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.     

Relationship Between Biological Control, Incidence of Hypovirulence, and Diversity of Vegetative Compatibility Types of Cryphonectria parasitica in France

ABSTRACT In France, chestnut blight, caused by Cryphonectria parasitica, has been controlled since 1974 in orchards, but never in coppice forests, by releasing hypovirulent strains infected with CHV1 hypovirus. We tested the hypothesis that this biological control (BC) has lead to a decrease in blight severity, spread of hypovirulence, and change in C. parasitica populations. The low severity of chestnut blight was confirmed in the six regions studied (subdivided into zones). The remission of cankers was associated with the presence of white isolates presumed to be hypovirulent. These two parameters were also correlated, at the zonal level, to the frequency of sites where BC was used. However, the estimates of the natural background level of hypovirulence, independent of BC, ranged from 4% in forests in Dordogne to 60% in orchards in Lozère. Differences in the rate of hypovirulent isolates among regions were consistent with the diversity of vegetative compatibility (VC) types in populations of C. parasitica. The highest VC-type diversity and mean allelic diversity for known vegetative incompatibility (vic) genes were observed in Dordogne. We showed that the current diversity of VC types in populations of C. parasitica was lower than in 1981. We found 30 VC types among 1,113 isolates of C. parasitica. Ten VC types were incompatible with known EU testers, suggesting that one additional vic gene or allele at one of the six vic loci known should be present in Europe.     

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.     

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.     

Low diversity of vegetative compatibility types and mating type of Cryphonectria parasitica in the southern Balkans

The diversity of vegetative compatibility (vc) types and mating type was estimated in populations of the chestnut blight fungus, Cryphonectria parasitica , throughout Macedonia and from selected areas in Greece. Nearly all of the 786 isolates (94%) from Macedonia were in a single vc type, EU-12; all 379 isolates from Greece were EU-12. Only six of 20 populations in Macedonia had more than one vc type. The diversity of vc types in the most diverse populations of Macedonia was comparable with the least diverse populations found previously in Italy. All but six of the 313 isolates assayed had the same mating type, MAT-1 , and no perithecia of Cryphonectria parasitica were observed in any population. These results lead to the conclusion that sexual reproduction does not occur in these populations. The lack of vc type diversity may indicate a high potential for the spread of hypoviruses and successful biological control with transmissible hypovirulence. However, if sexual reproduction should occur in Macedonian populations, up to 32 vc types would be possible by recombination among vegetative incompatibility loci.     

Factors influencing growth,sporulation and virus transfer in <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> isolates from Castilla and León (Spain)

Biological control with Cryphonectria hypovirus CHV1 of the chestnut blight, caused by the fungus Cryphonectria parasitica, has reduced the impact of the disease in Europe. The virus reduces the virulence of the fungus so that it causes non-lethal cankers, thus enabling the chestnut trees to overcome the disease. The virus can be transmitted horizontally by hyphal anastomosis or vertically to the conidia. In this study, we investigated growth and sporulation of the fungus as well as rates of horizontal transmission of the virus at different temperatures. We used fungal isolates of the vegetative compatibility types (vc types) that are most prevalent in Castilla and León (central northern Spain) to evaluate the effects of fungal strain on the parameters tested. In addition, we infected four isolates of C. parasitica with hypovirus subtypes CHV1-F1 and CHV1-I, to determine the influence of virus subtype on growth, sporulation and virus transfer. We assessed growth of fungal colonies and horizontal transmission of the virus at 15 °C and 25 °C. Colony growth was affected by an interaction between fungal isolates included in vc type EU1 or EU11 and virus at both 15 °C and 25 °C. However, horizontal transmission of the virus was only influenced by the fungal genotype of isolates included in vc type EU1 or EU11, and spore production was only affected by the virus subtype. Vertical transmission was also influenced by the fungal isolate and virus subtype. Growth of the fungal isolates varied depending on the virus subtype with which they were infected. This supports the theory that fungal host and virus subtype influence transmission and dissemination of hypovirulence. The fungal genotype affects colony growth and horizontal transmission of the virus. It is common to expect a good dissemination of the hypovirus with a low vc type diversity but the selection of the best combination of hypovirus and fungal isolate is crucial for the success of biological control not only for small areas but in larger chestnut populations as well.     

High vegetative compatibility diversity of Cryphonectria parasitica infecting sweet chestnut (Castanea sativa) in Britain indicates multiple pathogen introductions

Chestnut blight, caused by Cryphonectria parasitica, was identified in Devon, UK, in December 2016. Intensive surveys detected the disease at further sites in Devon (seven), Berkshire (one), Dorset (one), Derbyshire (four) and a cluster of eight sites in southeast London. Over 570 survey samples were tested, and 227 were positive for C. parasitica by isolation and real-time PCR. A total of 227 isolates were tested for mating type, and 197 screened for vegetative compatibility group (VCG) and compared with VCGs known from mainland Europe. The same isolates were also screened for the presence of Cryphonectria hypovirus 1 (CHV-1). Eleven VCGs were identified within the UK population. Five corresponded to already known European VCGs but six were unique. The European VCGs mainly came from the Devon, Dorset, Berkshire and Derbyshire disease outbreaks, whilst unique VCGs were almost exclusively from the southeast London cluster. Both mating types were detected, but only one mating type was present at each site, with the exception of a single Devon site. Perithecia of C. parasitica were never observed at any site. CHV-1 was found in seven isolates from three different locations and was always subtype-I, which has limited hypovirulence. Therefore, although CHV-1 is associated with C. parasitica at some outbreaks, it probably has limited impact on virulence. The diversity of VCGs and their distribution at outbreak sites, together with findings of CHV-1, suggests C. parasitica has been introduced to the UK multiple times over at least two decades through international plant trade.     

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.     

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.     

Saprophytic Activity and Sporulation of Cryphonectria parasitica on Dead Chestnut Wood in Forests with Naturally Established Hypovirulence

ABSTRACT Sustainable biological control of the chestnut blight fungus Crypho-nectria parasitica with hypovirulence depends on the production and dissemination of hypovirus-infected propagules of the pathogen. We investigated the ability of C. parasitica to sporulate and produce hypo-virus-infected spores on recently dead chestnut wood in coppice stands in southern Switzerland where hypovirulence has been naturally established. The number and type (active, inactive, or none) of cankers was assessed on experimentally cut and stacked stems, firewood stacks, and natural dead wood. Hypovirus-free and hypovirus-infected strains readily survived for more than 1 year in the chestnut blight cankers of the stacked stems. Sporulation of C. parasitica was observed on the surface of preexisting inactive and active cankers, as well as on newly colonized bark areas and was significantly more abundant than on comparable cankers on living stems. On all types of dead wood, we observed more stromata with perithecia than with pycnidia; however, a large proportion of the stromata was not differentiated. All perithecia examined yielded only hypovirus-free ascospores. The incidence of pycnidia that produced hypovirus-infected conidia ranged from 5% on natural dead wood to 41% on the experimental stacks. The mean virus transmission rate into conidia was 69%. Our study demonstrates a considerable saprophytic activity of C. parasitica on recently dead chestnut wood and supports the hypothesis of a role of this saprophytic phase in the epidemiology of hypovirulence.     

Characterization ofCryphonectria parasitica isolates collected from Aydın province in Turkey

The major chestnut-growing areas in Aydın Province, which yield nearly 35% of Turkey’s production, were examined for the presence of chestnut blight and bark samples were collected from the canker formations. Generally virulent type cankers were observed. Of 31 villages, 22 were found to be contaminated with the disease. A total of 97Cryphonectria parasitica (Murrill) M.E. Barr. isolates were characterized for the presence of potential hypovirulence by looking at cultural characteristics on media and virulence in cv. ‘Golden Delicious’ apple fruit. The isolates were placed into four groups based on pigmentation and abundance of pycnidia formation. Isolates produced lesions of various sizes on apple fruits but lesion size was not correlated with cultural characteristics typical to hypovirulent isolates. Among 97 isolates, ten possessing the characteristics associated with hypovirulence were assayed for the presence of dsRNA. Among these isolates only one, coded M1-3, was found to contain dsRNA. No dsRNA was detected in the single conidium isolates derived from M1-3. All of the isolates were screened for vegetative compatibility and two European vc types, EU-1 and EU-12, were detected. This low vc-type diversity in the region may indicate a high potential for spread of transmissible hypovirulence. http://www.phytoparasitica.org posting May 30, 2008.     

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.     

Hypovirulence detected in Brazilian isolates of Cryphonectria cubensis

A single 3 kb segment of double-stranded (dsRNA) was present in three of 30 Brazilian isolates of Cryphonectria cubensis . These dsRNA-containing isolates showed morphological characteristics suggestive of hypovirulence and were significantly less virulent than dsRNA-free isolates. One isolate, however, with morphological characteristics suggestive of hypovirulence, showed reduced virulence, but was free from dsRNA. Conversion of virulent isolates with normal morphology to a morphology associated with hypovirulence was achieved by pairing hypovirulent and virulent isolates of the same vegetative compatibility group (VCG). This suggests that dsRNA can be transmitted to isolates of the same vegetative compatibility group by hyphal anastomosis. Converted isolates exhibited the same hypovirulence-associated traits as those of the original dsRNA-containing hypovirulent isolates. These studies suggest that a single 3 kb segment of dsRNA alters both morphology and virulence by conferring hypovirulence on the pathogen; the first such report for Brazilian isolates of C. cubensis .     

低毒病毒CHV1-CN280生防潜力的初步研究

前期研究表明,分离自中国的低毒病毒CHV1-CN280与其它多数低毒病毒相比具有更高的水平传播能力。本研究中,我们对CHV1-CN280的田间定殖能力及其它生防性状进行了评估。与其它两个模式低毒病毒相比,CHV1-CN280对栗疫菌Cryphonectria parasitica的毒力显著弱于典型的烈性低毒病毒CHV1-EP713,而更接近于典型的温和低毒病毒CHV1-Euro7。强致病性的栗疫菌感染CHV1-CN280后,成为典型的低毒菌株,不再对板栗植株造成严重危害,但是与感染典型烈性低毒病毒CHV1-EP713的菌株相比,寄主真菌在板栗枝条上仍保持了一定的定殖能力,其菌丝生长速率和分生孢子产量较高。初步的田间试验表明,在人工接种两年后,CHV1-CN280在田间成功定殖,并已扩散到田间自然存在的、与人为释放的低毒菌株不亲和的其它营养体亲合群的栗疫菌菌株中。结果表明,低毒病毒CHV1-CN280对栗疫病具有非常好的生物防治潜力。     

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.     

Incidence and Diversity of Double-Stranded RNAs Occurring in the Chestnut Blight Fungus, Cryphonectria parasitica, in China and Japan

ABSTRACT Isolates of the chestnut blight fungus, Cryphonectria parasitica, were randomly sampled from 10 subpopulations in China and 8 subpopulations in Japan and screened for the presence of double-stranded (ds) RNA using an immunoblot procedure with a monoclonal antibody specific for dsRNA. The overall incidence of dsRNA in C. parasitica was 2 and 6% in China and Japan, respectively, much lower than the 28% found previously in North American populations. Genetic relatedness of dsRNAs within and among populations in China and Japan was examined using RNA-RNA hybridizations with labeled-dsRNA probes. The majority of Chinese and Japanese dsRNAs were members of a single hybridization group, related to Cryphonectria hypovirus 1 (CHV1) from Europe, and are referred to as CHV1-type dsRNAs. No evidence was obtained for genetic differentiation between CHV1-type dsRNAs sampled in China and Japan. Five Japanese isolates contained two genetically distinct dsRNAs. The larger segments (approximately 12 kilobases [kb]) were members of the CHV1 hybridization group, while the smaller segments (approximately 3 kb) did not hybridize with any known dsRNA from C. parasitica including the 2.7-kb dsRNA from isolate NB631 from New Jersey or dsRNA from isolate RC1 from Michigan. Two small dsRNA segments (approximately 1.8 and 2 kb) from one isolate sampled from Liaoning Province in northeastern China did not hybridize with any of the dsRNA probes tested including several described dsRNAs of similar size from C. parasitica in North America. Three dsRNAs from Anhui Province, China, hybridized to Cryphonectria hypovirus 2 (CHV2)-specific probes and are thus referred to as CHV2-type dsRNAs. Sequence analysis of 1,627 base pairs of these three CHV2-type dsRNAs from Anhui revealed that they were identical to each other in the region sequenced and very closely related to CHV2-NB58, isolated from New Jersey. We speculate that CHV2-NB58 may have been introduced into North America from this part of China. This is the first record of a North American C. parasitica dsRNA that is genetically related to a dsRNA from Asia.     

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.     

Diversity of Hypoviruses and Other Double-Stranded RNAs in Cryphonectria parasitica in North America

ABSTRACT Double-stranded (ds) RNAs in Cryphonectria parasitica were randomly sampled from nine subpopulations in North America using an antibody-based detection system for dsRNA. dsRNA was detected in 166 (28%) of a total of 595 C. parasitica isolates sampled by immunoblotting. Incidence of dsRNA infection within subpopulations ranged from 0% in samples from New Hampshire and Ontario to 100% in County Line, MI. Most of the dsRNAs sampled were approximately 9 to 13 kb in size. dsRNAs from 72 isolates analyzed by probing Northern blots with (32)P-labeled dsRNAs were in one of three hybridization groups. One hybridization group was widespread throughout eastern North America, being found in New York, New Jersey, Maryland, West Virginia, Kentucky, and Michigan. These dsRNAs hybridized to dsRNA from the previously described C. parasitica isolate SR2 from Maryland and are referred to as SR2-type dsRNAs. The second hybridization group was found almost exclusively in Michigan. The Michigan dsRNAs cross-hybridized to Cryphonectria hypovirus 3-GH2 (CHV3-GH2) and are referred to as CHV3-type dsRNAs.One dsRNA sampled from Kentucky hybridized to CHV3-type dsRNAs from Michigan. This dsRNA was probably derived from a fungal isolate that had been intentionally released for biological control at this same site 10 years previously and had become established in Kentucky. The third hybridization group was found only in New Jersey. These dsRNAs were much smaller than all other dsRNAs (3 and 5 kb) and were found in all 11 isolates that were probed; two of these isolates also had SR2-type dsRNA in mixed infections. None of the North American dsRNAs hybridized to CHV1 from Europe, even though CHV1 has been released in numerous locations in eastern North America for biological control of chestnut blight. Similarly, no dsRNAs hybridized to CHV2-NB58, a hypovirus found previously in New Jersey. Mixed infections of SR2-type and CHV3-type dsRNAs were found in 13 of 15 isolates from Frankfort, MI, while another nearby subpopulation (County Line) was infected with only CHV3-type dsRNAs. The distribution of dsRNA hybridization groups in C. parasitica thus presents a mixed picture, since one hybridization group is widespread, whereas two others are primarily restricted to smaller geographic areas.     

Comparison of soil and corn kernel Aspergillus flavus populations: evidence for niche specialization

Aspergillus flavus is considered a generalist-opportunistic pathogen, but studies are beginning to show that A. flavus populations have strains specific to various hosts. The research objective was to determine whether A. flavus soil populations consist of solely saprophytic strains and strains which can be facultatively parasitic on corn. A. flavus was isolated from both corn kernels and soil within 11 Louisiana fields. Sixteen vegetative compatibility groups (VCGs) were identified among 255 soil isolates. Only 6 of the 16 VCGs were identified in the 612 corn isolates and 88% of corn isolates were in two VCGs, whereas only 5% of soil isolates belonged to the same two VCGs. Isolates were characterized for aflatoxin B1 production and sclerotial size. A random subset of the isolates (99 from corn and 91 from soil) were further characterized for simple-sequence repeat (SSR) haplotype and mating type. SSR polymorphisms revealed 26 haplotypes in the corn isolates and 78 in the soil isolates, and only 1 haplotype was shared between soil and corn isolates. Corn and soil populations were highly significantly different for all variables. Differences between corn and soil populations indicate that some soil isolates are not found in corn and some isolates have become specialized to infect corn. Further understanding of A. flavus virulence is important for development of resistant hybrids and for better biological control against toxigenic A. flavus.     

Hypovirus Transmission to Ascospore Progeny by Field-Released Transgenic Hypovirulent Strains of Cryphonectria parasitica

ABSTRACT Strains of the chestnut blight fungus, Cryphonectria parasitica, have been genetically engineered to contain an integrated full-length cDNA copy of the prototypic virulence-attenuating hypovirus CHV1-EP713. Unlike natural hypovirulent C. parasitica strains, these transgenic hypovirulent strains are able to transmit virus to ascospore progeny under laboratory conditions. This ability provides the potential to circumvent barriers to cytoplasmic virus transmission imposed by the fungal vegetative incompatibility system. During July 1994, transgenic hypovirulent strains were introduced into a Connecticut forest site (Biotechnology Permit 94-010-01). Subsequent analysis of the release site confirmed hypovirus transmission from transgenic hypovirulent strains to ascospore progeny under field conditions. Additionally, it was possible to recover transgenic hypovirulent strains from the test site as long as 2 years after the limited, single-season release. Evidence also was obtained for cytoplasmic transmission of transgenic cDNA-derived hypovirus RNA, including transmission to mycelia of a virulent C. parasitica canker after treatment with conidia of a transgenic strain. Finally, a transgenic hypovirulent strain was recovered from a superficial canker formed on an untreated chestnut tree. Genetic characteristics of the recovered strain suggested that the canker was initiated by an ascospore progeny derived from a cross involving an input transgenic hypovirulent strain. The durability of a molecular marker for field-released cDNA-derived hypovirus RNA is discussed.