Genetic relatedness among dsRNAs from different isolates ofDiscula destructiva

The genetic relationships among double-stranded RNAs (dsRNAs) from 76 isolates ofDiscula destructiva obtained from different geographic locations from New York to Alabama were studied by dot-blot hybridization. The dsRNA segments, identified by agarose gel electrophoresis, varied in size and number. Probes were constructed from total dsRNA from six isolates with different dsRNA profiles and of different geographic origins. Each probe hybridized with dsRNA from ca. 62% of the isolates under the high-stringency washing conditions used. No major differences in the percentage of the total isolates that hybridized with each probe were observed. These results suggest a recent common origin of these isolates ofD. destructiva.     

High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae

Abstract Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species.     

Multiple double-stranded RNA segments are associated with virus particles infecting Trichomonas vaginalis.

Previous studies demonstrated that some isolates of the sexually transmitted protozoan Trichomonas vaginalis are infected with a nonsegmented, double-stranded RNA (dsRNA) virus. A reexamination of the total dsRNA extracted from several virus-harboring isolates indicated the presence of at least three dsRNAs with sizes ranging from 4.8 to 4.3 kbp. The double-stranded nature of each of the three segments was determined by hybridization experiments using riboprobes of opposite polarities obtained from cDNA generated to each of the segments. All three segments were present in agar clones originating from single organisms of T. vaginalis isolates, suggesting that the three segments were not the result of a mixed population of trichomonads harboring different sizes of dsRNA. The three segments were associated with CsCl-purified virus particles, as evidenced by electron microscopy, and RNAse treatment of the preparation containing virus particles did not destroy the dsRNAs. Finally, the individual dsRNA segments were purified for use as probes to determine whether the three dsRNAs shared any sequence homology. Each end-labeled dsRNA segment did not cross-hybridize to any of the other two segments, a finding consistent with the hybridization of labeled cDNAs to only the segments from which they were derived. These results show that the coding capacity of the dsRNA virus may be at least three times greater than that estimated earlier and illustrates further the complexity of this virus-parasite interrelationship.     

Dynamics of double-stranded RNA segments in a Helicobasidium mompa clone from a tulip tree plantation

Eighty-three isolates of the violet root rot fungus, Helicobasidium mompa, were collected in a tulip tree plantation and analyzed for the dynamics of double-stranded (ds) RNA for five years. They were divided into eight mycelial compatibility groups (MCGs). Prevalent MCGs 60 and 68 included 61 and 11 isolates, respectively. Electrophoretic profiles of dsRNA in the first year collection of MCG 60 contained no or a single large dsRNA (more than 10 kb) with or without small dsRNAs (ca. 2.0-2.5 kb). Additional dsRNA fragments, i.e., a middle dsRNA (ca. 8.0 kb) or another type of small dsRNAs, became evident within MCG 60 isolates with time. Northern hybridization revealed the relatedness of all large and middle dsRNA fragments within MCG 60 but small fragments of dsRNA were variable. Large dsRNA fragment differed from that in other MCGs even in the same field. Correlation between specific dsRNA fragments and hypovirulence was not observed. Possible explanations for the accumulation of dsRNA fragments during the growth of disease patch by MCG 60 are discussed in terms of their internal changes such as evolution of novel dsRNA fragments from pre-existing viruses or fungal genomic DNA and horizontal transmissions.     

豇豆单孢锈菌中病毒样颗粒中双链RNA的存在

从北京西郊清华园附近田间豇豆上采集的豇豆单孢锈菌(Uormyces vignal Barcl)夏孢子。萌发后提取双链RNA,电泳分析可测出300—8000碱基对的三组双链RNA。从萌发的孢子中通过差迷离心提取病毒样颗粒,可获得二种类型的病毒样颗粒,一种直径为35—40nm的等轴颗粒。另一种为长短不等的棒状颗粒,用提纯物提取核酸电泳分析与直接从孢子中提取的双链RNA有相同的核酸带,从而证明这些双链RNA存在于病毒样颗粒中。     

Incidence of dsRNA Mycoviruses in a Collection of Aspergillus fumigatus Isolates

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.     

Detection of double stranded RNA in phytopathogenic <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> causing charcoal rot in <Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>

One hundred one isolates of Macrophomina phaseolina from various hosts and eco-geographical locations were employed for elucidating relationships among genetic diversity and virulence. Highly pathogenic, moderately pathogenic, and hypovirulent cluster bean specific isolates were identified. In order to correlate respective phenotypes of plant pathogenic fungus multiple and complex patterns of dsRNA elements were analyzed. Double-stranded ribonucleic acids (dsRNA) are ubiquitous in all major groups and most of them have vast potential as biological control agents for fungi. Rate of virulence and its further association could ascertain by host plant and their fungal genotypes. Variability of the fungal genotypes decides the link between the complexity of dsRNA with different variants and the change in virulence pattern. Double-stranded RNA was identified in approximately 21.7% of M. phaseolina isolates from charcoal rot infected cluster bean varieties. After recurrent laboratory transfer on culture media, the preponderance of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to cluster bean. Macrophomina has successfully showed diversified and reproducible banding profile in dsRNA containing/free isolates. This is the first report of hypovirulence and detection of dsRNA in Macrophomina phaseolina isolates of cluster bean origin.     

Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum

We have discovered and analysed two novel, linear extrachromosomal double-stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum , a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L-dsRNA) had homology with viral RNA-dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activated c-June NH₂ terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C . parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.     

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis

Mitochondria from the green alga Bryopsis sp. very often contained a 4.5 kb double-stranded RNA (dsRNA) at a defined level. Complementary DNA probes derived from the mitochondrial dsRNA hybridized with none of the algal chloroplast dsRNAs of 1.7 to 2.2 kb, but did hybridize with a similar-sized dsRNA among several dsRNAs from the mitochondria of B. maxima. Sequence analysis of the mitochondrial dsRNA from Bryopsis sp. revealed only two large, overlapping, open reading frames (ORFs) on one strand if UGA was taken as a non-termination codon, suggesting the independent phylogenetic evolution of the mitochondrial dsRNA. Consensus sequence for RNA-dependent RNA polymerase was found within the longer ORF (2472 bp) of the dsRNA. The overlapping 52 bp of the ORFs in different reading frames is suggestive of the occurrence of a -1 ribosomal frameshift in the mitochondrial translation system. The observed simple genetic structures suggest that the algal mitochondrial dsRNA might be deficient in a gene for movement from cell to cell in host plants and, hence, has a plasmid-like nature that is distinct from that of infectious plant viruses. The nature and origin of the endogenous dsRNAs of various sizes and their relationships are discussed.     

Viruslike particles in tentoxin-producing strains of Alternaria alternata.

Double-stranded (ds) RNAs associated with viruslike particles have been found in six isolates of Alternaria alternata which produce tentoxin. Isolates had from one to three dsRNAs ranging in size from 1.0 to 5.1 kilobase pairs. In two isolates the dsRNAs were associated with 30-nm particles. No dsRNA was detected in any of six other tentoxin-producing isolates or nine isolates which did not produce tentoxin.     

Double-stranded RNA and virus-like particles in the edible basidiomycete Flammulina velutipes (Enokitake)

One of the commercial strains of Flammulina velutipes was analyzed for the presence of double-stranded RNA (dsRNA) elements to examine the underlying mechanism of strain degeneration. As a result, two dsRNA elements sized 1.9 and 1.8 kb were detected in mycelium derived from spontaneously brown-colored fruit body. They were not detected in the normal strains or in fruiting-impaired degenerative isolates. The dsRNAs were not in the nuclear or mitochondrial fractions, but were located in the cytoplasmic fraction. The presence of virus-like particles of ca. 50 nm diameter associated with the dsRNAs was confirmed by electron microscopic observation.     

Detection of Australian Field Isolates of Citrus Tristeza Virus by Double-Stranded RNA Analysis

Double-stranded RNA analysis indicated that widespread latent infection by citrus tristeza virus (CTV) occurs naturally in a citrus variety collection at Merbein, Australia. Detection was based on the presence of the putative replicative form (RF) of the virus (ca 19. 5 kilobase pairs) and the presence or absence of three previously known putative subgenomic dsRNAs with molecular sizes of ca 3. 1,1.7 and 0.8 kilobase pairs. With the exception of some citrus relatives normally used as rootstocks, the dsRNAs were readily detectable in all the trees tested. Variability was noticed among the dsRNAs profiles of individual seedling trees of Microcitrus australasica var. sanguinea. By contrast, dsRNA patterns of trees of open-pollinated Ellendale tangor were indistinguishable. A number of dsRNA bands found in West Indian lime infected with CTV were not detected when this isolate was graft-inoculated to healthy sweet orange seedlings. A simple method for enhancing the sensitivity of detection of the CTV dsRNA bands by silver staining is described which involves incubating the gels in RNase A prior to staining; this allowed detection of the putative CTV RF in only 40 mg of green hark material.     

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.     

A viral double-stranded RNA up regulates the fungal virulence of Nectria radicicola

Double-stranded RNAs (dsRNAs) are widespread in plant pathogenic fungi, but their functions in fungal hosts remain mostly unclear, with a few exceptions. We analyzed dsRNAs from Nectria radicicola, the causal fungus of ginseng root rot. Four distinct sizes of dsRNAs, 6.0, 5.0, 2.5, and 1.5 kbp, were detected in 24 out of the 81 strains tested. Curing tests of individual dsRNAs suggested that the presence of 6.0-kbp dsRNA was associated with high levels of virulence, sporulation, laccase activity, and pigmentation in this fungus. The 6.0-kbp dsRNA-cured strains completely lost virulence-related phenotypes. This 6.0-kbp dsRNA was reintroduced by hyphal anastomosis to a dsRNA-cured strain marked with hygromycin resistance, which resulted in the restoration of virulence-related phenotypes. These results strongly suggest that 6.0-kbp dsRNA up regulates fungal virulence in N. radicicola. Sequencing of several cDNA clones derived from 6.0-kbp dsRNA revealed the presence of a RNA-dependent RNA polymerase (RDRP) gene. Phylogenetic analysis showed that this gene is closely related to those of plant cryptic viruses. Biochemical analyses suggested that the 6.0-kbp dsRNA may regulate fungal virulence through signal-transduction pathways involving cyclic AMP-dependent protein kinase and protein kinase C.     

Genetic,morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii

In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.     

Presence and distribution of double-stranded RNA elements in the white root rot fungus Rosellinia necatrix

Double-stranded (ds)RNA of various types was detected in 65 (21.8%) of 298 isolates from vegetative hyphae of Rosellinia necatrix by electrophoresis, but dsRNA was not detected from 39 ascosporic isolates. There were 45 distinct dsRNA profiles in the 65 isolates: they varied in the number of electrophoretic bands from 1 to 12 and in size from less than 1000 bp to more than 10 kbp. Each dsRNA profile was unique to each locality. dsRNAs having the same profiles were restricted to isolates of the same mycelial compatibility groups (MCG) from the same trees, with an exception where different profiles were detected in different isolates of the same MCGs. Received: May 7, 2001 / Accepted: September 5, 2001     

栗疫病菌的营养体亲和性基因和dsRNAs对病毒传播的影响

研究了栗疫病菌[Cryphonectria parasitica(Murr.)Barr]的营养体亲和性基因及dsRNA病毒对菌株间病毒特征传播的影响。试验选用已知4个VC基因座位的15个Vc基因型菌株和3种dsRNA病毒,通过含病毒菌株与野生型菌株的配对培养,将病毒逐个转入不同Vc基因型菌株。将不同Vc基因型的含病毒菌株与具特定VC基因差异的野生型菌株配对培养,根据培养两周后野生型菌株培养性状的改变与否,统计菌株间的病毒传播率。结果表明,各个VC基因对菌株间病毒传播的影响不同;存在2个VC基因差异的菌株间病毒传播率低于相差1个VC基因的：病毒在相差1个VC基因的菌株间的传播多具单向性：不同类型的病毒在菌株间的传播率也有差异。     

Presence of double-stranded RNA and virus-like particles in Rhizopus isolates

Fungal isolates belonging to four Rhizopus species were screened for the presence of double-stranded RNA (dsRNA) molecules. Five (two R. stolonifer, two R. microsporus, and one R. oryzae) of the 27 isolates examined harboured such genetic elements. Electrophoresis of the nucleic acids revealed five RNA patterns, with 1-5 discrete dsRNA bands. The molecular sizes corresponding to these bands were 2.2-14.8 kb. Gel electrophoresis of purified virus-like particles (VLPs) indicated only one capsid of similar size in all virus-harbouring strains; when investigated by electron microscopy, they were found to be polyhedral VLPs 40 nm in diameter. In one of the R. microsporus isolates an uncapsidated large dsRNA segment (14.8 kb) was observed. No phenotypic differences were observed between uninfected and virus-harbouring Rhizopus isolates.     

The enigma of double-stranded RNA (dsRNA) associated with mushroom virus X (MVX)

New variants of pathogenic fungal viruses are emerging and they are enigmatic in revealing their molecular identity and of their origin. Double-stranded RNAs, some in non-encapsidated forms are increasingly becoming causal agents for sporadic diseases and are consistently associated with a complex profile of dsRNAs, presumably of (multiple) viral origin present in the host while the same are conspicuously absent in healthy (looking) counterparts. The emergence of an unusual Agaricus bisporus mushroom 'patch disease' first reported in 1996, later termed as 'mushroom virus X' (MVX), exhibited a wide range of symptoms (e.g. barren patches beside healthy looking mushrooms, arrested pins, premature opening, brown, off-colour and distortions in shape). A variable compendium of novel 26 (dsRNA) elements, ranging in sizes between 20.2kb to 0.64kb, several of them (-17/26) in non-encapsidated form have been shown to occur in the diseased mushroom fruiting bodies and are thought to comprise multiple viruses. Ten years on, this devastating disease is now more widespread and prevalent in a number of European countries (e.g. The Netherlands, Ireland) ranging from occasional to severe outbreaks leading to crop losses. Impressive data has been accumulated on the symptoms, but the potential aetiological sources, biochemical and molecular characterizations corresponding to the symptoms vis-a-vis MVX linked dsRNAs still remain either elusive or unclear. We have overviewed mainly the molecular findings of research groups working on MVX in these countries together with our own work on MVX in Northern Ireland. To date, the results reviewed suggest that with the exception of 4 low molecular weight dsRNA bands (sizes 2.0. 1.8, 0.8 and 0.6 kb) which consistently were found synchronous to mushroom off-colour/browning symptoms in the UK and The Netherlands, other individual MVX dsRNAs or their banding patterns clearly lack credible relationship with other symptoms of the MVX disease complex. The issues in the molecular characterisation of the MVX dsRNAs include the disparate results on the molecular sequences obtained for some of these by the different research groups, the varying molecular methods or approaches adopted by them for deciphering the nucleotide sequences of the novel dsRNAs that are different from previously encountered mushroom viruses. The future outlook and general consensus among mushroom researchers worldwide is for an urgent need to recruit international taskforce and re-focus on clarifying the symptom vis-a-vis dsRNAs in the enigmatic MVX disease complex. As crossing the cellular membrane is a key step to infection process, we have also attempted to draw parallels with other viruses in terms of the potential cell entry mechanisms for MVX dsRNAs. In the light of MVX disease and A. bisporus being a commercial crop worldwide in agri-food markets, and taking cue from its nearest Basidiomycete model mushroom, Coprinus cinereus whose genome mapping is completed, we also propose that it may be timely for the international research groups to renew efforts to prop up a network for sequencing the host A. bisporus mushroom genome (-38 MB) for a better understanding of host-pathogen relationships.