New detections of chestnut blight in Great Britain during 2019–2020 reveal high Cryphonectria parasitica diversity and limited spread of the disease

Cryphonectria parasitica was detected for the first time in the United Kingdom in 2011. A 2017–2018 survey detected the disease at different sites in Berkshire, Derbyshire, Devon, Dorset and London, while the present study comprises the results of the 2019–2020 survey with new findings and additional sites in the United Kingdom (Berkshire, Buckinghamshire, Cornwall, Derbyshire, Devon, London, West Sussex) and in Jersey, reflecting the progressive detection of more infected trees. A total of 189 samples were collected from 52 sites, and 123 samples tested positive both by quantitative real-time PCR and/or isolation from 43 sites. A total of 115 isolates were tested for mating type, vegetative compatibility group (VCG) and Cryphonectria hypovirus 1 (CHV-1). Twelve VCGs were identified, with four of them being first records in United Kingdom. The highest diversity of VCGs was detected in Devon followed by West Sussex while London and Derbyshire presented the lowest. Both mating types were detected (41% MAT-1 and 59% MAT-2), and no heterokaryons were detected. Perithecia of C. parasitica were not observed at any site during this survey. CHV-1 was detected in three isolates in very low concentration from three different locations in London and was always the unmutated subtype I haplotype E-5. A greater diversity of VCGs at outbreak sites compared with previous surveys, combined with their scattered distribution and the slow spread of the pathogen, supports the hypothesis that this disease has been introduced through imports over time from Europe.     

Characterization of hypovirulent isolates of the chestnut blight fungus, Cryphonectria parasitica from the Marmara and Black Sea regions of Turkey

Chestnut blight caused by the introduced fungus Cryphonectria parasitica has been responsible for the decline of Castanea sativa in Turkey since the 1960s. In this study, 72 C. parasitica isolates were recovered from the Marmara and Black Sea regions of Turkey showing white or cream-coloured culture morphology and were subjected to various tests to determine if they were infected by Cryphonectria hypovirus 1 (CHV-1). The vast majority of the isolates (69 out of 72) were vc type EU-1. Both mating types were found among a subsample of the isolates. The hypovirus was detected in 55 isolates by dsRNA extraction and/or virus specific RT-PCR on total RNA extracts. All but one isolates showed no or only weak phenol oxidase activity on agar medium containing tannic acid, typical of CHV-1 infected isolates. Through sequencing of a specific region of the hypovirus genome, we found that 24 hypovirus isolates belonged to the CHV-1 subtype I and six to the CHV-1 subtype F2. The distribution of the two CHV-1 subtypes in Turkey showed a clear geographic pattern. CHV-1 subtype I was only detected in the Marmara and western Black Sea region, whereas subtype F2 was restricted to the eastern part of the Black Sea region. The effectiveness of 23 hypovirulent isolates was tested against a virulent isolate on 2–3 years old chestnut sprouts. Ten hypovirulent isolates, all infected by CHV-1 subtype I, prevented canker development by more than 80 % suggesting that they might be suitable for biological control of chestnut blight in Turkey.     

Horizontal transmission of hypoviruses between vegetative compatibility types of <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> in Macedonia

Biological control of chestnut blight with hypovirulence depends on the successful transmission of hypoviruses between individuals of the chestnut blight fungus, Cryphonectria parasitica. Vegetative incompatibility inhibits horizontal virus transmission, but not completely. In an effort to assess the potential for the spread of hypoviruses in the Republic of Macedonia, we studied the transmission of Cryphonectria hypovirus 1 (CHV-1) among the five observed vegetative compatibility (vc) types of C. parasitica. One fungal isolate of each vc type was infected with CHV-1 and was paired in vitro with isolates of all other vc types for a total of 20 combinations of virus donors and recipients, and 250 replicate trials per combination. Virus transmission was scored after 7 days as successful if the recipient isolate took on an unpigmented culture phenotype typical of virus infection. Transmission occurred at high frequencies between some pairs of vc types, but in <1% of the trials for 10 of the 20 combinations of donors and recipients. Asymmetric transmission was observed between some vc types that had different alleles at vegetative incompatibility loci vic1 or vic7; i.e., transmission occurred at high frequencies in one direction, but very low frequencies between the same pair of isolates in the opposite direction. The expected virus transmission, calculated as the average transmission predicted for any two randomly chosen individuals from a population, was highly negatively correlated to vc type diversity. Results for isolates of C. parasitica from Macedonia were similar to those from Italy, but less transmission was generally observed. Differences in genetic background effects on transmission may vary among different populations even when isolates differ by the same vic alleles.     

Diversity and spatial distribution of vegetative compatibility types and mating types of <Emphasis Type="BoldItalic">Cryphonectria parasitica</Emphasis> in the Aydın Mountains,Turkey

In this study, the population structure of the chestnut blight fungus Cryphonectria parasitica in the Aydın Mountains was investigated to make inferences about fungal reproduction and population diversity. A total of 213 C. parasitica isolates from eight subpopulations were used to determine vegetative compatibility (vc) and mating types of the population. Furthermore geostatistical analysis was performed to define the spatial structure of the population. The results showed that the isolates were vegetatively compatible with the European vc types of either EU-1 or EU-12. Both vc types were found in almost all subpopulations, but their frequencies varied depending on location. The results of a PCR assay showed that both mating types of C. parasitica (MAT-1 and MAT-2) exist in the population. MAT-1 comprised 65% of the total isolates, and the ratio of mating types was significantly skewed from 1:1. Genotyping based on combined vc and mating type data revealed four genotypes: EU-1/MAT-1 (28.6%), EU-1/MAT-2 (34.7%), EU-12/MAT-1 (36.2%) and EU-12/MAT-2 (0.5%). Geostatistatical analysis indicated that vc types, mating types and vc/mating genotypes were spatially autocorrelated and clustered in their distributions. Results suggested that C. parasitica could have a clonal population structure that is generated by asexual reproduction. Low vc-type diversity suggests that the C. parasitica population in the Aydın Mountains may be highly suitable to hypovirus invasion, thereby providing a high potential for successful biological control. However, co-occurrence of sexually compatible strains of EU-1 and EU-12 at the same locations in close proximity creates a high risk of increase in vc-type diversity.     

Chestnut blight fungus in Croatia: diversity of vegetative compatibility types,mating types and genetic variability of associated Cryphonectria hypovirus 1

In order to improve understanding of its diversity, 338 isolates of Cryphonectria parasitica, the causal agent of chestnut blight, were sampled from 10 chestnut populations throughout chestnut‐growing coastal and continental areas of Croatia. Eighteen vegetative compatibility (VC) types were identified. The VC type EU‐1 was the most widespread, comprising 42·9% of the isolates, followed by EU‐2 (21%) and EU‐12 (14·2%). In respect to the occurrence of the main VC types, the C. parasitica populations in Croatia combined features of both northwestern and southeastern European populations. Perithecia and mating‐type ratios of approximately 1 : 1 were found in all populations, suggesting that sexual reproduction of the fungus is common in Croatia. Natural hypovirulence was also evident in all populations, with incidence of hypovirus‐infected isolates ranging from 12·7% in Istria‐Buje to 66·6% in the continental part of the country. A total of 36 hypovirus‐infected isolates sampled throughout Croatia were analysed in ORF‐A and ORF‐B by RT‐PCR/RFLP analysis. All viral isolates belonged to the Italian subtype of Cryphonectria hypovirus 1 (CHV‐1) and were closely related to the isolates found in other European countries. The RFLP patterns found were also identical or similar to the patterns of three isolates collected in Croatia 22 years ago, suggesting a slow evolution of the hypovirus.     

Multilocus PCR assay reveals high diversity of vegetative compatibility types in populations of Cryphonectria parasitica in Croatia

The ascomycete fungus Cryphonectria parasitica, causal agent of chestnut blight, is probably one of the best known invasive fungal pathogens in forests of Europe and North America. Mycovirus that reduces virulence of C. parasitica can be used as a biocontrol agent of the chestnut blight. However, anastomosis‐mediated virus transmission is limited by a vegetative (in)compatibility (vc) system involving at least six known diallelic vic genetic loci. This study looked at vegetative compatibility (vc) diversity in two populations of C. parasitica in Croatia. For that purpose, a PCR assay was validated and implemented using already known/published and newly designed primers for amplification of six known vic loci. The vc genotypes determined by PCR for 158 C. parasitica isolates investigated in this study were in complete agreement with the vc genotypes determined by pairwise co‐culturing of the same isolates, revealing the specificity and accuracy of the PCR‐based molecular vic genotyping assay. Twenty‐six unique vc genotypes were found among 158 isolates, and 19 vc types per population, which makes Croatian C. parasitica populations among the most diverse in Europe regarding the number of vc types and genetic diversity. Low values of multilocus linkage disequilibrium suggest sexual reproduction as a major contributor to high C. parasitica genetic diversity in studied populations.     

Clonal population structure and introductions of the chestnut blight fungus, <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>, in Asturias,northern Spain

To understand the history of introductions of the chestnut blight fungus, Cryphonectria parasitica, in the Principality of Asturias in northern Spain, we conducted an extensive survey of chestnut blight and collected C. parasitica from 216 sites. All 778 isolates were assayed for vegetative compatibility (vc) type, whereas a subsample of 301 isolates was assayed for mating type, and 189 isolates were genotyped at 16 microsatellite markers. We found low diversity for all markers. Nearly all isolates (95%) were compatible with vc type EU-1 and had the same microsatellite multilocus haplotype, or differed from the most common type by mutation at one locus. Approximately 5% of the isolates were vegetatively compatible with EU-13 and only two isolates (< 1%) were compatible with EU-3; five different microsatellite haplotypes were found among isolates in these latter two vc types. The overall mating-type ratio was 218 MAT-1: 81 MAT-2, with both mating types represented in each of the three vc types. Microsatellite haplotypes based on ten markers used in France showed that most isolates in Asturias were either identical to or only one marker different from one of the seven most common genotypes in France, RE103. Based on these ten markers alone, the population of C. parasitica in Asturias, would appear to have been founded by a single genotype from the C1 lineage (to which RE103 belongs) found in eastern France and northern Italy. However, additional genotyping by vc types suggests the introduction of multiple genotypes, with different vc types. The exact source for introduction into Asturias cannot be determined without additional genotyping of isolates from other locations. Regardless of their origin, the low diversity of vc types makes this population ideal for deploying hypovirulence because there will be few barriers for virus transmission between individuals.     

Identification and pathogenicity of Cryphonectriaceae species associated with chestnut canker in China

Cryphonectriaceae species cause serious canker diseases on chestnut, oak and eucalypt trees. Recently, canker symptoms with typical orange fruiting bodies were observed on Chinese chestnut and oak trees in Hebei, Hubei, Shaanxi and Shandong Provinces in China. In the present study, isolates of these fungi were identified based on phylogenetic and morphological evidence, and their pathogenicities were tested on detached chestnut (Castanea mollissima) branches. DNA sequence comparisons of the internal transcribed spacer (ITS) regions and two regions of β-tubulin (TUB1/TUB2) indicate that these isolates represent five species in the Cryphonectriaceae, viz. Cryphonectria japonica, Cryphonectria parasitica, Aurantiosacculus castaneae sp. nov., Cryphonectria neoparasitica sp. nov. and Endothia chinensis sp. nov. The sexual morph of Aurantiosacculus is discovered for the first time and can be distinguished from the other genera in Cryphonectriaceae by dark brown ascospores and tubiform appendages at both ends. Cryphonectria neoparasitica sp. nov. is different from the other Cryphonectria species by its aseptate ascospores. Endothia chinensis sp. nov. is the sole species in Endothia infecting the host genus Castanea. Additionally, it is much smaller than E. gyrosa and narrower than E. singularis in ascospores. The inoculation results showed that these five Cryphonectriaceae fungi isolated from chestnut or oak are all pathogenic to tested chestnut branches. Cryphonectria parasitica appears to be the most aggressive fungus, followed by C. neoparasitica sp. nov., C. japonica, E. chinensis sp. nov. and A. castaneae sp. nov.     

Population Structure and Disease Development of Cryphonectria parasitica in European Chestnut Forests in the Presence of Natural Hypovirulence

ABSTRACT The Cryphonectria parasitica populations in two 6-year-old European chestnut (Castanea sativa) coppices were investigated in southern Switzerland over a period of 4 years. Occurrence of white isolates indicating an infection with Cryphonectria hypovirus, vegetative compatibility groups (VCGs), hypovirulence conversion capacity, and mating types were used to characterize the populations. Sampling of randomly chosen cankers in the first year yielded 59% white isolates in one and 40% in the other population. The distribution of the VCGs and mating types was similar among white and orange isolates, indicating a homogeneous infection of the two populations by the hypovirus. Fourteen VCGs were found in the first population, 16 VCGs in the second. Altogether, 21 VCGs were determined. The same three VCGs dominated in both populations, comprising more than 60% of all isolates. Several VCGs were represented only by white isolates. Five of the six most common VCGs were clustered in two hypovirulence conversion groups, with almost 100% hypovirus transmission within each cluster. Repeated sampling of the same cankers in 1990, 1992, and 1994 did not reveal an increase of white isolates. The portion of blighted stems rose from 37% to about 60% in both plots within 4 years. In this time, chestnut blight killed 15% and competition an additional 21% of the sprouts. Predominantly, sprouts with low diameters at breast height were killed. The growth rate of new cankers was high in their first year and decreased gradually in the following years. A role of hypovirulence in the decline of disease severity was evident since (i) cankers yielding white isolates grew slower and killed considerably fewer sprouts than cankers with orange isolates; and (ii) the majority of the cankers yielded white isolates at least once during the 4-year observation period.     

Chestnut blight in Yugoslavia1

The present state of chestnut blight caused by the fungus Cryphonectria parasitica in Yugoslavia is presented. Canker disease is present all over the country wherever chestnut stands occur. Field observations have shown the presence of abnormal disease symptoms at some localities. Isolates of C. parasitica from these sites varied widely in cultural characteristics and pathogenic properties, some showing the characteristics of hypovirulent strains.     

Cryphonectria parasitica vegetative compatibility type analysis of populations in south-western France and northern Spain

A comprehensive study of the population biology of Cryphonectria parasitica , the causal agent of chestnut blight, is required to understand the spread of the epidemic in Europe and its natural regulation by the Cryphonectria hypovirus-1 (CHV-1). With this objective in mind, the diversity in vegetative compatibility (vc) types and mating types of C. parasitica populations was assessed in 43 chestnut sites in western France and northern Spain. Isolates were sampled in three regions along the Pyrénées mountains (Hautes Pyrénées, Pyrénées Atlantiques and Navarra) and in two regions north of the Pyrénées (Landes and Dordogne). There were 61 vc types observed in 682 isolates sampled. Ninety-five isolates (14%) could not be assigned to any one of the previously known European vc types. The finding of 47 incompatible vc types confirmed that the genetics of vegetative compatibility could not be totally accounted for by six diallelic vic genes. The two idiomorphs of the mating type gene were detected in all regions, indicating that sexual reproduction could occur within and between vc types. In all regions except Dordogne, C. parasitica populations were dominated either by the vc type EU-66 or EU-72. Neither vc type has been reported elsewhere in Europe, which suggests that two different introductions of C. parasitica have occurred in the study area. In Dordogne, populations were dominated by EU-33 and EU-2, and to a lesser extent EU-66 and EU-72. The low diversity in vc types for most of the C. parasitica populations provides good opportunities for natural regulation in forest coppices and for biocontrol with CHV-1 in orchards.     

Diversity of vegetative compatibility types and mating types of Cryphonectria parasitica in Slovenia and occurrence of associated Cryphonectria hypovirus 1

Cryphonectria parasitica, the causal agent of chestnut blight, has been present in Slovenia since at least 1950. To improve understanding of its diversity, 254 isolates of the fungus from 11 Slovenian populations were sampled. Fifteen vegetative compatibility (vc) types were identified. The dominant vc type was EU‐13, comprising 40·1% of all isolates tested, followed by EU‐1 (19·7%), EU‐2 (12·2%) and EU‐12 (9%). The vc type diversity in the most diverse population sampled in Slovenia was higher than in the populations found previously in northern Italy and Croatia. Both mating types and perithecia were observed in surveyed populations. Natural hypovirulence was found in six out of seven populations tested, with frequencies ranging from 72·2% in the population sampled near the Croatian border to 11·1% in the population sampled near the Austrian border. All identified hypoviral isolates (21) belonged to the Italian subtype of Cryphonectria hypovirus 1 and were closely related to the hypoviruses found in other European countries. Despite the high vc type diversity, incidence of hypovirulence was also high, indicating widespread natural biological control of the disease.     

Mixed inoculum for the biological control of chestnut blight1

The vegetative compatibility of four hypovirulent isolates of Cryphonectria parasitica was checked with 35 virulent isolates obtained from chestnut samples collected in many regions of Italy. Artificial inoculation tests confirmed the effectiveness of a mixture prepared with hypovirulent isolates. This was packed in squeezing tubes, to simplify the biological control of chestnut blight in practice.     

Vegetative compatibility groups in Colletotrichum coccodes from Turkey and their aggressiveness to potato

Black dot, caused by Colletotrichum coccodes, is a common disease of potato in Turkey, affecting tuber quality and yield. The objectives of the current study were to characterize vegetative compatibility groups (VCGs) of C. coccodes isolates from three regions in Turkey, and to assess the correlation between VCGs and aggressiveness of isolates on potato. A total of 147 C. coccodes isolates were recovered from plants showing typical black dot symptoms on stolons, roots and stems. The frequency of nitrate non‐utilizing (nit) nit1/nit3 and NitM phenotypes were 79% and 21%, respectively. Complementation between nit mutants of the isolates and eight European/Israeli EU/I‐VCG tester isolates was used to characterize the VCGs. Amongst the tested isolates, 33.3% were assigned to EU/I‐VCG6, 21.8% to EU/I‐VCG8, 15.7% to EU/I‐VCG4. EU/I‐VCG1, EU/I‐VCG3, EU/I‐VCG5 and EU/I‐VCG7 were classified at 1.4%, 3.4%, 4.8% and 5.4%, frequency, respectively. No isolate was assigned to EU/I‐VCG2 group, while 21 isolates (14.3%) were not assigned to any of the EU/I‐VCGs. The pathogenicity tests indicated significant differences in aggressiveness of the isolates with respect to sclerotia density on potato tissues. The highest densities of sclerotia on roots and crown were obtained with EU/I‐VCG6 isolates and the lowest with EU/I‐VCG1, EU/I‐VCG3 and EU/I‐VCG5 isolates. The results demonstrate that there is significant VCG diversity among C. coccodes isolates from potato plants in Turkey.     

Elevated mitochondrial alternative oxidase activity in dsRNA-free, hypovirulent isolates of Cryphonectria parasitica

In Cryphonectria parasitica, the causal agent of chestnut blight, hypovirulence is commonly associated with the presence of cytoplasmically transmissible, double-stranded RNA (dsRNA). Recently, hypovirulent isolates of C. parasitica that do not have detectable levels of dsRNA have been obtained from healing cankers on chestnut trees. In these isolates, most of the respiratory activity is cyanide-resistant and salicylhydroxamate-sensitive, indicating that the mitochondrial alternative oxidase was induced. In contrast, the respiration of virulent isolates from the same trees and most of the dsRNA-containing hypovirulent isolates from a variety of geographical locations was cyanide sensitive. In all of these isolates, the alternative oxidase could be induced by growing them in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis. The attenuated virulence and altered respiratory phenotypes could be transferred from the dsRNA-free hypovirulent isolates to virulent strains through hyphal anastomosis. Some of the dsRNA-free hypovirulent isolates grew more slowly than virulent strains. These results indicate that, in Cryphonectria, cytoplasmically transmitted hypovirulence may have more than one cause, and that mitochondrial mutations sometimes may be involved in the generation of this phenotype.     

Genetics of resistance to downy mildew in Brassica oleracea and breeding towards durable disease control for UK vegetable production

Downy mildew resistance was previously identified from screening a Brassica oleracea collection against two standard UK isolates of Hyaloperonospora parasitica. Sources of resistance were chosen from this material and developed further in this study by generating doubled haploid (DH) and inbred lines. Seedlings from the new lines were tested for resistance to a larger collection of H. parasitica isolates collected in 2001–2002 and 2007–2008 from the main broccoli and cauliflower production regions of the UK. Three lines (derived from borecole or summer cabbage) were broadly resistant to the pathogen isolates. Three of the remaining lines exhibited strong isolate‐specific resistance; several examples of weak or basal level of resistance to some isolates were observed. A new H. parasitica variant collected in 2008 was virulent in the broadly resistant lines, but was avirulent in a line with narrow specificity of resistance. The F₂ and BC₁ seedlings derived from outcrossing each of the three broadly resistant lines to susceptible broccoli and cauliflower lines segregated in a manner indicating that the resistance was controlled by a single dominant gene. No susceptibility was observed amongst F₂ seedlings derived from intercrossing the three resistant lines, indicating that they all share the same or closely linked broad‐spectrum resistance gene(s). DH lines were produced from F₁ plants, and resistant plants were further backcrossed to produce broccoli and cauliflower‐like lines that could be useful pre‐breeding material. A combination of resistance from lines with broad and narrow specificity is recommended for controlling downy mildew in UK brassica production.     

Genetic diversity of Fusarium oxysporum and related species pathogenic on tomato in Algeria and other Mediterranean countries

In order to characterize the pathogen(s) responsible for the outbreak of fusarium diseases in Algeria, 48 Fusarium spp. isolates were collected from diseased tomato in Algeria and compared with 58 isolates of Fusarium oxysporum originating from seven other Mediterranean countries and 24 reference strains. Partial sequences of the translation elongation factor EF‐1α gene enabled identification of 27 isolates as F. oxysporum, 18 as F. commune and three as F. redolens among the Algerian isolates. Pathogenicity tests confirmed that all isolates were pathogenic on tomato, with disease incidence greater at 28°C than at 24°C. All isolates were characterized using intergenic spacer (IGS) DNA typing, vegetative compatibility group (VCG) and PCR detection of the SIX1 (secreted in xylem 1) gene specific to F. oxysporum f. sp. lycopersici (FOL). No DNA polymorphisms were detected in the isolates of F. redolens or F. commune. In contrast, the 27 Algerian isolates of F. oxysporum were shown to comprise nine IGS types and 13 VCGs, including several potentially new VCGs. As none of the isolates was scored as SIX1+, the 27 isolates could be assigned to F. oxysporum f. sp. radicis‐lycopersici (FORL). Isolates from Tunisia were also highly diverse but genetically distinct from the Algerian isolates. Several Tunisian isolates were identified as FOL by a PCR that detected the presence of SIX1. The results show that isolates from European countries were less diverse than those from Tunisia. Given the difference between Algerian populations and populations in other Mediterranean countries, newly emergent pathogenic forms could have evolved from local non‐pathogenic populations in Algeria.     

The effect of Cryphonectria parasitica on water relations of American chestnut

Chestnut blight has commonly been regarded as a phloem disease due to conspicuous stem cankers that result from infection by the fungal pathogen Cryphonectria (Endothia) parasitica. Stomatal conductance (g_s), transpiration rate (E) and leaf water potential were measured throughout the day on leaves distal to naturally-occurring virulent (sunken bark with abundant stromata) or hypovirulent (swollen bark lacking stromata) cankers and cankers induced by inoculation with virulent or hypovirulent strains of C. parasitica. Relative to control stems, hydraulic conductivity (K_h), g_s and E were reduced significantly (α = 0·05) for both natural virulent cankers, and cankers that were induced by a virulent strain. These effects were less pronounced for both natural and induced hypovirulent cankers. Isolation experiments revealed that the percentage of xylem tissue chips yielding C. parasitica was greater for virulent than for hypovirulent cankers. The data provide evidence that the localized presence of C. parasitica in cankers of American chestnut results in stomatal closure, possibly as a direct result of xylem dysfunction.     

Characterization of Pyricularia grisea in the United States Using Independent Genetic and Molecular Markers

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.