dsRNA-free Isolates Research Articles

Description, Distribution, and Relevance of Viruses of the Forest Pathogen Gremmeniella abietina.

The European race of the ascomycetous species Gremmeniella abietina (Lagerberg) Morelet includes causal agents of shoot blight and stem canker of several conifers in Europe and North America, which are known to host a diverse virome. GaRV6 is the latest and sixth mycovirus species reported within G. abietina. Before its description, one victorivirus and one gammapartitivirus species were described in biotype A, two mitoviruses in both biotypes A and B and a betaendornavirus in biotype B. Possible phenotypic changes produced by mycoviruses on G. abietina mycelial growth have been reported in Spanish mitovirus-free and GaRV6-hosting G. abietina isolates, which had higher growth rates at the optimal temperature of 15 °C, but no other major differences have been observed between partitivirus-like dsRNA and dsRNA-free isolates. In this review, we reappraise the diversity of viruses found in G. abietina so far, and their relevance in clarifying the taxonomy of G. abietina. We also provide evidence for the presence of two new viruses belonging to the families Fusariviridae and Endornaviridae in Spanish isolates.

Effect of Double-Stranded RNAS on Virulence and Deoxynivalenol Production of Fusarium Graminearum Isolates

Various viral genomes from plant pathogenic fungi have been reported. The most common of these genomes found are dsRNA which in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. In this study 33 Iranian isolates of Fusarium graminearum, were examined for dsRNA infection. In 12 of these isolates, more than three viral fragments measuring 0.9, 1.2, 3, 3.2 and 5 kb were detected. The presence and nature of dsRNA was confirmed by RNaseA and DNaseI treatments. To obtain dsRNA-free isolates, hyphal tip cultures of six isolates were transferred to PDA containing different concentra- tions of cycloheximide. Three hyphal tip cultures were confirmed to be dsRNA-free. Mycelial growth in dsRNA-containing isolates was less than that of their counterpart cured isolates with no dsRNA. Meanwhile, no significant difference was observed for colony morphology, pigmentation and conidia production of viruliferous and cured isolates. However, the disease severity of the dsRNA- containing isolates was significantly (p < 0.01) less than that of the dsRNA-free isolates on susceptible wheat in greenhouse. Deoxyni- valenol (DON) production by dsRNA-free and dsRNA-containing isolates was confirmed using HPLC analysis. The range of DON production levels for dsRNA-free and dsRNA-containing isolates varied from 0.07 to 1.6 ppm and 0.06-0.4 ppm, respectively. A very significantly reduced level of DON, up to 50%, was detected in dsRNA-containing derivatives compared to the dsRNA-free isolates.

Double-stranded RNA mycoviruses inFusarium culmorumandFusarium graminearumisolates

The presence of double-stranded RNA (dsRNA) elements in Fusarium culmorum and F. graminearum isolates of different origin, and the possible effect of these elements on pathogenicity and toxin production of the isolates were examined. Altogether 40 F. culmorum and 38 F. graminearum isolates were involved in this study, together with F. cerealis and F. pseudograminearum strains. Double-stranded RNA elements indicative of mycovirus infection were detected for the first time in 5 F. culmorum isolates. The isolates originated from the United States, The Netherlands, Australia and Israel. The dsRNA nature of the fragments was proved by RNase, DNase and S1 nuclease treatments. The sizes of the dsRNA elements varied between 0.6–3.95 kbp. A dsRNA element of 3.0 kbp in size was also detected in a F. graminearum isolate came from South Africa. None of the Central-European isolates examined were found to carry such elements. We also examined the mycotoxin producing abilities and pathogenicity of the dsRNA infected isolates. Four of the mycovirus infected F. culmorum isolates produced deoxynivalenol and zearalenone (chemotype I), while one of the isolates came from the USA produced nivalenol and fusarenone X (chemotype II). The dsRNA-containing F. graminearum isolate produced deoxynivalenol and zearalenone. In general, dsRNA-containing isolates were found to be as pathogenic to two wheat cultivars as dsRNA free isolates. RAPD analysis and sequence analysis of a putative reductase gene fragment indicated that dsRNA-containing isolates are scattered among dsRNA free isolates. Further work is in progress in our laboratory to characterize these dsRNA elements by sequence analysis.

Comparison between Rosellinia necatrix isolates from soil and diseased roots in terms of hypovirulence

The white root rot fungus, Rosellinia necatrix, is a devastating soil-borne pathogen of many plant species. Biocontrol with the hypovirulence factor is promising, but disease symptoms, signs or culture morphology of the pathogen cannot be reliably used as markers for hypovirulence in this fungus. We attempted to obtain hypovirulent isolates from soil rather than from diseased roots, based on the hypothesis that hypovirulent isolates were more likely to persist in soil as saprobes. Sixteen isolates, belonging to eight mycelial compatibility groups (MCGs), were obtained from soil in two active and one abandoned Japanese pear orchards. Comparison of these isolates based on clonality revealed that six MCGs were commonly recovered from both diseased roots and soil and two MCGs exclusively from soil. No MCG was found in more than one orchard. With two exceptions, isolates within the same MCG were similar in virulence, competitive saprophytic ability (CSA) and mycelial growth rate whether or not they carried dsRNA. The two exceptional isolates recovered from soil had multiple dsRNA segments that caused hypovirulence, weakened CSA and restricted mycelial growth on nutrient-rich media. They belonged to different MCGs, each including dsRNA-free isolates. Isolates from soil contained various dsRNAs (44%), including the hypovirulence factor, more frequently than isolates from diseased roots in the same fields (25%), which is much higher than the proportion of isolates with dsRNA from diseased roots (19%) in a total of 424 isolates from Japan examined so far. These results suggest that isolation of R. necatrix from soil is an effective method to obtain isolates with dsRNAs, including the hypovirulence factor.

Detection of double-stranded RNA in Macrophomina phaseolina

One hundred ten isolates of Macrophomina phaseolina, the causal agent of charcoal rot of many different plant species, were obtained from Mexico, Somalia, and several locations in the United States, and analyzed for the presence of double-stranded RNA (dsRNA). Twenty-one isolates from the United States and Mexico contained dsRNA elements. The sizes of the dsRNAs ranged from ca 0.4 kbp to 10 kbp and the number of dsRNAs present among the isolates ranged from one to ten. Attempts to detect virus particles by sucrose gradient centrifugation were unsuccessful. Three isolates having dsRNA and three dsRNA-free isolates were evaluated for virulence on sorghum at two different field locations and for mycelial growth in PDA and V8 agar. For both locations, the isolates that had dsRNA were significantly less virulent (P < 0.05) than the dsRNA-free isolates. Differences between locations were not significant (P < 0.05). The contrasts between means of isolates with and without dsRNA suggest a strong association between the presence of dsRNA and a reduction in both mycelial growth and the capacity to cause disease. These results suggest an association between the presence of dsRNA and hypovirulence in the fungus.

Double-stranded RNA and virus-like particles in the grass endophyte Epichloë festucae

Two double-stranded ribonucleic acid (dsRNA) elements were detected in an isolate of the clavicipitaceous grass endophyte Epichloe festucae obtained from an asymptomatic Festuca rubra plant. No dsRNA was detected in three other isolates of E. festucae. The size of the two dsRNA elements isolated from mycelium was of 5.2 and 3.2 kbp. Hybridization experiments with a cDNA probe complementary to the 5.2 kbp dsRNA showed that there was no sequence similarity between the dsRNA elements. Isometric virus-like particles of about 50 nm in diameter were observed by electron microscopy in sucrose-gradient purified virus preparations from the isolate containing dsRNA. The 5.2 kbp dsRNA element was present in the partially purified virus preparations, suggesting that this element is encapsidated. There was no difference with respect to growth rate or colony morphology between the dsRNA containing and dsRNA-free isolates. The presence of dsRNA and virus-like particles suggests the presence of a mycovirus in the Vit5 strain of E. festucae.

Diversity of double-stranded RNA viruses within populations of entomopathogenic fungi and potential implications for fungal growth and virulence

Naturally occurring isolates as well as several culture collection isolates of the entomopathogenic fungi, Metarhizium anisopliae, Beauveria bassiana, and Metarhizium flavoviride were assessed for the presence of double-stranded RNA (dsRNA). DsRNA was present in the mycelium of 28 of 73 isolates (38.4%) of M. anisopliae and 2 of 12 isolates (16.7%) of B. bassiana collected from southern Ontario soil samples. DsRNA was also present in mycelium of 2 of 3 isolates of M. anisopliae and 5 of 6 isolates of M. flavoviride obtained from culture collections. DsRNA banding patterns were variable and, in total, eighteen dsRNA banding patterns were identified, containing from one to eight bands ranging from 5.2 to 0.5 kbp in size. Partially purified virion preparations from selected dsRNA-containing isolates provided similar banding patterns to dsRNAs extracted from mycelium. Comparison of 12 dsRNA-containing isolates with 10 dsRNA-free isolates indicated dsRNAs were not responsible for the large variation in growth rate, conidiospore production, and insect virulence observed in M. anisopliae. However, a comparison between two isogenic strains, one with dsRNA and one without, showed a significantly increased growth rate and conidiospore production on potato dextrose agar (PDA) amended with dodine, as well as significantly increased virulence, in the dsRNA-free isolate. These results indicate that dsRNA infections may not be entirely latent in these fungi.

Presence of double-stranded RNA and virus-like particles in Botrytis cinerea

Double-stranded RNA (dsRNA) was demonstrated in mycelial extracts in 143 of 200 isolates of Botrytis cinerea from a range of hosts in New Zealand. The dsRNA profiles differed widely between isolates in the number, size and relative concentration of dsRNA species present. Virus-like particles (VLPs) were observed by electron microscopy in partly purified virus preparations in three of five dsRNA-containing isolates examined. Morphological types present included isometric particles of varying size classes (approx. 30, 35, 40 and 45 nm) and bacilliform particles (approx. 25 × 63 nm). Such particles were not observed in five isolates apparently lacking dsRNA but flexuous rods with a modal length of approx. 720 nm were present in one. Total RNA extraction of partly purified VLP preparations gave similar profiles to those obtained from mycelial dsRNA extractions of the same isolates, suggesting that the VLPs represent encapsidated dsRNA. Comparison of 12 dsRNA-containing and 12 dsRNA-free isolates indicated slight differences between the two groups in radial growth rate, virulence on bean leaves and sclerotial number but the differences were minor and the ranges overlapped.

Ultrastructure of a hypovirulent isolate of Sclerotinia sclerotiorum containing double-stranded RNA

Sclerotia and hyphae of hypovirulent double-stranded RNA (dsRNA)-containing and virulent dsRNA-free isolates of Sclerotinia sclerotiorum were examined to describe and compare their subcellular structures using light and electron microscopy. Light microscopic analysis of virulent and hypovirulent tissues indicated that, in general, tissues from both isolates were similar. However, sclerotia of the hypovirulent isolate contained fewer protein bodies than those of the virulent isolate and both hyphae and sclerotia of the hypovirulent isolate displayed a more granular appearance throughout the cytoplasm. Electron microscopic analysis revealed that the granular appearance was associated with the presence of many small, double membrane bodies within all cells of the hypovirulent isolate. Comparable structures were not observed in any tissues examined from the virulent isolate. The average diameter of the double membrane bodies was 70 nm and they often were observed in continuum with the nuclear envelope. Particles representative of a mycovirus were not observed in any virulent or hypovirulent tissues. The results indicate that double-stranded RNA and hypovirulence in S. sclerotiorum is not associated with a typical mycovirus but may represent an unencapsidated mycovirus or viral-like agent packaged as double membrane bodies.