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.     

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.     

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.     

Diversity of Cryphonectria parasitica in western Spain and identification of hypovirus‐infected isolates

Isolates of the chestnut blight fungus Cryphonectria parasitica were obtained from 44 localities in four provinces in Western Spain and characterized for vegetative compatibility (vc) types, mating types and the presence of Cryphonectria hypovirus 1 (CHV1). Among the 1232 isolates recovered from chestnut blight cankers, 11 vc types were identified: five known vc types included in EU1 to EU74 (EU1, EU11, EU12, EU28 and EU66) and six unknown vc types (CL4, CL5 CL6, CL8, CL9 and CL10). The number of vc types found per province varied between two and seven. The vc type EU11 was present in all provinces and accounted for 48.9% of all isolates. EU1 was detected in three provinces and accounted for 39.1% of the isolates. The vc types EU12, EU66, CL5 and CL6 were present in one or two provinces and comprised between 2.4% and 3% of the isolates. The other vc types were represented by only one or very few isolates. The mating type MAT‐1 was largely dominant in the provinces Leon and Avila, while both mating types MAT‐1 and MAT‐2 were found in Salamanca and Zamora. Fourteen hypovirus‐infected C. parasitica isolates were found, nine were in vc type EU1 and five in EU11, and they were detected only in the province León. All isolates analysed contained the French hypovirus subtype CHV1‐F1.     

Effects of root damage associated with Phytophthora cinnamomi on water relations,biomass accumulation,mineral nutrition and vulnerability to water deficit of five oak and chestnut species

The effects of root damage associated with Phytophthora cinnamomi on water relations, biomass accumulation, mineral nutrition and vulnerability to water deficit were investigated in pedunculate oak (Quercus robur), red oak (Quercus rubra) and holm oak (Quercus ilex) saplings over two years. Comparison was made with sweet chestnut (Castanea sativa), a susceptible species to infection by P. cinnamomi, and with a resistant hybrid chestnut (Castanea crenata × C. sativa). Trees were inoculated in 1998 and were subjected to water shortage in 1999. All inoculated sweet chestnuts died before the application of water shortage. Hybrid chestnut, pedunculate oak and red oak displayed low root susceptibility to P. cinnamomi. In these species, water relations, aerial growth and mineral nutrition were slightly affected by inoculation. By contrast, holm oak was the most susceptible oak species to P. cinnamomi as inoculated well‐watered trees displayed the highest root loss (67%) and a 10% mortality. Root loss was associated with a decrease in predawn leaf water potential, a 61% reduction in stomatal conductance, a 55% reduction in aerial biomass, a decrease in leaf carbon isotope discrimination and reduced leaf N and P contents in comparison with controls. In hybrid chestnut and pedunculate oak, water shortage resulted in a similar decrease of predawn leaf water potential, stomatal conductance and aerial biomass in inoculated and non‐inoculated trees. In red and holm oaks, soil volumetric water content of inoculated trees subjected to water shortage remained high. The effects observed in those trees were similar to those of inoculated well‐watered trees and were probably the result of root infection only.     

Cryphonectria parasitica in Portugal: diversity of vegetative compatibility types,mating types,and occurrence of hypovirulence

Cryphonectria parasitica, the causal agent of chestnut blight, has been responsible for the decline in chestnut in Portugal for the last two decades. In order to improve understanding of C. parasitica diversity, a total of 617 isolates from all affected chestnut‐growing areas in continental Portugal, Madeira and the Azores archipelagos were studied. Nine vegetative compatibility (vc) types were identified among the isolates. EU‐11 was the most widespread vc type comprising 80.2% of the isolates, followed by EU‐12 (7.1%) and EU‐66 (6.6%). Two of the Portuguese vc types could not be assigned to a known European vc type. The diversity of vc types was low in the Portuguese populations of C. parasitica, but comparable with other areas where C. parasitica was introduced recently. The frequent occurrence of perithecia and both mating types of C. parasitica indicates that sexual reproduction of the chestnut blight fungus is common in Portugal. One C. parasitica isolate from Trás‐os‐Montes showed a white culture morphology and contained dsRNA, indicating the presence of hypovirulence in this area.     

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.     

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.     

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.     

Identification and geographic distribution of vegetative compatibility types of Cryphonectria parasitica and occurrence of hypovirulence in Greece

Castanea sativa, one of the most precious forest trees in Greece, is affected by the spread of chestnut blight all over the country. Identification and mapping of the vegetative compatibility (vc) types of the fungus Cryphonectria parasitica was carried out and the occurrence of hypovirulence was checked. In 11 chestnut populations distributed all over Greece, 627 isolates were obtained and their vc type was determined. Four vc types were identified by means of tester isolates: EU‐12 was found to be the dominant vc type reaching a percentage of 88%, with EU‐2 (6%), EU‐10 (3%) and EU‐1 (2%) following. EU‐12 was found in all 11 populations investigated whereas EU‐2 was isolated from five populations and EU‐10 and EU‐1 were found only in one population each. Natural hypovirulence was also found in five populations of 11 examined. Perithecia were not found in either population, a finding supported by the fact that all Greek isolates examined belonged to a single mating type, MAT‐1. The low diversity in vc types, the absence of the perithecial stage and the occurrence of natural hypovirulence in some areas support the feasibility of successful application of biological control on the national level in Greece.     

Involvement of Phytophthora spp. in chestnut decline in the Black Sea region of Turkey

Chestnut blight caused by Cryphonectria parasitica is a serious disease of Castanea sativa in the Black Sea region of Turkey. During disease surveys, dieback and decline symptoms were observed on trees without apparent blight and ink disease symptoms. Black necroses, similar to those caused by Phytophthora infections, were noted on some of the chestnut coppices and saplings in one nursery in Ordu and led to an investigation into this disease complex. Only symptomatic plants showing dieback symptoms were investigated. Soil samples together with fine roots were collected from two directions, north and north‐east, approximately 150 cm away from the main stems. Phytophthora spp. were baited with young chestnut leaves. Three Phytophthora spp., P. cambivora, P. cinnamomi and P. plurivora, were identified from 12 soil samples collected from 73 locations, while from the nurseries, only P. cinnamomi was obtained. Phytophthora cinnamomi was the most common species, obtained from seven locations in five provinces and from four nurseries having similar symptoms mentioned above in different locations. Phytophthora cambivora and P. plurivora were less frequently obtained, from three to two stands, respectively. Phytophthora cinnamomi and P. cambivora were the most aggressive species when inoculated at the stem base on 3‐year‐old chestnut saplings, killing six saplings of eight inoculated in 2 months. The three Phytophthora species were first recorded on chestnut in Black sea region of Turkey with the limited samples investigated in a large area about 150 000 ha chestnut forest.     

Chestnut blight (Cryphonectria parasitica) north of the Swiss alps

Chestnut blight (Cryphonectria parasitica) was found in several stands in the Swiss plateau. At each site only one or two compatibility (vc) groups were present. The vc-group I was the most common. At a heavily infested site also young oaks (Quercus petraea) were infected.     

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.     

Susceptibility of the Quercus rubra root system to Phytophthora cinnamomi; comparison with chestnut and other oak species

The susceptibility of oak seedlings (Quercus palustris, Quercus robur, Quercus rubra) and chestnut seedlings (Castanea sativa) to Phytophthora cinnamomi was tested. The dynamics of infection was examined in plant material raised in a rhizotron. In the oak species, primary root tissues were susceptible whereas secondary cortical tissues showed some resistance to P. cinnamomi. Secondary cortical tissues of the tap root in C. sativa were susceptible. Inoculations with P. cinnamomi were performed both in situ and on excised roots of mature Q. rubra. In both cases, the resistance of Q. rubra roots and shoots was negatively correlated with diameter at the inoculation point. Small roots (l–5-cm diameter) were resistant, whereas collar and trunk were susceptible. In contrast to oak, small excised roots of mature C. sativa (0.7–2-cm diameter) were susceptible to P. cinnamomi. This may explain why P. cinnamomi does not induce a decline of the attacked oaks, but rather a trunk canker.     

The role of Spulerina simploniella in the spread of chestnut blight

The bark miner Spulerina simploniella (Lepidoptera: Gracilariidae) was found in coppice chestnut (Castanea sativa) forests in Greece but was not found in chestnut orchards. Its larvae mine under the thin periderm of young trees, 4–10 years old, while the stem bark is still smooth. Under normal conditions it does not cause any damage to the trees. However, when chestnut blight caused by Cryphonectria parasitica is present in the area, the insect may be an agent of disease spread. Experiments revealed that spraying of 23 pupation sites with a C. parasitica conidiospore suspension caused canker formation at a rate of 100% in the coppice chestnut forests of Mount Athos, North Greece. It is believed that rain during the pupation period (approximately May 23 to June 15) may deposit conidiospores on the freshly exposed phloem and cause cankers. This bark miner has been detected in several parts of Greece, however, always in intensively managed chestnut coppice forests.     

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.     

Roe deer (<Emphasis Type="Italic">Capreolus capreolus</Emphasis> L.) browsing effects and use of chestnut and Turkey oak coppiced areas

Introduction   

Roe deer (Capreolus capreolus) browsing pressure on vegetative regeneration of Turkey oak (Quercus cerris) and chestnut (Castanea sativa) and roe deer use of coppiced areas were investigated.     

Cryphonectria hypovirus 1 (CHV‐1) survey reveals low occurrence and diversity of subtypes in NE Spain

Cryphonectria parasitica (Murrill.) Barr is a necrotrophic fungus that affects European chestnut populations. In Catalonia (NE Spain), this fungus is widely spread affecting chestnut stands. In addition to vegetative compatibility (VC) types of the fungus, knowledge of the Cryphonectria hypovirus (CHV) occurrence is important to implement a biocontrol programme. We confirmed the presence of hypoviruses in several chestnut subpopulations of Catalonia, and we determined the VC types of CHV‐infected isolates. We also studied the nucleotide sequences of these hypoviruses from a variable region located in the hypovirus ORF‐A. Our results show a low occurrence of hypoviruses throughout C. parasitica populations in Catalonia, except for a few localized stands. From 312 sampled cankers, we obtained 179 white, pale orange or rickety isolates. In 35 of them, we detected CHV‐1. Infected isolates belonged to 5 of the 7 sampled subpopulations. We found 12 CHV‐1 haplotypes, based on the nucleotide sequence analysis. Most of the hypoviruses have the same nucleotide sequence or show high homology at nucleotide level with isolates previously included in CHV‐1 subtype‐I. However, we found that 3 haplotypes share at least 97% of their ORF‐A nucleotide sequence with CHV‐1 subtype‐E. Phylogenetic analysis grouped these 3 isolates in a different cluster than the other hypoviruses. These results suggest that multiple introductions of CHV‐1 have occurred recently in Catalonia. Dominant VC types of hypovirus‐infected isolates are EU‐1, EU‐2 and EU‐5, which are similar to those previously reported in this region.     

Late leaf development in pedunculate oak (Quercus robur): An antiherbivore defence?

Abstract

Two phenological forms of the pedunculate oak co-occur in the same habitats throughout the species range: the early trees (Quercus robur var. praecox) develop leaves up to 5 weeks before the late ones (Quercus robur var. tardiflora). This study tests the idea that late leaf flushing serves as an antiherbivore defence, i.e. late trees, which develop leaves asynchronously with eclosion of folivorous caterpillars, avoid the costs of defoliation, which could offset the costs of a later onset of the growing season. Effects of folivorous caterpillars foraging on oaks were observed in 1998–2006 in remnants of primeval temperate lowland forest preserved in the Bia?owie?a National Park (eastern Poland). Observations covered trough and outbreak years of the major defoliator, the winter moth [Operophtera brumata L. (Geometridae)]. In seven out of nine seasons, including all peak caterpillar years, the amount of frass produced by folivorous caterpillars on late trees (n=8–18) was significantly (up to 7.1 times) lower than on the early ones (n=12–32). Assessment of the degree of defoliation in 2002–2006 showed that the late oaks were visibly defoliated only during a caterpillar peak (2003), while the early trees were affected in all years and in 2003 almost completely lost their leaves. The results confirm the effect of late budburst on lowering herbivore damage, and give support to the idea that late leaf flushing acts as an antiherbivore defence.     

Epidemiological role of strains of Cryphonectria parasitica isolated from hosts other than chestnut

Cryphonectria parasitica is not common in the forests of the Emilia Romagna region of Italy on hosts other than chestnut. Three isolates with morphological cultural characteristics of C. parasitica were collected: two from infected bark of Quercits pubescens (EQ1 and EQ2) and one from Carpinus betulus (EC1). Pathogenicity tests on chestnut and crosses in vitro with strains of C. parasitica of different mating types confirmed that EQ1 and EQ2 are C. parasitica isolates, whereas EC1 seems to belong to a different species. Progeny analysis of crosses with the isolate EQ1 and a strain from chestnut showed the formation of new vegetative compatibility groups. Therefore hosts other than chestnut can contribute to increasing pathogen variability through recombination of the v-c genes.