Mechanisms and metabolomics of the host–pathogen interactions between Chestnut (Castanea species) and Chestnut blight (Cryphonectria parasitica)

Chestnut blight is a stem‐girdling disease of Castanea caused by the fungal pathogen Cryphonectria parasitica. Chestnut blight affects all Castanea species to some degree. In Asian species, chestnut blight is a commercially relevant disease which primarily affects nut production. In American and European species, chestnut blight has caused significant declines in wild populations and continues to negatively affect nut production in the European chestnut (C. sativa). Despite the profound effect of this disease in the Castanea genus, very little is known concerning the factors involved in the host–pathogen interaction between C. parasitica and its Castanea hosts. This review summarizes information on known mechanisms and metabolites involved in the host–pathogen interaction and contributes original information on the pathogen in relation to susceptible and putatively resistant genotypes with a view to furthering research that will promote a better understanding of this devastating disease and enable its control.     

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.     

Variations in Disease Resistance of Chestnut Cultivars against Chestnut Blight Fungus,Cryphonectria parasitica

INTRODUCTION Chestnut trees cultivated worldwide are planted as fruit tree in Korea and the area is increasing annually. In the 1970s, chestnut trees were enormously planted in many provinces in Korea. However,chestnut blight occurred and increased due to lack of disease management and the aging of the trees. Blight caused by Cryphonectria parasitica is a kind of important disease and occurred in oak and maple. American chestnut is more susceptible to this pathogen than Korean or Japane…     

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.     

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.     

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.     

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.     

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.     

Rindenkrebs der Eßkastanie (Cryphonectria parasitica) in Südwestdeutschland

Chestnut blight (Cryphonectria parasitica) in South-west Germany . In 1992 and 1993, chestnut blight (Cryphonectria parasitica) was found in three stands in the south-west of Germany. Each of the three stands has only one compatibility group (vc-group). Two of them (97 and 75 Isolates) belong to the vc-group I. They are compatible with the Swiss vc-group I. The 43 Isolates from the third stand were not compatible with the most common Swiss vc-groups (vc-group I-V). In the German vc-group I, one hypovirulent strain was found.     

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.     

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.     

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。     

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.     

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.     

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.     

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     

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.     

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.