Diversity of Cryphonectria parasitica in callused chestnut blight cankers on European and American chestnut

Infection of American and European chestnuts with the chestnut blight fungus Cryphonectria parasitica results in the formation of cankers, lesions caused by the growth of mycelia within bark tissue of the host plant. Infection of the fungus with Cryphonectria hypovirus 1 (CHV‐1) results in conversion of the mycelial phenotype from virulent to hypovirulent, thus allowing production of callus around cankers as a reaction by infected trees, rendering active into inactive cankers. In this study, we sampled one USA and six European chestnut stands and assessed frequency of hypovirulent C. parasitica and diversity of vegetative compatibility (vc) types present in calluses and randomly sampled cankers. Callused cankers on C. dentata at West Salem in the USA yielded significantly more hypovirulent C. parasitica isolates compared with four sampled populations on C. sativa, while all six sampled European populations did not show any statistically significant differences among themselves. We observed no correlation between hypovirulence frequencies in randomly sampled cankers and calluses, as well as no correlation of C. parasitica vc type diversity in calluses and residential populations of the fungus. Furthermore, even though we have observed calluses with more than one vc type, they do not occur regularly. Even when present in C. parasitica populations with high vc type diversity, no more than three different vc types were observed in a single callus.     

Characteristics of the Cryphonectria parasitica isolated from Quercus in Slovakia

The occurrence of chestnut blight (Cryphonectria parasitica) on oaks in mixed chestnut‐oak forests was studied in 2003–2008 in Slovakia. Infections on living Quercus trees were found at four of seven localities. The disease incidence on oaks ranged from 1.3% to 15.8%. The symptoms on infected oaks were similar to those on chestnut, but less conspicuous. Cankers of C. parasitica were found only on Quercus robur and Q. petraea. A total of 22 isolates of C. parasitica, all virulent, were isolated. Each site yielded only a single vc type (EU12 or EU13). Field inoculation experiments on chestnuts with seven strains of C. parasitica from oaks and an isolate from Castanea sativa showed no differences in virulence. On Quercus robur stems, the cankered area was significantly smaller than on C. sativa and the cankers developed very slowly.     

Spread of white hypovirulent strains of Cryphonectria parasitica on grafted American chestnut trees exhibiting a high level of blight control

In 1982 and 1983, natural blight cankers, located in a zone extending from the ground to 183 cm on the main stem of grafted American chestnut trees, were inoculated with a mixture of dsRNA-containing, white (European) and pigmented hypovirulent strains of Cryphonectria parasitica (H-inoculated zone). In 1996, white isolates (34% of 156 C. parasitica isolates) were recovered from superficial cankers throughout the grafts and as far as 564 cm from the H-inoculated zone. Lloyd's index of patchiness value (1.36) indicated that white isolates were slightly aggregated in cankers on the grafts. Forty-five percent of 95 C. parasitica isolates, recovered 5–50 months after inoculating the grafts with pigmented virulent strain WK, were white and some converted strain WK to the white phenotype in vitro. All cankers and bark cores yielding only pigmented isolates, vegetatively compatible with virulent strain WK, had superficial necrosis 5 and 11 months after inoculation with strain WK. All white isolates of C. parasitica assayed contained a 12.7 kbp dsRNA in high concentrations, and were hypovirulent in pathogenicity trials. Twenty-eight vegetative compatibility groups were identified among 65 pigmented graft area isolates; none of 48 pigmented isolates contained dsRNA. In addition to host resistance factors, spread of white strains may be responsible for the unusually high level of blight control on the grafts.     

Frequencies and spatial patterns of white hypovirulent and pigmented strains of Cryphonectria parasitica within blight-controlled cankers on grafted American chestnut trees 15–16 years after inoculation

The frequencies and spatial patterns of white and pigmented strains of Cryphonectria parasitica were investigated within cankers in a zone on grafted American chestnut trees inoculated with white (European) and pigmented hypovirulent strains (H-inoculated zone) 15–16 years earlier. Six 7 × 7 lattice plots (each 17.8 × 17.8 cm) were established on cankers in the H-inoculated zone of the grafts. Assays of 49 bark cores per lattice indicated that 35.3% of 306 C. parasitica isolates recovered from the six lattice plots were white. The white isolates had a random pattern, potentially favorable to biocontrol, within the highly superficial cankers, based on join-count statistics of the six lattice plots. Pigmented isolates dominated the C. parasitica population, and virulence trials on American chestnut sprouts indicated 36% of the pigmented isolates from the H-inoculated zone were hypovirulent and 27% were virulent. Most (84.3%) pigmented isolates in a bark core could not be converted to the white phenotype in vitro by white isolates in the same bark core. Five of six lattice plots had a random pattern of pigmented isolates, based on join-count statistics. The sixth lattice plot was composed of an aggregate of 36 lattice cells (area = 232 cm²) containing 12 pigmented vegetative compatability (vc) groups of C. parasitica, which were interwoven in the lattice as a mosaic of thread-like forms, blocks, or ‘islands’ 32 cm² or less in area for each vc group. Hypotheses are advanced to explain why virulent pigmented strains persist in blight-controlled cankers of the H-inoculated zone but do not kill the vascular cambium.     

Distribution of chestnut blight and diversity of Cryphonectria parasitica in chestnut forests in Bulgaria

We surveyed chestnut stands at 18 sites in 11 locations in Bulgaria in 2005 and 2007 for the presence of chestnut blight. We found chestnut blight in seven locations (Belogradchik, Berkovitsa, Brezhani, Barziya, Govezhda, Petrich and Petrovo) but not in four others (Tsaparevo, Kresna, Dupnica and Botevgrad). We successfully isolated Cryphonectria parasitica from cankers on 606 trees with symptoms of chestnut blight and assayed them for vegetative compatibility (vc) types and mating type. Three vc types were identified among the 606 isolates; all three were among the European vc types with known vegetative incompatibility (vic) genotypes. Vc type EU‐12 was the most common, representing 80% of the isolates, and was found at all locations with blight, with the exception of Belogradchik in north‐west Bulgaria, where all isolates were vc type EU‐2. Only one population (Barziya) had more than one vc type, with a combination of EU‐12 and EU‐10 in almost equal frequencies. Similarly, the diversity of mating types was very low. All but three of 536 isolates assayed were in mating‐type MAT‐1; MAT‐2 was only found in one population in the north‐west (Berkovitsa). We inspected 671 bark samples from chestnut blight cankers with stromata of C. parasitica and found perithecia in only 33, of which 28 were from Berkovitsa where MAT‐2 was present. We did not detect hypoviruses in any of the 270 isolates screened using the standard double‐stranded RNA extraction protocol. Similar to results from previous studies in south‐eastern Europe, the diversity of vc types and mating type of C. parasitica in Bulgaria is low, and reproduction of the fungus is mainly asexual. Unfortunately, naturally occurring hypovirulence was not detected. Nevertheless, we observed a small number of superficial cankers typical of those caused by C. parasitica isolates infected with a hypovirus.     

Incomplete movement of Cryphonectria hypovirus 1 within a vegetative compatibility type of Cryphonectria parasitica in natural cankers on grafted American chestnut trees

American chestnut trees, grafted in 1980 from large survivors, were inoculated in 1982 and 1983 with four white (European) hypovirulent strains of Cryphonectria parasitica, infected with C. hypovirus 1 (CHV1); this hypovirus has been shown to be capable of moving rapidly within the mycelium of a vegetative compatibility (vc) type of C. parasitica in blight cankers. Using a 49‐cell lattice plot, 17.8×17.8 cm, the spatial patterns and frequencies of white and pigmented isolates and white and pigmented vc types were investigated within superficial cankers on the grafts located outside the hypovirulent‐strain‐inoculated zone. Four of six cankers assayed contained white isolates, and three of the four had random spatial patterns of white isolates, based on join‐count statistics. Vc tests, using pigmented isolates and pigmented single‐spore colonies of white isolates, indicated that the majority of white and pigmented isolates recovered from each of two cankers assayed were in one vc type. White and pigmented lattice‐plot cells of the same vc type were frequently in contact with each other, indicating incomplete movement of CHV1 within a vc type. Nine and 10 vc types were found in the two cankers; it is hypothesized that small, white vc type areas in each canker may be a source of CHV1 transmission to the major vc types. Based on join‐count statistics, the spatial pattern of the single, major vc type in one canker was non‐random (aggregated), whereas the other canker had a random major vc type pattern. White and pigmented in vitro variants (sectors) of C. parasitica, that resemble white and pigmented in vivo variants in spatial contact and vc compatibility, were intermediate hypovirulent and virulent on forest American chestnuts, and dsRNA positive and negative, respectively. Incomplete movement of CHV1 within a vc type could be a major cause of the prevalence of pigmented isolates in superficial cankers on chestnut trees.     

Long‐term field study of transgenic hypovirulent strains of Cryphonectria parasitica in a forest setting

Transgenic hypovirulent strains of Cryphonectria parasitica, the chestnut blight fungus, engineered to contain a chromosomally integrated full‐length infectious cDNA copy of virulence‐attenuating hypoviruses, differ from natural hypovirulent strains in the ability to transmit hypoviruses to ascospore progeny and with 100% efficiency through asexual spores. We report the results of a long‐term field study that examined whether these properties result in enhanced hypovirulence establishment, dissemination and persistence under field conditions. Informed by previous field results using a severe hypovirus, this study that employed 144 American chestnut trees was designed to provide improved inoculum formulation and delivery and to include the use of a mild hypovirus isolate (less debilitating) CHV‐1/Euro7 in an attempt to increase dissemination. Isogenic transgenic hypovirulent (TG), non‐transgenic cytoplasmic hypovirulent (CH) or virus‐free virulent (V) treatment strains were applied to artificially initiated and natural C. parasitica cankers three times each year for 7 years. Reservoirs of treatment inoculum also were initiated and refreshed annually for the first 6 years of the study. Sampling of 111,000 individual ascospores from 4,500 perithecia confirmed hypovirus‐containing spermatia successfully transmitted TG hypoviruses to ascospore progeny under field conditions. Surprisingly, TG ascospore progeny were recovered 3 years after the last annual application of treatment inoculum. Repeated sampling of over 440 cankers revealed dissemination of both CH and TG hypovirulent strains. However, no significance differences in establishment or dissemination were observed for the two hypovirulent strains. The results are discussed in terms of the contribution of ascospore progeny to infection, competition by endemic virulent C. parasitica, size of inoculated trees and the biological control potential of TG hypovirulent strains.     

Informative value of canker morphology on the presence or absence of virus infection in chestnut blight cankers

Chestnut blight destroyed the native chestnut forests in North America and also severely affected the European chestnut trees after its introduction in the 20th century. The ascomycete fungus Cryphonectria parasitica is responsible for this serious disease and causes lethal bark cankers on susceptible chestnut trees. In Europe, however, an infection of C. parasitica with Cryphonectria hypovirus 1 (CHV‐1) causes hypovirulence in C. parasitica and reduces the severity of the disease. Hypovirulence biologically controls chestnut blight in many regions to date. In this study, our goal was to determine morphological canker characteristics that are indicative of virus presence or absence in C. parasitica. We investigated 677 chestnut blight cankers from seven different geographic locations across Europe. For each canker, we assessed canker length, stem encircling, canker depth, presence of sporulation, canker activity and virus infection. We statistically analysed the informative value of these morphological characteristics for the presence or absence of CHV‐1. However, we did not find reliable indicators. Our logistic regression analysis revealed that virus infection of C. parasitica is not clearly related to canker morphology. This implies that fungal isolations from chestnut blight cankers and assessments in the laboratory are required to determine infection with CHV‐1 unequivocally.     

Low genetic diversity but frequent sexual reproduction of the chestnut blight fungus Cryphonectria parasitica in Azerbaijan

Chestnut blight was first recorded in Azerbaijan on the native European chestnut (Castanea sativa) in 2004, and since then, the disease is expanding in the country. In this study, chestnut blight was detected in seven of eight forest districts that were inspected. To characterize the local population structure of the chestnut blight fungus, 199 Cryphonectria parasitica isolates were collected and assessed for vc type, mating type and microsatellite genotype. Among these isolates, one dominant vc type was detected comprising 93% of the isolates. Six additional vc types were identified among the other isolates. The microsatellite analysis revealed a very similar pattern with 96.5% of the isolates belonging to the same multilocus genotype. Both mating types of C. parasitica were present in all seven districts with a mating type ratio not different from 1:1 in five districts. In accordance with the occurrence of both mating types, sexual fruiting bodies (perithecia) of C. parasitica were found in all districts with an overall prevalence on 20.6% of the cankers. The dominant vc type and microsatellite genotype in Azerbaijan do not occur in Europe, but have previously been found to be widespread in neighbouring Georgia. Our study reveals that sexual reproduction in the invasive C. parasitica population in Azerbaijan is frequent, although the population shows a low genetic diversity. This could favour the biological control of chestnut blight using hypovirulence, which so far does not seem to be present in Azerbaijan.     

Eutypella canker of maple: first report from Germany and situation in Austria

Eutypella canker of maple, caused by Eutypella parasitica (which is native to North America), is reported for the first time from Germany. From 2013 to 2015, this perennial canker disease was recorded on 105 maple trees in Munich. Six maple species were affected: Acer pseudoplatanus, A. campestre, A. platanoides, A. cappadocicum, A. heldreichii ssp. trautvetteri and A. hyrcanum. Occurrence on the latter three species represents new host records for E. parasitica. In Austria, Eutypella canker was newly discovered on two trees at a second locality in 2011, and it is now known to occur on seven A. pseudoplatanus trees at two localities, which are separated nearly 150 km. A. pseudoplatanus was the most frequent host of E. parasitica in Munich and Austria, which is in agreement with previous studies in Europe. The identity of the causative pathogen as E. parasitica was verified by ITS rDNA sequencing of fungal cultures obtained from cankers in Munich and at both Austrian localities. The presence of large and old cankers in both countries suggests that introduction of E. parasitica dates back a long time, probably several decades. The new records of Eutypella canker in Germany and Austria show that the disease is more widely distributed in central Europe than previously recognized.     

Chrysoporthe puriensis causing canker and mortality in Pleroma mutabile in the Atlantic Forest,Brazil

Fungi of the Cryphonectriaceae family are globally known to be tree pathogens. In Brazil, several species of Chrysoporthe have been found causing stem and branch cankers in Pleroma (= Tibouchina) spp. Recently, Chrysoporthe puriensis was described as a new species causing stem and branch cankers in Pleroma granulosum, Pleroma candolleanum, and Pleroma heteromallum, all native species of the Melastomataceae family. During an investigation to collect isolates of the Cryphonectriaceae family in an important Brazilian biome, the Atlantic Forest, in Serra do Mar, structures typical of Chrysoporthe species were found in a different host, Pleroma mutabile. Fruiting bodies present in the bark of these trees were collected and isolated. The isolates were submitted on morphological characterization and phylogenetic analyses of the internal transcribed spacer (ITS) and β-tubulin gene regions using Maximum likelihood, Bayesian inference and Maximum parsimony methods. The isolates collected, for this study, were identified as C. puriensis. Pathogenicity tests on seedlings of Pleroma species and Eucalyptus clones revealed C. puriensis can infect and cause canker in these plant species as mortality. The results demonstrate the importance of delimiting the C. puriensis collection range to track its dissemination in other hosts. No host specificity was observed in the inoculation tests, suggesting this is an important finding, the pathogen causes diseases and mortality in several plants of the Atlantic Forest. Additionally, the pathogen can affect others hosts, such as Eucalyptus clones in commercial plantations.     

Chestnut blight in south‐western Germany: multiple introductions of Cryphonectria parasitica and slow hypovirus spread

Cryphonectria parasitica was discovered in the sweet chestnut forests of south‐western Germany in 1992. Two main areas affected by chestnut blight were discerned, one to the east and one to the west of the Rhine valley. The occurrence of the fungal pathogen with respect to vegetative compatibility (vc) type and hypovirulence was analysed by sampling chestnut blight cankers between 1992 and 2010. Among 368 C. parasitica isolates sampled in south‐western Germany, 9 different vc types were found. East of the Rhine valley, EU‐2 is the most widespread vc type. In addition, two isolated forest areas infected with C. parasitica of the vc types EU‐14 and EU‐28 were detected. West of the Rhine valley, C. parasitica of the vc type EU‐65 was repeatedly isolated from an infection focus, the spread of which was successfully suppressed for several years by sanitation measures. Since 2003, additional outbreaks of C. parasitica belonging to the vc types EU‐2 and EU‐5 were detected in the vicinity. Several other vc types (EU‐1, EU‐12, EU‐33 and one vc type incompatible with any of the 74 European testers) were identified on isolated trees mainly in urban areas across the study area and were subsequently eradicated. The spatial and temporal distribution of the different vc types indicates at least nine different introductory events of C. parasitica into south‐western Germany. Natural hypovirulence was only found in the infection area in Baden‐Württemberg. A total of four hypovirulent isolates of the vc type EU‐2 were obtained, one in 1992 and three in recent years. The four hypoviruses were genetically closely related and belonged to the Spanish/German subtype of Cryphonectria hypovirus 1. As the different vc types in south‐western Germany occur mostly in spatially separated, single stands, the introduction of hypovirulence as biological control is expected to be effective.     

Dieback and decline of wild almond (Amygdalus scoparia Spach) in the Harat protected forest of Yazd Province,Iran

Wild almond (Amygdalus scoparia) is a dominant shrub species in mountain forests of the Irano‐Turanian region. Dieback and decline symptoms of wild almond shrubs were first observed in Harat protected forest (Yazd, Iran) in the autumn of 2014. Since then, the incidence and severity of the disease have increased. To study the aetiology and estimate losses from the disease, field studies were conducted in 2016 and 2017. For this purpose, eight stands were selected, in which 24%–99% of the trees were wild almond in their species composition. In total, 50 cankers and 50 infected twigs of wild almond shrubs were collected. Fungal isolates were identified based on morphological and cultural characteristics, as well as sequence data of ITS1 + 5.8S + ITS2 rDNA. Pathogenicity of fungi was checked on detached shoots and evaluated after 40 days by measuring the discoloured lesion length at the inoculation site. Among 681 wild almond shrubs checked, 84.44% showed decline symptoms with different severities; only 15.56% did not show observable disease symptoms. The most frequent colonizers of infected tissues taken from cankers were Wilsonomyces carpophilus (56%), Thyrostroma cornicola (40%) and Collophorina paarla (30%). Infected twigs were extensively colonized by W. carpophilus (36%), Th. cornicola (24%) and Ulocladium consortiale (24%). Other species, such as Endoconidioma populi, Microsphaeropsis olivacea, Trichoderma asperellum, Paecilomyces formosus, Saccothecium rubi, Preussia sp. and Chaetomium globosum, had lower isolation frequencies. W. carpophilus, Th. cornicola, C. paarla and U. consortiale were found as pathogens on detached wild almond shoots. Based on the frequency of the isolates and the pathogenicity tests, four fungi, such as W. carpophilus, Th. cornicola, C. paarla and U. consortiale, are considered serious contributing agents playing a significant role in the dieback and decline of wild almond. All isolated species are reported for the first time on the wild almond shrubs of the world.     

Variation in virulence of Cryphonectria hypovirus 1 in Macedonia

We investigated variation in virulence of Cryphonectria hypovirus 1 (CHV‐1) to the chestnut blight fungus, Cryphonectria parasitica, in Macedonia by inoculating chestnut stems in the field. We inoculated trees with two isolates of C. parasitica, each infected with one of five isolates of CHV‐1, four of which were the same for both fungal isolates. Two virus isolates, [Sk28] and [Sk47], were significantly more virulent than the others when compared in the same fungal host isolates, as measured by reduced canker growth and increased callus formation. Mycelial growth rate in vitro was weakly correlated to canker growth or callus formation and is therefore not a reliable predictor for virulence. We found significant fungus × virus interactions for canker growth and callus formation, which seems due mainly to one virus isolate. Significant interactions were not expected because the two fungal host isolates are members of the same clone that is dominant in Macedonia and most of southeastern Europe. Phenotypic variation for response to viruses, therefore, is greater than variation revealed by the genetic markers used to define clones. More than half of the trees inoculated with virus‐free controls were dead within 2 years, and the 30% still alive after 5 years had cankers with extensive callus formation, indicating that natural virus transmission had occurred after inoculation. In contrast, only 2% of the trees inoculated with virus‐infected isolates were dead after 5 years. Hypoviruses naturally occurring in Macedonia reduce canker development and tree mortality similarly to those in other parts of southern Europe, and therefore, may have good potential for biological control of chestnut blight.     

Vegetative compatibility and conversion to hypovirulence among Italian isolates of Cryphonectria parasitica

A total of 850 virulent isolates of Cryphonectria parasitica were isolated from natural cankers on European chestnut trees growing in various Italian regions. Vegetative-compatibility (v-c) group membership was tested using the merge-barrage method. In all, 19 vegetative-compatibility groups were found, and, for each of these, a representative isolate (v-c tester) was identified; 49% of the isolates were compatible with the tester of only one v-c group, whereas 51% were compatible with the testers of more than one v-c group; 3% of the isolates showed compatibility with seven v-c groups. The coincidence between vegetative incompatibility and failure of conversion occurred in only a few cases; 42% of the converts were stable with respect to their morphological characteristics and presence of cytoplasmic ds-RNA. The results obtained in Italy suggest that, for an efficient biocontrol programme, it is preferable to evaluate the tendency of a population to be converted rather than evaluating the v-c group membership.     

Establishment and dissemination of hypovirulent strains of Cryphonectria parasitica in Greece

Chestnut blight caused by Cryphonectria parasitica has been one of the major reasons for the decline of chestnut cultivation in Greece over the last 50 years. A previous detailed study of the vc types of the fungus has revealed only four vc types in the entire country, those of EU‐1, EU‐2 and EU‐10 with the dominant being EU‐12 counting for 88% of the isolates. As the loss in orchard trees reached in some chestnut producing prefectures over 35% of the trees, the decision was taken to apply biological control on a nationwide scale. The project was implemented in 17 prefectures during the period 2007–2009. Hypovirulent (hv) strains of C. parasitica which were infected by the CHV‐1 subtype I (Italian subtype) viruses which occur naturally in Greece were used. The necessary hypovirulent inoculum was prepared at the Forest Research Institute during the period 2007–2009 on a large scale, however, with laboratory care. Three million inoculations were made around accessible developing cankers over three consecutive years by trained personnel. An extensive evaluation conducted in 2011 in the 12 of the 17 treated prefectures, where no natural hypovirulence had been found before, demonstrated not only the successful establishment of hypovirulence but also profound dissemination and healing of non‐inoculated cankers. The extent of dissemination varied significantly among sampling plots and among prefectures. The conclusion is drawn that CHV‐1 subtype I has successfully established and spread into chestnut orchards and coppice forests resulting in a gradual decline of chestnut blight.