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Department of Plant Pathology, Cornell University, Ithaca, New York 14853-4203
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We found high diversity of vegetative compatibility (vc) types in native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China; almost every isolate was in a unique vc type. In Japan we found 71 vc types in a sample of 79 isolates pooled from six populations. Within two populations in China, all isolates (n = 28 and 11) had unique vc types; we found 15 vc types among 25 isolates in a third Chinese population where multiple isolates were collected from some trees. None of the isolates from China and only three isolates in the 71 vc types from Japan were compatible with any of 64 vc type testers from Europe, which have known vegetative incompatibility genotypes. To our knowledge this is the first report of vc type diversity for C. parasitica in Japan or of any comparisons of vc types between Asia and Europe. The most significant result of this survey is the identification fungal isolates for expanding knowledge of the genetics of vegetative incompatibility.
Key words: genotypic diversity, heterokaryon incompatibility, hypovirulence, vegetative incompatibility
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Anagnostakis 1982, MacDonald and Fulbright 1991, Heiniger and Rigling 1994, Milgroom and Cortesi 2004). This phenomenon is known as hypovirulence because, unlike most fungal viruses that have no effect on their hosts, some viruses in the chestnut blight fungus, Cryphonectria parasitica, reduce its virulence to chestnut trees. Viruses that cause hypovirulence are in the family Hypoviridae and are known commonly as hypoviruses (Hillman and Suzuki 2004, Nuss et al 2005). Despite repeated attempts hypovirulence has failed to control chestnut blight in eastern North America (MacDonald and Fulbright 1991, Milgroom and Cortesi 2004). Hypoviruses released in North America have not become established, presumably because their spread is inhibited by vegetative incompatibility (Anagnostakis 1987, MacDonald and Fulbright 1991). Because transmission of hypoviruses is restricted by vegetative incompatibility in C. parasitica (Anagnostakis 1983, Liu and Milgroom 1996, Cortesi et al 2001) they are less likely to spread (or to spread more slowly) in populations that have a high diversity of vc types.
Although C. parasitica is native to east Asia and first was found respectively in North America and Europe in 1904 and 1938 (Anagnostakis 1987), little is known about vegetative incompatibility in native populations. We are aware of only one study on the diversity of vc types in Asian populations (Wang et al 1991). In contrast numerous surveys of vc type diversity have been conducted in Europe and North America (for examples see Cortesi et al 1998, Robin and Heiniger 2001, Sotirovski et al 2004). However the greatest diversity of vc types observed in C. parasitica is in Asia. Wang et al (1991) found 131 vc types in a sample of 219 C. parasitica isolates from Jiangsu and Anhui provinces in eastern China. Comparable sampling in Europe has yielded far fewer vc types. For example Cortesi et al (1996) found 20 vc types among 716 isolates collected throughout Italy and Sotirovski et al (2004) found one vc type among 379 isolates from Greece and five vc types among 786 isolates from the Republic of Macedonia. The lack of information about C. parasitica from Japan is striking; we know of no published data on vc type diversity in C. parasitica in Japan, and yet Japan is the most likely source of introduction of C. parasitica into Europe and North America (Anagnostakis 1992, Milgroom et al 1996).
Here we report a survey of vc types of C. parasitica in Japan and China. Our first objective was to estimate the diversity of vc types within local populations of C. parasitica. We studied local diversity, rather than regional scale diversity as in Wang et al (1991), because it relates directly to the transmission of hypoviruses and biological control within populations. Our second objective was to compare vc types found in Asia to those with known vegetative incompatibility (vic) genotypes (Cortesi and Milgroom 1998), many of which are found in Europe and North America (Cortesi et al 1998, Milgroom and Cortesi 1999, Robin et al 2000).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). We assayed 79 C. parasitica isolates from six populations in Japan and 66 isolates from three populations in eastern China for vegetative incompatibility (TABLE I, FIG. 1). Populations are defined as comprising the C. parasitica individuals inhabiting a relatively small geographic location; sampling for each population usually was restricted to a single orchard (or forest stand) of Japanese chestnut (Castanea crenata) or Chinese chestnut (Castanea mollissima). In addition to the 1992 collections we assayed vc types for isolates within two populations in Japan, Kukizaki and Chudai, sampled in 1998 (Liu et al 2003). Each location sampled was typically 1–2 ha or smaller. Because the incidence of chestnut blight was low in all locations we sampled stromata from every tree showing canker symptoms or signs of C. parasitica. Only one isolate was sampled per tree, except in Taian, Shandong Province, China, where one collector sampled multiple cankers on some trees.
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. Isolates were grown on malt agar, then pairs of small cubes of agar (ca. 3 mm on a side) with mycelium were placed on PDAg medium (Powell 1995, Cortesi and Milgroom 1998). Plates were incubated at 27 C in the dark 5–6 d before scoring. The presence of a barrage (zone of dead cells) and/or discoloration in the medium at the interface of colonies was interpreted as evidence of vegetative incompatibility.
To determine the number of vc types within samples we paired isolates in all combinations. Pairwise comparisons also were made among 79 isolates from six populations in Japan but not among isolates from different populations in China. The diversity of vc types in each population was estimated by comparing richness, evenness and total diversity (Grünwald 2003). The richness component of diversity is the observed number of vc types in each population. Genotypic diversity was estimated with the Shannon-Wiener index: H' = – 
pi ln pi, where pi is the frequency of the ith vc type (Shannon and Weaver 1949). We also used the genotypic diversity index of Stoddart and Taylor (1988): G = 1/pi2; this index also can be calculated as G = 1/( [(fx)(x/n)2], where n is the sample size, and fx is the number of genotypes observed x times. For evenness, we used the index described by Grünwald et al (2003): E5 = (G – 1)/(eH' – 1).
All vc types found also were tested against 64 vc type testers, EU-1 through EU-64, which represent all possible vc types defined by the six polymorphic vegetative incompatibility (vic) loci identified in Europe (Cortesi and Milgroom 1998). Many of these 64 vc types occur naturally in Europe (Cortesi et al 1998, Robin et al 2000) and in North America (Milgroom and Cortesi 1999).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In Japan all isolates had unique vc types within four populations and only one vc type was found twice in each of four other populations. The evenness index (E5) was 1.0 or close to 1.0 for all populations. Five vc types were found in more than one population, giving a total of 71 vc types when 79 isolates were compared from six pooled Japanese populations (TABLE I).
High diversities were found also within populations in China (TABLE I). All isolates had unique vc types within two of the three populations assayed. The third population (Taian, Shandong Province) was less diverse, with 15 vc types found among 25 isolates: one vc type was found in eight isolates, three were found twice, and 11 were found only once. Unlike the other populations, multiple isolates were collected from some trees; isolates with identical vc type or RFLP genotype have been recovered frequently when sampled from the same tree in North America (Milgroom et al 1991, Milgroom and Lipari 1995). Although no comparisons were made among populations in China, the maximum number of vc types from these samples would be 54 if no vc type were found in more than one population.
All 71 vc types found in six pooled populations in Japan and 54 isolates in three populations in China were paired with tester isolates for European vc types EU-1 to EU-64. Only three of the 71 vc types from Japan were compatible with European vc types. Isolate JA12 (Chiyoda, Ibaraki Prefecture) and isolate JA100 (Sanwa, Ibaraki Prefecture) were compatible respectively with EU-15 and EU-18. Isolate JA17 (Chiyoda, Ibaraki Prefecture) previously was reported as being compatible with EU-24 (Cortesi and Milgroom 1998). None of the 54 Chinese isolates tested was compatible with any European vc type testers.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). To our knowledge this is the first report of vc type diversity in C. parasitica from Japan. We also confirmed the findings that vc type diversity in China is high (Wang et al 1991). Our results provide a more fine-scale perspective than known previously because we sampled more intensively than Wang et al (1991) within single orchards to estimate diversity locally. Wang et al (1991) assessed diversity regionally by pooling a few isolates from each of many orchards in two provinces. In both Japan and China our sample sizes were too small to assess the full extent of vc type diversity because the same vc type was rarely found twice. Comparing isolates in all combinations requires a large number of vc tests; e.g. testing the 79 isolates from Japan in all combinations required more than 3000 tests ([79 x 78]/2 = 3081). Therefore further increasing sample sizes was not practical for this type of survey. Nonetheless these populations of C. parasitica in Japan and China have the highest diversity of vc types (to our knowledge) of any populations studied to date.
Without empirical data we can only speculate on the significance of vc type diversity to virus transmission in Japan and China. The presence of Cryphonectria hypovirus 1 (CHV-1) and other viruses in these populations (Peever et al 1998, Liu et al 2003) suggests that virus transmission might occur between vc types despite the high diversity. Although virus incidence was low in all populations sampled, at least one virus-infected isolate was found in six of the nine populations sampled for vc types, all except Soya/Sanwa and Morioka in Japan and Taian in China. Liu et al (2003) similarly concluded that CHV-1 was transmitted between C. parasitica and another Cryphonectria species found on chestnut trees in Japan (now known to be C. nitschkei [Myburg et al 2004]) even though it occurred only infrequently in the laboratory. Because vc type barriers to virus transmission are not absolute either in the laboratory (Anagnostakis 1983, Liu and Milgroom 1996, Cortesi et al 2001) or in the field (Double 1982, Carbone et al 2004), transmission might occur at high vc type diversities if given enough encounters over a long time. Therefore the presence of viruses in populations with high vc type diversity is not contradictory, assuming that hypo-viruses have been associated with C. parasitica for a sufficiently long time for them to spread by rare transmission events. On the other hand hypoviruses could be maintained in populations by vertical transmission (via conidia) during clonal reproduction. However this would depend on asexual reproduction in C. parasitica, but the high diversity of vc types argues against clonality as a dominant reproductive mode, except perhaps for spread of C. parasitica to multiple cankers within a tree (as in Taian, Shandong Province). Moreover perithecia of C. parasitica have been observed in the field in Japan and the outcrossing rate was high (Marra et al 2004), including in Chudai where all 28 isolates were in unique vc types. Therefore we speculate that recombination plays an important role in maintaining vc type diversity in these populations and at least some horizontal transmission must occur.
The lack of concordance between vc types found in Europe and Asia has several implications for the genetics of vegetative incompatibility in C. parasitica. First, it is clear that vc type testers representing all 64 possible genotypes for the six polymorphic vic loci found in Europe (Cortesi and Milgroom 1998) are not useful for analyzing vegetative incompatibility genetically in Asia because we cannot infer vic genotypes. Although this type of analysis was highly informative in Europe and North America (Milgroom and Cortesi 1999, Robin et al 2000), analyses of population structure cannot be made from this survey of vc types in Japan and China and therefore cannot be compared to those done on RFLPs (Milgroom et al 1996). Second, Japanese isolate JA17 previously was shown to be compatible with European vc type EU-24 and when crossed with an Italian isolate the progeny were in the 32 predicted vc types, demonstrating that the same vic genes control vegetative incompatibility in Japan as in Europe (Cortesi and Milgroom 1998). This same cross was used for linkage mapping and finding markers linked to vic genes (Kubisiak and Milgroom 2006). Third, in Japan only three of 71 vc types in C. parasitica were compatible with the 64 vc type testers from Europe, leaving 68 vc types with unknown vic genotypes; in China none of the 54 isolates were compatible with the European vc type testers. The large number of vc types in Asia that are not found among the 64 European testers could be explained in two ways. Multiple alleles at some vic loci, some of which are not found in Europe, would increase the number of possible vc types. On the other hand, there also might be additional vic loci that are polymorphic in Asia but monomorphic in Europe. Only two alleles per locus have been found so far for the six vic loci identified in C. parasitica (Cortesi and Milgroom 1998), and multiple vic alleles are uncommon among ascomycetes, although they have been found (Dales et al 1993, Saupe and Glass 1997). In other ascomycetes whose genetics of vegetative incompatibility have been studied thoroughly, larger numbers of incompatibility loci have been found; e.g. 17 in Podospora anserina and 11 in Neurospora crassa (reviewed in Glass et al 2000). Therefore we consider it more likely that additional vic loci are polymorphic in these C. parasitica populations as predicted also for North America and Europe (Milgroom and Cortesi 1999, Robin et al 2000). Assuming only two alleles per locus, at least two additional vic loci must be polymorphic in Japan to obtain 68 additional vc types (one more polymorphic vic locus in addition to the six already known would result in 64 new vic genotypes). As found with RFLP markers (Milgroom et al 1996) we expect to find more polymorphic vic loci and more vic alleles in Asia than in founder populations in Europe and North America, resulting in greater vc type diversity overall. However the genetics underlying differences in vc type diversity between Asia and Europe cannot be determined without further genetic analysis.
Results of this survey will affect future research directions on vegetative incompatibility in C. parasitica. The identification of a large number of vc types will aid in studying the genetics of vegetative incompatibility, and especially for identifying new vic loci and/or alleles.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544 
2 Corresponding author. FAX: +1-607-254-6448. E-mail: mgm5{at}cornell.edu
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