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In order to understand the pathogenicity differentiation of rice blast fungus (Pyricularia oryzae Cavara), a total of 206 isolates of P. oryzae were collected from three Japonica rice regions in Jilin Province, northeast China. Pathogenicity test showed that the reaction pattern of 25 monogenic differential varieties (MDVs) of rice (Oryza sativa L.) demonstrated a wide pathogenic diversity among the isolates. Those MDVs harbor 23 resistance (R) genes with the susceptible variety Lijiangxintuanheigu (LTH) as control. Virulent isolates of MDVs harboring R genes Pish, Pit, Pia, Pii, Pik-s, Pik, Pita (two lines), and Pita-2 (two lines) had high frequencies ranging from 80 to 100%, to MDVs harboring R genes Pib, Pi5(t), Pik-m, Pi1, Pik-h, Pik-p, Pi7(t), Piz, Piz-5, and Piz-t showed intermediate frequencies ranging from 40 to 80%, and to MDVs with R genes Pi3, Pi9(t), Pi12(t), Pi19(t) and Pi20(t) presented low frequencies ranging only from 0 to 40%. The U-i-k-z-ta pattern of race-named criteria categorized the 206 isolates into 175 races. Sub-unit U73 for Pib, i7 for Pi3 and Pi5(t), k177 for Pik-m/Pik-h/Pik-p, z17 for Pi9(t), and ta332 for Pi20(t) were crucial on pathogenic differences in regions. Twenty-seven standard differential blast isolates (SDBIs) were selected to characterize resistance in rice accessions. This study could help to build a durable identification system against blast in the Japonica rice area of northeast China and enhance our understanding of the differentiation and diversity of blast races in the world. [ABSTRACT FROM AUTHOR]

Rice (Oryza sativa L.) is a staple food in many countries around the world, particularly in China. The production of rice is seriously affected by the bacterial leaf streak and rice blast, which can reduce rice yield or even cause it to fail to be harvested. In this study, susceptible material 58B was edited by CRISPR/Cas9, targeting a target of the Pi21 gene and a target of the effector-binding element (EBE) of the OsSULTR3;6 gene, and the mutants 58b were obtained by Agrobacterium-mediated method. The editing efficiency of the two targets in the T0 generation was higher than 90.09%, the homozygous mutants were successfully selected in the T0 generation, and the homozygous mutation rate of each target was higher than 26.67%. The expression of the edited pi21 and EBE of Ossultr3;6 was significantly reduced, and the expression of defense responsive genes was significantly upregulated after infected with rice blast. The lesion areas of rice blast and bacterial leaf streak were significantly reduced in 58b, and the resistance of both was effectively improved. Furthermore, the gene editing events did not affect the agronomic traits of rice. In this study, the resistance of 58b to rice blast and bacterial leaf streak was improved simultaneously. This study provides a reference for using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) to accelerate the improvement of rice varieties and the development of new materials for rice breeding. [ABSTRACT FROM AUTHOR]

The rice blast disease (caused by Magnaporthe oryzae) is a devastating disease in China. Understanding the molecular mechanisms of interaction for the cognate avirulence (AVR) gene with host resistance (R) genes, as well as their genetic evolution is essential for sustainable rice production. In the present study, we conducted a high-throughput nucleotide sequence polymorphism analysis of the AVR-Pi9 gene that was amplified from the rice-growing regions of the Yunnan Province in China. We detected the presence of seven novel haplotypes from 326 rice samples. In addition, the sequences of AVR-Pi9 were also obtained from two non-rice hosts, Eleusine coracana and Eleusine indica. The sequence analysis revealed the insertions and deletions in the coding and non-coding regions of the gene. The pathogenicity experiments of these haplotypes on previously characterized monogenic lines showed that the newly identified haplotypes are virulent in nature. The breakdown of resistance was attributed to the development of new haplotypes. Our results suggest that the mutation in the AVR-Pi9 gene is an alarming situation in the Yunnan province and thus needs attention. [ABSTRACT FROM AUTHOR]

Traditional agrosystems, where humans, crops and microbes have coevolved over long periods, can serve as models to understand the ecoevolutionary determinants of disease dynamics and help the engineering of durably resistant agrosystems. Here, we investigated the genetic and phenotypic relationship between rice (Oryza sativa) landraces and their rice blast pathogen (Pyricularia oryzae) in the traditional Yuanyang terraces of flooded rice paddies in China, where rice landraces have been grown and bred over centuries without significant disease outbreaks. Analyses of genetic subdivision revealed that indica rice plants clustered according to landrace names. Three new diverse lineages of rice blast specific to the Yuanyang terraces coexisted with lineages previously detected at the worldwide scale. Population subdivision in the pathogen population did not mirror pattern of population subdivision in the host. Measuring the pathogenicity of rice blast isolates on landraces revealed generalist life history traits. Our results suggest that the implementation of disease control strategies based on the emergence or maintenance of a generalist lifestyle in pathogens may sustainably reduce the burden of disease in crops. [ABSTRACT FROM AUTHOR]

Rice is the most important crop for worldwide consumers. The water utilization of rice planting is more than 50% of agricultural water in China, and it is necessary to breed water-saving and drought-resistant rice. The rice variety Dianheyou 615 can be planted in the paddy and upland fields, which satisfies rice production farmers in mountainous regions of Yunnan. We aimed to explore the variations in Magnaporthe oryzae isolates collected from Dianheyou 615 planted in paddy or upland fields. Through pathogenicity tests, we found that most isolates had the highest pathogenicities, but there were no significant differences between the paddy and upland isolates. By a combination of monogenetic and elite rice lines, with a further resistance assessment, the monogenetic lines with Pi9, Diantun 506, and Lvhan 1 displayed better resistances. Moreover, we re-sequenced 15 isolates to explore their genetic variations. Our results showed that the source of the upland isolates may have been the offspring of the paddy isolates, but there were many genes with specifically found SNPs in two populations that would develop subdivisions after long-time planting. Overall, we compared the pathogenicities and genetic variations in blast isolates from the planting of Dianheyou 615 in paddy and upland fields, which provided references for the influence of the planting environment on population subdivisions. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae is known for its genetic diversity and pathogenic variability, leading to rapid breakdown of resistance in rice. Incorporating multiple broad-spectrum blast resistance genes into rice cultivars would extend disease resistance longevity. Effective resistance breeding in rice therefore requires continual enrichment of the reservoir of resistance genes and alleles. We conducted a large-scale screen of rice blast resistance in about 2 000 rice accessions. Among them, 247 accessions showed at least medium resistance to the natural infection of rice blast and 7 novel Pik alleles were identified from them. Variations in gene sequences were then correlated with the phenotypic trait to enable the identification of favorable alleles. Among the seven novel Pik alleles, the resistant rate of Pik-R0/ME/7017 donors was greater than 80%, and the disease score was less than 3. Through molecular marker-assisted backcross breeding, we successfully transferred the three Pik alleles, Pik-R0/ME/7017 , into an elite cultivated line Kongyu 131 to obtain BC 3 F 2 lines, which showed enhanced resistance to rice blast compared with the recurrent parent. Assessment of these near-isogenic lines in the greenhouse using 31 isolates of M. oryzae from Heilongjiang Province of China revealed that the resistant levels of the BC 3 F 2 lines with Pik-R0/ME/7017 were significantly higher than those of the established cloned resistance genes Pik-m and Pi1. Exploring such alleles will enrich our gene library for resistance to rice blast. [ABSTRACT FROM AUTHOR]

Summary: Because of the frequent breakdown of major resistance (R) genes, identification of new partial R genes against rice blast disease is an important goal of rice breeding. In this study, we used a core collection of the Rice Diversity Panel II (C‐RDP‐II), which contains 584 rice accessions and are genotyped with 700 000 single‐nucleotide polymorphism (SNP) markers. The C‐RDP‐II accessions were inoculated with three blast strains collected from different rice‐growing regions in China. Genome‐wide association study identified 27 loci associated with rice blast resistance (LABRs). Among them, 22 LABRs were not associated with any known blast R genes or QTLs. Interestingly, a nucleotide‐binding site leucine‐rich repeat (NLR) gene cluster exists in the LABR12 region on chromosome 4. One of the NLR genes is highly conserved in multiple partially resistant rice cultivars, and its expression is significantly up‐regulated at the early stages of rice blast infection. Knockout of this gene via CRISPR‐Cas9 in transgenic plants partially reduced blast resistance to four blast strains. The identification of this new non‐strain specific partial R gene, tentatively named rice blast Partial Resistance gene 1 (PiPR1), provides genetic material that will be useful for understanding the partial resistance mechanism and for breeding durably resistant cultivars against blast disease of rice. [ABSTRACT FROM AUTHOR]

DNA test is broadly used in diagnosis of crop disease and identification of genetic modified organism (GMO) in agriculture. However, rapid, low-cost, user-friendly, and field-deployable DNA test method is still limit. Recently, the RNA programmable nuclease of CRISPR/Cas is engineered as a new nucleic acid detection platform, but their application in plant remains to explore. In this study, we evaluated the Cas12a-based DNA detection for crop disease diagnosis and GMO test. A total of 14 crRNAs were designed to target two Magnaporthe oryzae genes and a synthetic Cry1C gene which encodes Bacillus thuringiensis δ-endotoxin and has been used to develop transgenic rice cultivar (Bt-rice) in China. Using a fluorescent reporter, targeted genes were easily detected by LbCas12a after recombinase polymerase amplification (RPA) for all crRNAs, despite that the signal strength varied 2–3-folds between different crRNAs. We further combined the filter paper-based DNA extraction and lateral flow assay (LFA) with RPA-Cas12a for DNA detection. This optimized Cas12a diagnostics method is carried at body temperature and does not require extra instrument except filter paper and LFA strip. Our data show that rice blast pathogen and Bt-rice were efficiently identified from leaf disc samples using this optimized DNA test method with highly active crRNAs. Moreover, LbCas12a exhibited variable nuclease activities on different targets; therefore, highly active crRNA is critical for successful DNA test using Cas12a and LFA. Owing to its simplicity, efficiency, and low-cost, DNA test using CRISPR/Cas12a would be easily applied in field for crop disease diagnosis and GMO administration. [ABSTRACT FROM AUTHOR]

Rice blast is a very serious disease caused by Magnaporthe oryzae , which threatens rice production and food supply throughout the world. The avirulence ( AVR ) genes of rice blast are perceived by the corresponding rice blast resistance ( R ) genes and prompt specific resistance. A mutation in AVR is a major force for new virulence. Exploring mutations in AVR among M. oryzae isolates from rice production fields could aid assessment of the efficacy and durability of R genes. We studied the probable molecular-evolutionary patterns of AVR-Pib alleles by assaying their DNA-sequence diversification and examining their avirulence to the corresponding Pib resistance gene under natural conditions in the extremely genetically diverse of rice resources of Yunnan, China. PCRs detected results from M. oryzae genomic DNA and revealed that 162 out of 366 isolates collected from Yunnan Province contained AVR-Pib alleles. Among them, 36.1-73.3% isolates from six different rice production areas of Yunnan contained AVR-Pib alleles. Furthermore, 36 (28.6%) out of 126 isolates had a transposable element (TE) insertion in AVR-Pib , which resulted in altered virulence. The TE insertion was identified in isolates from rice rather than from Musa nana Lour. Twelve AVR-Pib haplotypes encoding three novel AVR-Pib variants were identified among the remaining 90 isolates. AVR-Pib alleles evolved to virulent forms from avirulent forms by base substitution and TE insertion of Pot2 and Pot3 in the 5' untranslated region of AVR-Pib . These findings support the hypothesis that functional AVR-Pib possesses varied sequence structures and can escape surveillance by hosts via multiple variation manners.

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most destructive plant diseases in the world and a serious threat to global food security. Biofungicide physcion, extracted from roots of Chinese traditional herb rhubarb, has been commercialized to control cucumber powdery mildew ( Podosphaera fuliginea) in China as a new active and environmentally-friendly ingredient. This study investigated its bioactivity on rice blast and impact on the infection of M. oryzae. Physcion was effective on M. oryzae with 50% inhibition concentration of 18.19 mg/l on the fungus mycelium growth in vitro and provided 86 and 98% control efficacy in vivo at the concentrations of 40 and 60 mg/l, respectively. It inhibited the initial infection of M. oryzae by inhibiting growth of mycelia, the germination of conidia and the formation of appressoria. Moreover, it inhibited the secondary infection of the fungus at the concentration of 40 mg/l, but had no influence at the low concentrations of 1.25-20 mg/l. So physcion might be a promising natural plant extract that is worthy of being explored to apply on the control of rice blast. [ABSTRACT FROM AUTHOR]

Excavation of resistance genes is one of the most effective and environment-friendly measures to control the devastating rice disease caused by Magnaporthe oryzae. Many resistance genes have been mapped and characterized in the last century. Nevertheless, only a few of the total resistance genes could be really applied in the rice breeding program. Huazhan (HZ) is a new native rice restorer line developed in China and widely used in hybrid rice in recent years. HZ and its crossed combinations usually show a broad spectrum of resistance against rice blast in different rice ecosystems in China. Dissection of the genetic background of HZ is very useful for its further application. In this study, a combined method based on bulked segregation analysis (BSA) and specific length amplified fragment sequencing (SLAF-seq) was used to identify blast resistance gene(s) in HZ. A total of 56,187 SLAFs labels were captured and 9051 polymorphic SLAFs markers were analysed and procured in this study. One trait associated with candidate resistance genes region on chromosome 12 overlapping 10.2–17.6 Mb has been identified, in which 10 NBS-LRR (nucleotide-binding site-leucine-rich repeat) coding genes were used as resistance gene candidates. Our result indicated that SLAF-seq with BSA is a rapid and effective method for initial identification of blast resistance genes. The identification of resistance gene in HZ will improve its molecular breeding and resistance variety application. [ABSTRACT FROM AUTHOR]

Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production, in this study, on the basis of detection arid verification of the genotypes of 22 rice varieties harboring known blast resistance genes (Pi-ta and Pi-b) and blast susceptibility genes (pi-ta and pi-b), two multiple PCR systems for these genes were established by using the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta arid pi-b. Iri addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resistance in rice breeding. [ABSTRACT FROM AUTHOR]

Sensitivity to azoxystrobin and kresoxim-methyl of 80 single-spore isolates of Magnaporthe oryzae was determined. The EC50 values for azoxystrobin and kresoxim-methyl in inhibiting mycelial growth of the 80 M. oryzae isolates were 0.006-0.056 and 0.024-0.287 µg mL−1, respectively. The EC50 values for azoxystrobin and kresoxim-methyl in inhibiting conidial germination of the M. oryzae populations were 0.004-0.051 and 0.012-0.105 µg mL−1, respectively. There was significant difference in sensitivity to azoxystrobin or kresoxim-methyl between the tested isolates representing differential sensitivity to carbendazim ( MBC) and kitazin P ( IBP); however, there was no correlation between this difference in sensitivity to azoxystrobin or kresoxim-methyl and sensitivity to MBC or IBP, indicating that there was no cross-resistance between azoxystrobin or kresoxim-methyl and MBC or IBP. In the protective and curative experiments, kresoxim-methyl exhibited higher protective and curative activity than azoxystrobin when applied at 150 and 250 µg mL−1 accordingly, while azoxystrobin exhibited stronger inhibitory activity against M. oryzae isolates than that of kresoxim-methyl in the in vitro test. The results of field experiments also suggested that both azoxystrobin and kresoxim-methyl at 187.5 g.a.i. ha−1 gave over 73% control efficacy in both sites, exhibiting excellent activity against rice blast. Taken together, azoxystrobin and kresoxim-methyl could be a good substitute for MBC or IBP for controlling rice blast in China, but should be carefully used as they were both at-risk. [ABSTRACT FROM AUTHOR]

In order to determine the resistance of leading rice varieties to rice blast in Guangxi, three natural induction rice blast nurseries were established in Cenxi, San-jiang and Yongfu. The resistance of the 45 leading rice varieties in Guangxi to rice blast was determined under conditions of spray inoculation and natural induction at seedling stage. The results showed that resistance frequencies of the 45 leading rice varieties ranged from 8.33% to 94.44%. Among the 45 tested varieties, there were 8 varieties of which the resistance frequencies were all higher than 80%, accounting for 17.78%; there were 11 varieties of which the resistance frequencies ranged from 50% to 80%, accounting for 24.44%; the resistance frequencies of the remaining varieties were all lower than 50%, accounting for 57.78% of the total tested varieties. The two-consecutive-year determination results showed none of the 45 leading rice varieties showed resistance to rice blast. In 2010, there were 26, 12 and 7 varieties that were moderately sensitive, sensitive and greatly sensitive to rice blast respectively, accounting for 57.78%, 26.67% and 15.56% of the total tested varieties. In 2011, there were 16, 23 and 12 varieties that were moderately sensitive, sensitive and greatly sensitive to rice blast respectively, accounting for 35.56%, 51.11% and 13.33%. The overall resistance of the 45 leading rice varieties trended to be decreased year by year. So in rice production, they should be selected properly. [ABSTRACT FROM AUTHOR]

[Objective] This study aimed to explore rice germplasm resources resistant to rice blast and provide theoretical basis for the modification of rice blast-resistant germplasm resources. [Method] The resistance of 120 rice materials to rice blast was identified at three experimental areas. [Result] The resistance of 120 experimental materials was at grade 0-9 to panicle blast in Hainan Province, at grade 0-9 to seedling blast in Fujian Province and at grade 3-9 to panicle blast in Guangxi Zhuang Autonomous Region. Analysis of correlations between agronomic traits and rice blast resistance indicated that the resistance of experimental materials could not be simply determined based on agronomic traits. Furthermore, 61 published SSR makers closely linked to rice blast were selected and synthesized for genotypic analysis of 120 experimental materials. The results of cluster analysis preliminarily divided 120 germplasms into resistant varieties and susceptible varieties. [Conclusion] These rice germplasm resources are of important value for breeding new materials resistant to rice blast. [ABSTRACT FROM AUTHOR]

Abstract: Strain BC79, isolated from primeval forest soil in Qinling, Mountains, China, was identified as Bacillus methylotrophicus based on morphological, biochemical, physiological and chemotaxonomic analyses as well as phylogenetic 16S rDNA sequencing data. This strain was able to suppress mycelial growth and conidial germination of numerous plant pathogenic fungi in dual cultures on solid media. For exploring potential biocontrol activity, we assessed fermentation conditions for studying B. meth1ylotrophicus BC79. The active substance of BC79, phenaminomethylacetic acid, was extracted by TLC and HPLC, and identified as the strongest inhibitory substance described in B. methylotrophicus. Experiments in a greenhouse showed that application of BC79 culture filtrates 24 h before inoculation of Magnaporthe oryzae, the causal agent of rice blast, had 89.87% biocontrol efficiency. B. methylotrophicus BC79 colonized rice plant tissues and at 10 days after filtrate application, its population in leaves (1.65 × 108 CFU/g) was much larger than in stems (6.78 × 107 CFU/g) or roots (3.56 × 107 CFU/g). Field trials indicated that BC79 culture filtrate (4000 g/667 m2) showed the highest efficiency for M. oryzae, with 84.8% biocontrol effect, followed by of 15% phenaminomethylacetic acid extract (75.5%) and 20% tricyclazole (76.1%). Seedling and post-transplant stages were the best periods to apply BC79 for control of rice blast. The B. methylotrophicus BC79 strain hence has enormous potential as an agricultural agent for biocontrol of rice blast. [Copyright &y& Elsevier]

Abstract: Race-specific resistance and field resistance of 30 rice blast resistance monogenic lines derived from different resources were evaluated. The spectra of resistance to 163 Magnaporthe grisea isolates collected from indica rice in Guangdong Province, China ranged from 0.6% to 89.6%. Most of the monogenic lines showed a narrow resistance spectrum and high susceptibility in rice blast area, whereas the lines with Pik h and Pi1(t) had the broad resistance spectra of 89.6% and 82.2% respectively, showing a high and stable blast resistance in fields. According to the cluster analysis of specific resistance to 163 blast isolates tested, the 30 monogenic lines were divided into 15 groups, and based on the principal factor analysis, nine kinds of race-specific resistance were identified. Pik, Piz 5 , Pi9 and Pish can be used as candidate resistance genes for rice breeding since their specific resistance differed from those of the backbone parents in Guangdong, China. Gene pyramiding of Pik h [or Pi1(t)], Pi9 (or Piz 5 ) and Pish (or Pita 2 ) will be effective to obtain broad-spectrum blast resistance in rice breeding program in Guangdong, China. The strategies for studying and application of rice blast resistance genes were discussed. [Copyright &y& Elsevier]

A rice isolate of Magnaporthe grisea collected from China was avirulent on rice cvs. Hattan 3 and 13 other Japanese rice cultivars. The rice cv. Hattan 3 is susceptible to almost all Japanese blast fungus isolates from rice. The genetic basis of avirulence in the Chinese isolate on Japanese rice cultivars was studied using a cross between the Chinese isolate and a laboratory isolate. The segregation of avirulence or virulence was studied in 185 progeny from the cross, and monogenic control was demonstrated for avirulence to the 14 rice cultivars. The resistance gene that coresponds to the avirulence gene Avr-Hattan 3) is thought to be located at the Pik locus, Resistance and susceptibility in response to the Chinese isolate in F3 lines of a cross of resistant and susceptible rice cultivars were very similar to the Pik tester isolate, Ken54-20. Random amplified polymorphic DNA markers and restriction fragment length polymorphism markers from genetic maps of the fungus were used to construct a partial genetic map of Avr-Hattan 3. We obtained several flanking markers and one co-segregated marker of Avr-Hattan 3 in the 144 mapping population. [ABSTRACT FROM AUTHOR]

Background: Effective management of rice blast, caused by the fungus Magnaporthe oryzae, requires an understanding of the genetic architecture of the resistance to the disease in rice. Rice resistance varies with M. oryzae strains, and many quantitative trait loci (QTLs) affecting rice blast resistance have been mapped using different strains of M. oryzae from different areas. However, little is known about the genetic architecture of rice resistance against the M. oryzae population in Hunan Province, which is a main rice production area in South China. Results: In this study, we used three isolates from Hunan Province and the rice diversity panel 1 to perform a genome-wide association study (GWAS) of blast resistance in rice. A total of 56 QTLs were identified. One of the QTLs is localized with the resistance gene Pik locus which confers resistance to all three isolates. Genomic sequence analysis of the resistant cultivars led to the identification of a new Pik allele, which we named Pikx. Yeast two-hybrid and co-immunoprecipitation assays between AvrPiks and Pikx confirmed that Pikx is a new allele at the Pik locus. Conclusions: Our GWAS has identified many new blast resistance QTLs. The identified new Pik allele Pikx will be useful for breeding cultivars with high resistance to blast in Hunan and other South China provinces. Further research on the relationship between AvrPiks and Pikx will provide new insights into the molecular mechanism of rice resistance to M. oryzae. [ABSTRACT FROM AUTHOR]

The identification and utilization of broad-spectrum resistance genes is the most effective and economical strategy of controlling rice blast disease. Kangfeng B (KFB), an elite Chinese rice cultivar, has been shown to exhibit broad-spectrum resistance to 53 isolates of Magnaporthe oryzae, the rice blast causative agent, from different regions of China. In this study, we identified a dominant blast-resistance gene at the Pi2/Pi9 locus from cultivar KFB, through genetic analysis and physical mapping. Allele-specific marker-based assessment revealed that Pi2, Pi9, and Piz-t are not the blast-resistance genes in KFB. By combining bulked segregation analysis (BSA) and recessive class analysis (RCA), the blast-resistance gene was fine-mapped to an approximately 249-kb interval between markers InDel-22 and Rm7213 on chromosome 6. Three bacterial artificial chromosome (BAC) clones spanning the region were identified. This region contains 19 predicted genes, including 7 nucleotide binding site–leucine-rich repeat (NBS-LRR) genes at the Pi2/Pi9 locus in japonica cv. Nipponbare genome. Further sequence comparison of the four functional NBS-LRR genes revealed that NBS-LRR2 and NBS-LRR4, as evidenced by their allelic/orthologous to Pi9 or Pi2, had significant differences of 9 to 43 and 14 to 48 amino acids in KFB, respectively, unlike the other known Pi2/Pi9 alleles, suggesting that KFB carries a hitherto undocumented member of the Pi2/Pi9 multigene family. It was tentatively designated as Pi-kf2(t). Our results provide essential information for the isolation of the Pi-kf2(t) gene and will facilitate both map-based cloning and marker-assisted selection of this gene for rice blast-resistance breeding. [ABSTRACT FROM AUTHOR]