Search

Characterization of the Cryphonectria parasitica population was initially done by a phenotypical assessment of 40 in vitro grown isolates obtained from 52 healing cankers collected from eight important chestnut-growing provinces of Turkey. The results of Bavendamm test, often correlated indirectly to hypovirulence, suggested 31 possibly hypovirulent and 9 virulent isolates. PCR tests amplified two regions of ORFs A and B of Cryphonectria hypovirus 1 (CHV-1) from 36 of 40 isolates. The PCR test confirmation was more sensitive than the Bavendamm test. Partial ORFA sequencing revealed 36 CHV-1 haplotypes belonging to Italian subtype (I), with all hypovirulent isolates being of EU-1 vc type. The CHV-1 from 10 native EU-1 isolates were first transferred to six European vc type testers, EU-2, EU-3, EU-5, EU-7, EU-26, and EU-44, having heteroallelism at one vic locus. The presence of the vic locus difference generally reduces virus transmission. The easiest and highest frequency virus transfer was obtained by vic4 and vic6 allelic differences, while the differences vic2 and vic7 made the transfer more challenging. Finally, in this study we successfully transferred CHV-1 to an EU-1 isolate obtained from the Bursa province to an EU-12 European tester isolate. [ABSTRACT FROM AUTHOR]

A wide diversity of mycoviruses has been reported from Botrytis species, some with the potential to suppress the pathogenic abilities of this fungus. Considering their importance, this study was devised to find potential hypovirulence-associated mycoviruses found in Botrytis cinerea strains isolated from Pakistani strawberry fields. Here we report the complete genome characterization of two fusariviruses co-infecting a single isolate of phytopathogenic fungus B. cinerea (Kst14a). The viral genomes were sequenced by deep sequencing using total RNA fractions of the Kst14a isolate. The identified viruses were tentatively named Botrytis cinerea fusarivirus 9 (BcFV9) and Botrytis cinerea fusarivirus 3a (BcFV3a). Both viruses had a single-segmented (ssRNA) genome having a size of 6424 and 8370 nucleotides encoding two discontinuous open reading frames (ORFs). ORF-1 of both mycoviruses encodes for a polyprotein having a conserved domain of RNA-dependent RNA polymerase (RdRP) and a helicase domain (Hel) which function in RNA replication, while ORF2 encodes a hypothetical protein with an unknown function, respectively. Phylogenetic analysis indicated that BcFV9 made a clade with the genus Alphafusarivirus and BcFV3a fall in the genus Betafusarivirus in the family Fusariviridae. To our knowledge, this is the first report of two fusariviruses identified in isolates of B. cinerea from Pakistan. Both mycoviruses successfully transfected to a compatible strain of B. cinerea (Mst11). A comparison of virus-free (VF) and virus-infected (VI) isogenic lines showed the presence of these viruses was causing hypovirulence in infected strains. Virus-infected strains also had a small lesion size while testing the pathogenicity via apple assay. [ABSTRACT FROM AUTHOR]

Plant pathogenic fungi pose a significant and ongoing threat to agriculture and food security, causing economic losses and significantly reducing crop yields. Effectively managing these fungal diseases is crucial for sustaining agricultural productivity, and in this context, mycoviruses have emerged as a promising biocontrol option. These viruses alter the physiology of their fungal hosts and their interactions with the host plants. This review encompasses the extensive diversity of reported mycoviruses, including their taxonomic classification and range of fungal hosts. We highlight representative examples of mycoviruses that affect economically significant plant-pathogenic fungi and their distinctive characteristics, with a particular emphasis on mycoviruses impacting Sclerotinia sclerotiorum. These mycoviruses exhibit significant potential for biocontrol, supported by their specificity, efficacy, and environmental safety. This positions mycoviruses as valuable tools in crop protection against diseases caused by this pathogen, maintaining their study and application as promising research areas in agricultural biotechnology. The remarkable diversity of mycoviruses, coupled with their ability to infect a broad range of plant-pathogenic fungi, inspires optimism, and suggests that these viruses have the potential to serve as an effective management strategy against major fungi-causing plant diseases worldwide. [ABSTRACT FROM AUTHOR]

Cryphonectria hypovirus 1 (CHV1), as a biological agent, has a great potential for controlling of chestnut blight. CHV1 infects Cryphonectria parasitica, the causal agent of chestnut blight, via horizontal transmission, subsequently weakening the pathogen's virulence. C. parasitica has a vegetative incompatibility system that restricts the transmission of CHV1 between different vc types, but not completely. This study aimed to determine the horizontal transmission of CHV1 between the vc types related to the C. parasitica population in Aydın Province. The two CHV1 infected isolates, Zgl709, and Fr22, which belong to EU-1 and EU-12 vc types, respectively, were used as the virus donor isolates in the horizontal transmission test. Thirty offspring single ascospore isolates, representing 16 vc types, which were obtained from the sexual cross between a local EU-1 and a local EU-12 isolate, served as the recipients. CHV1 was transmitted 100% if the donor and the recipient were from the same vc type. The average transmission rate between the donor EU-1 (Zgl709) and the recipients from different vc types was 13.6%. Fourteen recipient isolates from 8 vc types (EU-3, EU-4, EU-7, EU-8, EU-25, EU-26, EU-28, and EU-30) were successfully converted to hypovirulent by the donor EU-1. The average transmission rate between the donor EU-12 (Fr22) and the recipients from different vc types was 7.5%. Only 5 recipient isolates from 4 vc types (EU-4, EU8, EU-29, and EU-30) were converted to hypovirulent by the donor EU-12. There was no transmission of CHV1 between EU-1 and EU-12, which are the two dominant vc types present in Aydın Province. [ABSTRACT FROM AUTHOR]

Botrytis cinerea is a phytopathogenic fungus that belongs to the family Sclerotiniaceae in the Ascomycota group. This pathogen infects economically important crops and is responsible for substantial preharvest and postharvest losses each year worldwide. Mycoviruses are commonly associated with Botrytis cinerea isolates. In this work, we report the hypovirulence potential of an endornavirus harboured by the Kst31c isolate of Botrytis cinerea collected from a strawberry field in Pakistan. Our analysis revealed the presence of a positive-sense single-stranded RNA (+ ssRNA) mono-segmented virus in this isolate. Next-generation sequencing of total RNA harvested from Kst31c revealed the presence of an endornavirus with a genome length of 13,765 nt comprising a single open reading frame (ORF) encoding a polyprotein of 4,546 amino acids. The identified polyprotein includes four conserved domains: a viral methyltransferase, an RNA-dependent RNA polymerase 2, and the two putative viral helicases (DExDc-like and Hel-1). No cysteine-rich regions were identified in this virus. Phylogenetic analysis placed it in a clade within the genus Betaendornavirus. The current virus was tentatively named Botrytis cinerea endornavirus 4 (BcEV4). A comparison of virus-free and virus-infected isogenic lines showed that BcEV4 was associated with hypovirulence, slow growth, and altered phenotype. Furthermore, these results were also confirmed by pathogenicity testing via an apple assay. The virus can be tested as a potential biocontrol agent against Botrytis cinerea infections. [ABSTRACT FROM AUTHOR]

Laccase3 is an important virulence factor of the fungus Cryphonectria parasitica. Laccase3 gene (lac3) transcription is induced by tannic acid, a group of phenolic compounds found in chestnut trees, and its induction is regulated by the hypovirus CHV1 infection. CpHsp24, a small heat shock protein gene of C. parasitica, plays a determinative role in stress adaptation and pathogen virulence. Having uncovered in our previous study that transcriptional regulation of the CpHsp24 gene in response to tannic acid supplementation and CHV1 infection was similar to that of the lac3, and that conserved phenotypic changes of reduced virulence were observed in mutants of both genes, we inferred that both genes were implicated in a common pathway. Building on this finding, in this paper we examined whether the CpHsp24 protein (CpHSP24) was a molecular chaperone for the lac3 protein (LAC3). Our pull-down experiment indicated that the protein products of the two genes directly interacted with each other. Heterologous co-expression of CpHsp24 and lac3 genes using Saccharomyces cerevisiae resulted in more laccase activity in the cotransformant than in a parental lac3-expresssing yeast strain. These findings suggest that CpHSP24 is, in fact, a molecular chaperone for the LAC3, which is critical component of fungal pathogenesis. [ABSTRACT FROM AUTHOR]

Botrytis cinerea is a ubiquitous plant pathogenic fungus, causing gray mold disease on many economically important crops, including apple. The present investigation was carried out to characterize a hypovirulent strain AppleBc.07 of B. cinerea collected from an apple orchard located in Yantai City, Shandong Province, China. A comparison with the reference strain of B. cinerea B05.10 suggested that the isolated strain AppleBc.07 was B. cinerea consisting of gray-white, circular, fluffy pattern mycelia and black sclerotia. BLASTn analysis showed that the sequences of internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) were 99–100% identical to the corresponding sequences of other B. cinerea strains, and the phylogenetic analysis based on concatenated G3PDH, HSP60, and RPB2 sequences also indicated that strain AppleBc.07 was clustered with reference strains of B. cinerea. Moreover, it was also found that the strain AppleBc.07 rarely infect apples, tomato, rapeseed, and pepper, whereas the reference strain B05.10 isolates aggressively infect those. Interestingly, no viral dsRNA was speculated in AppleBc.07 strain, postulated that it was a hypovirulent strain, which might be responsible to reduce production of laccase and the limited number of infection cushion (IC) formation. Furthermore, the IC formation genes (Bciqg1, Bcmsb2, and Bcsac1) in AppleBc07 showed alternative expression that could be another possible reason behind this hypovirulence. To the best of our knowledge, this is the first report of a natural occurrence of a hypovirulent B. cinerea strain from apple in China. [ABSTRACT FROM AUTHOR]

Many Mediteranean isolates of Rosellinia necatrix, causing root rot in avocado, have previously been screened for viruses. Among them is isolate Rn459 that has been shown to be infected by at least three viruses such as Rosellinia necatrix partitivirus 10 (RnPV10), Rosellinia necatrix hypovirus 2 (RnHV2), Rosellinia necatrix fusagravirus 1 (RnFGV1), and Rosellinia necatrix virga-like virus (RnVLV). Here, we attempted to eliminate the viruses by hyphal tip cultures to examine their effect on colony growth in vitro and virulence on avocado plants. The obtained fungal strain termed, Rn459_PV10F/VLVF, which was confirmed to be cured of RnPV10 and RnVLV, but still retaining RnHV2, manifested a phenotype different from the original Rn459. Colony growth comparison showed that Rn459_PV10F/VLVF grew faster than the original Rn459 isolate and the virulence on avocado plants of this Rn459_PV10F/VLVF strain was higher than the original Rn459 strain. These combined results suggest that RnPV10 and RnVLV, alone or together, contribute to confer hypovirulence on the R. necatrix isolates. [ABSTRACT FROM AUTHOR]

The invasive ascomycete Hymenoscyphus fraxineus is the causative agent for ash dieback on the European species Fraxinus excelsior and Fraxinus angustifolia, and there is concern that it is going to replace the native, closely related and nonpathogenic Hymenoscyphus albidus. Fungal management in forests is limited, and alternative approaches for control are needed. Within the scope of the project “FraxForFuture”, several strategies are being investigated. One idea comprises the use of a viral hyperparasite, which can induce a reduced virulence in the fungal host H. fraxineus in an antagonist-like system. This phenomenon, the reduction of fungal virulence by a viral infection, is known as hypovirulence, and a similar method has already been established to control the Chestnut Blight in Europe. We examined 34 isolates of H. fraxineus for both their virulence and presence of a viral infection. Although a predominant number of isolates were found to be infected with Hymenoscyphus mitovirus 1 (HfMV1), no additional viruses were detected, and our data did not indicate a link to reduced virulence. The search for a viral infection was extended to one isolate of H. albidus in which we found and characterized a novel mycovirus. Based on phylogenetic analysis and sequence properties, it was assigned to the genus Victorivirus in the family of Totiviridae and was tentatively denominated as Hymenoscyphus albidus victorivirus 1. This novel and native mycovirus might be suitable for inducing hypovirulence in H. fraxineus as a biocide. [ABSTRACT FROM AUTHOR]

Abstract: This paper reports brief systematization of the current knowledge of the biology of mycoviruses, viral morphology, and genetics in particular as well as characteristics of the virus transmission and infection symptoms in fungal cells. The mechanisms involved in antiviral defense in the members of different classes of fungi are discussed. Insights into the role of hypovirulent mycoviruses in the biotechnological control of phytopathogenic fungi are provided. [ABSTRACT FROM AUTHOR]

Biological control with Cryphonectria hypovirus CHV1 of the chestnut blight, caused by the fungus Cryphonectria parasitica, has reduced the impact of the disease in Europe. The virus reduces the virulence of the fungus so that it causes non-lethal cankers, thus enabling the chestnut trees to overcome the disease. The virus can be transmitted horizontally by hyphal anastomosis or vertically to the conidia. In this study, we investigated growth and sporulation of the fungus as well as rates of horizontal transmission of the virus at different temperatures. We used fungal isolates of the vegetative compatibility types (vc types) that are most prevalent in Castilla and León (central northern Spain) to evaluate the effects of fungal strain on the parameters tested. In addition, we infected four isolates of C. parasitica with hypovirus subtypes CHV1-F1 and CHV1-I, to determine the influence of virus subtype on growth, sporulation and virus transfer. We assessed growth of fungal colonies and horizontal transmission of the virus at 15 °C and 25 °C. Colony growth was affected by an interaction between fungal isolates included in vc type EU1 or EU11 and virus at both 15 °C and 25 °C. However, horizontal transmission of the virus was only influenced by the fungal genotype of isolates included in vc type EU1 or EU11, and spore production was only affected by the virus subtype. Vertical transmission was also influenced by the fungal isolate and virus subtype. Growth of the fungal isolates varied depending on the virus subtype with which they were infected. This supports the theory that fungal host and virus subtype influence transmission and dissemination of hypovirulence. The fungal genotype affects colony growth and horizontal transmission of the virus. It is common to expect a good dissemination of the hypovirus with a low vc type diversity but the selection of the best combination of hypovirus and fungal isolate is crucial for the success of biological control not only for small areas but in larger chestnut populations as well. [ABSTRACT FROM AUTHOR]

Hypovirulent strains of the chestnut blight fungus Cryphonectria parasitica have not been registered in the autonomous region of Castilla y León (Spain), except in the province of León. In this laboratory-based study, we analyzed the rates of horizontal transmission of hypovirus CHV1 subtype F1, isolated from chestnut stands in León. We tested the conversion capacity of the six vegetative compatibility (vc) types of C. parasitica isolates most commonly distributed in Castilla y León (EU1, EU11, EU12, EU66, CL5 and CL6). We investigated conversion rates of virulent isolates into hypovirulent isolates between pairings of isolates of the same vc type (EU1, EU11 and CL6) and also tested cross conversion rates between isolates of different vc types (EU1, EU11 and CL6 as donors and EU12, EU66, CL5 and CL6 as recipients). We carried out the hypovirus transmission assay with 1700 pairings, of which 700 had donor and recipient isolates of the same vc type and 1000 pairings of isolates had different vc types. Our results show that the conversion frequency to hypovirulent isolates was significantly affected by the vc type, the genotype of isolates with the same origin (province) and the interaction between both factors. In the conversion between isolates of the same vc type, the conversion rates were better with EU1 (ranging between 56 and 94 %) than with EU11 (varying from 4 to 58 %). In the cross conversion between donor and recipient isolates of different vc types, only CL5 and CL6 recipients were converted and the conversion of recipient isolates of EU12 and EU66 failed in all cases. For CL5 as recipient isolate, the conversion rates were similar with EU11 and CL6 as the donor isolates. Recipient isolates of CL6 had good conversion with donor isolates from the same vc type (CL6) and in cross conversion the results were better with donor EU1. Fungal isolates from chestnut stands in León displayed the best conversion rates, followed by those from Zamora, Salamanca and Ávila when the donor and the recipient isolates were of the same vc type. The fungal isolates appear to have a strong influence on the transmission rate of the hypovirus, at least between strains isolated from chestnut stands in Castilla y León. EU1 isolates were more susceptible to conversion than the isolates from EU11. The results highlight the differences in hypovirus transmission, regarding vc types and the genotype of isolates from the same province, when donor and recipient isolates are of the same vc type. Further transmission assays would be useful to determine why hypovirus transmissions, when compared to the hypovirus transmission of other European assays, have different conversion rates with fungal isolates from Castilla y León. [ABSTRACT FROM AUTHOR]

A collection of clinical and environmental isolates of the opportunistic human pathogen, Aspergillus fumigatus, were screened for the presence of mycoviruses and 6.6 % of 366 isolates contained dsRNA segments ranging in size from ~1.0 to 4.0 kbp. The dsRNAs were categorised into three different groups comprising bipartite dsRNAs, quadripartite dsRNAs, representative isolates of which have both been sequenced, and an uncharacterised mycovirus, whose genome apparently consists of four dsRNAs 1-2.5 kbp in size. Here, we describe dsRNA incidence in the A. fumigatus isolates examined, their provenance and also note that on occasion individual isolates were infected with two groups of different dsRNAs. [ABSTRACT FROM AUTHOR]

In this study, the population structure of the chestnut blight fungus Cryphonectria parasitica in the Aydın Mountains was investigated to make inferences about fungal reproduction and population diversity. A total of 213 C. parasitica isolates from eight subpopulations were used to determine vegetative compatibility (vc) and mating types of the population. Furthermore geostatistical analysis was performed to define the spatial structure of the population. The results showed that the isolates were vegetatively compatible with the European vc types of either EU-1 or EU-12. Both vc types were found in almost all subpopulations, but their frequencies varied depending on location. The results of a PCR assay showed that both mating types of C. parasitica ( MAT-1 and MAT-2) exist in the population. MAT-1 comprised 65% of the total isolates, and the ratio of mating types was significantly skewed from 1:1. Genotyping based on combined vc and mating type data revealed four genotypes: EU-1/MAT-1 (28.6%), EU-1/MAT-2 (34.7%), EU-12/MAT-1 (36.2%) and EU-12/MAT-2 (0.5%). Geostatistatical analysis indicated that vc types, mating types and vc/mating genotypes were spatially autocorrelated and clustered in their distributions. Results suggested that C. parasitica could have a clonal population structure that is generated by asexual reproduction. Low vc-type diversity suggests that the C. parasitica population in the Aydın Mountains may be highly suitable to hypovirus invasion, thereby providing a high potential for successful biological control. However, co-occurrence of sexually compatible strains of EU-1 and EU-12 at the same locations in close proximity creates a high risk of increase in vc-type diversity. [ABSTRACT FROM AUTHOR]

A DnaJ-like gene, Cpdj1, a molecular chaperone and regulator of Hsp70 in Cryphonectria parasitica, was characterized. The protein product of Cpdj1 gene consists of 379 amino acids with a predicted molecular mass of 40.6 kDa and a pI of 7.79. The deduced protein sequence revealed preservation of the conserved hall-mark J-region and exhibited high homolo y to all known DnaJ-like proteins. Disruption of the Cpdj1 gene resulted in slow growth and produced colonies characterized by retarded growth and deep orange color. Accordingly, reduced virulence of the Cpdj1-null mutant was observed. This reduced growth rate was magnified when the Cpdj1-null mutant was cultured under heat-stress conditions. Reduced conidiation was also observed in the Cpdj1-null mutant, indicating that Cpdj1 gene, although not essential for cell viability, is required for appropriate cellular processes including growth and sporulation. Northern analysis showed that Cpdj1 was constitutively expressed, and when the culture was subject to high temperature, a strong induction of the transcript was observed. No significant difference in the expression and induction pattern of Cpdj1 was observed between virus-free EP155/2 and virus-infected hypovirulent UEP1 strains. However, further severe defects in mycelia rowth and conidiation were observed in the hypovirus-infected Cpdj1-null mutant suggesting that the presence of Cpdj1 is required for mycelia growth and sporulation of the hypovirus-infected strain. [ABSTRACT FROM AUTHOR]

Cryparin is an abundant cell-wall-associated hydrophobin of Cryphonectria parasitica. Although cryparin is encoded as a single copy gene, it is the most abundant protein produced by this fungus when grown in liquid culture. Studies to characterize the transcriptional regulatory element(s) found that the fragment between nt −188 and the start codon was the minimal but sufficient promoter element for expression of the cryparin gene. To explore the possibility of using this small fragment as a minimal core promoter, three different chimeric reporter genes were constructed using a bacterial hygromycin B resistance gene ( hph), an inducible laccase of C. parasitica, and glucose oxidase of Aspergillus niger to examine the promoter properties of the fragment. When using C. parasitica as an expression host, the 188-bp fragment functioned as a promoter for the expression of all three reporter genes. Moreover, a high level of expression was further confirmed by measuring the relative amount of transcripts of hph and an internal control gene, glyceraldehyde-3-phosphate dehydrogenase, using quantitative real-time polymerase chain reaction. The 188-bp fragment also showed promoter activity in other fungi, Gibberella zeae, A. niger, and Aspergillus nidulans, which suggests that this fragment can be applied as a strong core promoter for heterologous gene expression in various fungi. [ABSTRACT FROM AUTHOR]

Cryparin, encoded as a single copy gene (Crp) of the chestnut blight fungus Cryphonectria parasitica, is the most abundant protein produced by this fungus. However, its accumulation is decreased remarkably in C. parastica strains containing the double-stranded (ds) RNA virus Cryphonectria hypovirus 1. To characterize the transcriptional regulatory element(s) for strong expression and viral regulation, promoter analysis was conducted. Serial deletion of the Crp promoter region resulted in a step-wise decrease in promoter activity, indicating a localized distribution of genetic elements in the cryparin promoter. Promoter analysis indicated two positive and a repressive c/s-acting elements. Among them, the promoter region between nt -1,282 and -907 appeared to be necessary for hypoviral-mediated down-regulation. An electrophoretic mobility shift assay (EMSA) on the corresponding promoter region (-1,282/-907) indicated two regions at (-1,257/-1,158) and (-1,107/-1,008) with the characteristic AGGAGGA-N42-GAGAGGA and its inverted repeat TCCTCTC-N54-TCCTCCT, respectively, appeared to be specific binding sites for cellular factors. [ABSTRACT FROM AUTHOR]

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 97 Cryphonectria 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. [ABSTRACT FROM AUTHOR]

Fusarium graminearum causes a serious scab disease of small grains in Korea. The nucleotide sequence of the genomic RNA of a double-stranded RNA (dsRNA) virus, Fusarium graminearum virus-DK21 (FgV-DK21), from F. graminearum strain DK21, which is associated with hypovirulence in F. graminearum, was determined and compared to the genome sequences of other mycoviruses, including Cryponectria hypoviruses. The FgVDK21 dsRNA consists of 6,624 nucleotides, excluding the 3'-terminal poly(A) tail. The viral genome has 53- and 46-nucleotide 5' and 3' untranslated regions (UTRs), respectively, and five putative open reading frames. A phylogenetic analysis of the deduced amino acid sequence of ORF1, which encodes a putative RNAdependent RNA polymerase, and those of other mycoviruses revealed that this organism forms a distinct virus clade with other hypoviruses, and is more distantly related to other mycoviruses (3.8 to 24.0% identity). However, pairwise sequence comparisons of the nucleotide and deduced amino acid sequences of ORFs 2 through 5 revealed no close relationships to other protein sequences currently available in GenBank. Analyses of RNA accumulation by Northern blot and primer extension indicated that these putative gene products are expressed from at least two different subgenomic RNAs (sgRNAs), in contrast to the cases in other hypoviruses. This study suggests the existence of a new, as yet unassigned, genus of mycoviruses that exhibits a potex-like genome organization and sgRNA accumulation. [ABSTRACT FROM AUTHOR]

The hyphal tip was isolated from 13 weakly or moderately virulent strains of Helicobasidium mompa to remove double-stranded (ds) RNAs and demonstrate their role as the hypovirulence factor. All of 829 hyphal tip subcultures retained dsRNAs. However, strain v670 containing two large fragments (≫10 kb) and one small fragment (ca. 2.3 kb) of dsRNA lost the largest fragment in 3 of 63 subcultures analyzed. One of the three subcultures (v670hti) was used to inoculate carrots to regain virulence compared to the parental strain v670. When isolate v670hti was paired with v670, the largest fragment was reintroduced to v670hti, and its virulence was diminished. Northern blot analysis with two probes hybridizing dsRNA fragments in most H. mompa strains revealed that the largest fragment involved in hypovirulence was different from two other fragments that are common in Japan. These results indicate that the largest dsRNA fragment in strain v670 is associated with hypovirulence in H. mompa. [ABSTRACT FROM AUTHOR]

A double-stranded (ds)RNA, designated as M2, is associated with hypovirulence, conversion of the quinic acid pathway from inducible to constitutive and downregulation of the shikimic acid pathway in the Rhizoctonia solani culture Rhs 1A1. In this study, we report that in the virulent, M2-lacking isolate Rhs 1AP, which is isogenic to Rhs 1A1, quinic acid reduces virulence dramatically and induces synthesis of an M2-encoded polypeptide and its respective mRNA. The full-length sense strand of M2 is detected in untreated Rhs 1AP only after a second 30-cycle amplification, using nested primers. Quinate-induced Rhs 1AP contains low concentrations of both full-length sense and complementary strand of M2. The quinic acid-induced hypovirulence in Rhs 1AP cannot be overturned by the end-product of the shikimic acid pathway, chorismic acid, which enhances the virulence of Rhs 1AP dramatically when used alone. In addition to its apparent applications, this study confirms the strong association between the M2 dsRNA and hypovirulence in R. solani. [ABSTRACT FROM AUTHOR]

We reported previously that a 3.6-kb double-stranded RNA, designated as M2, is associated with hypovirulence in the Rhizoctonia solani isolate Rhs 1A1 and proposed that the M2-encoded putative polypeptide A (pA) might interfere with the regulation of the quinate and shikimate pathways. In this study, Western blot analysis showed that a protein band of the predicted size (83 kDa) binds antibodies specific to a pA epitope and is detectable in M2-containing but not in M2-lacking cultures. A mRNA, associated with Rhs 1A1 polysomes immunoprecipitated with anti-pA antibodies, has a sequence basically identical to that of the sense-strand of M2. The normally inducible quinate pathway was constitutively expressed, whereas the shikimate pathway was down-regulated in the M2-containing, hypovirulent Rhs 1A1. Finally, the relative concentration of phenylalanine, precursor of the virulence determinant phenylacetic acid, was correlated with the degree of pathogenicity in the virulent Rhs 1AP but not in the hypovirulent Rhs 1A1. [ABSTRACT FROM AUTHOR]

Twelve double-stranded (ds) RNA segments were detected from a hypovirulent strain W370 of the white root rot fungus Rosellinia necatrix. The estimated molecular weights ranged from 0.41×106 to 2.95×106. Full length cDNA clones for eight segments were obtained. Northern blot analysis suggested that each segment was genetically unique. The nucleotide sequences of eight full length dsRNA segments were determined. One long open reading frame was found in each segment. Conserved sequences at the 5′-end (5′-ACAAUUU-3′) and at the 3′-end (5′-UGCAGAC-3′) were identified in all eight segments. Segment-specific panhandle structures, formed by inverted terminal repeats, were also found in all segments. Comparative analyses of the predicted translational products of eight dsRNA segments showed that the deduced amino acid sequence partially matched those of the Reoviridae family members: Colorado tick fever virus, Nilaparvata lugens reovirus, and rice black streaked dwarf virus. The results suggested that W370 dsRNA is derived from a new member of the family Reoviridae detected in fungus. [ABSTRACT FROM AUTHOR]

In the chestnut-blight fungus Cryphonectria parasitica, a plasmid, pCRY1, occurs in the mitochondria of several strains isolated at various locations in the northeastern United States and Canada. The monomer of this plasmid is a 4.2-kb circular double-stranded DNA that has no detectable sequence homology with the 160–kb mitochondrial DNA of Ep155, a standard virulent laboratory strain of C. parasitica. The circular nature and oligomeric characteristics of the plasmid were deduced from the heterogeneous size of plasmid DNA molecules as detected by one- and two-dimensional gel-electrophoresis, the nature and alignment of restriction fragments, and the lack of detectable termini in the nucleotide sequence. The cytoplasmic location of the plasmid was deduced from its co-purification with mitochondria, uniparental (maternal) transmission in sexual crosses, dissociation from the nuclei of the donor strain during its horizontal transfer between vegetatively compatible strains through hyphal anastomoses, and mitochondrial codon usage (UGA=Try). The pCRY1 plasmid contains a long open reading frame that is transcribed and potentially encodes a unique 1214 amino-acid, B-family DNA polymerase similar to those encoded by the LaBelle and Fiji circular mitochondrial plasmids of Neurospora. In this subgroup of proteins, the DTD motif characteristic of B-family DNA polymerases is replaced by TTD. Amino-acid motifs related to those that are characteristic of the 3′→5′ exonuclease domains of B-family DNA polymerases have been located in the amino-terminal portion of the proteins. A comparison of isogenic plasmid-free and plasmid-containing cultures indicates that pCRY1 is an infectious agent that effects a reduction in the pathogenicity of some, but not all, strains of C. parasitica. [ABSTRACT FROM AUTHOR]

In the chestnut-blight fungus, Cryphonectria parasitica, a cytoplasmically transmissible (infectious) form of hypovirulence is associated with mitochondrial DNA (mtDNA) mutations that cause respiratory deficiencies. To facilitate the characterization of such mutations, a restriction map including the probable location of 13 genes was constructed for a relatively well-characterized virulent strain of the fungus, Ep155. The physical map is based on the order of all fragments generated by cleavage of the mtDNA by the PstI restriction endonuclease and includes some of the cleavage sites for HindIII, EcoRI, and XbaI. It was constructed from hybridization patterns of cloned mtDNA fragments with Southern blots of mtDNA digested with the four restriction enzymes. On this map, the probable locations of genes commonly found in the mitochondrial genomes of ascomycetes were determined by low-stringency hybridization of cloned Neurospora crassa mitochondrial gene probes to Southern blots of C. parasitica mtDNA. The data indicate that the mtDNA of strain Ep155 is a circular molecule of approximately 157 kbp and ranks among the largest mitochondrial chromosomes observed so far in fungi. The mtDNAs of 11 different C. parasitica isolates range in size from 135 to 157 kbp and in relatedness from 68 to 100 percent, as estimated from restriction-fragment polymorphisms. In addition to the typical mtDNA, the mitochondria of some isolates of the fungus contain double-stranded DNA plasmids consisting of nucleotide sequences not represented in the mtDNA of Ep155. [ABSTRACT FROM AUTHOR]

Movement via somatic fusion and inheritance of a small mitochondrial double-stranded (ds) RNA element was examined in Cryphonectria parasitica. The 2.7-kb dsRNA from the C. parasitica strain NB631 encodes a putative RNA-dependent RNA polylmerase when the mitochondrial code (UGA = Trp) is invoked. All progeny from asexual spores (conidia) of strain NB631 examined for dsRNA contained the 2.7-kb element. Unlike other C. parasitica dsRNAs, which are cytoplasmic, the dsRNA in strain NB631 was transmitted through the sexual cycle (ascospores) if the strain containing the element acted as the female in crosses. Movement of the 2.7-kb dsRNA was also observed through hyphal anastomosis. Transfer by anastomosis was accompanied by mitochondrial movement and recombination of the mitochondrial genome as determined by RFLP analysis. In control pairings between isolates lacking dsRNA, mitochondrial movement and recombination were also observed. Transfer by anastomosis allowed the generation of infected and uninfected isogenic lines, and permitted us to evaluate the effects of the dsRNA element on virulence of the host. Bark virulence assays on American chestnut suggest that NB631 dsRNA decreases the virulence of C. parasitica, but not to the level associated with members of the Hypoviridae. [ABSTRACT FROM AUTHOR]