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.     

Comparison of vegetative compatibility types in Italian and Swiss subpopulations of Cryphonectria parasitica

Tester isolates of 20 vegetative compatibility (vc) types from 11 Italian subpopulations and tester isolates of 26 vc types from five Swiss subpopulations of Cryphonectria parasitica were compared by two different vc test methods. A total of 31 different vc types was identified; 15 vc types were common to both countries, five Italian vc types were not found in Switzerland and 11 Swiss vc types were not found in Italy. These 31 vc types were labelled with the acronym EU, followed by progressive numbers and may constitute the base for a common European nomenclature. The vc type EU-2 was the most common vc type in both countries and dominated in all Swiss and in seven Italian subpopulations. The vc types EU-1 and EU-5 were found primarily in northern Italy and Switzerland, while EU-10 and EU-12 were most common in southern Italy. The diversity of vc types was greater in the subpopulations of Switzerland and northern Italy than in the subpopulations of southern Italy. The geographical distribution of vc types in Europe should therefore be taken into consideration for biological control measures of chestnut blight and quarantine regulations.     

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.     

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.     

Diversity of vegetative compatibility types,distribution of mating types and occurrence of hypovirulence of Cryphonectria parasitica in chestnut stands in NW Spain

An extensive survey on chestnut stands in NW Spain was carried out to study the population biology of Cryphonectria parasitica in order to design future biological control programmes of chestnut blight based on the field introduction of native hypovirulent strains. Five hundred and thirty-nine Cryphonectria parasitica isolates, belonging to five populations (A Coruña, Lugo, Ourense, Pontevedra and León), were obtained. The diversity of vegetative compatibility was very low. Six vc types were found, each population showing only from 2 to 4 vc types. Two vc types were the known European testers EU1 and EU66, and the other four were undescribed vc types that were named E1, E2, E3 and E4. E1 was the dominant vc type in Lugo and Ourense populations, and EU1 in Pontevedra and León. Hypovirulent isolates (a total of 15) were only detected in the León population, all but one being compatible with the vc type EU1. Hypovirulent strains belonged to the French subtype F1 of the Cryphonectria hypovirus-1 (CHV1).     

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.     

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.     

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.     

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.     

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.     

栗疫病菌弱毒性菌株的筛选及其dsRNA的转化稳定性

为获取栗疫病生物防治的基础信息,本研究根据菌株在PDA培养基上培养7天后的菌丝生长速度、分生孢子形成能力等培养特性,从韩国国立山林科学院树木病理研究室保藏的60个栗疫病菌菌株中筛选了2个弱致病力菌株,进行dsRNA检测、弱致病力菌株和强致病力菌株间的细胞融合试验。结果表明:2个弱致病力菌株(KCP-135和KCP-136)中均检测到了dsRNA,弱毒性菌株KCP-22和其他19个强毒性菌株之间的菌落形成明显的隔离带并沿着隔离带产生分生孢子,没有明显的细胞融合现象,而弱毒性菌株KCP-22和强毒性菌株KCP-9之间的菌落则呈现了显著的细胞融合现象,而且其细胞融合菌株的培养特性和转化dsRNA数量均发生了变异。     

栗疫菌弱毒力特征的传递及其影响因素

栗疫菌 (Cryphonectriaparasitica)弱毒力 (hypovirulence)的本质是菌体中含有一种弱毒性病毒 (cryphonectriahypovirulenceviruses ,CHV)dsRNA(Choietal,1992 ;Morrisetal,1979)。栗疫菌的弱毒力菌株 (H菌株 )一般都具有将强毒力 (virulent)菌株 (V菌株 )转化为弱毒力菌株的能     

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.     

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.     

细菌嗜铁素、DAPG和PCA对板栗疫病菌的抑制作用

为了探讨生防细菌防治板栗疫病的潜力,测定了恶臭假单胞杆菌(Pseudomonas putida)WCS358r菌株及其嗜铁素缺失突变体JM218,转基因菌株WCS358::phl和WCS358::phz对板栗疫病菌生长和分生孢子萌发的抑制作用。在KB和板栗枝条韧皮部抽提液培养基(BA)中,WCS358r对病菌生长和分生孢子萌发具有较强的抑制作用,而JM218几乎没有抑制效果;在培养基中加入200μmol/L FeCl3后,WCS358r对病菌的抑制能力显著降低,证明WCS358r产生的嗜铁素是抑制菌丝生长和分生孢子萌发的重要因子。在PDA培养基中,WCS358r对病菌无抑制效果。在上述三种培养基中,产生抗生素2,4-二乙酰间苯三酚(DAPG)的转基因菌株WCS358::phl与原始菌株WCS358r相比,抑菌效果明显增强,而产生酚嗪-1-羧酸(PCA)的WCS358::phz的抑菌能力无明显提高。培养基中添加铁对WCS358::phl的抑菌活性无影响,但能降低WCS358::phz对板栗疫病菌的抑制效果。由此说明,嗜铁素和DAPG对抑制板栗疫病菌生长和孢子萌发具有重要作用,而PCA的抑菌效果不明显。     

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.     

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.     

几种杀菌剂树干注药对板栗疫病的防治效果

通过室内抑菌及田间树干注药药效试验,探讨戊唑醇、烯唑醇、己唑醇、甲霜灵等几种杀菌剂对板栗疫病的防治效果.结果表明,室内戊唑醇、烯唑醇、己唑醇、甲霜灵等几种药剂对板栗疫病病原菌菌丝生长均有一定的抑制作用,其EC50在(0.24～65.64) mg/L之间,其中戊唑醇的抑制作用最强,其EC50为0.24 mg/L,其次为烯唑醇和己唑醇,EC50分别为0.28 mg/L和0.29 mg/L.戊唑醇对板栗疫病病原菌孢子毒力最高,EC50为0.67 mg/L.田间树干注药,10％戊唑醇注干杀菌剂对板栗疫病的防治效果最佳,按树干地径1 mL/cm的注药量防效83.85％,可有效控制板栗疫病的危害,其次为10％烯唑醇和10％己唑醇,防效依次为73.19％和71.03％.     

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.