A Satellite RNA of Ophiostoma novo-ulmi Mitovirus 3a in Hypovirulent Isolates of Sclerotinia homoeocarpa

ABSTRACT Two genetically distinct double-stranded RNA (dsRNA) elements were identified in hypovirulent isolates of Sclerotinia homoeocarpa, the causal agent of dollar spot of turfgrass. The large dsRNA (L-dsRNA) was consistently present in all hypovirulent isolates, whereas the small dsRNA (S-dsRNA) was found only in some hypovirulent isolates. Virulence comparisons revealed that there was no significant difference between isolates containing one or both dsRNAs. Therefore, the L-dsRNA appears to be the genetic determinant of hypovirulence, while the S-dsRNA is not essential for hypovirulence in S. homoeocarpa. The L-dsRNA in hypovirulent isolate Sh12B of S. homoeocarpa was previously characterized as a fungal mitochondrial virus and designated Ophiostoma novo-ulmi mitovirus 3a-Sh12B (OnuMV3a-Sh12B) because it was conspecific with O. novo-ulmi mitovirus 3a-Ld from O. novo-ulmi, the causal agent of Dutch elm disease. In the present study, the nucleotide sequences of the S-dsRNAs (738 to 767 nucleotides) in hypovirulent isolates Sh12B, Sh279B, and Sh286B were determined. Nucleotide sequence analysis indicated that the S-dsRNA was not derived from the OnuMV3a dsRNA and it could not encode an RNA-dependent RNA polymerase. These results are consistent with biological data that the S-dsRNA was always associated with the L-dsRNA and was never found independently. Therefore, the S-dsRNA can be regarded as a satellite RNA of OnuMV3a in S. homoeocarpa. Northern blotting analysis indicated that nucleic acid extracts from isolate Sh12B of S. homoeocarpa contained more single (+) stranded RNA than dsRNA for this satellite RNA. The 5'- and 3'-terminal sequences of the positive strand of the S-dsRNA each could be folded into a stem-loop structure and the terminal 21 nucleotides were complementary to each other, potentially forming a panhandle structure.     

Hypovirulence detected in Brazilian isolates of Cryphonectria cubensis

A single 3 kb segment of double-stranded (dsRNA) was present in three of 30 Brazilian isolates of Cryphonectria cubensis . These dsRNA-containing isolates showed morphological characteristics suggestive of hypovirulence and were significantly less virulent than dsRNA-free isolates. One isolate, however, with morphological characteristics suggestive of hypovirulence, showed reduced virulence, but was free from dsRNA. Conversion of virulent isolates with normal morphology to a morphology associated with hypovirulence was achieved by pairing hypovirulent and virulent isolates of the same vegetative compatibility group (VCG). This suggests that dsRNA can be transmitted to isolates of the same vegetative compatibility group by hyphal anastomosis. Converted isolates exhibited the same hypovirulence-associated traits as those of the original dsRNA-containing hypovirulent isolates. These studies suggest that a single 3 kb segment of dsRNA alters both morphology and virulence by conferring hypovirulence on the pathogen; the first such report for Brazilian isolates of C. cubensis .     

Elevated mitochondrial alternative oxidase activity in dsRNA-free, hypovirulent isolates of Cryphonectria parasitica

In Cryphonectria parasitica, the causal agent of chestnut blight, hypovirulence is commonly associated with the presence of cytoplasmically transmissible, double-stranded RNA (dsRNA). Recently, hypovirulent isolates of C. parasitica that do not have detectable levels of dsRNA have been obtained from healing cankers on chestnut trees. In these isolates, most of the respiratory activity is cyanide-resistant and salicylhydroxamate-sensitive, indicating that the mitochondrial alternative oxidase was induced. In contrast, the respiration of virulent isolates from the same trees and most of the dsRNA-containing hypovirulent isolates from a variety of geographical locations was cyanide sensitive. In all of these isolates, the alternative oxidase could be induced by growing them in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis. The attenuated virulence and altered respiratory phenotypes could be transferred from the dsRNA-free hypovirulent isolates to virulent strains through hyphal anastomosis. Some of the dsRNA-free hypovirulent isolates grew more slowly than virulent strains. These results indicate that, in Cryphonectria, cytoplasmically transmitted hypovirulence may have more than one cause, and that mitochondrial mutations sometimes may be involved in the generation of this phenotype.     

Hypovirulence and Double-Stranded RNA in Botrytis cinerea

ABSTRACT Twenty-one strains of Botrytis cinerea isolated from 13 species of plants grown in China were compared for pathogenicity on Brassica napus, mycelial growth on potato dextrose agar, and presence of double-stranded (ds)RNA. The results showed that the strain CanBc-1 was severely debilitated in pathogenicity and mycelial growth, compared with the 20 virulent strains. A dsRNA of approximately 3.0 kb in length was detected in CanBc-1 and 4 hypovirulent single-conidium (SC) isolates of CanBc-1, but was not detected in the 20 virulent strains of B. cinerea and 4 virulent SC isolates of CanBc-1. Results of the horizontal transmission experiment showed that the hypovirulent trait of CanBc-1 was transmissible and the 3.0-kb dsRNA was involved in the transmission of hypovirulence. Analysis of a 920-bp cDNA sequence generated from the 3.0-kb dsRNA of CanBc-1 indicated that the dsRNA element was a mycovirus, designated as B. cinerea debilitation-related virus (BcDRV). Further analyses showed that BcDRV is closely related to Ophiostoma mitovirus 3b infecting O. novo-ulmi, the causal agent of Dutch elm disease. Mitochondria and cytoplasm in hyphal cells of CanBc-1 became degenerated, compared with the virulent isolate CanBc-1c-66 of B cinerea. This is the first report on the occurrence of Mitovirus-associated hypovirulence in B. cinerea.     

Isolation and identification of hypovirulent Rhizoctonia spp. from soil

Two-hundred and forty-eight isolates of Rhizoctonia spp, were obtained from 13 locations in Gifu Prefecture in Japan using the plant debris particles isolation, colonization of bait tissue, and soil-clump plating methods. Of the isolates, 143 were binucleate Rhizoctonia spp., 60 were R. solani and 45 were R. zeae. Three isolates of R. solani and 54 of binucleate Rhizoctonia spp, were hypovirulent on radish, whilst all isolates of R. zeae were highly virulent, Hypovirulent strains were isolated most frequently by the plant debris particles isolation method, Hypovirulent isolates of R. solani belonged to anastomosis group 4, whilst the hypovirulent binucleate Rhizoctonia isolates belonged to AG A, AG Ba, AG G, and AG O.
Thirty-two isolates of Rhizoctotria spp, selected for hypovirulence on radish were tested on cucumber in vitro. Only five binucleate Rhizoctonia isolates and one R. solani isolate were hypovirulent on both species, and these isolates were also hypovirulent on seven other crop species. Cucumber showed wide variation in disease susceptibility to different isolates but hypovirulent isolates exhibited a consistent reaction on five different host cultivars, Pathogenicity tests using cucumber grown in soil also showed consistent reactions with isolates selected either for hypovirulence or virulence. The results support the use of cucumber in bioassays for identifying hypovirulent isolates of binucleate Rhizoctonia spp.     

Characterization ofCryphonectria parasitica isolates collected from Aydın province in Turkey

The major chestnut-growing areas in Aydın Province, which yield nearly 35% of Turkey’s production, were examined for the presence of chestnut blight and bark samples were collected from the canker formations. Generally virulent type cankers were observed. Of 31 villages, 22 were found to be contaminated with the disease. A total of 97Cryphonectria parasitica (Murrill) M.E. Barr. isolates were characterized for the presence of potential hypovirulence by looking at cultural characteristics on media and virulence in cv. ‘Golden Delicious’ apple fruit. The isolates were placed into four groups based on pigmentation and abundance of pycnidia formation. Isolates produced lesions of various sizes on apple fruits but lesion size was not correlated with cultural characteristics typical to hypovirulent isolates. Among 97 isolates, ten possessing the characteristics associated with hypovirulence were assayed for the presence of dsRNA. Among these isolates only one, coded M1-3, was found to contain dsRNA. No dsRNA was detected in the single conidium isolates derived from M1-3. All of the isolates were screened for vegetative compatibility and two European vc types, EU-1 and EU-12, were detected. This low vc-type diversity in the region may indicate a high potential for spread of transmissible hypovirulence. http://www.phytoparasitica.org posting May 30, 2008.     

苹果褪绿叶斑病毒生物学及生化特性研究

 对从苹果和扁桃上获得2个苹果褪绿叶斑病毒的分离物ACLSV-C和ACLSV-B的主要生物学和生化特性进行了比较。人工接种5科19种草本植物,发现两者均能侵染苋色藜(Chenopodium amaranticolor)、昆诺藜(Ch.quinoa)和西方烟(Nicotiana occidentalis),产生局部侵染斑和系统褪绿斑。但症状反应存在差异,后者在这3种植物上引起叶片反卷等较强症状反应,还可潜伏侵染笋瓜(Cucurbita maxima cv.Buttercup Burgess)。经SDS-聚丙烯酰胺凝胶电泳结果显示,ACLSV-B衣壳蛋白的迁移率较ACLSV-C快。两者的RNA分子量及双链RNA数量无明显差异。根据已报道的核苷酸系列设计合成引物,采用PCR法检测ACLSV分离物,均获得特异性扩增产物。     

Inoculation of Lupinus luteus with White Root Rot Fungus, Rosellinia necatrix, to Estimate Virulence

An inoculation method using Lupinus luteus was developed for estimating virulence of isolates of the white root rot fungus, Rosellinia necatrix Prillieux. Fungal cultures grown on pieces of mulberry twigs were placed in contact with the hypocotyl of 3-week-old seedlings growing in pots of soil. Disease development was uniform and reproducible in repeated experiments. Of 24 isolates with double-stranded RNA, eight were weakly virulent. This method is useful throughout the year for estimating virulence of many isolates of the fungus and for screening for hypovirulent isolates. Received 2 March 2001/ Accepted in revised form 25 May 2001     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001     

Relationship Between Biological Control, Incidence of Hypovirulence, and Diversity of Vegetative Compatibility Types of Cryphonectria parasitica in France

ABSTRACT In France, chestnut blight, caused by Cryphonectria parasitica, has been controlled since 1974 in orchards, but never in coppice forests, by releasing hypovirulent strains infected with CHV1 hypovirus. We tested the hypothesis that this biological control (BC) has lead to a decrease in blight severity, spread of hypovirulence, and change in C. parasitica populations. The low severity of chestnut blight was confirmed in the six regions studied (subdivided into zones). The remission of cankers was associated with the presence of white isolates presumed to be hypovirulent. These two parameters were also correlated, at the zonal level, to the frequency of sites where BC was used. However, the estimates of the natural background level of hypovirulence, independent of BC, ranged from 4% in forests in Dordogne to 60% in orchards in Lozère. Differences in the rate of hypovirulent isolates among regions were consistent with the diversity of vegetative compatibility (VC) types in populations of C. parasitica. The highest VC-type diversity and mean allelic diversity for known vegetative incompatibility (vic) genes were observed in Dordogne. We showed that the current diversity of VC types in populations of C. parasitica was lower than in 1981. We found 30 VC types among 1,113 isolates of C. parasitica. Ten VC types were incompatible with known EU testers, suggesting that one additional vic gene or allele at one of the six vic loci known should be present in Europe.     

Interspecific Transmission of Double-Stranded RNA and Hypovirulence from Sclerotinia sclerotiorum to S. minor

ABSTRACT Interspecific transmission of a hypovirulence-associated double-stranded RNA (dsRNA) and hypovirulent phenotype was attempted from hypovirulent isolate Ss275 of Sclerotinia sclerotiorum to five virulent isolates of S. minor. dsRNA and the hypovirulent phenotype were successfully transmitted to one of the five isolates, Sm10. Three putative converted isolates of Sm10 were slow growing and developed atypical colony morphologies characteristic of the hypovirulent phenotype. These isolates were assayed for virulence and produced significantly smaller lesions than isolate Sm10 on detached leaves of Romaine lettuce. One of these putative converted isolates, designated Sm10T, was tested to confirm interspecific transmission of dsRNA. In northern hybridizations, dsRNA isolated from Sm10T hybridized with a digoxigenin-labeled cDNA probe prepared from dsRNA isolated from Ss275. Random amplified polymorphic DNA analysis confirmed that isolate Sm10T was derived from Sm10 and not from Ss275 or a hybrid of the two species. The dsRNA and hypovirulent phenotype were subsequently transmitted intraspecifically from Sm10T to Sm8. To our knowledge, this is the first report of interspecific transmission of dsRNA and an associated hypovirulent phenotype between fungal plant pathogens by hyphal anastomosis.     

Hypovirulence and Double-Stranded RNA in Sclerotinia homoeocarpa

ABSTRACT One hundred and thirty-two isolates of Sclerotinia homoeocarpa, the causal agent of dollar spot of turfgrass, were evaluated for virulence on swards and detached leaves of creeping bentgrass and for the presence of double-stranded RNA (dsRNA). In at least four isolates, the hypovirulent phenotype was associated with the presence of specific segments of dsRNA. In addition, these hypovirulent isolates often grew slowly on potato dextrose agar (PDA), formed thin colonies with atypical colony margins, and failed to produce typical black stroma. The hypovirulent phenotype and dsRNA were transmitted from hypovirulent isolate Sh12B to virulent isolate Sh48B, and the converted isolate was hypovirulent and contained dsRNA. The hypovirulent phenotype and dsRNA also were transmitted to at least four other isolates of the pathogen, including the fungicide-resistant, dsRNA isolate KY-7. Converted isolates of KY-7 developed the hypovirulent phenotype, grew on fungicide-amended medium, and contained dsRNA. Subcultures of hypovirulent isolate Sh12B that did not contain dsRNA were obtained through curative treatment using cycloheximide-containing medium and heat. Cured subcultures grew faster on PDA, had more typical colony morphologies, were more virulent on bentgrass leaves, and did not contain dsRNA. No cured subcultures were obtained from hypovirulent isolate Sh09B. Isolates regenerated from protoplasts of hypovirulent isolate Sh12B were not cured, remained hypovirulent, and contained dsRNA. Transmission of hypovirulence and dsRNA in S. homoeocarpa has potential as a novel approach to the management of dollar spot of turfgrass.     

Hypovirulence-Associated Double-Stranded RNA from Sclerotinia homoeocarpa Is Conspecific with Ophiostoma novo-ulmi Mitovirus 3a-Ld

ABSTRACT The nucleotide sequence of the hypovirulence-associated double-stranded RNA (dsRNA) in hypovirulent isolate Sh12B of Sclerotinia homoeocarpa, the causal agent of dollar spot of turf grass, was determined. This large dsRNA (L-dsRNA) is 2,632 bp long and is A and U rich (61.0% A+U residues). One strand of this dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 720 amino acids. This ORF contains 12 UGA codons, predicted to encode tryptophan in ascomycete mitochondria, and has a codon bias typical of mitochondrial genes, which is consistent with a mitochondrial localization of this dsRNA. The amino acid sequence contains conserved motifs typical of RNA-dependent RNA polymerases (RdRps). Sequence analyses of the nucleotide and RdRp-like protein revealed that the L-dsRNA is homologous with previously characterized mitochondrial viruses and dsRNAs from other phytopathogenic fungi, and shares 92.4% nucleotide and 95.1% amino acid sequence identities with the Ophiostoma novo-ulmi mitovirus 3a-Ld from Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. The results indicate that these two dsRNAs are conspecific. This is the first report that a hypovirulence-associated dsRNA virus naturally occurs in two taxonomically distinct fungi, and indicates that horizontal transmission of this dsRNA virus may have occurred between these fungi.     

Effect of Diaporthe RNA virus 1 (DRV1) on growth and pathogenicity of different <Emphasis Type="Italic">Diaporthe</Emphasis> species

A 4.1 kbp positive-strand RNA virus known as Diaporthe RNA virus 1 (DRV1) occurs in hypovirulent, non-sporulating isolates of the fungal pathogen Diaporthe perjuncta. A full-length cDNA clone of DRV1 was developed and RNA transcribed from the cDNA clone used to transfect different Diaporthe spp. The transfected species included three D. ambigua isolates and an unidentified Phomopsis asexual state of a Diaporthe sp. Successful transfections were confirmed using RT-PCR. Although the in vitro-transcribed positive sense single-stranded RNA used for transfection included vector sequences at both ends, the genomes of progeny virus from DRV1-transfected isolates were free of the vector sequences. Transfection resulted in morphological changes in these fungal pathogens. However, the presence of DRV1 did not reduce growth rate in two of the three D. ambigua or the Phomopsis sp. significantly. Pathogenicity studies showed that the transfected isolates have reduced aggresiveness.     

Characterization of hypovirulent isolates of the chestnut blight fungus, Cryphonectria parasitica from the Marmara and Black Sea regions of Turkey

Chestnut blight caused by the introduced fungus Cryphonectria parasitica has been responsible for the decline of Castanea sativa in Turkey since the 1960s. In this study, 72 C. parasitica isolates were recovered from the Marmara and Black Sea regions of Turkey showing white or cream-coloured culture morphology and were subjected to various tests to determine if they were infected by Cryphonectria hypovirus 1 (CHV-1). The vast majority of the isolates (69 out of 72) were vc type EU-1. Both mating types were found among a subsample of the isolates. The hypovirus was detected in 55 isolates by dsRNA extraction and/or virus specific RT-PCR on total RNA extracts. All but one isolates showed no or only weak phenol oxidase activity on agar medium containing tannic acid, typical of CHV-1 infected isolates. Through sequencing of a specific region of the hypovirus genome, we found that 24 hypovirus isolates belonged to the CHV-1 subtype I and six to the CHV-1 subtype F2. The distribution of the two CHV-1 subtypes in Turkey showed a clear geographic pattern. CHV-1 subtype I was only detected in the Marmara and western Black Sea region, whereas subtype F2 was restricted to the eastern part of the Black Sea region. The effectiveness of 23 hypovirulent isolates was tested against a virulent isolate on 2–3 years old chestnut sprouts. Ten hypovirulent isolates, all infected by CHV-1 subtype I, prevented canker development by more than 80 % suggesting that they might be suitable for biological control of chestnut blight in Turkey.     

Behavior of Ralstonia solanacearum Race 3 Biovar 2 During Latent and Active Infection of Geranium

ABSTRACT Southern wilt of geraniums (Pelargonium hortorum), caused by the soilborne bacterium Ralstonia solanacearum race 3 biovar 2 (R3bv2), has inflicted significant economic losses when geranium cuttings latently infected with this quarantine pest were imported into the United States. Little is known about the interaction between R. solanacearum and this ornamental host. Using UW551, a virulent R3bv2 geranium isolate from a Kenyan geranium, we characterized development of Southern wilt disease and R3bv2 latent infection on geranium plants. Following soil inoculation, between 12 and 26% of plants became latently infected, carrying average bacterial populations of 4.8 x 10(8) CFU/g of crown tissue in the absence of visible symptoms. Such latently infected plants shed an average of 1.3 x 105 CFU/ml in soil run-off water, suggesting a non-destructive means of testing pools of asymptomatic plants. Similarly, symptomatic plants shed 2 x 10(6) CFU/ml of run-off water. A few hundred R. solanacearum cells introduced directly into geranium stems resulted in death of almost all inoculated plants. However, no disease transmission was detected after contact between wounded leaves. Increasing temperatures to 28 degrees C for 2 weeks did not convert all latently infected plants to active disease, although disease development was temperature dependent. Holding plants at 4 degrees C for 48 h, a routine practice during geranium cutting shipment, did not increase frequency of latent infections. R. solanacearum cells were distributed unevenly in the stems and leaves of both symptomatic and latently infected plants, meaning that random leaf sampling is an unreliable testing method. UW551 also caused potato brown rot and bacterial wilt of tomato, surpassing race 1 strain K60 in virulence on tomato at the relatively cool temperature of 24 degrees C.     

Molecular Identification of Oat Mosaic Virus as a Bymovirus

A partial sequence of Oat mosaic virus (OMV) has been obtained for four isolates of the virus from four European countries. This represents the first available sequence data for this important disease of winter-sown oats. The longest clone of 1699 nucleotides was obtained from infected English oats using a degenerate primer, designed to members of the Potyviridae family. Alignment of the predicted amino acid sequence with members of the Potyviridae showed closest identity with viruses of the Bymovirus genus. The predicted amino acid sequence has one open reading frame corresponding to part of the NIb and capsid protein, with a 3 untranslated region of 351 nucleotides, followed by a poly(A) tail. PCR primers were designed to the coat protein and NIb gene of members of the Bymovirus genus and used to obtain partial sequences of 1441 nucleotides at the 3 end of infected oats from both Wales and France. A specific primer set designed to the English isolate was used to generate a product of 701 nucleotides from OMV-infected oat leaves from Ireland. All four isolates are highly conserved at the amino acid level.The first two authors contributed equally to the work     

核盘菌中dsRNA种类及其与致病力的关系

 对源于我国黑龙江省佳木斯市同一块茄子田的14个核盘菌(Sclerotinia sclerotiorum)菌株及其中1个菌株Ep-1PNA5的2个衍生菌株的致病力和菌丝中的双链RNA(dsRNA)进行了分析。在马铃薯琼脂培养基(PDA)上对5个弱致病型核盘菌菌株(Ep-1PD、Ep-1PI、Ep-1PL、Ep-1PM、Ep-1PN)异常性状(菌丝生长慢,且异常分枝)的传染特性进行了测定。结果表明:在离体油菜叶片上,16个供试核盘菌菌株中,7个属于强致病类型,7个属于弱致病类型,2个属于中等致病类型。从10个核盘菌菌株的菌丝中检测到dsRNA因子,并可分成3类dsRNA电泳谱型。第一类dsRNA谱型只含有7.4kb大小的dsRNA分子,3个强致病型核盘菌菌株属于这种谱型;第二类dsRNA谱型含有2种dsRNA分子,大小分别为6.4kb和7.4kb。6个弱致病型菌株属于这种谱型;第三类dsRNA谱型只含有6.4kb大小的dsRNA分子。1个弱致病类型菌株属于这种谱型。从另外6个核盘菌菌株的菌丝中没有检测到任何dsRNA因子,其中4个菌株属于强致病型菌株,2个菌株属于中等致病型。可见,6.4kb大小的dsRNA因子与核盘菌弱致病性状密切相关。5个弱致病型核盘菌菌株(Ep-1PD、Ep-1PI、Ep-1PL、Ep-1PM、Ep-1PN)的异常性状可以通过菌丝接触传染给一些强致病型核盘菌菌株,使其菌丝生长变慢及分枝异常。结果还表明:这些弱致病型核盘菌异常性状的传染具有菌株特异性。