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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Plants for planting; indirect evidence for the movement of a serious forest pathogen, <Emphasis Type="Italic">Teratosphaeria destructans</Emphasis>, in Asia

Fungal diseases caused by native pathogens and pathogens introduced with planting stock have a significant impact on exotic plantation forestry in the tropics. Teratosphaeria destructans (formerly Kirramyces destructans) is a serious pathogen causing leaf, bud and shoot blight diseases of Eucalyptus spp. in plantations in the sub-tropics and tropics of south-east Asia. This pathogen was first discovered in Indonesia in 1995 and has subsequently spread to Thailand, China, Vietnam and East Timor. The biology, ecology and genetics of this important pathogen have not been explored yet. The objective of this study was, thus, to determine the genetic diversity and movement of T. destructans throughout south-east Asia using multi-gene phylogenies and microsatellite markers. Out of nine gene regions only two microsatellite markers detected a very low nucleotide polymorphism between isolates; seven other gene regions, ITS, β-tubulin, EF1-α, CHS, ATP6 and two microsatellite loci, reflected genetic uniformity. The two polymorphic molecular markers resolved six haplotypes among isolates from Indonesia and only a single haplotype elsewhere in Asia. The low diversity observed among isolates in the region of the first outbreak is as expected for a small founder population. The spread of a single clone over large distances throughout the region supports the hypothesis of spread via the human-mediated movement of germplasm.     

Altered Phenotypic Response to Peronospora parasitica in Brassica juncea Seedlings Following Prior Inoculation with an Avirulent or Virulent Isolate of Albugo candida

A study was made of the biological interactions between an isolate of Peronospora parasitica compatible with Brassica juncea and two isolates of Albugo candida either incompatible or compatible with the host species. Prior inoculation with the incompatible isolate of A. candida induced resistance to subsequently inoculated P. parasitica. The degree of resistance was proportional to the zoosporangia concentration of the incompatible isolate and induced resistance was more marked in the cotyledon receiving the inducing inoculum compared to the opposite cotyledon and subsequently emerging true leaves that had not been pre-inoculated. Induction of resistance was also observed if the incompatible isolate of A. candida and P. parasitica were co-inoculated simultaneously. However, the effect was greater the longer the interval between inoculations, up to a period of 4 days. When the incompatible isolate of A. candida was inoculated 4h after P. parasitica, there was no marked effect on resistance to the latter. In contrast, prior inoculation with the compatible isolate of A. candida increased susceptibility to P. parasitica inoculated subsequently. However, pre- or co-inoculation with P. parasitica suppressed the development of the compatible isolate of A. candida. A spectrum of responses was observed when one cotyledon was inoculated simultaneously with both the incompatible and compatible isolates of A. candida and followed subsequently with P. parasitica after different time intervals. In such combinations, a transition was observed in the host response to P. parasitica from induced resistance/reduced susceptibility, which increased up to 24h following a simultaneous inoculation with incompatible + compatible isolates of A. candida to an almost neutral reaction after 72h to induced susceptibility after 96h. This range of altered responses appeared to reflect the outcome of the differing kinetics and counter-effects of resistance and susceptibility induction.     

First record of Dryocosmus kuriphilus in Greece

After the first introduction of chestnut gall wasp, Dryocosmus kuriphilus, in Italy, it has spread rapidly in several other European countries, probably largely due to trade in chestnut plants. Despite the measures taken to prevent further spread of this pest to other countries, D. kuriphilus was detected in Greece for the first time in 2014. However, surveys have shown that the pest is still of very limited distribution in Greece. Having faced the damage caused by chestnut blight (Cryphonectria parasitica) over the last 50 years, the production of chestnuts in Greece is now seriously threatened by chestnut gall wasp, which has already caused extensive losses in chestnut production of other countries. In order to minimize the impact of chestnut gall wasp, control measures are being designed involving the release of the parasitoid Torymus sinensis.     

Population structure and genetic diversity suggest recent introductions of Dothistroma pini in Slovakia

Dothistroma pini is one of two pathogens causing Dothistroma needle blight (DNB), a foliar disease of pines. The species was redefined in 2004 and subsequently recorded in several European countries. In Slovakia, the first report of the pathogen was in 2013. In this study, the population structure, genetic diversity, and reproductive mode of 105 isolates collected from 10 localities and seven hosts were determined in Slovakia. Species-specific mating type markers, ITS haplotype determination, and 16 microsatellite markers were used to characterize and genotype the isolates. Overall, 15 unique multilocus haplotypes (MLHs) based on microsatellite markers and three ITS haplotypes were identified. Three independent methods (DAPC, STRUCTURE, EDENetwork) separated the isolates into two distinct population clusters corresponding with ITS haplotypes. A high level of clonality was recorded suggesting that conidia are the primary source of pathogen dispersal. The low genetic diversity, predominantly asexual reproductive mode of the pathogen, and the fact that most isolates were collected from introduced tree species and native species in artificially planted urban greenery, supports the hypothesis that D. pini has been recently introduced into Slovakia.