates. Two-month old Nipponbare and transgenic plants were inoculated with Xoo strain J22 (0.5 OD at 590 nm). Three-week old wild type Nipponbare and transgenic plants were inoculated with four M. oryzae isolates. Preliminary disease evaluation showed that OX Spin3 transgenic plants conferred enhanced resistance to both Xoo and M. oryzae isolates. In contrary, no significant difference was observed between the wild type and the RNAi transplants. In addition, the OX transgenic plants had a 2.2 times higher PAL activity than that of the wild type. These results suggest that Spin3 might play a role in defense response to rice pathogens.

Keywords: SPIN3, RNAi , Overexpression, Agrobacterium-mediated transformation, Resistance




4


Host Resistance, QTLs and Defense Gene Identification and Characterization




Molecular Evolution of Rice Oxalate Oxidases--Candidate Genes for Quantitative Resistance to Rice Blast


G. Carrillo1, P. Goodwin2, M. Reveche1, J.E. Leach3, H. Leung1 and

C.M. Vera Cruz1


1 International Rice Research Institute, DAPO Box 7777, Metro Manila Philippines;

2 University of Guelph, Guelph, N1G 2W1, Canada;

3 Colorado State University,Fort Collins, 80523-1177, USA.


Oxalate oxidases in cereals have been implicated to play a role in defense response to pathogen infection. While many studies have focused on the rapid evolution of major R genes involved in pathogen recognition, relatively little is known about the molecular evolution of defense genes in plant-pathogen coevolution. Here, we analyzed the molecular changes in members of rice Oxo genes mapped to chromosome 3 that are associated with resistance to blast. There are four tandemly duplicated oxalate oxidases (OsOxo) in chromosome 3 as well as 70 related sequences forming the cupin superfamily of proteins in the rice genome. These genes exhibit > 90% similarity at the nucleotide and amino acid level. EST clones were identified only for OsOxo1, OsOxo3 and OsOxo4. Expression analysis using resistant and susceptible advance backcross lines of Vandana x Moroberekan showed that only OsOxo4 is expressed during rice-Magnaporthe grisea interaction. Analyses of the OsOxo from 62 rice cultivars show that synonymous substitution rates often exceed nonsynonymous rates suggesting that purifying selection is the major factor maintaining Oxo protein homogeneity. The average frequency of SNPs was one per 86, 32, 114 and 32 bp across the coding region for OsOxo1, OsOXo2, OsOxo3, and OsOxo4, respectively. Haplotype and nucleotide diversities were moderate with an average of Hd = 0.755 ?0.031 and = 0.004 ?0.015, respectively. The results obtained from phylogenetic analysis reveal that every Oxo gene is distinct from each other despite the high similarity such that genes from all the 62 cultivars cluster accordingly. However, within the gene cluster, there was no distinct grouping corresponding to rice isozyme groups. OsOxo1 and OsOxo4 were most homogeneous among the 62 cultivars with only 1 and 5 amino acid changes, respectively, across the coding region. For OsOxo2, two insertion sites result in a stop codon in 11 cultivars. A transposon-insertion in the 5’end of the 1kb-upstream region of OsOxo2 is common in all Aus/Boro tested. There was no clear association between SNPs and reaction to rice blast, but this may be due to epistatic effect of major resistance genes present in the cultivars. Overall, our data suggest that, contrary to the divergent evolution of R-genes, oxalate oxidases in rice are under purifying selection. This observation is consistent with the presumed roles of oxalate oxidases in general defense against pathogens.




Construction of Near-isogenic Isolates of Magnaporthe grisea for Identification of Blast Resistance Genes


Yueqiu He1,2, Huiping Zhou2, Yixin Wu2, Shunde Li3, Gento Tsuji4 and

Yasuyuki Kobo4


1 Faculty of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, China;

2 Faculty of Plant Protection, YAU, Kunming 650201, China;

3 Agriculture Bureau of Yuxi City, Yunnan Province, China;

4 Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522, Japan


Rice blast is a destructive disease worldwide. Deployment of resistant cultivars is the most effective control although other methods may reduce its damage. Resistance (R) genes of rice and avirulence genes of the pathogen, Magnaporthe grisea determine whether the method is successful. R genes are identified by isolates of the pathogen and vice versa, avirulence genes by rice genes according to gene-for-gene hypothesis. Because R genes are identified in different laboratories with different isolates, an R gene is referred to different names or different R genes are designated to the same name. Therefore, experimental data are difficult to be used directly between laboratories, and new R genes are identified difficultly in a sense, especially, a new R gene should be designated only after a lot of allelic tests have been done, which are backbreaking jobs.

We used an isolate, CY2, avirulent to 500 cultivars including Lijiangxituanheigu (LTH), a Japonica cultivar, originated from Lijiang, Yunnan Province with non-R gene considered universally, the CO39 near-isogenic lines bred by the International Rice Research Institute, the LTH near-isogenic lines bred by Ling and commercial and traditional cultivars. We mutated CY2 with Agrobacterium tumefaciens C58C1 and transposon, pBIG2RHPH2 harboring the hygromycin B gene (HPH) and obtained more than 8000 mutants screened by hygromycin medium. Inoculation of the mutants onto 42 cultivars known R genes verified that the mutants also mutated in pathogenicity. We isolated the pathogenic mutants by collection of typical lesions from rice leaves and single-spore-isolation procedure. Sixty pathogenic mutants were obtained, which overcame 20 R genes. Some mutants mutated in an avirulence locus, some mutated in more than one locus. Around 50% isolates mutated in avirulence Pi-ta2 locus, it seemed the mutation has “hot spot” by Agrobacterium tumefaciens mediated transformation. Southern blotting indicated that most of mutants have one copy of the transposon insert, some have two or three inserts. Because the pathogenic mutants come from a common isolate, CY2, they have the same genetic background except for the parts of DNA fragment disruption by transposon insert, they can be used to identify R genes of rice and save a lot of labor and money with fine accuracy.


This work was funded by the National “863”Program2002AA245041),the Natural Science Foundation Program 30260006and the“948” Program of Ministry of Agriculture(2006-G61.




A New Member of the Blast Resistance Gene Family Pi2/Pi9 was Identified in the Indica Rice Cultivar 28-zhan


Xiaoyuan Zhu 1,3,* , Shen Chen 1, Jianyuan Yang 1, Shaochuan Zhou 2,

Ling Wang 3, Liexian Zheng 1, Qinghua Pan 3,*


1 Plant Protection Research Institute;

2 Rice Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 50640, China;

3 Laboratory of Plant Resistance and Genetics, College of Resources and Environmental Sciences, South China Agricultural University, Guangzhou 510642, China

*Corresponding author: Zhu Xiaoyuan, E-mail:gzzhuxy@tom.com; Pan Qinhua , E-mail: panqh@ scau.edu.cn.


28-zhan is an indica cultivar (cv.), which has been widely used as the major blast resistant donor in rice breeding program in Guangdong province China. Most of the cvs derived from this donor show the broad-spectrum and stable resistance to rice blast fungus. To identify the resistance (R) gene(s) controlling the broad-spectrum resistance, two isolates, GD288 and GD193 is coercive to the Pi9 carrier and the Pi2/Piz-t carriers, respectively, were selected for gene mapping using 292 recombinant inbred lines (F8 RILs) derived from a cross between Lijiangxintuanheigu (LTH) and 28-zhan.The consistent phenotypes and segregation ratio (1R:1S) were/was observed in the RIL population when challenged with both isolates, indicating that the same R gene was detected in the cv. 28-zhan. The R gene locus was defined into the region located the Pi2/Pi9 family on the clone AP005659 based on the linkage analysis using PCR-based markers. Since the R gene in cv. 28-zhan was identified using the Pi2 family-compatible isolates, it may be a new member of the Pi2/Pi9 family, and is, therefore, designated as Pi42(t).


This project was supported by grants from Guangdong Natural Sciences Foundation (04101156) and National “863” Project (2006AA100101)




Genetic Diversity in Malaysian Traditional Rice Genotypes with Resistance to Blast, as Revealed by

Microsatellite Markers


Chee Fong Tyng1, Kalai Vani A/P Maniam2, Nurulashikin Binti Abd Hadi2 and Mariam Abd Latip1


1 School of Sustainable Agriculture

2 School of Science and Technology, Universiti Malaysia Sabah, Locked Bag 2073

88999 Kota Kinabalu Sabah, Malaysia

Email: ftchee@ums.edu.my


Rice blast, caused by the fungal pathogen Pyricularia grisea, has been one of the most serious diseases affecting rice growing regions around the world. Development of blast resistant cultivars has always been a high priority objective in rice breeding programmes. In Malaysia, several traditional rice genotypes have been found to be resistant to blast disease. This study was carried out to examine the genetic variation in eight blast resistant traditional varieties using microsatellite markers. Twenty one rice microsatellite markers were randomly chosen for analyses, and of these, 17 showed polymorphic bands (81%). The highest allele frequency (1.000) was detected at markers RM207, RM230, RM247 and RM313, while the lowest allele frequency was shown by marker RM21 (0.1250). A total of 44 alleles were detected among the eight rice samples, and the number of alleles ranged from one to four with a mean 2.0952. Mean heterozygosity and genetic distance obtained from this study were 0.0238 and 0.6546, respectively. A dendrogram generated using NTSYS showed three major clusters with no specific classification. This finding showed a low DNA variation among the samples but nonetheless this information could be useful for future studies in identification of markers associated with blast disease in the plants.




Analysis of Resistance Genes to Blast in Known Korean Germplasm including Weedy and Land Race Rices


Young-Chan Cho1*, Soon-Wook Kwon1, Ji-Ung-Jeong1, Jae-Hwan Roh1, Myung-Kyu Oh1, In-Bae Choi1, Jong-Seong Jeon3, Im-Soo Choi1, Kishirod Jena2, Yeon-Gyu Kim1, Tae-Soon Kwak3, Sae-Jun Yang1, Young-Tae Lee1


1. National Institute of Crop Science, RDA, Korea,

2. IRRI-Korea Office, NICS, RDA, Korea,

3. Kyung-Hee Univ., Yongin, Korea,

4. Sangji Univ., Wonju, Korea

Corresponding Author: Tel. 031-290-6666, E-mail: yccho@rda.go.kr


Rice blast continues to be a potentially devastating disease of rice, affecting yield and decreasing its quality. It is necessary to look for novel resistance gene(s) for blast that can express a broad spectrum of resistance in diverse environmental conditions. In this study, we have made an attempt to search for the new gene/QTL from Korean weed rice germplasm, GL33. A major QTL qBL4 was mapped to a spanning 1.3cM region at RM5586-RM6679 on chromosome 4, and explaining 43.5-53.1% and 26.1-28.1% of total phenotypic variation for blast nursery test and isolate inoculation by the GL33 allele, respectively. This major QTL was designated as Pi41(t) gene. Two SSR markers RM5586 and RM6679 were landed at a BAC clone OSJNBb0012E08, and RM5586 was specified to the locus Os04g32940 including the LRR family protein related to disease resistance gene through BLASTN analysis. We were developed a few candidate R gene markers based on the sequence downloaded from the RGP web site for fine-scale mapping the Pi41(t) locus. A few promising BC3F2 lines based on backcross method to develop Pi41(t)(QTL)-NIL were selected and would be evaluated for the resistance effect to diverse spectrum of blast in the field and by isolate inoculation.




Identification of Genes for Durable Blast Resistance Using an Advanced Backcross Population in Rice


Hong-Guang Ju1, Ju-Won Kang1, Jae-Hwan Roh2, In-Seok Oh2, Seong-Sook Han3, Sang-Nag Ahn1*


1 College of Agricultule & Life Sci, Chungnam National University, Daejeon, Korea

2 National Institute of Crop Science, RDA, Suwon, Korea

3 National Institute of Agriculture Biotechnology, RDA, Suwon, Korea

Corresponding Author: Tel. 042-821-5728, e-mail: ahnsn@cnu.ac.kr


Rice blast disease caused by the filamentous ascomycete fungus Magmaporthe grisea is one of the most destructive diseases of rice worldwide. In this study, we have made an attempt to search for the new genes/QTL from an African upland cultivar, Moroberekan. A total of 117 BC3F5 advanced backcross lines were developed from a cross between Ilpumbyeo as a recurrent parent and Moroberekan. 134 SSR markers were used to map and characterize quantitative trait loci (QTLs) for traits related to blast resistance, agronomic performance and grain quality. QTL analysis identified 34 significant QTLs for 19 traits with a range of 1- 4 QTLs detected per trait. Three major QTLs for the blast resistance in the blast nursery test were detected on chromosomes 4, 5, and 7. The percentage of phenotypic variation associated with single QTLs for blast reaction at the nursery ranged from 9.3 to 35.9%. Based on the agronomic performance, blast reaction and grain quality, 27 BC3F5 lines were selected and tested for the durability using a newly designed method (Hahn, in preparation). Ten of the lines showed similar level of durable resistance to the check cultivar, Palgongbyeo. Also, five promising lines nearly isogenic to Ilpumbyeo except for blast resistance were evaluated at the preliminary yield trial. These lines with enhanced blast resistance did not show any differences from the recurrent parent, Ilpumbyeo in terms of days to heading, culm length, grain weight, amylose content and other grain quality traits. The lines will be subjected to rigorous evaluation for traits of economic importance such as quality in multi-location trials to provide the foundation for variety release in the future.

To develop SSR markers linked to a blast resistant QTL detected on chromosome 4, ten each resistant and susceptible RILs were screened with SSR markers located near the detected QTL. Two markers RM3843, RM5709 covering 1.7cM were tightly linked to the gene and would be useful in MAS program. To isolate defense genes in Moroberekan, a novel DEG gene fishing system was employed. In the analysis, a number of candidate defense genes were identified. The result revealed that many rice genes are up-regulated and down-regulated in response to rice blast. To develop a rice cultivar with enhanced blast resistance, some of defense genes have been transformed or are currently being transformed into Dongjinbyeo via Agrobacterium mediation.





Identification of Blast Resistance Genes in IRRI-bred Rice Varieties by Segregation Analysis Based

on a Differential System


Nobuya Kobayashi1, Leodegario A. Ebron1, Daisuke Fujita1, and Yoshimichi Fukuta2


1 International Rice Research Institute (IRRI), Los Baños, Philippines, E-mail: NK: n.kobayashi@cgiar.org, Email: LE: l.ebron@cgiar.org, DF: d.fujita@cgiar.org; n.kobayashi@cgiar.org

2 Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Japan. E-mail: zen@jircas.affr.go.jp


A differential system for rice blast disease consists of standard blast isolates (Pyricularia grisea Sacc.) and differential rice varieties (Oryza sativa L.) with known resistance genes. Monogenic lines for blast resistance have been newly developed at the International Rice Research Institute (IRRI) as international differential varieties and differential blast isolates from the Philippines were selected. Using a differential system based on the gene-for-gene theory, blast resistance genes were estimated in the IRRI-bred elite rice varieties. At least seven kinds of resistance genes -- Pi20, Pita, Pik* (one of the Pik allele genes except Pik-s), Pik-s, Pib, Piz-t, and Pii or Pi3 -- were estimated in 42 rice varieties on the basis of reaction patterns to 14 standard blast isolates. These were compared with those of the blast monogenic lines. To confirm this gene estimation, genetic analysis was done using segregating populations derived from crosses between the IRRI varieties and a susceptible Indica-type variety, CO 39. The BC1F2 populations (with CO 39 as recurrent parent) were segregated for reaction to the specific standard isolates. Furthermore, the estimated genes were confirmed by allelism test against the blast resistance monogenic lines. As a result of the segregation analysis of 10 of 42 IRRI-bred varieties, seven genes -- Pi20, Pita, Pik*, Pia, Pib, Pik-s -- and Piz-t, were identified. Genes Pia, Pib, Pik-s, and Piz-t were not estimated by reaction patterns to the blast isolates but identified by genetic analysis in some varieties. The effectiveness of the differential system, which is based on conventional methods and which does not require advanced facilities, is discussed as a fundamental tool to provide essential information to develop breeding programs for blast resistance.




A Review of Rice Blast Resistance Genes and QTLs.

Elsa Ballini1, Jean-Beno Morel1, Brigitte Courtois2, Jean-Loup Notteghem1, Didier Tharreau1


1 UMR BGPI, CIRAD-INRA-SupAgro.M, TA A54/K, 34398 Montpellier Cedex, France. ballini@ensam.inra.fr

2 UMR DAP, CIRAD, TA A96/03, 34398 Montpellier Cedex, France.

E-mail :ballini@supagro.inra.fr


With the completion of rice and Magnaporthe oryzae genome sequences, rice blast disease has strengthened its position as a model to study plant pathogen interactions in monocotyledons. The genetic study of blast resistance in rice is an old story as first studies were established as early as 1917 in Japan. Despite these long lasting studies, examples of varieties with durable resistance are poorly documented. The lack of durably resistant varieties is, to some extent, due to our limited knowledge of resistance mechanisms. A rising number of blast resistance genes and Quantitative Trait Loci have been described and some of them were characterized during the last 20 years. Can we go now a step further to better understand genetics of blast resistance by combining all of these results? Is this knowledge appropriate and sufficient to improve breeding for durable resistance? By curating bibliographics references we identified 85 blast resistance genes and ca. 350 QTLs that we mapped on the rice genome. These data provide a useful update on blast resistance genes and new insights to formulate hypothesis about the molecular function of blast QTLs with special emphasis on QTLs for partial resistance. All these data are available on the OrygenesDB database (http://orygenesdb.cirad.fr/index_fr.htm).




Cloning and Sequencing Homologs of Pi9 from Oryzae sativa Cultivars Based on Allelism Analysis


Xionglun Liu1#,Jun Wu 1#, Yajun Hu 1#, Suhua Wang 1, Jingling Liu 1, Nan Jiang 1, Jia Gao 1, Liangying Dai 1, and Guo-Liang Wang 1,2*


1 Rice Genomics Laboratory, Hunan Agricultural University, Changsha, Hunan 410128, China

2 Department of Plant Pathology, the Ohio State University, Columbus OH 43210 USA

*Correspondence author: wang.620@osu.edu


To map and clone novel broad-spectrum rice blast resistance genes, we introduced 22 resistant rice lines to Magnaporthe oryzae from different countries. Resistance- spectra were proved quite different among these lines by inoculation test using 36 M. oryzae isolates collected from 13 countries, and 7 of them showed high resistance to most tested isolates and became candidate broad-spectrum resistant lines.

To test resistance gene allelism between above candidate lines and the cloned broad-spectrum resistance gene Pi9, we constructed seven F2 populations using the candidate lines and Pi9 donor line 75-1-127. F2 population was inoculated with M. oryzae isolates which are avirulent to both parents, and recombination ratio was used to analyze the allelism of new resistance genes and Pi9. Results showed that 4 out of the 7 candidate lines possess resistance alleles of Pi9 which harbors at the Pi9/Pi2 locus on chromosome six. We then cloned and sequenced a 2.8kb homolog across the Pi9 NBS-LRR domains from the genome of these 4 resistant lines, based on Pi9 gene molecular biology information. All 4 sequences showed high homology with Pi9 gene, including a conserved NBS domain and a specific LRR domain. Farther analysis will help us to understand the molecular evolutional mechanism of broad-spectrum rice blast resistance genes. Cloning and characterization of full-length novel resistance genes from these candidate rice lines are in progress.


Keywords: rice blast, resistance alleles, homologs, Pi9


Acknowledgements:This project was supported by NSFC(30571063), “973”Project (2006CB101904), Ph.D. Programs Foundation of Ministry of Education of China 20060537004),Hunan Natural Science Foundation(06JJ10006),and Scientific Research Fund of Hunan Provincial Education Department (05B028)

# These authors made equal contribution to this research




5


Use of Host Resistance, Screening and Breeding Strategies




Studies on the Incidence, Interaction and Yield Loss Due to Blast Disease and Molecular Characterization of

Resistance Genotypes in Bangladesh


M. A. Latif, M. S. Kabir, M. S. Mian and M. A. Hossain


Plant Pathology Division, Bangladesh Rice Research Institute (BRRI), Gazipur-1701, Bangaldesh. E-mail: alatif1965@yahoo.com


A survey was undertaken to determine the incidence and severity of blast disease in the seedbed of irrigated rice of southern Bangladesh during 2003-2004. A total of 88 seedbeds under two different seedbed conditions (dry and wet seedbed) were observed. Incidence and severity of blast was higher in dry seedbed compared to wet seedbed. The variety, BRRI dhan29 showed the highest severity and incidence followed by BRRI dhan28, Purbachi, BR16, Anamika (an Indian variety) BRRI dhan36 and BR14. In interaction study, it was observed that ufra (a nemic disease) enhances the incidence of blast disease. Plant analysis revealed that total nitrogen content increased in ufra-infected plant. That total nitrogen content might influence blast disease. Yield loss was determined in susceptible variety, BRRI dhan28. About 60% yield loss occurred due this disease in the farmer’s field. Molecular characterizations of 14 blast resistant genotypes including resistant and susceptible checks were done. Three VNTR primers viz. MR: GAGGGTGGCGGTTCT, RY : CAGCAGCAGCAGCAG, GF: TCCTCCTCCTCCTCC showed polymorphism among the resistant and susceptible genotypes. But no specific marker(s) among the three primers linked to the blast resistant or susceptible genotypes as preliminary studies reported.




Development of Mass Screening Method for Rice Panicle Blast in the Greenhouse


Jae-Hwan Roh1, In-Seok Oh1, Seong-Sook Han2, and Byung-Ryun Kim3


1 National Institute of Crop Science, Suwon, 441-857, Korea

2 National Institute of Agricutural Sci. & Tech., Suwon, 441-857, Korea

3 Chungnam Agricultural Research and Extension Services, Yesan, Chungnam 340-861, Korea


Rice blast disease caused by the fungal pathogen Margnaporthe grisea is one of the most serious and widespread disease of rice worldwide. Through the screening the resistant of leaf blast in rice breeding lines, most of rice cultivars were bred as a resistant rice cultivar against rice blast. But panicle blast is more dangerous than leaf blast in rice production. Up to now, nursery test, seedling test, greenhouse test, and polycyclic test were only used for screening the resistant against rice leaf blast but field screening was only used for screening the resistant both of leaf and panicle blast. Screening for panicle blast in the greenhouse is not established using different ecotype rice cultivars.

In this study, a mass screening method for panicle blast in greenhouse condition was developed. Optimum inoculation time and sowing time in different ecotype rice cultivars to synchronize the heading time were established. Through this screening method, resistant characteristics of panicle blast in rice cultivars were classified into resistance or susceptible effectively and it will be seriously aimed to develop resistant rice cultivars to rice blast diseases.




A New Screening Method for Evaluation of Durability against Rice Blast Diseases in Rice Cultivars


J.H.Roh1, S.S.Han2, Y.C.Cho1, I.S. Oh1, C.V. Cruz2, and H.leung2


1 National Institute of Crop Science, Suwon, 441-857, Korea

2 National Institute of Agricultural Science & Technology, Suwon, 441-857, Korea

3 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines


Rice blast disease caused by Margnaporthe grisea is one of the most serious and widespread disease of rice worldwide. Although resistant cultivars is considered as most effective and economical way of controlling blast disease, the resistant rice cultivars against rice blast sometimes breakdown in a few years after releasing to the farmers. Up to now, blast nursery, seedling test by inoculation, and field test were used for screening blast resistance in rice cultivars and breeding lines. But a resistance cultivar developed through these screening methods was not characterized it’s durability against rice blast disease.

Sequential planting method was developed to predict the durable resistance to rice blast diseases in rice. Tested rice cultivars showed different resistant characteristics were classified into three types of resistant reaction. Resistant type I was showed less than 20% of DLA (diseased leaf area) from 1st planting to last planting stably. Two rice cultivars classified into type I have been showed low disease occurrence and sustainable field resistance during the last 20 years in farmers' field. Resistant type II was showed less than 40% of DLA until 2nd and 3rd planting time but later showed higher than 40% DLA. Resistant type III was showed more than 40% of DLA from 1st planting to last planting continuously. Rice cultivars showed highly susceptible reactions in the farmer's field when the environmental factors are favorable for disease occurrence were classified into type III.

Results from the sequential planting method in greenhouse were significantly similar to the farmers' field data, suggesting that the current sequential planting method is effective to evaluate durability of rice blast resistance.




Study on Compare of Two Set of Differential Varieties to Magnaporthe grisea in Yunnan


Jin-bin Li 1*Cheng-yun Li 2Yan Chen 2Cai-lin Lei 3, Zhong-zhuan Ling 3


1 Yunnan Academy of Agricultural Sciences, Agricultural Environment and Resources Research Institute, Kunming, China, 650205;

2 The Ministry of Education Key Laboratory for Agricultural Biodiversity and Pest Management, Yunnan Agricultural University, Kunming, China, 650201;

3 Institute of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciencs, Beijing 100081)

*Email: kmlijinbin@yahoo.com


Two hundred sixty-four Magnaporthe grisea monoconidial isolates collected from 24 counties belong to 3 rice regions of Yunnan Province were identified using monogenic lines (IRBL) of IRRI which was developed using susceptible variety Lijiangxintuanheigu(LTH) as recurrent parent, hold same resistance gene set ( Hereafter IRBL lines) with Japanese differential varieties. Differentiate ability of IRBL lines to Yunnan isolates was compared with that of Japan differential varieties. The result showed that the differential ability of IRBL lines was better than Japan differential varieties to rice blast fungus colleted from Yunnan. These monoconidial isolates were divided into 96 races based on the reaction of IRBL lines, while they were divided into 71 races based on Japan differential varieties. Furthermore, some isolates appearance faint lesions on differential varieties from Japan, but it is legible on IRBL lines. The result indicated that differential varieties with same genetic background is better than that of complex background.

Key wordsMagnaporthe grisea; Race; differential lines; Monogenic resistant lines




Blast Field Resistant Cultivars Utilized in Arkansas for Record Rice Yields


Lee, F.N., Cartwright, R.D., Wilson, C.E., Jr. and Lee, J.D.


Rice (Oryza sativa L.) is a critical commodity in the Arkansas agricultural economy. A sustained period of historic per-acre rice production was realized for Arkansas during years 2001 through 2006. Modern cultivars grown during this period possessed a very high yield potential packaged with multiple desirable agronomic characteristics. Control of rice blast, incited by Magnaporthe grisea Cav., using flood-induced field resistance was a key component of the agronomic package.

Although cultivars with the efficacious Pi-ta gene and other R-genes were available, blast susceptible cultivars were planted to more than 80% of Arkansas production acres during 2001?006. In the absence of effective R-genes, producers relied upon cultivar field resistance for blast control. On the whole, cultivars utilized during 2001- 2006 were susceptible to panicle blast when growing in drought stressed blast field nurseries and were rated as being moderately susceptible (MS) to very susceptible (VS) to rice blast. These susceptible cultivars typically exhibited a high degree of flood-induced blast field resistance in disease test plots and in production fields, especially when growers were careful about field selection and irrigation schedules. Field resistance was impacted by cultural practices, primarily fertility and irrigation, and were supplemented with fungicides when cultivars were mismanaged.

Flood-induced blast field resistance occurs with depletion of root zone dissolved oxygen to establish anaerobic conditions. The anaerobic environment defines availability and form of nutrients associated with blast susceptibility, influences production of hormones mediating disease resistance mechanisms, and induces morphological modifications that facilitate oxygen transport to the roots and restricts pathogen growth. Flood-induced blast resistance is cumulative with duration and depth of flood and in many susceptible cultivars is comparable to that expressed by major R-genes. To date, some degree of flood-induced field resistance has been detected in all flooded rice cultivars inoculated with a virulent race.

Historically, field resistant cultivars such as Starbonnet and Cypress have performed exceptionally well in Arkansas. The very durable Starbonnet was planted to 42 to 65 % of Arkansas production from 1969 through 1984. Cypress was planted to 15 to 39 % of Arkansas rice production during 1994 through 2000. In inoculated greenhouse tests, Starbonnet and Cypress were susceptible to common blast races occurring in Arkansas. However blast was rarely observed in either cultivar unless production fields were significantly drought stressed.




Association of Pathotypes and Broad-spectrum Resistance Genes Against Rice Blast Disease in Korean Germplasm


Jae-Hwan Roh2, C.V. Cruz3, and H.Leung3, Se-Weon Lee1 and Seong-Sook Han1


1 National Institute of Agricultural Science and Technology, Suwon, 441-707 Korea

2 National Institute of Crop Science, Suwon, 441-857 Korea

3 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines


Broad-spectrum resistance against multiple rice blast pathogen populations is an important goal for Korean rice breeding program due to the high degree of pathogenic variation in the rice blast fungus caused by Magnaporthe grisea. Using a sequential planting technique, we characterized Korean germplasm and identified a small set of varieties expressing quantitative resistance in both Korea and Philippines, suggesting that the resistance is broad spectrum. The quantitative resistance as measured by sequential planting technique appeared to be correlated with durability of resistance based on the historical performance of the varieties in Korea. Advanced backcross lines derived from donor germplasm possessing properties of durable blast resistance were used to identify candidate defense genes. Graphical genotypes of advanced BC lines with differential resistance were produced. Common and contrasting introgressed chromosomal segments from the donor lines were identified in the genetic backgrounds of the recurrent parents. 11 candidate genes from 22 Korean and donor germplasm were sequenced for blast resistance. The genes have >90 identity and reveal a modest level of SNPs across germplasm. This suggests that SNP marker for functional alleles may be developed for MAS application in the future.




A Survey of Rice Blast Resistance in Landrace and Wild Relatives of Rice as a Hidden Source of Resistance


Saengchai Sriprokhon1, Chanakarn Wongsaprom1, Tanee Sreewongchai1, Apichart Vanavichit1,2, Theerayut Toojinda2, and Pattama Sirithunya3


1 Rice Gene Discovery, National Center for Genetic Engineering and Biotechnology (BIOTEC), Kasetsart University, Kamphangsaeng, Nakornpathom, 73140, Thailand.

2 Agronomy Department, Kasetsart University, Kamphangsaeng Campus, Nakornpathom, 73140, Thailand.

3 Rajamangala University of Technology Lanna, Science and Agricultural Technology faculty , Chiengmai, 50000, Thailand.

Emailsaengchaisri@hotmail.com


Rice blast, caused by Pyricularia grisea, is one of the most devastate diseases of rice which costs millions dollar each year due to its infection. Resistance cultivar is a mean protection from severe losses although pathogen usually adapts itself and is able to attack new release cultivars in short period of time. Therefore, search for new source of resistance is necessary, especially from landrace and wild rice that live with the pathogen for a long times. In this study, one hundred and seventy eight landrace cultivars gathered from northern Thailand and thirty six accession numbers from four species of wild relatives of rice, Oryza rufipogon, O. officinalis, O. spontaneous and O. nivara, collected throughout the country were investigated their resistances to leaf with four isolates. After comparison with 9 Near-Isogenic Lines (NILs), the results showed that up to one fourth of landrace rice cultivar and several accession numbers of wild relatives of rice gave resistance to blast. Clustering analysis suggested that eight accession of wild rice possess resistant genes for residing in the same group with NILs carraying Pi-z, Pi-2 (C101A51) and Pi-1 (C101LAC) and one accession of O. rufipogon from southern of Thailand offered resistance to blast superior to some NILs. Interesting, seven accessions from O. rufipogon and O. nivara might carrying new source of resistance genes.

This result suggested that Thailand wild relatives of rice and landrace cultivars are very interesting to discover novel resistant gene in order to employ as a resistant source although a limited number of material was screened.




Marker-Assisted Selection (MAS) to Improve a Broad Spectrum Leaf Blast Resistance in the Glutinous

Jasmine Rice RD6


Chanakarn Wongsaprom 1, Tanee Sreewongchai 1, Saengchai Sriprokhon 1, Apichart Vanavichit 1,2, Theerayut Toojinda 2, and Pattama Sirithunya 3


1 Rice Gene Discovery, National Center for Genetic Engineering and Biotechnology (BIOTEC), Kasetsart University, Kamphangsaeng, Nakornpathom, 73140, Thailand.

2 Agronomy Department, Kasetsart University, Kamphangsaeng Campus, Nakornpathom, 73140, Thailand.

3 Rajamangala University of Technology Lanna, Science and Agricultural Technology faculty , Chiengmai, 50000, Thailand.


Blast disease caused by the fungus Pyricularia grisea is one of the most devastating rice disease in Thailand and worldwide. RD6 is the most popular glutenous rice cultivar occupied about 40% the total area of the rainfed lowland in the North and Northeast of Thailand. RD6 is very susceptible to leaf and neck blast diseases. Board-spectrum and durable resistance are one of breeding targets for rainfed lowland breeding program. An improved variety ?ao Hawm Nin (JHN) is an excellent source of a broad-spectrum resistance because it is resistance to all except one group of pathogen population in Thailand. Two genomic regions harboring a cluster of QTL were identified for a broad-spectrum resistance (BSR) in Jao Hawm Nin (JHN) using 13 blast isolates. They were located on the rice chromosome 1 and 11. JHN contributed resistant alleles in all of the QTL loci.

To improve a broad spectrum leaf and neck blast resistance in RD6, Marker Assisted Selection (MAS) was used to select individual carrying two clusters of QTL. Four generations of backcrossing was applied to recover a RD6 genetic background.

Twenty-six plants of BC4F1 were selected that carry two clusters of QTL and self-pollinated until BC4F4. Twenty-two plants homozygous were selected two clusters QTL of chromosome 1 and 11. Furthermore, they were screened leaf blast with eight isolates blast, high virulent from diverse of Thailand. Finally, they were highly resistant with Thai blast isolates that will be a new cultivar of glutinous jasmine rice, high quality and durable blast resistance in Thailand.




DNA Marker Analysis of Blast Resistance Genes Pib and Pita in IRRI-bred Rice Varieties Comparing with Gene Estimation by a Differential System


Daisuke Fujita1, Leodegario A. Ebron1, Nobuya Kobayashi1 and Yoshimichi Fukuta2


1 International Rice Research Institute (IRRI), Los Baños, Laguna, Philippines. E-mail:Fujita:d.fujita@cgiar.org,Ebron:l.ebron@cgiar.org,Kobayashi:n.kobayashi@cgiar.org

2 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1, Ohwashi, Tsukuba, Ibaraki 305-8686, Japan. E-mail: zen@jircas.affrc.go.jp


The rice blast resistance genes are important in improvement programs of rice (Oryza sativa L.). The DNA markers linked to resistance genes are a powerful tool to detect the presence of genes and are widely used to select breeding materials through marker-assisted selection. This study was conducted to evaluate the detection ability of DNA markers for rice blast resistance genes, Pib and Pita, in IRRI-bred rice varieties. Forty-two Indica-type varieties, which have been previously analyzed for the presence of Pib and Pita by conventional genetic analysis using a differential system involving standard blast isolates (Pyricularia grisea Sacc) from the Philippines, were tested. To estimate the presence of Pib and Pita, PCR-based dominant markers previously reported by Wang et al. (1999) and Jia et al. (2002) were used. The DNA markers, Sub3-5 (Pib), YL153/YL154, YL155/YL87 and YL100/YL102 (Pita), have been developed on the basis of sequence information of Pib and Pita. The target DNA fragments of Pib using Sub3-5 were amplified in 40 varieties but not in 2 varieties. Also, the target DNA fragments of Pita using YL153/YL154 and YL155/YL87 were amplified in 28 varieties but not in 14 varieties and those using YL100/YL102 were amplified in 26 varieties but not in 16 varieties. The results of DNA marker analysis of 42 IRRI-bred rice varieties were compared with previous gene estimation of Pib and Pita by the differential system. In 6 varieties, detection of DNA fragments for Pib by marker analysis did not correlate with estimation by the differential system. On the other hand, detection of DNA fragments for Pita corresponded well with estimation by the differential system excluding 2 varieties. The use of DNA markers enabled the detection of Pita in most IRRI-bred rice varieties. These suggest that the efficiency of detecting blast resistance genes through use of DNA markers depend on the rice variety and the DNA markers. The proper markers for the Pita gene provide a basis for stacking other blast resistance genes into high-yielding and good-quality advanced breeding rice lines.




Resistance of Rice Cultivars Induced by Inoculation of an Avirulent Isolate of Magnaporthe grisea with

Two Avirulence Genes


Nobuko Yasuda, Masako Tsujimoto Noguchi, and Yoshikatsu Fujita


Rice Disease Resistance Research Team, National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba 305-8666, Japan; E-mail: yasuda@affrc.go.jp


Resistance reaction of rice cultivars induced by avirulent isolate of the blast fungus Magnaporthe grisea cause various degrees of lesions (from large brown flecks to nearly invisible lesions). We studied relationship between lesion types of resistance in rice and the avirulence genes of the blast fungus. We previously identified four avirulence genes in M. grisea isolates by genetic analysis of progeny from crosses between isolates with differing pathogenicity. Using progeny known to contain a certain avirulence gene, we demonstrated that the type of resistance lesion produced in rice by an avirulent isolate. The rice cultivar ‘Miyamanishiki’, which contains resistance genes Pia and Pii, produced minute brown flecks in response to the blast isolate Y93-165g-1, which is known to contain the avirulence gene AvrPia, whereas ‘Miyamanishiki’ produced large brown flecks in response to Y93-164a-1, which contains AvrPii. The blast isolate AI33, which is the progeny of a cross between Y93-165g-1 and Y93-164a-1, was shown to contain both AvrPia and AvrPii. This isolate caused minute brown flecks on ‘Miyamanishiki’ with results similar to those produced by inoculation with Y93-165g-1. Thus, the types of resistance lesions triggered by the two avirulence genes resembled those caused by one or the other of the two avirulence genes. We also studied relationship between the strength of the resistance induced by pre-inoculation with the avirulent isolate and the avirulence genes of the blast isolate. The strength of the resistance induced by pre-inoculation with an avirulent isolate increases with larger resistance lesions. The strength of resistance induced by the isolate with two avirulence genes was comparable to that induced by an isolate with the avirulence gene that produced the smaller effect.




Genetic Characterization of Universal Differential Varieties’ Sets Developed under the IRRI-Japan Collaborative Research Project


Donghe Xu1, Nobuya Kobayashi2, Mary Jeanie T. Yanoria2, Aris Hairmansis3, Yoshimichi Fukuta1

1 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1, Ohwashi, Tsukuba, Ibaraki 305-8686, Japan. E-mail: zen@jircas.affr.go.jpxudh@jircas.affrc.go.jp

2 International Rice Research Institute (IRRI). DAPO Box 7777, Metro Manila, Philippines. E-mail: Kobayashi: n.kobayashi@cgiar.org, Yanoria: m.yanoria@cgiar.org

3 Indonesian Center for Rice Research. JL. Raya Muara No.25A Ciapus Bogor, West Java, Indonesia. E-mail: a.hairmansis@yahoo.com


The International Rice Research Institute (IRRI) and the Japan International Research Center for Agricultural Sciences (JIRCAS) have developed four kinds of differential sets of varieties: monogenic lines (MLs) with a Japonica-type variety Lijianxintuanheigu (LTH) genetic background; near isogenic lines (NILs) with three different genetic backgrounds, (LTH, Indica type variety CO 39; and a universal susceptible line US-2) under the IRRI-Japan Collaborative Research Project. These target 24 kinds of resistance genes: Pia, Pib, Pii, Pik, Pik-h, Pik-m, Pik-p, Pik-s, Pish, Pita, Pita-2, Pit, Piz, Piz-5(Pi-2), Piz-t, Pi1, Pi3, Pi5, Pi7, Pi9, Pi11(t), Pi12, Pi19 and Pi20, and the MLs have been distributed to more than 15 countries. To characterize these chromosome components and confirm the introgression of chromosome segments harboring resistance genes, the graphical genotypes were investigated using 162 simple sequence repeat (SSR) markers distributed on whole rice genome chromosomes. These chromosome components of the three sets’ NILs were more uniform than those of MLs when compared to the corresponding recurrent parent. Several introgression segments, which corresponded to the locations of blast resistance genes, were also confirmed. Almost all the MLs were developed by backcrossing one or two times, while some lines were backcrossed three to five times with the recurrent parent, LTH. The restoration of genomic chromosomes of 31 MLs to LTH ranged from 50 to 90.0%, averaging 77.3%. All LTH, CO 39, and US-2 NILs were developed by backcrossing six times with each recurrent parent. The frequencies of genome restoration to the parent in 34 LTH NILs ranged from 75.6% to 96.9%, averaging 90.6%, lower than theoretical value of 99%. The 31 CO 39 NILs showed greater than 90% genome restoration to the recurrent parent, averaging 97.3%. The 16 US-2 NILs were closely similar to those of the recurrent parent: these genome restorations ranged from 88.9% to 98.8%, averaging 94.6%. Four resistance genes, Pik, Pik-h, Pi1, and Pi7 in the LTH NILs confirmed introgression by co-segregation analysis with DNA markers of F3 family lines derived from the crosses between the LTH NILs and the recurrent parent. Genetic characterizations of four kinds of universal differential sets of varieties were carried out using DNA markers, and the introgression of several resistance genes was confirmed by co-segregation analyses using DNA markers. This information on DNA markers linked with resistance genes is potentially very useful for marker-assisted selection (MAS) in the breeding program, since the differential varieties can be applied as gene sources.




The Problems of Rice Production in the Federal Capital Territory Abuja Nigeria


Michael Adedotunoke


PRESIDENT AGRIC LINK MULTIPURPOSE COOPERATIVE SOCIETY LIMITED P.O. BOX 11611, GARKI ABUJA NIGERIA.

Email: agriclinkcooperative@yahoo.com


The rural farmers in the Federal Capital Territory face a lot of problems in the areas of planting, harvesting and the Technology invovle in the postharvesting and processing with harversation and preservation of Rice.

This paper give detail information on the various problems and the infect of this in the production methodology and Marketing oppournity of Rice Cultivation in the Federal Capital Territory.

In addition how the Interntional Countries could help to solve the various problems and the solution that are needed.




Lesion-based Analysis of Leaf Blast Suppression in Mixture of Rice Cultivar and a Resistant Near-isogenic Line


Taketo Ashizawa, Masashi Sasahara, Atsushi Ohba, Takeshi Hori, Kouji Ishikawa, Yukio Sasaki, Tomohisa Kuroda, Ryoei Harasawa, Kaoru Zenbayashi-Sawata, Shinzo Koizumi


National Agricultural Research Center 1-2-1 Inada, Joetsu, Niigata 943-0193, Japan

Email: toketa@affrc.go.jp


Leaf blast suppression in multilines was evaluated based on the number of susceptible lesions observed in a pure stand of susceptible rice cultivar Sasanishiki, 1:1 and 1:3 mixtures of Sasanishiki and a resistant near-isogenic line, Sasanishiki BL4 or BL7 from 1998 to 2001. The number of lesions first observed in fields in the 1:1 and 1:3 mixtures were close to theoretical numbers calculated using the number of lesions observed in the pure stands and the ratios of the susceptible Sasanishiki in the mixtures. The ratio of the number of lesions in the 1:1 and 1:3 mixtures to the number in the pure stand was 0.29 and 0.09, respectively. The relationship between these ratios and the ratios of susceptible Sasanishiki in mixtures was defined in an equation to estimate the degree of leaf blast suppression. Validation studies for the ratios of the number of lesions in the 1:1 and 1:3 mixtures to the number in the pure stand were conducted in two different locations showed that the ratios are almost acceptable. The calculated autoinfection to alloinfection ratio was 1.3 and 1.4 in the 1:1 and 1:3 mixtures, respectively, suggesting that the calculated ratio will affect the degree of leaf blast suppression. Thus, predictors were obtained to estimate leaf blast suppression for effective blast control in multilines.




Validation of Linked Molecular Markers for Effective Blast Resistance Genes in Rice


Thippeswamy Sanka 1*, Kancharla Nagesh1 and Chintala Sadasiva Reddy2


1 Barwale Foundation, # 8-2-703, A.G. Heights, Road No. 12, Banjara Hills, Hyderabad 500 034, Indial;

2 Directorate of Rice Research, Rajendranagar, Hyderabad 500 0036, India.

*Corresponding author, email:thippeswamy@barwalefoundation.org


Rice blast caused by the heterothallic ascomycete fungus Magnaporthe grisea (anamorph: Pyricularia grisea Sacc.) is the most widespread and devastating disease of rice, this disease has become a major constraint in augmenting the yields worldwide and can cause yield loss as high as 70% in some areas of Asia. Exploitation of host plant resistance has been most effective and environmentally sound strategy for the management of disease. However, it is known that resistant cultivars that carry single genes for resistance break down within a few years after their release. Identification of effective resistance (R) genes and their pyramiding seems promising to provide a broad spectrum and durable resistance. Combining several genes and monitoring their presence is difficult by conventional methods because of their interaction effects. Mapping blast resistance genes with closely linked molecular markers has made it possible to confirm the presence of a given gene in a segregant with multiple genes.

To date, approximately 50 Pi genes (blast R genes) have been described and mapped in the rice genome. The present study was undertaken to identify effective blast resistance gene/s and validate their linked markers. A set of 152 genotypes that included near-isogenic lines (NILs), resistant donors, popular varieties and parents of commercial hybrids were screened against the most virulent blast race (C-8) of India. Three major genes (Pi-1, Pi-2 and Pi-Kh) showed resistance while the gene Pi-9 was susceptible. Among the combination of genes, Pi-1+Pi-2, Pi-1+Pi-4 and Pi-1+Pi-2+Pi-4 combination were highly resistant as compared to Pi-2+Pi-4. Twenty nine reported molecular markers linked to the three major genes (Pi-1, Pi-2 and Pi-Kh) were validated using 23 genotypes which included NILs, popular varieties, maintainer and restorer lines of commercial hybrids. Four SSR markers for Pi-1, three for Pi-2 and one for Pi-Kh were validated across the genotypes and rest of the markers did not validate. We are in the process of validating such markers in segregating populations and use them in our gene pyramiding programmes to improve popular rice varieties and hybrids for durable blast resistance.

Keywords: Molecular markers, Pyricularia grisea, Oryza sativa.




Selection of Breeding Lines for Diverse Blast Resistance


Santoso1, Anggiani1, E. Lubis1, Suwarno1 and C.M. Vera Cruz2


1 Indonesian Center for Rice Research (ICRR), Jln. Raya No. 9 Sukamandi Subang, West Java, Indonesia 41256.

2 International Rice Research Institute (IRRI), DAPO Box 777, Metro Manila, Philippines.


Many blast resistant varieties have been released; however the resistance was quickly breakdown due to the dynamic of the pathogen population. Genetic diversity of rice could be needed to improve blast resistance. The objective of the study was to select breeding lines with different blast resistance. A total of 465 breeding lines derived from different crosses were used as material for the selection. The breeding lines were artificially inoculated with 5 major races of blast pathogen. The selected lines having different pattern of blast resistance were than inoculated with other 17 pathogen races. The result indicated the breeding lines have diverse blast resistance. Out of 465 lines tested against 5 blast races, five lines were resistant to all of blast races. More lines were resistant to 4 or less number of blast races. Considering the sources and the patterns of the resistance, 60 lines were selected and than tested against 17 blast races. Among the lines tested 11 lines were resistant to 6 races and 10 lines were resistant to 8 races. Considering the reaction to different pathogen races, 20 lines with different resistant pattern were selected.




Development and characterization of blast resistance using differential varieties in rice

——A blast research network for stable rice production


Yoshimichi Fukuta1, Nobuya Kobayashi2, Casiana M. Vera Cruz2


1 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1, Ohwashi, Tsukuba, Ibaraki 305-8686, Japan. E-mail: zen@jircas.affr.go.jp

2 International Rice Research Institute (IRRI). DAPO Box 7777, Metro Manila, Philippines.E-mail:Kobayashi:

Email: n.kobayashi@cgiar.org, Vera Cruz: c.veracruz@cgiar.org


The Japan International Research Center for Agricultural Sciences (JIRCAS) and the International Rice Research Institute (IRRI) are organizing a collaborative study, begun in 2006, focusing on rice blast resistance. The aim is to develop and apply a universal differential system, designed to strengthen the sustainability of rice production systems against disease. A differential system is a basic tool for understanding host-pathogen interactions that consist of rice varieties, each ideally carrying a single gene for blast resistance, and blast isolates differing in their corresponding avirulence/virulence genes. They are classified based on the specificity of the reaction between the particular differential variety and differential blast isolates, and can be used to identify resistance gene(s) in the varieties and avirulence/virulence genes in the blast isolates. Seven institutes have so far joined this research project: the China National Rice Research Institute (CNRRI) and Institute of Crop Sciences (ICS) in China, the Cuu Long Delta Rice Research Institute (CLRRI) in Vietnam, the Philippines Rice Research Institute (PhiliRice) in the Philippines, the Indonesian Center for Rice Research (ICRR) and the National Nuclear Energy Agency, Center for Research and Development of Isotopes and Radiation Technology (CRDIRT) in Indonesia, and the National Institute of Agrobiological Sciences (NIAS) in Japan. The National Institute of Crop Science (NICS) in Korea is attending as an observer. Based on collaborations between pathologists and rice breeders, the following projects are planned: (1) diversity research into blast pathogens, (2) development of differential systems for blast resistance, and (3) diversity research and identification of novel resistance genes in each region and globally as part of a collaborative effort with all participating scientists. This networked research configuration should enable the discovery of the relationship and differentiation between blast races and resistance genes and lead to the development of a universal differential system. The characterization of novel resistance genes and differential isolates, development of a new designation system of blast race, and conservation of blast isolates are anticipated. Although this network research project as yet has few members and is targeting only East and Southeast Asian countries, it is an open collaboration. It is the first attempt to develop a universal differential system that can act as a common tool and language that links breeders, geneticists, and pathologists.




Selection, Evaluation and Utilization of Rice Germplasm Resources Resistant to Magnaporthe grisea


Fu Huang 1, Rong Xie 2, Cheng-yuan Liu 2, Yan Zheng 1, Shi-qi Zhao 1,

Fu-ling Peng 1, Xue-mei Zhang 1, Cong Luo 1, Hua-zhi Ye 1


1 Agricultural College, Sichuan Agricultural University, Yaan 625014Sichuan, China;

2 Rice and Sorghum Research Institute, Sichuan Academy of Agricultural Sciences, Luzhou 646100, Sichuan, China


Rice blast, caused by Magnaporthe grisea (Hebert) Barr, is one of the most devastating rice diseases all through the world. It has been proved that breeding and planting disease-resistant varieties is the most economical and efficient means to control this disease. Screening and utilization of rice germplasm resources resistant to rice blast were the important foundation for the breeding of resistant varieties. This study systematically assessed the blast resistance, main agronomic traits, grain quality, and the restoring or mantaining characteristics of 97 rice germplasm resources. The results showed that the blast resistance of the 97 rice germplasm resources was high or medium and their main agronomic traits varied significantly: heading date from 99 to 132 daysplant height ranged from 89.6 to 149.2cm, effective panicle number each plant ranged from 4 to 13.1, 1000-grain weight ranged from 18.50 to 42.50g, spikelet number in each panicle ranged from 97 to 268, seeds setting rate ranged from 45.83% to 93.90%the grain weight in each plant ranged from 15.28 to 32.58gsuggesting the abundance of genetic diversity in the tested germplasm resources. Among them, 37 germplasm resources have restoring ability to infertile lines; 9 germplasm resources have maintaining ability to infertile lines; The milling and appearance quality of 6 rice germplasm resources reached the third grade of the national high quality rice standards and that of 4 resources reached the second grade, which are invaluable resources for breeding new resistant restoring, infertile lines and high-yield, high quality resistance hybrid combinations. IRAT144 , a resistant rice line with large grain introduced from IRRI in early 90s last century, was crossed with N56, an F5 breeding line resulting from hybridization between Minghui63, an Indica restoring line with high combining ability, and 02428, an Japonica wide-compatibility line. A new heavy-panicle and resistant restoring line Luhui 1345 was constructed in 2002 and get the national patent in 2003 (CNA20020267.7). A new high-yield, high quality and resistance hybrid rice combination D62A/Luhui 1345 was constructed using the restoring line Luhui1345 and infertile line D62A. In 2006 this combination has been approved by Chongqing Crop Variety Approval Committee.

Key words: Rice; germplasm resources; Blast resistance; Agronomic traits; Evaluation; Utilization




Construction of Monogenic Differentials of Magnaporthe grisea in GuangdongSouth China


Jian-yuan Yang 1, Qi-yun Yang 1, Shen Chen 1, Cai-lin Lei 2, Jiu-lin Wang 2,

Zhong-zhuan Ling 2, Xiao-yuan Zhu 1*

1 The Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;

2 Institute of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciences, Beijing 100081, China

*Corresponding author: Zhu Xiaoyuan, E-mail: gzzhuxy@tom.com


Rice blast, caused by the fungus Magnaporthe grisea, is the most devastating rice disease worldwild. Races analysis is the fundamental work for the application of host resistance which is an effective and economic method for the disease controlling. 24 monogenic lines from International Rice Research Institute (IRRI) and 6 near isogenic lines (NILs) from Chinese Academy of Agricultural Sciences (CAAS) were used in the present study, we tried to construct a new set of monogenic differentials of Magnaporthe grisea instead of 7 Chinese differentials which was released in 1980s and was no good for differentiating the pathogencity of blast fungus from the indica rice in Guangdong, South China.

30 monogenic lines were inoculated with 146 single spore cultivated isolates which were selected from 1200 isolates collected in Guangdong, South China. The value of resistance spectrum (no. of isolates incompactible with a tested line/no. of total tested isolates) of monogenic lines against the tested isolates is from 0 to 88.5%. Of the 30 tested lines, 13 lines including IRBLt-K59 (Pi-t), F98-7 (Pi-km), IRBLa-C (Pi-a(2)), F129-1 (Pi-kp(C)), IRBLa-A (Pi-a(2)), IRBLks-F5 (Pi-ks(1)), F124-1 (Pi-ta(C)), IRBL12-M (Pi-12), IRBLb-B (Pi-b), IRBL19-A (Pi-19), IRBLks-S (Pi-ks(2)), IRBLta-CP1 (Pi-ta(2)) and IRBL3-CP4 (Pi-3) showed quite susceptible to almost the isolates inoculated. Those lines were excluded in the construction of monogenic differentials.

17 monogenic lines of which resistance spectrum value is higher than 10% were used for further analysis. Taking the resistant pattern (0 as resistant, 1 as susceptible) of 17 monogenic lines against 146 tested isolates as variance, factor analysis with principal component method of the variance matrix had been conducted. Nine groups explaining 80.4% (Table 1) of the total variance were abstracted by the SPSS11.0 software analysis. They were listed as follows: 1. IRBLkp-K60 (Pikp), IRBLk-Ka (Pik), F80-1 (Pik(C)), IRBL7-M (Pi7(t)); 2. IRBLi-F5 (Pii), IRBL3-CP4 (Pi3), IRBL5-M (Pi5(t)); 3. F128-1 (Pita2), F145-2 (Pib(C)); 4. IRBL9-W (Pi9); 5. IRBLsh-S (Pish(1)); 6. IRBLz-Fu (Piz); 7. IRBLkh-K3 (Pikh); 8. IRBL1-CL (Pi1); 9. IRBLz5-CA (Piz5). The cluster analysis indicated that the pattern of the resistant and susceptible responses of monogenic lines in the same group were quite similar by bootstrap analysis (by wintboot software developed by IRRI). Nine monogenic lines were selected from each group based on their contribution of variance, they are IRBLkp-K60 (Pikp), IRBLi-F5 (Pii), F128-1 (Pita2), IRBL9-W (Pi9), IRBLsh-S (Pish(1)), IRBLz-Fu (Piz), IRBLkh-K3 (Pikh), IRBL1-CL (Pi1), RBLz5-CA (Piz5 ),Those lines were recommended to be blast monogenic differentials in Guangdong, South China.

This work is supported by National “863” Project (2001AA2410111) and Guangdong Sciences Foundation (04101156)




Improving Resistance of Super-hybrid Rice Lines to Magnaporthe oryzae Using the Broad-spectrum

Resistance Gene Pi9


Xionglun Liu1, Sujun Pan1, Jun Wu1, Jia Gao1,Xiaoshan Zeng1, Suhua Wang1, Yajun Hu1,Jingling Liu1, Yuese Ning1,Ling Huang1,Nan Jiang1,Ling Liu1,Liangying Dai1*, and Guoliang Wang1,2*


1 Rice Genomics Laboratory, Hunan Agricultural University, Changsha, Hunan 410128, China

2 Department of Plant Pathology, the Ohio State University, Columbus OH 43210 USA

* Correspondence authors:daily@hunau.net; wang.620@osu.edu


Pi9 was introgressed from the wild rice Oryza minuta into the breeding line IR31917 and confers broad-spectrum resistance to diverse rice blast isolates. This gene has been cloned by a map-based cloning strategy and encodes a NBS/LRR protein. To improve the resistance of super-hybrid rice to the fungal pathogen Magnaporthe oryzae, we started to introduce the new resistance gene into indica super-hybrid rice parents via Agrobacterium-mediated transformation with hygromycin marker or marker-free selection. An efficient transformation system has been established for most of the indica rice lines we have tested. Twenty-four elite indica super-hybrid rice parents including six male-sterility lines and eighteen restorers were transformed with the Pi9 construct. Fifteen lines were successfully transformed and more than two hundred transgenic T1 lines were obtained. Subsequently, 71 transgenic T3 lines and 150 transgenic T4 lines were bred from them assisted by histochemistry, PCR and RT-PCR analyses. We also obtained the results about the major factors affecting the transformation efficiency of the indica and japonica cultivars. Interestingly, the indica cultivars R288, R996, 1701 and C815s showed similar transformation efficiency as the japonica cultivar Nipponbare using our optimized medium formula.

Most transgenic lines and their hybrids showed significantly improved resistance to M. oryzae isolates. Inheritance and compatibility analyses of some elite transgenic lines and their hybrids are in progress. The resistant transgenic lines from this study will provide new parental lines for a stable super-hybrid rice production.


Keywords: rice blast, resistance improvement, super-hybrid rice, Pi9


Acknowledgements: This project was supported by NSFC(30571063 ,30470990), “948” Project(2006-G61), Hunan Natural Science Foundation(06JJ10006),and Scientific Research Fund of Hunan Provincial Education Department (05B028)




6


Epidemiology, Disease Management and Control Methods




Screening of Antifungal Activities of Endophytic Fungi Against Magnaporthe grisea


Chu-long Zhang 1, Ying-yin Pan 2, Fu-cheng Lin 1


1 Institute of Biotechnology, Zhejiang University, Hangzhou, 310029, China;

2 City College, Zhejiang University, Hangzhou, 310015


To the 56 strains of endophytic fungi isolated from Taxus mairei, the antifungal activities against Magnaporthe grisea strain GUY11 was tested. The results revealed that 16 strains (about 28.6%) of the crude fermentation broths in the total presented significant activity with ID50 above 10 which inhibited the spores of GUY11 to germinate and also with ID50 above 50 which inhibited GUY11 to form appressorium. It is showed that active strains mostly distributed in Paecilomyces sp. (9 strains) and Trichoderma sp. (2 strains). The results also showed that inhibition activities of different endophytic fungi vary between spore germination and appressorium form, inhibiton activities of appressorium form greater than spore germinatoin to the crude fermentation broths of three strains F0767, F1021 and F1231 which belong to Paecilomyces sp., they represent greater exploiture potential act as biochemicals, so further search active constituent in fermentation broths is necessary.

Key words: endophytic fungi, Magnaporthe grisea, antifungal activities




Enhancing the Blast Resistance Using

Interplanting Methods


In-Seok Oh1, Jae-Hwan Roh1, Byung-Ryun Kim3, Myeong-Ki Kim1and

Seong-Sook Han2


1 National Institute of Crop Science, Suwon, 441-857, Korea

2 National Institute of Agricutural Sci. & Tech., Suwon, 441-857, Korea

3 Chungnam Agricultural Research and Extension Services, Yesan, Chungnam 340-861, Korea


Interplanting involves growing two different cultivars in an area at the same time. This method of planting is adapted to chemical-free control of rice blast disease. In this study, mixed interplanting was investigated at Hongsung country in Chungnam province. Mixed interplantiing is growing two cultivars together in no distinct row arrangement. Interplanting results, enhanced blast resistance was not appeared in leaf blast at the maximum tillering stage. However, two rice cultivars, Samdukbyeo and Gopumbyeo complex that are cultivated mix interplanting were enhanced the resistant response with 50.8% of panicle and neck blast. Rice cultivars, Hopyeongbyeo and Nampyeongbyeo were decreased panicle and neck blast at two ratio of mixed interplanting with 48.6 and 61.8%. Hopyeongbyeo and Nampyeongbyeo complex that are cultivated mix interplanting were highly enhanced blast resistance. Furthermore, the yields and qualities of mix interplanting cultivars were better than single planting cultivars. Results of this study suggest that interplanting methods have an effect on the panicle blast resistance and rice grain qualities.




Detection of Leaf Rust Infection by Chlorophyll Fluorescence and Photographic

Imaging in Wheat Leaves


Zsuzsanna Deák1, Mária Csősz2, László Purnhauser2 and Imre Vass1


1 Institute of Plant Biology, Biological Research Center, Szeged, Hungary

2 Cereal Research Non-Profit Company, Szeged

Email: Hungaryimre@brc.hu


We used the combination of high resolution of digital photography and chlorophyll fluorescence imaging to study the development of leaf rust infection in leaves of wheat varieties carrying different types of resistance genes. In the sensitive LR26 wheat line the infection caused the development of necrotic spots and the appearance of spores. Together with these effects the so called variable chlorophyll fluorescence was decreased showing the decrease of photosynthetic activity in the infected area. This demonstrates that the efficiency of photosynthesis is decreased not only due to pigment loss, but also due to damage of photosynthetic centers. The presence of resistance genes caused two main types of protective effects. LR9 (and to a smaller extent LR1) protected the leaves against loss of photosynthetic activity and development of necrosis as well as of spores. In the presence of LRW strong necrosis was induced concomitant with decreased variable fluorescence, but sporulation was prevented. Other resistance genes caused intermediate behavior. In case of the Alc and Tc resistance genes the loss of variable fluorescence was accompanied by the increase of the basic fluorescence level arising from the photosynthetically inactive pigment bed. This phenomenon indicates formation of free chlorophylls, which probably participate in the development of necrosis via a photodynamic effect leading to the production of reactive oxygen species, singlet oxygen in particular, as part of the resistance mechanism. Our results show that high resolution chlorophyll fluorescence imaging is a sensitive tool for detection of photosynthetic activity changes induced by leaf damaging fungi. The method can be applied to rice and other cereals, which are prone to infection by leaf damaging fungi.



This work was supported by the Economic Competitiveness Operational Program (GVOP- 3.1.1-2004-05-0096/3.0)




Occurrence, Epidemiology and Management of Rice Blast Disease in Western Ghat Region of Tamil Nadu, India


R.Rabindran*, S.Bharathi, S.Robin, S.Suresh, R.Samiyappan, V.Prakasam, K.Mohanasundram, K.Thiayagarajan and T.S.Raveendran


Centre for Plant Breeding and Genetics and,Centre for Plant Protection Studies Tamil Nadu Agricultural University, Coimbatore-641 003 Tamil Nadu, India.

* Correspondence Author: Email: rabinr2003@yahoo.com ,rabinr1956@gmail.com


Rice blast, caused by Pyricularia grisea (Cooke) Sacc. (teleomorph, Magnaporthe grisea (T.T. Hebert) Yaegashi & Udagawa), has the potential to cause severe crop losses in rice where environmental conditions are favorable for disease development. Survey of ten years data in western districts of Tamil Nadu (India) viz., Coimbatore, Erode, Dharmapuri, Krishnagiri,Salem, Namakkal, Karur, Dingigul, Madurai, Theni, Sivagangai, Ramanathapuram, Thoothukudi, Thirunelveli, Kanyakumari, Rajapalayam and Uthagamandalam indicated that the disease is rampant in all the areas whenever susceptible variety was cultivated in the late Kharif (Rainy season) and Rabi seasons (Winter season). The virulence of blast was monitored using various accessions and Recombinant inbred lines and Near isogenic lines at Paddy breeding station Coimbatore & Hybrid Rice Evaluation centre, Gudalur (Uthagamandalam Dt). The data indicates that the accession christesed as BL122 (Pi -1 & Pi -2 gene Coimbinations) recorded resistant reaction against blast at both the location. In addition IR64 and Tetep also recorded resistant reaction (under field conditions) under Coimbatore conditions, while at Gudalur the accession C 101 Lac, C 101 A51, C 105 TTP-4-L23, RIL 10, USEN, Tadukan, IR 64, Tetep, Rasi and CO 39 recorded zero grade under field conditions. Two sets of new chemicals were tested against blast under field conditions during 2005 -06 & 2006 -07 (Late Kharif). The results revealed that during both years Kasu – B3SL @ 2.5 ml/lit was effective in managing the epidemics.

Linear correlation coefficients were worked out to find out the relationship between the blast incidence scores in two varieties of rice (IR 50 and TN 1) and weather variables (Maximum, minimum temperature, relative humidity and rainfall) using a sub-set of 100 observations recorded over a period of six years [ from 1999-2003 & 2005-2007 ( 2003-2004 drought year)]. Generally, the two rice varieties covariate (r = 0.80**) each other with respect to susceptibility to blast incidence (p<0.05). Among the predictors, maximum temperature showed strong negative relationship (-0.58) with intensity of blast incidence (p < 0.05) both in IR 50 and TN1. However, the influence of minimum temperature was relatively less prominent (r = - 0.47). Alternatively, relative humidity has a positive relationship with blast intensity score and was strong in IR 50 (r = 0.54) compared to TN1. The relationship between rainfall and disease incidence was marginal. The preliminary analysis could yield an appropriate prediction scheme to explain the blast intensity using maximum temperature and relative humidity.




Introduction on Hunan Identification Center

for Rice Blast Disease


Fanghua Xiao


Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha Hunan.China


Hunan Academy of Agricultural Sciences established “Hunan Identification Center for Rice Blast Disease”in 1994. It is the professional institutions studying rice blast disease, and Hunan Plant Protection Institute is responsible for its concrete implementation. In 2006, the relevant leaders and experts commissioned by the National Agricultural Technology Extention Center agreed that "Identification Center" became a "National Identification Base for Rice Blast Disease Area Test ".

Hunan Identification Center for Rice Blast Disease” reposed on Meishuidong village, Gaoqiao town, Taojiang County. “Identification Center” is located in the transition area from Xuefeng mountain to the Dongting Lake, north Hunan ,112 ° 03'E , 28 ° 22 'N, belonging to subtropical monsoon climate. Leaf wetness from dew was kept for 12-14h , and the sunlight was less than 6-7h during the epidemic periods of rice blast.These conditions were very favorable for Pyricularia grisea infection, reproduction and expansion. The results by Professor Shen Ying showed that Pyricularia grisea was very rich in races composition in Taojiang blast nursery including ZA, ZB, ZC, ZD, ZE, ZF, ZG groups, and ZB was dominant group. The tests in several years suggested that resistance to rice blast in Taojiang nursery had obvious differences, and the identification results could meet with production. "Identification Center" is the ideal blast nursery.

The main research achievements in “Hunan Identification Center for Rice Blast Disease” are as follows: 1. Two National Natural Science Fund "Research on Exploration and Use of Durable Blast Resistance Gene in Rice (1990-1996) were made by Second Prize of Scientific Progress in Hunan Province in 2000. 2. Blast resistance identification and sceening in rice varieties containng Regional Trial of New Varieties of 1000-2000 and low-generation 8000-17000 materials provided by breeding sector was completed in Taojiang nursery every year. 3. Three anti-drought and disease-resistant rice varieties were screened from 638 foreign anti-drought varieties. "Research on anti-drough screening and ecological adaptation of rice varieties" was made by Third Prize of Scientific Progress in Hunan Province in 2004. 4. Rice variety 143 screened in many years had strong resistance to rice blast, and the achievement was registered in Hunan Office of Science and Technology. 5. “Monograph of Research on Resistance to Rice Blast Disease”was published and 38 papers were published on domestic and foreign academic journals, 4 papers among them were made as excellent academic paper.




Induction of Leaf Blast Resistance in Rice by Avirulent Isolates of M. oryzae


Filippi, MC.

Embrapa CP 179 Brazil.

Email: cristina@cnpaf.embrapa.br


We investigated the best concentration and period for induction of resistance in rice leaves. Plants were sprayed with an avirulent isolate (inducer) at three different concentrations 24, 48, and 72 hours prior to inoculation of virulent isolate. The induction of resistance was observed on the leaf area affected and lesion type.




Identification of M. grisea Gene Specific for the

Penetration Process


Filippi, MC


Embrapa CP 179. Brazil.

Email:cristina@cnpaf.embrapa.br


We aimed to elucidate the role of Pth1 gene in appressorium maturation. pth1 conidia have a delay in appressorium formation. Typical lesions formed after wound inoculation suggesting that the disruption of pth1 gene does not affect host colonization. We propose that pth1 functions to target proteins for degradation.




Late Submissions


High-resolution mapping of the rice blast resistance gene Pik-h into the Pik gene cluster with HEGS, using newly isolated differentiating blast race


Xin Xu1, 2, 4, N. Hayashi1, CT. Wang4, H. Kato3, T. Fujimura2, S. Kawasaki1 *


1 NIAS, Kannon-dai 2-1-2, Tsukuba, Ibaraki, 305-8602 Japan

2 University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8572 Japan

3 NICS, Kannon-dai 2-1-18, Tsukuba, Ibaraki, 305-8518 Japan

4 South-Central University for Nationalities, Wuhan, 430074, P. R. China

*E-mail: kawasa@nias.affrc.go.jp


The wide-spectrum rice blast resistance gene, Pik-h, considered to be a member of the Pik cluster, is present in several rice indica cvs, and confers resistance even to rice blast mutants that have overcome the other Pik genes. Although the loss of a Pik-h-differentiating mutant isolate has long hindered R-gene’s accurate characterization, we have isolated new natural Pik-h-differentiating isolates, and confirmed the authenticity of the IRRI “monogenic” line IRBLkh-K3, and then fine-mapped Pik-h in the Pik cluster region, within 290 kb in the Nipponbare genome sequence, using 709 susceptible individuals selected from the 3090 siblings of a cross IRBLkh-K3×CO-39; the narrowest delineation among the Pik group genes reported to date. Even in the susceptible Nipponbare’s genome region, there were 6 NBS-LRR type candidates of the Pik-h gene counterpart, in the predicted more than 40 ORFs, excluding transposon-related ones. Interestingly, this Pik-h region is narrowly apart from the recently identified Xa4 and Xa26 bacterial bright resistance genes, considered also to be in the Pik cluster. The closest seven recombination events were found only in the marginal districts of the Pik-h region. The inner district of about 200 kb with 4 co-segregating markers was strongly suppressed in recombination, suggesting vigorous genome mobilization. Mapping was performed in a short period without compromise, using the HEGS (high-efficiency genome scanning) high-density lane electrophoresis system. The claim of “Pik-h cloning” by Sharma et al. (2005) without any gene verification by the differentiating races or complementation data, and mapping it far from the Pik cluster, may require strict re-examination.




Exploiting transcript profiling data to decipher rice – Magnaporthe grisea compatibility mechanisms


Judith hirsch, Virginie pacaly, Didier tharreau, Jean-Loup notteghem and Jean-Benoît morel

UMR BGPI, INRA-CIRAD-SupAgro, TA 54 / K, Campus International de Baillarguet,

34398 Montpellier Cedex 5


hirsch@supagro.inra.fr


Whereas the molecular events associated with disease resistance are largely studied, those occurring in susceptible plants are poorly known, especially in Monocots. We chose the rice/Magnaporthe grisea system to explore the molecular modifications that are induced in susceptible plant tissues following pathogen attack. A truly compatible interaction (between O. sativa cultivar Nipponbare and M. grisea isolate FR13) at time points where infection occurs without visible symptoms was studied. Changes in rice transcript levels at 3 and 4 days post inoculation were measured using Affymetrix chips. In addition to the up-regulation of a large number of defence-related genes, this analysis revealed new features indicating extensive reprogramming of host gene expression. Microarray expression data were confirmed for 35/38 genes using quantitative RT-PCR, thus demonstrating the robustness of these transcript profiling experiments. This comprehensive survey of global gene expression in response to colonization by a virulent fungal isolate constitutes an important resource that should help us to further our understanding of the rice blast disease process.




Genome analysis on Magnaporthe oryzae Korean strain KJ201


Yoonhee Kim1, Hyejeong Kim1, Jinsoo Kim2, Miyeon Jeong2, Soyoung Park2, Suyoung

Kim2, Soonok Kim3, Yong-Hwan Lee3, Seong Sook Han4, Jae Hwan Roh5 and Woobong

Choi1,2

1 Department of Biomaterial Control, Dongeui University, Busan 614-714, Korea,

2 Department of Biotechnology and Bioengineering, Dongeui University, Busan 614-714,

Korea,

3 School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea,

4 Division of Plant Pathology, National Institute of Agricultural Science and Technology,

Suwon 441-707, Korea,

5 Environment and Biotechnology Division, National Institute of Crop Science, Suwon

441-857, Korea

Magnaporthe oryzae is a causal pathogen of the rice blast, the most destructive disease of

rice worldwide. This ascomycete fungus has been intensively studied as a model

organism in plant-pathogen interactions. Recently, the genome sequence of M. oryzae 70-

15 was published by International Rice Blast Consortium. This led us to investigate

comparative and functional analysis of this pathogen at genome-wide level. As a first step,

sequencing project of M. oryzae Korean strain KJ201 was launched to face practical

interests on diversity of plant pathogen. We constructed a fosmid library with a copy

number controllable pCC1 vector. Over 13,000 end reads from 6,637 fosmid clones were

generated and anchored on genome sequence of strain 70-15. Currently, 9,793 end reads,

in which 2,684 clones with both ends and 1,757 clones with one end matched, are aligned

to the sequence of strain 70-15. With following criteria for match regions: matching

length longer than 90% of reads length, matching score larger than 50, and matching

percentage larger than 95% , about 90% genome coverage was achieved and coverage

plot was presented for each chromosome. For better alignment, in-house python-based

sequence analysis program, Match_manager, was developed and Cross_match

(http://www.phrap.org) was integrated as a key analyzer. In the next step, shotgun

sequencing of fosmid clones was conducted for fine scale comparative analysis of

chromosome 7 that is well defined in genome sequencing of strain 70-15. Totally 119

fosmid clones were pooled in region and analyzed. With the recent development in

sequencing technology, another round of whole genome analysis using 454 Life Science

Genome Sequencer FLX is being performed. The results from these combined analyses

will be presented.