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.     

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.     

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

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

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

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

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.     

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.     

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.     

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.     

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.     

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.     

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     

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.     

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。     

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.     

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.     

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.