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ABSTRACT A total of 540 isolates of Pyricularia grisea from rice in the United States were examined for vegetative compatibility, MGR586 DNA fingerprint diversity, and mating type based on hybridization with the mat1-1 and mat1-2 sexual mating type alleles. The collections contained both archived and contemporary field isolates representative of the known MGR586 lineages and races that occur throughout the United States. Complementary nitrate nonutilizing (nit) or sulfate nonutilizing (sul) mutants were used to assess vegetative compatibility in P. grisea. There was a complete correspondence between vegetative compatibility groups (VCGs), MGR586 lineage, and mating type among 527 contemporary isolates (collected between 1991 and 1997) from Arkansas, Louisiana, Missouri, Mississippi, and Texas; all isolates in MGR586 lineages A, B, C, and D belonged to VCGs US-01, US-02, US-03, and US-04, respectively. In addition, all isolates tested in VCGs US-01 and US-04 had the mat1-1 mating type allele whereas those in VCGs US-02 and US-03 had the mat1-2 allele. The strict association of independent markers during this sample period was consistent with a strictly asexual mode of reproduction. However, examination of archived isolates collected in the 1970s and 1980s and contemporary isolates revealed an incongruent relationship between the independent markers. MGR586 C and E isolates were vegetatively compatible which indicated that multiple robust MGR586 delineated lineages could be nested within certain VCGs. Although isolates in lineages C and E were vegetatively compatible, they were of opposite mating type. Several hypotheses, including recombination, could account for the incongruence between the various markers. Among the eight MGR586 lineages (A through H) that occur in the United States, all isolates in lineages A, D, E, G, and H had the mat1-1 allele, whereas isolates in lineages B, C, and F had the mat1-2 allele. Nit mutants can be recovered relatively easy from P. grisea and should allow large numbers of individuals within a population to be assessed for vegetative compatibility. VCGs may prove to be an effective multilocus marker in P. grisea. Thus, VCGs should be a useful means for characterizing genetic structure in populations of the rice blast fungus worldwide, provide a useful genetic framework to assist in interpreting molecular population data, and may provide insight into potential sexual or asexual recombination events.

ABSTRACT Thirty isolates of P. griseacollected from rice during a blast epidemic in 1995 in the high (1,800 to 2,600 m) and middle (1,200 to 1,800 m) elevations of Bhutan and 80 isolates collected from one rice cultivar from two high- and two mid-elevation sites in 1996 were analyzed for virulence. Differential varieties were indica CO39, with five near-isogenic lines (NILs) for resistance genes in the genetic background of CO39, and japonica Lijiangxintuanheigu (LTH), with five NILs for LTH. Twelve selected Bhutanese landraces also were studied. In addition, 10 blast nurseries consisting of the NIL sets, important local landraces, and representatives of international differential groups were established in the 1996 and 1997 growing seasons in the mid- and high-elevation agroecological zones. The 110 isolates were differentiated into 53 pathotypes based on the 2 NIL sets. Thirteen isolates were avirulent on all of the NILs but were compatible with some landraces. Several isolates were able to attack one of the NILs of CO39 but not CO39. These results strongly suggest that both CO39 and LTH possess previously unidentified resistance. The landraces were not uniform in their reactions to the isolates. When a reaction index taking into account all individual plant reactions was used, isolates that had been assigned to the same pathotype could be further differentiated, indicating that the NIL sets could not completely discriminate virulences in Bhutanese P. grisea populations. In the trap nurseries, disease was always present in the middle elevations, but disease was very low during July 1996 in the high elevations and only present during August and September 1997. Almost all varietal groups were more frequently attacked in the middle than in the high elevations, indicating that the virulence spectrum is wider and the conduciveness of the environment is greater in the middle elevations. Landraces from the high elevations were most susceptible, followed by international differential groups 7 and 8. The results suggest that selection has yielded landraces with more complete and complex resistance in the more disease-conducive mid-elevation environment. At the same time, the pathogen population also possesses a wider virulence spectrum in that environment.

Five hundred twenty-seven isolates of Pyricularia grisea were collected from trap rice cultivars of indigenous and exotic origin across three seasons at five sites in Thailand. Single conidium isolates were inoculated onto 15 rice lines near-isogenic (NILs) for resistance genes, one recurrent parent, and two local cultivars. One hundred seventy-five pathotypes were identified, of which 160 were represented by fewer than eight isolates. Predicted pathotype number was estimated at greater than 450 for the study region. Significant differences in pathotype diversity were detected across sites, seasons, and among isolates collected from exotic versus indigenous hosts. Isolates and pathotypes with greater numbers of virulence genes (as inferred from compatibility with NILs) were less common than those with fewer virulence genes. Analysis of virulence distributions among isolates grouped according to their MGR586 DNA-fingerprint similarities (i.e., "lineages") also showed that, for the most commonly represented lineages, isolates with fewer virulence genes predominated. Lineages represented by one or a few isolates had greater numbers of virulence genes. Lower frequency of recovery of isolates with accumulated virulence genes is consistent with an associated fitness penalty. Resistance genes Pi 1, Pi z-5, and Pi ta 2 were broadly effective across this population, compatibility with Pi 1 and Pi z-5 was very rare, and no isolate combined compatibility with both genes. Well-represented (more than 20 isolates) MGR586 lineages showed specific incompatibilities with some NILs, but these were restricted to Pi 1 and Pi z-5. No combination of resistance genes would confer resistance across all lineages.

Sexual fertility and mating type distribution of Magnaporthe grisea field isolates collected in Thailand were analyzed from sites previously found to harbor diverse populations of the pathogen. Three hundred forty-one single conidium isolates of M. grisea collected from five sites in north, northeast, and central Thailand were evaluated for in vitro sexual fertility and mating type by pairing with strains of known mating type. Most isolates (67%) were infertile when crossed with the hermaphrodite tester strains; but fertile isolates of each mating type that yielded viable ascospores were detected in all sites from the northeastern and northern regions. MAT1-2 predominated over MAT1-1 in bioassay mating type. Male fertility (female sterility) predominated in fertile MAT1-1 (50 to 75%) and MAT1-2 (50 to 85%) isolates from all locations in Thailand; however, hermaphroditic and/or female fertile isolates were also detected in all but one site. Fertility, as determined by perithecia density, was low (<10 perithecia cm -2 ) for most isolates, although a few produced in excess of 20 perithecia cm -2 .

The population genetics of Magnaporthe grisea, the rice blast pathogen, were analyzed in a center of rice diversity (the Uttar Pradesh hills of the Indian Himalayas) using multilocus and single-, or low-copy, DNA markers. Based on DNA fingerprinting with the multilocus probe MGR586 and single-locus probes, 157 haplotypes clustered into 56 lineages (at >/=70% MGR586 band similarity, each with unique single-locus profiles) and high diversity indices were detected among 458 isolates collected from 29 sites during 1992-1995. Most valleys sampled had distinct populations (73% of the lineages were site specific) with some containing one or a few lineages, confirming the importance of clonal propagation, and others were very diverse. Widely distributed lineages suggested that migration occurs across the region and into the Indo-Gangetic plains. Repeated sampling at one site, Matli, (170 isolates, 1992-1995) yielded 19 lineages and diversity significantly greater than that reported from similar samples from Colombia and the Philippines. Analysis of allelic associations using pairwise comparisons and multilocus variance analysis failed to reject the hypothesis of gametic phase equilibrium. The Matli population shifted from highly diverse in 1992 to almost complete dominance by one lineage in 1995. Such population dynamics are consistent with recombination followed by differential survival of clonal descendants of recombinant progeny. At another site, Ranichauri, population (n = 84) composition changed from 2 to 11 lineages over 2 yr and yielded additional evidence for equilibrium. Sexually fertile and hermaphrodite isolates of both mating types were recovered from rice in both Matli and Ranichauri. We demonstrate that Himalayan M. grisea populations are diverse and dynamic and conclude that the structure of some populations may be affected to some extent by sexual recombination.

ABSTRACT DNA samples from Magnaporthe grisea isolates were fingerprinted by using repetitive element-based polymerase chain reaction (rep-PCR) with two outwardly directed primer sequences from Pot2, an element found in approximately 100 copies in the fungal genome. Variable length fragments, defining the sequences lying between these elements, were generated, and fingerprint patterns specific for individual strains were established. "Long PCR" conditions, including higher pH (9.2) and increased extension time (10 min) were used to amplify DNA fragments ranging from 400 bp to longer than 23 kb. Polymorphisms specific to M. grisea strains were generated, allowing inference of their genetic relationships. Segregation analysis was used to confirm single-locus inheritance for the fragments amplified by rep-PCR. Cluster analysis revealed robust groupings that corresponded to previously determined MGR586 restriction fragment length polymorphism lineages of the rice-infecting strains of the pathogen. We have also demonstrated the utility of rep-PCR to differentiate isolates that infect rice from those that infect nonrice hosts. DNA fingerprinting by Pot2 rep-PCR provides an efficient means to monitor the population dynamics of the blast pathogen. Because of the method's low cost and ease in application, it is now feasible to conduct large-scale population studies to understand the impact of host genotypes on pathogen evolution.

The heterothallic ascomycete, Magnaporthe grisea, is the blast pathogen of rice and about 50 other grasses, and has potential for sexual and asexual reproduction. In most populations, data from mating type, fertility assays, and genotypic diversity strongly suggest that the pathogen is asexual. However, parasexual recombination cannot be ruled out. Chromosome length polymorphisms and translocations may prevent successful meiosis in most populations. Pathogens of millets and some grasses growing with rice appear to be largely genetically isolated, though some gene flow may occur. Sexual fertility has repeatedly been reported in rice pathogens from mountainous regions of South and East Asia. Several means by which sexual fertility may be lost in an agricultural setting are advanced.

ABSTRACT We applied DNA markers to determine whether parasexual recombination may contribute to the extreme genetic diversity and variability observed in Magnaporthe grisea, the causal agent of rice blast disease. Dispersed repetitive elements and mapped, low-copy restriction fragment length polymorphism (RFLP) probes were used to detect transfers of DNA between cultured isolates of M. grisea. Low-copy RFLP probes also were used to detect putative recombinants among isolates from well-characterized field populations of the pathogen. Microscopic examination of tufted mycelium between cocultured isolates revealed frequent hyphal fusions. Hyphal tips and conidia were recovered without selection from tufted zones in two separate vegetative pairings involving isolates with dissimilar haplotypes, based on the repetitive element MGR586. Haplotypic changes were observed at a higher frequency in tuft derivatives than in subcultures of each isolate alone. From 136 tuft derivatives analyzed, 5 putative recombinant haplotypes were identified. Introgression was demonstrated with two independent repetitive elements, fosbury and MGR586, as probes on DNA digested with several restriction enzymes. Introgressions were characterized by addition of 1 to 10 MGR586 bands, and 1 to 3 fosbury bands from one parent into the background of the other. Polymorphic single-copy probes were used to analyze putative recombinants. One probe detected an introgression event as predicted by analysis with MGR586. To assess the possible role of parasexual recombination in field populations of the pathogen, isolates in the Philippines previously grouped based on DNA fingerprinting were analyzed with low-copy RFLP markers. Polymorphism in single-copy loci typically was seen between, but not within, putative pathogen lineages. One lineage (designated lineage 4), however, was polymorphic for several probes. For some isolates, alleles at these loci comigrated with alleles characteristic of other lineages, suggesting the transfer of DNA fragments between lineages. One isolate was apparently a merodiploid, carrying an allele typical of lineage 4 plus another allele characteristic of a different lineage. In a survey of isolates from the Indian Himalayas, a merodiploid also was found with single- or low-copy probes. Examination of MGR586 profiles of the putative recombinant and its putative donor strains showed the expected introgression of MGR586 bands. The detection of parasexual DNA exchanges in wild-type strains under unselected conditions and the existence of merodiploids in nature suggest that parasexual recombination occurs in field populations of M. grisea. This raises questions concerning exclusive clonality in the blast fungus.