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.     

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.     

Transmission of hypovirulence agents among some Cryphonectria parasitica strains from Italy

Six hypovirulent (H) strains of Cryphonectria parasitica collected in Italy were tested for dsRNA content, virulence, and conversion ability. Nine converts were also checked for dsRNA. In inoculation trials, H and converted (C) strains showed reduced pathogenicity. Most H and C strains were able to convert strains from various v-c groups. DsRNA banding patterns were similar in five of the H strains and in their converts, while the last one and its converts showed no evidence of dsRNA. The broad conversion ability of H strains seems to be responsible for the natural recovery of Italian chestnut trees.     

Growth and physiological responses of chestnut calli to crude extracts of virulent and hypovirulent strains of Cryphonectria parasitica

The effect of crude extracts (CEs) of virulent (V‐E4) and hypovirulent (H‐E13) Cryphonectria parasitica strains on growth and physiological activities of chestnut calli was investigated on cell cultures obtained from either a susceptible Castanea sativa or a resistant Castanea mollissima chestnut species. The V‐E4 CEs significantly reduced calli fresh weight in both species when used at 50 and 500 μg g^–1 and, in general, showed an inhibitory effect on either O₂ uptake and H⁺‐ATPase activity. Only the highest (500 μg g^–1) concentration of H‐E13 CEs had a detrimental effect on callus growth, whereas the lowest one (5 μg g^–1) induced a slight, but significant early increase in callus fresh weight of both genotypes. The O₂ uptake and H⁺‐ATPase activity were enhanced by the presence of H‐E13 CEs. The decrease of callus growth and physiological activities, caused by V‐E4 CEs, was attributed to toxic compounds produced by the C. parasitica virulent strain, whereas the stimulatory effects of H‐E13 CEs on the overall callus metabolism seemed to be due either to the lack or very reduced amount of phytotoxic compounds and to growth‐regulating substances produced only by the hypovirulent strain of the fungus.     

Growth and ultrastructural modifications to chestnut calli induced by culture filtrates of virulent and hypovirulent Cryphonectria parasitica strains

Callus cultures of two susceptible Castanea sativa cultivars, ‘Garrone rosso’ and ‘Clone 71’ were grown on culture medium supplemented to 50% with culture filtrates (CFs) from E4 virulent (E4-V) or E13 hypovirulent (E13-H) Cryphonectria parasitica strain, respectively. E13-H CFs caused a reduction in fresh weight and an increase in dry weight on calli of both genotypes. E4-V CFs did not induce any modification of these parameters compared with the control, but fresh weight of ‘Clone 71’ was reduced. Light and electron microscopy observation showed the presence of large osmiophilic aggregates in the vacuoles and, sometimes, the ruptured tonoplast in the E4-V treated calli. Calli grown on El 3-H CFs had a meristematic-like appearance, with small, scarcely vacuolated cells containing altered mitochondria and plasmalemma proliferations.     

Inoculation trials with hypovirulent strains of Cryphonectria parasitica1

Healing cankers were obtained in some artificial inoculation tests with combination of four hypovirulent strains of Cryphonectria parasitica which sporulated on the cankers.     

Spread of Cryphonectria hypovirus1 into 45 vegetative compatibility types of Cryphonectria parasitica on grafted American chestnut trees

A mixture of hypovirulent strains of Cryphonectria parasitica, including four white (European) strains infected with Cryphonectria hypovirus1 (CHV1), was used in 1982 and 1983 to inoculate natural blight cankers located within a zone ranging from the ground to 183 cm on grafted American chestnut trees. These four white strains belonged to three vegetative compatibility (vc) types. Using pigmented, single‐spore colonies from white isolates, 48 vc types were identified among 110 white isolates recovered in 1996, 1998, and 1999 from cankers located outside the inoculated zone. Twenty‐five of the 48 white vc types consisted of two or more isolates. The 25 major white vc types were vegetatively incompatible with all four of the original white hypovirulent strains, providing evidence for spread of CHV1 but not for spread of the original inoculated strains. Forty‐five vc types represent the minimum number of `new' vc types into which CHV1 had spread. The ratio of white vc types to white isolates tested (S/N) and Shannon diversity index were 0.436 and 3.64, respectively. The spatial pattern of white vc types on the grafts was found to be non‐random (p=0.019). White single‐spore colonies of white isolates were placed into four cultural morphology (CM) groups. The two largest groups contained 37 (CM group 3) and 33 (CM group 1) isolates. Single‐spore colonies from the original, white inoculated strain, Ep 49, were classified into CM groups 3 and 1, and colonies of Ep 51 W were classified into CM group 1.     

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.     

Growth performance and chestnut blight incidence (Cryphonectria parasitica) of backcrossed chestnut seedlings in surface mine restoration

Anthropogenic disturbances such as mining for coal have caused significant disturbance to the Appalachian forests of North America. Recovery of these disturbances is highly dependent on restoration methods that encourage natural succession. Unfortunately, current reclamation protocols have resulted in soil compaction and aggressive herbaceous groundcovers that impede the recovery of native trees. To overcome this, methods such as deep ripping and plow and disking were applied to a reclaimed mine land in Ohio, USA Plantings of pure American chestnut (Castanea dentata) and two seedling types (BC₁F₃ and BC₂F₃) bred for resistance to chestnut blight fungus (Cryphonectria parasitica) were assessed among different soil preparation treatments over five field seasons. Seedling mortality due to natural infection by chestnut blight was recorded and related to the disease resistance potential of the BC₁F₃ and BC₂F₃ seedlings. The growth and survival of chestnuts in plots that employed either ripping, plow disking, or the combination of the two methods were significantly greater than the control plots. After five seasons, differences existed among the soil treatments; plots that applied deep ripping had the highest survival and growth. When chestnut types were compared, pure American chestnut was the tallest. However, BC₂F₃ chestnut seedlings had the highest survival and lower disease incidence. Results suggest that employing deep ripping with backcrossed chestnut seedlings provides a method for establishing hardwood seedlings in soils impacted by surface mining. Planting methods that promote vigorous growth can be applied more broadly to other regions where anthropogenic disturbances create soil conditions that hinder seedling establishment.     

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.     

Natural spread of Cryphonectria parasitica and persistence of hypovirulence in three Italian coppiced chestnut stands

Three coppiced chestnut stands near Florence (Tuscany, Italy) were monitored for 15 years. Presence of blight, damage, evaluation of different types of cankers and their relationship with stand structure were assessed. Disease incidence ranged from 67% to 99% of infected shoots, and mortality caused by blight was between 6% and 12%. Canker types were defined in the study sites; healing and healed cankers represented 70–88% of the total infections recorded, whereas normal cankers represented 9–12.5%. In general, more than one infection was detected for each shoot, and on some trunks up to nine cankers were observed. During the entire investigation period, a constant trend of hypovirulence spread was observed that was symptomatic of a substantial stability of the phenomenon: most (63.5%) undifferentiated new infections evolved into healing and healed cankers, compared with 6.5% that developed into normal infections. Isolations confirmed that a mixed inoculum (normal, intermediate and white strains) of the pathogen was spreading in the coppices. Inocula produced by pycnidia from healing cankers were examined and tested in an artificial inoculation trial. dsRNA was detected in white strains isolated from healing cankers. In this natural condition and in the complete absence of silvicultural treatments, this inoculum seems capable of ensuring both the spread of hypovirulence and its persistence over the years. Data suggest that hypovirulence and its spread are complex phenomena based on the interaction between fungal inoculum, host resistance and site conditions.     

Synergistic effect of drought and chestnut blight (Cryphonectria parasitica) on growth decline of European chestnut (Castanea sativa)

The chestnut blight fungus [Cryphonectria parasitica (Murill) Barr] has threatened European chestnut stands (Castanea sativa Mill.) in the 20th century, but infected trees recovered because of the appearance of hypovirulent strains. However, within a dry inner Alpine valley (Italy), blight‐infected C. sativa showing various degrees of crown dieback and dead trees were found. We conducted a dendroecological analysis to retrospectively evaluate a possible synergistic effect of blight infection in the early 1970s and climate stress on the growth decline of C. sativa. In the Eisack Valley (Italy), where annual precipitation is <700 mm, increment cores were taken from blight‐infected C. sativa (n = 103) showing different levels of decline symptoms, i.e. extent of crown dieback (healthy, moderate, severe), and from dead trees. Ring width and basal area increment (BAI) chronologies were developed based on dendroecological methods. Growth–climate relationships were explored using response function analysis and Pearson correlation coefficients. Major findings of our study were: (i) C. sativa growth is limited by low precipitation from December to February and high temperatures in June, (ii) BAI of all vitality classes except healthy trees shows a decreasing trend since 1980; and (iii) a severe drought in 1996 accelerated growth decline and caused death of infected C. sativa individuals. Because of the strong influence of climate on radial tree growth within the study area and observed divergent growth trends in selected vitality classes after infestation by hypovirulent C. parasitica strains in the early 1970s, we conclude that although blight infection is a prerequisite for observed growth declines, soil water availability in the years of drought strongly affects susceptibility to tree death.     

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.     

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.     

Distribution and severity of damage by Cryphonectria parasitica in the chestnut stands in Guilan province,Iran

Chestnut blight caused by Cryphonectria parasitica has recently been reported from Guilan province, the only region with natural chestnut (Castanea sativa) stands in Iran. During the past few years, chestnut stands in Iran have been seriously threatened by the fungus, incidence of the disease is increasing and vast canopies are reduced to sprouts. As yet, there is no report on the disease distribution and severity in this region. Six sites from three main growing regions of chestnut in Guilan province were selected for investigation. We report occurrence and evaluation of the damage of the disease caused by C. parasitica. To evaluate the scale of damage, the investigated trees were classified into six categories based on the degree of crown damage, the number of canker wounds and the presence of the fungus. Index of health condition was calculated for all sites. During this study, a total of 250 isolates of Cryphonectria species were obtained, of which 232 isolates were C. parasitica and 18 were Cryphonectria radicalis. Castanea parasitica was observed in all regions. Index of health condition was scored from 0 to 6 with 0 being disease free and six being the most severe infection. Index results in the investigated sites varied between 0.69–5.45 and 0.93–5.55 for years 2006 and 2007, respectively. The highest damage was found in Doran (I_H = 5.55), which is located some 100 km away from Shahbalutmahalleh, the site with lowest damage (I_H = 0.93). This is the first extended report on aspects of chestnut blight in Iran.     

Vegetative compatibility types of Cryphonectria parasitica,causal agent of chestnut blight,in the Black Sea region of Turkey

Vegetative compatibility types (vc types) of 296 isolates of the chestnut blight fungus, Cryphonectria parasitica, were determined. The isolates had been obtained from 32 localities in 11 provinces in the Black Sea region of Turkey. Five vc types were detected: EU‐1, EU‐12, EU‐14, EU‐2 and EU‐5. The number of vc types found in single provinces varied between one and five. All of the five vc types were present only in the Kastamonu province. Vc type EU‐1 was detected in all the provinces. EU‐1 accounted for 90.8% of all isolates. Vc type EU‐12 was present in eight provinces and accounted for 6.8% of the isolates, whereas one or two isolates each of EU‐14, EU‐2 and EU‐5 were found in one or two provinces. Isolates possessing the white colony phenotype were considered to be hypovirulent. Hypovirulent isolates of each vc type were found, and they were detected in nine of 11 provinces.     

Characterization of Cryphonectria parasitica strains by random amplified polymorphic DNA (RAPD) technique and conventional methods

In recent years damage to Austrian Castanea sativa populations caused by Cryphonectria parasitica has increased. A total of 34 isolates out of 13 observation plots of this phytopathogen in the south-east of Austria were examined for laccase activity, virulence, number of vegetative compatibility groups and for the existence of hypovirulence associated dsRNA. Furthermore, the applicability of the random amplified polymorphic DNA (RAPD) technique for investigating population structure was tested. Small dsRNA fragments were found in two Austrian strains. The same two strains exhibited reduced virulence. Seven vegetative compatibility groups were found in the areas examined. RAPD analysis proved to be an efficient method for distinguishing between different C. parasitica genotypes.     

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.