Sensitivity of Globisporangium ultimum to the fungicide metalaxyl is enhanced by the infection with a toti-like mycovirus.

In recent years, numerous oomycete mycoviruses have been discovered; however, very few studies have focused on their effects on the host oomycete phenotype. In this study, we investigated the impact of toti-like Pythium ultimum RNA virus 2 (PuRV2) infection on the phytopathogenic soil-borne oomycete Globisporangium ultimum, which serves as a model species for Globisporangium and Pythium, specifically the UOP226 isolate in Japan. We generated a PuRV2-free isogenic line through hyphal tip isolation using high-temperature culture and subsequently compared the phenotypic characteristics and gene expression profiles of UOP226 and the PuRV2-free isogenic line. Our findings revealed that the metalaxyl sensitivity of UOP226 was greater than that of the PuRV2-free isogenic line, whereas the mycelial growth rate and colony morphology remained unchanged in the absence of the fungicide. Furthermore, transcriptome analyses using RNA-seq revealed significant downregulation of ABC-type transporter genes, which are involved in fungicide sensitivity, in UOP226. Our results suggest that PuRV2 infection influences the ecology of G. ultimum in agricultural ecosystems where metalaxyl is applied.

Mycelia-Assisted Isolation of Non-Host Bacteria Able to Co-Transport Phages.

Recent studies have demonstrated that phages can be co-transported with motile non-host bacteria, thereby enabling their invasion of biofilms and control of biofilm composition. Here, we developed a novel approach to isolate non-host bacteria able to co-transport phages from soil. It is based on the capability of phage-carrying non-host bacteria to move along mycelia out of soil and form colonies in plaques of their co-transported phages. The approach was tested using two model phages of differing surface hydrophobicity, i.e., hydrophobic Escherichia virus T4 (T4) and hydrophilic Pseudoalteromonas phage HS2 (HS2). The phages were mixed into soil and allowed to be transported by soil bacteria along the mycelia of Pythium ultimum. Five phage-carrying bacterial species were isolated (Viridibacillus sp., Enterobacter sp., Serratia sp., Bacillus sp., Janthinobacterium sp.). These bacteria exhibited phage adsorption efficiencies of ≈90-95% for hydrophobic T4 and 30-95% for hydrophilic HS2. The phage adsorption efficiency of Viridibacillus sp. was ≈95% for both phages and twofold higher than T4-or HS2-adsorption to their respective hosts, qualifying Viridibacillus sp. as a potential super carrier for phages. Our approach offers an effective and target-specific way to identify and isolate phage-carrying bacteria in natural and man-made environments.

Viral Interactions and Pathogenesis during Multiple Viral Infections in Agaricus bisporus.

Viral interactions during multiple viral infections were examined in Agaricus bisporus cultures harboring 9 viruses (comprising 18 distinct viral RNAs) by statistically analyzing their relative abundance in fruitbodies. Four clusters of viral RNA were identified that suggested synergism and coreplication. Pairwise correlations revealed negative and positive correlations between clusters, indicating further synergisms and an antagonism involving a group containing a putative hypovirus and four nonhost ORFan RNAs (RNAs with no similarity to known sequences) possibly acting as defective interfering RNAs. The disease phenotype was observed in 10 to 15% of the fruitbodies apparently randomly located among asymptomatic fruitbodies. The degree of symptom expression consistently correlated with the levels of the multipartite virus AbV16. Diseased fruitbodies contained very high levels of AbV16 and AbV6 RNA2; these levels were orders of magnitude higher than those in asymptomatic tissues and were shown statistically to be discretely higher populations of abundance, indicating an exponential shift in the replicative capacity of the virus. High levels of AbV16 replication were specific to the fruitbody and not found in the underlying mycelium. There appeared to be a stochastic element occurring in these viral interactions, as observed in the distribution of diseased symptoms across a culture, differences in variance between experiments, and a number of additional viruses undergoing the step-jump in levels between experiments. Possible mechanisms for these multiple and simultaneous viral interactions in single culture are discussed in relation to known virus-host regulatory mechanisms for viral replication and whether additional factors could be considered to account for the 1,000-fold increase in AbV16 and AbV6 RNA2 levels.IMPORTANCE How viruses interact in a multiple-virus infection was examined by quantifying the levels of 18 viral RNAs in fruiting cultures of the agriculturally cultivated fungus Agaricus bisporus and statistically analyzing and modeling their abundance. Synergistic, antagonistic, and neutral interactions occurred simultaneously in cultures. The viral RNAs were grouped into four clusters, each displaying similar relative abundance, and between clusters, further interactions were found with positive, negative, or no correlations. Mushroom fruitbodies showing disease symptoms were distributed apparently randomly across the culture. These symptoms were associated with the presence of viral RNAs from two different clusters at very high levels, 1,000-fold higher than asymptomatic fruitbodies. The role of viral interaction together with stochastic factors and the regulation of host antiviral defenses in pathogenesis are discussed.

FISHing in fungi: Visualisation of mushroom virus X in the mycelium of Agaricus bisporus by fluorescence in situ hybridisation.

Agaricus bisporus is a commercial mushroom crop susceptible to a disease caused by a complex of viruses known collectively as mushroom virus X (MVX). Symptoms of MVX include bare patches and mushroom cap discolouration (browning) in the fruiting bodies, phenotypes associated with the viruses AbV6 and AbV16, respectively. Limited understanding exists of the localisation and mobilisation of these viruses within the mycelium of A. bisporus. To this end, a non-destructive fluorescence in situ hybridisation (FISH) method was developed for in situ targeting of AbV6 and AbV16 in A. bisporus mycelium. An MVX strain associated with the bare patch disease phenotype revealed predominantly high signal towards the growing edges of cultures when probed for AbV6, with a 'halo-effect' of high signal intensity around putative vacuoles. An MVX strain associated with the browning disease phenotype showed high signal intensities within reticulating networks of hyphae in a highly compartmentalised manner when probed for AbV16. Localisation of the two viruses in MVX-infected cultures appears independent, as both viruses were found in completely discrete areas of the mycelium in differential patterns. FISH detected low level presence of the two viruses, AbV6 and AbV16 in a number of cultures which had tested negative for the viruses by RT-PCR. This suggests that FISH may be more sensitive at detecting viruses at low levels than molecular methods. This study demonstrates that FISH is a powerful tool in the field of mycovirology.

Multiple virus infections on Heterobasidion sp.

Heterobasidion viruses have previously been shown to affect each other's transmission and phenotypic effects on their hosts in a complex way. In this work, Heterobasidion parviporum strains hosting five coinfecting viruses simultaneously were constructed and used as donors in transmission experiments. They showed that viruses move more frequently between the mycelia of the same species than between the mycelia of H. parviporum and Heterobasidionannosum. One of the strains was used to show that coinfection of five viruses is relatively unstable in a natural environment and analyses of the growth rate and competitive ability of Heterobasidion strains hosting various virus combinations revealed that viral effects are not additive. The results also supported the view that the transmission of the promising virocontrol agent HetPV13-an1 may be enhanced by coinfecting viruses in the donor mycelium. However, its detrimental effects may be blocked by the presence of other viruses in the same mycelium. REPOSITORIES: GenBank accession number MN058080.

The PoV mycovirus affects extracellular enzyme expression and fruiting body yield in the oyster mushroom, Pleurotus ostreatus.

Isogenic virus-cured and virus-infected fungal strains were previously obtained and compared to investigate mycoviral diseases and, specifically, the influence of viral infection on the vegetative growth of Pleurotus ostreatus. The present study demonstrated that infection with mycovirus PoV-ASI2792 (PoV) caused phenotypic and physiological changes in fungal cells and mycelia. The microscopically determined growth rate of the virus-infected strain was lower than that of the virus-cured strain, due to the conglomerate phenomenon during the mycelial growth process. An exploration of the viral effects of PoV on fruiting bodies yield showed significantly lower than that on virus-cured P. ostreatus. A colorimetric assay of polyphenol oxidase activity in the strains showed very weak activity in the virus-infected strain. To estimate the activity levels of enzymes related to the growth and fruiting body formation, the relative expression levels of genes encoding various extracellular enzymes such as Carbohydrate-Active Enzymes (CAZymes) were measured by quantitative RT-PCR. The expression levels of the assayed genes were significantly lower in virus-infected than in virus-cured P. ostreatus. Together, these results indicate that PoV infection affects the spawn growth and fruiting body formation of P. ostreatus via decreased expression and activity of some extracellular enzymes including lignocellulolytic enzymes.

Characterization of Cronartium ribicola dsRNAs reveals novel members of the family Totiviridae and viral association with fungal virulence.

BACKGROUND: Mycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems. METHODS: Full-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression. RESULTS: In this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1-5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes. CONCLUSION: Phylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.

Challenges to elucidating how endornaviruses influence fungal hosts: Creating mycovirus-free isogenic fungal lines and testing them.

Determining roles of mycoviruses in fungal biology is complicated, especially when fungi are co-infected with multiple viruses. Genetically identical (isogenic) fungal lines that are infected by and not infected by viruses must be created and compared. Here, we study an isolate of Ceratobasidium sp., a fungus isolated from pelotons in roots of a wild terrestrial orchid. The fungal isolate was co-infected with three distinct endornaviruses, isolates of Ceratobasidium endonarvirus B (CbEVB), Ceratobasidium endonarvirus C (CbEVC) and Ceratobasidium endonarvirus D (CbEVD). An experiment to reveal natural distribution of the three mycoviruses within a fungal colony revealed no sectoring; they were all evenly distributed throughout the colony. Hyphal tipping and treatments with one of five antibiotics (kanamycin, streptomycin, cycloheximide, rifampicin and ampicillin) were applied in attempts to 'cure' fungal lines of one, two or three of the viruses present. Surprisingly, the three mycoviruses responded differentially to each curing approach. The isolate of CbEVC was eliminated upon treatment with cycloheximide, but not with kanamycin or streptomycin, whereas the isolate of CbEVD did not respond to cycloheximide. The isolate of CbEVB was eliminated upon all treatments. In some cases, a virus was undetectable by species-specific RT-PCR assay after treatment, but when the fungus was cultured for a period on non-selective medium, the virus was detected again. Effects of mycoviruses on growth characteristics of isogenic fungal lines on two nutrient media were studied. Co-infection by the three viruses reduced mycelial growth rate on both media. In contrast, some fungal lines infected with one or two mycoviruses grew more rapidly than virus-free lines.

Extreme Diversity of Mycoviruses Present in Isolates of Rhizoctonia solani AG2-2 LP From Zoysia japonica From Brazil.

Zoysia japonica, in Brazil, is commonly infected by Rhizoctonia solani (R. solani) in humid and cool weather conditions. Eight isolates of R. solani, previously identified as belonging to the AG2-2 LP anastomosis group, isolated from samples from large path symptoms, were collected from three counties in São Paulo state (Brazil) and investigated for the presence of mycoviruses. After detection of double-strand RNA (dsRNA) in all samples, RNA_Seq analysis of ribosomal RNA-depleted total RNA from in vitro cultivated mycelia was performed. Forty-seven partial or complete viral unique RNA dependent-RNA polymerase (RdRp) sequences were obtained with a high prevalence of positive sense ssRNA viruses. Sequences were sufficiently different from the first match in BLAST searches suggesting that they all qualify as possible new viral species, except for one sequence showing an almost complete match with Rhizoctonia solani dsRNA virus 2, an alphapartitivirus. Surprisingly four large contigs of putative viral RNA could not be assigned to any existing clade of viruses present in the databases, but no DNA was detected corresponding to these fragments confirming their viral replicative nature. This is the first report on the occurrence of mycoviruses in R. solani AG2-2 LP in South America.

Symptomatic plant viroid infections in phytopathogenic fungi.

Viroids are pathogenic agents that have a small, circular noncoding RNA genome. They have been found only in plant species; therefore, their infectivity and pathogenicity in other organisms remain largely unexplored. In this study, we investigate whether plant viroids can replicate and induce symptoms in filamentous fungi. Seven plant viroids representing viroid groups that replicate in either the nucleus or chloroplast of plant cells were inoculated to three plant pathogenic fungi, Cryphonectria parasitica, Valsa mali, and Fusarium graminearum By transfection of fungal spheroplasts with viroid RNA transcripts, each of the three, hop stunt viroid (HSVd), iresine 1 viroid, and avocado sunblotch viroid, can stably replicate in at least one of those fungi. The viroids are horizontally transmitted through hyphal anastomosis and vertically through conidia. HSVd infection severely debilitates the growth of V. mali but not that of the other two fungi, while in F. graminearum and C. parasitica, with deletion of dicer-like genes, the primary components of the RNA-silencing pathway, HSVd accumulation increases. We further demonstrate that HSVd can be bidirectionally transferred between F. graminearum and plants during infection. The viroids also efficiently infect fungi and induce disease symptoms when the viroid RNAs are exogenously applied to the fungal mycelia. These findings enhance our understanding of viroid replication, host range, and pathogenicity, and of their potential spread to other organisms in nature.

Viromes in Xylariaceae fungi infecting avocado in Spain.

Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem.

Transcriptional and Small RNA Responses of the White Mold Fungus Sclerotinia sclerotiorum to Infection by a Virulence-Attenuating Hypovirus.

Mycoviruses belonging to the family Hypoviridae cause persistent infection of many different host fungi. We previously determined that the white mold fungus, Sclerotiniasclerotiorum, infected with Sclerotinia sclerotiorum hypovirus 2-L (SsHV2-L) exhibits reduced virulence, delayed/reduced sclerotial formation, and enhanced production of aerial mycelia. To gain better insight into the cellular basis for these changes, we characterized changes in mRNA and small RNA (sRNA) accumulation in S.sclerotiorum to infection by SsHV2-L. A total of 958 mRNAs and 835 sRNA-producing loci were altered after infection by SsHV2-L, among which >100 mRNAs were predicted to encode proteins involved in the metabolism and trafficking of carbohydrates and lipids. Both S. sclerotiorum endogenous and virus-derived sRNAs were predominantly 22 nt in length suggesting one dicer-like enzyme cleaves both. Novel classes of endogenous small RNAs were predicted, including phasiRNAs and tRNA-derived small RNAs. Moreover, S. sclerotiorum phasiRNAs, which were derived from noncoding RNAs and have the potential to regulate mRNA abundance in trans, showed differential accumulation due to virus infection. tRNA fragments did not accumulate differentially after hypovirus infection. Hence, in-depth analysis showed that infection of S. sclerotiorum by a hypovirulence-inducing hypovirus produced selective, large-scale reprogramming of mRNA and sRNA production.

Differential Contribution of RNA Interference Components in Response to Distinct Fusarium graminearum Virus Infections.

The mechanisms of RNA interference (RNAi) as a defense response against viruses remain unclear in many plant-pathogenic fungi. In this study, we used reverse genetics and virus-derived small RNA profiling to investigate the contributions of RNAi components to the antiviral response against Fusarium graminearum viruses 1 to 3 (FgV1, -2, and -3). Real-time reverse transcription-quantitative PCR (qRT-PCR) indicated that infection of Fusarium graminearum by FgV1, -2, or -3 differentially induces the gene expression of RNAi components in F. graminearum Transcripts of the DICER-2 and AGO-1 genes of F. graminearum (FgDICER-2 and FgAGO-1) accumulated at lower levels following FgV1 infection than following FgV2 or FgV3 infection. We constructed gene disruption and overexpression mutants for each of the Argonaute and dicer genes and for two RNA-dependent RNA polymerase (RdRP) genes and generated virus-infected strains of each mutant. Interestingly, mycelial growth was significantly faster for the FgV1-infected FgAGO-1 overexpression mutant than for the FgV1-infected wild type, while neither FgV2 nor FgV3 infection altered the colony morphology of the gene deletion and overexpression mutants. FgV1 RNA accumulation was significantly decreased in the FgAGO-1 overexpression mutant. Furthermore, the levels of induction of FgAGO-1, FgDICER-2, and some of the FgRdRP genes caused by FgV2 and FgV3 infection were similar to those caused by hairpin RNA-induced gene silencing. Using small RNA sequencing analysis, we documented different patterns of virus-derived small interfering RNA (vsiRNA) production in strains infected with FgV1, -2, and -3. Our results suggest that the Argonaute protein encoded by FgAGO-1 is required for RNAi in F. graminearum, that FgAGO-1 induction differs in response to FgV1, -2, and -3, and that FgAGO-1 might contribute to the accumulation of vsiRNAs in FgV1-infected F. graminearumIMPORTANCE To increase our understanding of how RNAi components in Fusarium graminearum react to mycovirus infections, we characterized the role(s) of RNAi components involved in the antiviral defense response against Fusarium graminearum viruses (FgVs). We observed differences in the levels of induction of RNA silencing-related genes, including FgDICER-2 and FgAGO-1, in response to infection by three different FgVs. FgAGO-1 can efficiently induce a robust RNAi response against FgV1 infection, but FgDICER genes might be relatively redundant to FgAGO-1 with respect to antiviral defense. However, the contribution of this gene in the response to the other FgV infections might be small. Compared to previous studies of Cryphonectria parasitica, which showed dicer-like protein 2 and Argonaute-like protein 2 to be important in antiviral RNA silencing, our results showed that F. graminearum developed a more complex and robust RNA silencing system against mycoviruses and that FgDICER-1 and FgDICER-2 and FgAGO-1 and FgAGO-2 had redundant roles in antiviral RNA silencing.

Heterobasidion Partitivirus 13 Mediates Severe Growth Debilitation and Major Alterations in the Gene Expression of a Fungal Forest Pathogen.

The fungal genus Heterobasidion includes some of the most devastating conifer pathogens in the boreal forest region. In this study, we showed that the alphapartitivirus Heterobasidion partitivirus 13 from Heterobasidion annosum (HetPV13-an1) is the main causal agent of severe phenotypic debilitation in the host fungus. Based on RNA sequencing using isogenic virus-infected and cured fungal strains, HetPV13-an1 affected the transcription of 683 genes, of which 60% were downregulated and 40% upregulated. Alterations observed in carbohydrate and amino acid metabolism suggest that the virus causes a state of starvation, which is compensated for by alternative synthesis routes. We used dual cultures to transmit HetPV13-an1 into new strains of H. annosum and Heterobasidion parviporum The three strains of H. parviporum that acquired the virus showed noticeable growth reduction on rich culturing medium, while only two of six H. annosum isolates tested showed significant debilitation. Based on reverse transcription-quantitative PCR (RT-qPCR) analysis, the response toward HetPV13-an1 infection was somewhat different in H. annosum and H. parviporum We assessed the effects of HetPV13-an1 on the wood colonization efficacy of H. parviporum in a field experiment where 46 Norway spruce trees were inoculated with isogenic strains with or without the virus. The virus-infected H. parviporum strain showed considerably less growth within living trees than the isolate without HetPV13-an1, indicating that the virus also causes growth debilitation in natural substrates.IMPORTANCE A biocontrol method restricting the spread of Heterobasidion species would be highly beneficial to forestry, as these fungi are difficult to eradicate from diseased forest stands and cause approximate annual losses of €800 million in Europe. We used virus curing and reintroduction experiments and RNA sequencing to show that the alphapartitivirus HetPV13-an1 affects many basic cellular functions of the white rot wood decay fungus Heterobasidion annosum, which results in aberrant hyphal morphology and a low growth rate. Dual fungal cultures were used to introduce HetPV13-an1 into a new host species, Heterobasidion parviporum, and field experiments confirmed the capability of the virus to reduce the growth of H. parviporum in living spruce wood. Taken together, our results suggest that HetPV13-an1 shows potential for the development of a future biocontrol agent against Heterobasidion fungi.

Identification of Diverse Mycoviruses through Metatranscriptomics Characterization of the Viromes of Five Major Fungal Plant Pathogens.

UNLABELLED: Mycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant-pathogenic fungi, Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembled de novo, and contigs with predicted amino acid sequence similarities to viruses in the nonredundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages: Barnaviridae, Benyviridae, Chrysoviridae, Endornaviridae, Fusariviridae, Hypoviridae, Mononegavirales, Narnaviridae, Ophioviridae, Ourmiavirus, Partitiviridae, Tombusviridae, Totiviridae, Tymoviridae, and Virgaviridae More than half of the viral sequences were predicted to be members of the Mitovirus genus in the family Narnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae, Ophioviridae, and Virgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts. IMPORTANCE: Plant-pathogenic fungi reduce crop yields, which affects food security worldwide. Plant host resistance is considered a sustainable disease management option but may often be incomplete or lacking for some crops to certain fungal pathogens or strains. In addition, the rising issues of fungicide resistance demand alternative strategies to reduce the negative impacts of fungal pathogens. Those fungus-infecting viruses (mycoviruses) that attenuate fungal virulence may be welcome additions for mitigation of plant diseases. By high-throughput sequencing of the RNAs from 275 isolates of five fungal plant pathogens, 66 previously undescribed mycoviruses were identified. In addition to identifying new potential biological control agents, these results expand the grand view of the diversity of mycoviruses and provide possible insights into the importance of intracellular and extracellular transmission in fungus-virus coevolution.

Double-stranded RNA mycovirus infection of Aspergillus fumigatus is not dependent on the genetic make-up of the host.

Aspergillus fumigatus is a fungus that causes opportunistic infections in immunocompromised patients, with high morbidity and mortality. In its turn, A. fumigatus can become infected with mycoviruses. Most mycoviruses have a dsRNA genome and can cause fungal hypovirulence. For that reason, mycoviruses could theoretically be used as therapeutic tools to combat fungal infections. We determined if a certain genetic make-up of A. fumigatus was associated with the presence of mycoviruses in 86 clinical A. fumigatus isolates. Mycovirus screening was performed by isolating dsRNA from mycelial cultures using a Trizol/Chloroform method. The genetic relatedness of dsRNA infected A. fumigatus was determined by cell surface protein (CSP) typing and determination of the mating type. Sixteen (18.6%) of the 86 clinical A. fumigatus isolates contained dsRNA. The A. fumigatus collection could be divided into 11 different CSP types. DsRNA infected A. fumigatus isolates had similar CSP types as non-infected isolates. In both cases, the CSP types t01, t02, t03 and t04 were the most prevalent and the distribution comparable to the CSP types observed in other Dutch collections. Mating types MAT1-1 and MAT1-2 were evenly distributed among all A. fumigatus strains, regardless of CSP type. No difference was observed in mycovirus infections between MAT1-1 and MAT1-2 isolates. DsRNA mycovirus infections in A. fumigatus are not related to either CSP or mating type and therefore represent an interesting future therapeutic tool to combat fungal infections.

Virus community dynamics in the conifer pathogenic fungus Heterobasidion parviporum following an artificial introduction of a partitivirus.

Viruses infecting the conifer pathogenic fungus Heterobasidion annosum sensu lato are intracellular and spread via anastomosis contacts. In the laboratory, these viruses transmit readily even between somatically incompatible isolates, but their dispersal capacity in natural conditions has not been previously studied. We introduced a mycovirus to a heavily diseased forest site by inoculating Norway spruce stumps with heartrot decay using a mycelial suspension of Heterobasidion parviporum strain RT3.49C hosting the partitivirus strain HetRV4-pa1. The Heterobasidion population at the sample plot was screened for mycoviruses prior to and after the inoculation. Based on sequence analysis, the resident H. parviporum strains harbored six different strains of the virus species Heterobasidion RNA virus 6 (HetRV6) and one strain of HetRV4 prior to the inoculation. After three growth seasons, the inoculated H. parviporum host strain was not detected, but the introduced virus had infected two resident H. parviporum genets. The presence of a preexisting HetRV6 infection did not hinder spread of the introduced partitivirus but resulted in coinfections instead. The resident HetRV6 virus population seemed to be highly stable during the incubation period, while the single indigenous HetRV4 infection was not detected after the inoculation. In laboratory infection experiments, the introduced virus could be transmitted successfully into all of the resident H. parviporum genets. This study shows for the first time transmission of a Heterobasidion virus between somatically incompatible hosts in natural conditions.

A spectroscopy approach to the study of virus infection in the endophytic fungus Epichloë festucae.

BACKGROUND: In this work we propose a rapid method based on visible and near-infrared (Vis-NIR) spectroscopy to determine the occurrence of double-stranded RNA (dsRNA) viruses in Epichloë festucae strains isolated from Festuca rubra plants. In addition, we examined the incidence of infections by E. festucae in populations of F. rubra collected in natural grasslands of Western Spain. METHODS: Vis-NIR spectra (400-2498 nm) from 124 virus-infected and virus-free E. festucae isolates were recorded directly from ground and freeze-dried mycelium. To estimate how well the spectra for uninfected and infected fungal samples could be differentiated, we used partial least-squares discriminant analysis (PLS1-DA) and several data pre-treatments to develop calibration models. RESULTS: Applying the best regression model, obtained with two sampling years and using standard normal variate (SNV) combined with first derivative transformation to a new validating data set (42 samples), we obtained a correct classification for 75% of the uninfected isolates and up to 86% of the infected isolates. CONCLUSIONS: The results obtained suggest that Vis-NIR spectroscopy is a promising technology for detection of viral infections in fungal samples when an alternative faster approach is desirable. It provides a tool adequately exact and more time- and cost-saving than the conventional reference analysis.

Genomic characterization of a novel dsRNA virus detected in the phytopathogenic fungus Verticillium dahliae Kleb.

Four novel double-stranded RNA segments were detected in a Verticillium dahliae Kleb. strain (V. dahliae isolate 0-21), a causal fungal agent of Verticillium wilt disease of cotton. Each dsRNA genome segment contains a single large open reading frame (ORF) that encodes a distinctive protein with modest levels of sequence similarities to the corresponding putative proteins in the genus Chrysovirus. These include an RNA-dependent RNA polymerase (RdRp), a coat protein, an undefined replication-related protein and an ovarian tumor domain peptidase. Phylogenetic analysis of the four putative proteins unanimously indicated that they are evolutionarily related to viruses in Chrysovirus. The 5'- and 3'-untranslated regions of the four dsRNAs share highly similar internal sequence and contain conserved sequence stretches of UGAUAAAAAA(/U)UG(/U)AAAAA- (in the 5'-UTR) and -UUUACUACU (in the 3'-UTR), indicating that they have a common virus origin. Indeed, isometric virus-like particles (VLPs) with a diameter of approximately 34nm were extracted from the fungal mycelia, and the four dsRNA segments were also detected in the virus-like particle (VLP) fraction. These results suggest that the mycovirus with four different dsRNA genome segments from the fungal isolate 0-21 is a new member of the genus Chrysovirus. We named the virus Verticillium dahliae chrysovirus 1 (VdCV1).

A novel colony-print immunoassay reveals differential patterns of distribution and horizontal transmission of four unrelated mycoviruses in Rosellinia necatrix.

A colony-print immunoassay (CPIA) using an anti-dsRNA antibody was developed to visualize the distribution of four unrelated mycoviruses with dsRNA genomes, a partitivirus (RnPV1), mycoreovirus (RnMyRV3), megabirnavirus (RnMBV1), and an unidentified virus (RnQV1), in mycelia of the white root rot fungus, Rosellinia necatrix. CPIA revealed different distribution patterns within single colonies for each virus. Both RnPV1 and RnMBV1 were distributed throughout single colonies, RnMyRV3 was absent from some colony sectors, and RnQV1 exhibited varied accumulation levels between sectors. RnMyRV3 and RnQV1 were transmitted to the recipient virus-free colonies of virus-infected and virus-free colony pairs more slowly than were RnPV1 or RnMBV1. The presence of RnMyRV3 in recipient colonies restricted horizontal transmission of RnPV1 and RnMBV1. These results imply that one or more mechanisms are present in host-virus and virus-virus interactions that restrict the spread of viruses within and between colonies.