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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

Multi‐seasonal field release and spermatization trials of transgenic hypovirulent strains of Cryphonectria parasitica containing cDNA copies of hypovirus CHV1‐EP713

Three indigenous field strains of the chestnut blight fungus, Cryphonectria parasitica collected in 1996 from a test site in the Meshomasic State Forest, Connecticut, USA, were engineered to contain a chromosomally integrated full‐length infectious cDNA copy of the severe, virulence‐attenuating hypovirus CHV1‐EP713. These transgenic hypovirulent strains were introduced into the test site after it had been clear‐cut, as sprayed conidia in multiple applications over 4 years, beginning in 1997. Evidence was obtained for cytoplasmic transmission of cDNA‐derived hypovirus RNA and for recovery of transgenic strains from canker tissue of the spray‐treated trees. However, there was no indication of spread of transgenic strains or cDNA‐derived viral RNA to the control plot or outside the treatment plot. A single transgenic C. parasitica isolate was identified from a total of 156 isolates recovered from 1330 insects representing five insect orders. Only 2.6% of the 1082 C. parasitica bark isolates recovered during the course of this study were vegetatively compatible with the untransformed input strains. Transgenic ascospore progeny were produced by 40% of the perithecia collected in 1999 and by only 7.3% of the perithecia collected in 2000. A concurrent field release study performed in the Monongahela National Forest in West Virginia in 1998 and 1999 with transgenic hypovirulent strains constructed with the same CHV1‐EP713 cDNA‐containing plasmid identified seasonal application dates and delivery methods that resulted in very high levels of transgenic ascospore production. Results of the combined field release studies are discussed in terms of requirements for improved formulations and delivery methods and the importance of balancing ecological fitness and virulence attenuation.     

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.     

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.     

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.     

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.     

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.     

Determination of vc and mating types of Cryphonectria parasitica isolates by multiplex PCR and their genetic diversity in 13 chestnut-growing provinces of Turkey

Vegetative compatibility (vc) and mating types and genetic diversity of Cryphonectria parasitica isolates were determined using 183 isolates obtained from 215 infected chestnut trees growing in 13 provinces of Turkey. Based on the cultural aspects, 143 of these isolates were evaluated as virulent whereas the remaining 40 isolates were hypovirulent. When vc types of 183 isolates were classically differentiated, 135 of them matched to EU-1 (82.3%), 29 of them to EU-12 (17.6%) vc type, whereas 19 of them did not match to the two. When molecular vic markers were used, all the isolates were assigned to two EU vc types; 149 to EU-1 (81.4%) and 34 (18.5%) to EU-12. Of the majority of the isolates, 134 (73.2%) had mating-type MAT-1, while 44 (24%) isolates had MAT-2 and 5 (2.8%) isolates had both mating types. The population analysis based on two DNA marker systems, Inter-Primer Binding Site and Start Codon Targeted Polymorphism, showed no intraspecific genetic variation among the C. parasitica isolates. The prevalence of two dominant vc types revealed by this study shows that biological control with hypovirulent EU-1 and EU-12 isolates will be significant for the country. The results might be helpful to chestnut breeders carrying out resistance breeding studies to manage this disease based on hypovirulence attributed to Cryphonectria hypovirus 1.     

Seasonal effect on infection and development of lesions caused by Cryphonectria parasitica in Castanea sativa

Seasonal variation in the development of chestnut blight, caused by Cryphonectria parasitica, was investigated by inoculating in situ chestnut trees and in vitro excised chestnut segments, at either monthly or 3‐monthly intervals throughout 30 months. Inoculations were made with conidia and mycelium of a virulent isolate and with mycelium of a hypovirulent isolate. Conidial inoculations of living sprouts or excised segments between May and July resulted in the greatest incidence of infection whereas inoculations in autumn and winter, in vitro as well as in situ, did not reveal any visible disease. However, from these symptomless inoculated stems, C. parasitica was isolated 3 months after inoculation. Inoculations with the mycelium of the virulent isolate always resulted in lesions, except in January 1999, and the greatest rate of lesion development occurred for inoculations made in the spring and summer. There was a significant seasonal effect on lesion development. Lesions caused by the hypovirulent isolate, smaller than those caused by the virulent isolate, followed a similar seasonal pattern. The same seasonal variations were observed for inoculations in vitro of excised segments. Relative water content (RWC) of chestnut bark significantly varied with bark sampling date. The rate of lesion development in sprouts significantly correlated with average minimum (ATn) and maximum (ATx) temperatures and the sum of rainfall during inoculation period, with the rate of lesion development measured in excised segments 10 days after inoculation (R10d) and with RWC measured on the day of inoculation. In multiple regression models, variables ATx and R10d best explained variation in lesion development.     

Decline‐associated Phaeoacremonium species occurring on forest trees in the north of Iran

Between 2013 and 2015, several surveys were conducted throughout forest areas in the north of Iran with the aim of isolating and identifying fungi present on trees showing decline symptoms. Fungal species, isolated from symptomatic wood tissues of various tree genera, including Zelkova, Acer, Parrotia, Quercus, Diospyros, Fagus, Fraxinus, Populus, Pinus, Pterocarya, Gleditsia, Carpinus, Ulmus, Crataegus, Alnus, Mespilus, Punica, Cydonia, Cupressus and Prunus, were morphologically identified and characterized. A molecular identification based on comparisons of DNA sequences of the β‐tubulin gene allowed us to identify seven species belonging to Phaeoacremonium; namely, Phaeoacremonium parasiticum, Pm. alvesii, Pm. minimum, Pm. iranianum, Pm. scolyti, Pm. fraxinopennsylvanicum and Pm. croatiense were isolated and identified. Phaeoacremonium minimum was the most common species and isolated from Gleditsia caspica, Parrotia persica, Cydonia oblonga and Alnus glutinosa. Pathogenicity on selected plants using a mycelium plug inoculation method revealed that most of these species are pathogenic to all the tested trees, with Pm. parasiticum and Pm. minimum being the most pathogenic species. According to host plant species and geographical distribution, the majority of Phaeoacremonium species found represent new records. This is also the first report of Pm. croatiense in Iran and outside Croatia.