Characterization of a Novel Mycovirus from the Phytopathogenic Fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is, globally, one of the most economically important phytopathogenic fungi worldwide, causing the canker and dieback of fruit trees. An increasing number of viruses infecting B. dothidea have lately been reported, several of which could confer hypovirulence. In this study, isolated from strain ZM170285-1 of B. dothidea, a novel double-stranded RNA (dsRNA) mycovirus, tentatively named Botryosphaeria dothidea partitivirus 2 (BdPV2), was identified well. The BdPV2 harbored three dsRNA segments (1-3) with lengths of 1751, 1568, and 1198 bp, which encoded an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), and a hypothetical protein of unknown function, respectively. BLASTp searches revealed that the predicted protein sequences of dsRNA1 and dsRNA2 had the highest identities (74.95% and 61.01%) with the corresponding dsRNAs of Penicillium stoloniferum virus S (PsV-S), whereas dsRNA3 shared the highest identity (32.95%) with the dsRNA3 of Aspergillus ochraceous virus 1 (AoV1). Phylogenetic analysis indicated that BdPV2 belonged to the Gammapartitivirus genus and Partitiviridae family. To our knowledge, this is the first report of a Gammapartitivirus in B. dothidea.

Nine viruses from eight lineages exhibiting new evolutionary modes that co-infect a hypovirulent phytopathogenic fungus.

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation.

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.

Magnaporthe oryzae chrysovirus 1 strain D confers growth inhibition to the host fungus and exhibits multiform viral structural proteins.

A Japanese isolate of Magnaporthe oryzae is infected by Magnaporthe oryzae chrysovirus 1-D (MoCV1-D), which is classified in cluster II of the family Chrysoviridae. The genome of MoCV1-D consists of five dsRNAs. dsRNAs 1-4 show high identity with those of related MoCV1 viruses, whereas dsRNA5 shows relatively low identity and is sometimes deleted during virus propagation. MoCV1-D causes growth inhibition of its host fungus, and the protein encoded by its dsRNA4 impairs cell growth when expressed in yeast cells. It also causes abnormal pigmentation and colony albinization, and we showed that these phenotypes are associated with reduced accumulation of the melanin biosynthesis intermediate scylatone. MoCV1-D exhibits multiform viral structural proteins during prolonged culture. The original host isolate is co-infected with MoCV1-D, a victorivirus, and a partitivirus, and these mycoviruses are detected in cell-free supernatant fractions after prolonged liquid culturing. Hyphal fusion experiments demonstrated that MoCV1-D is transmissible via anastomosis.

Alphapartitiviruses of Heterobasidion Wood Decay Fungi Affect Each Other's Transmission and Host Growth.

Heterobasidion spp. root rot fungi are highly destructive forest pathogens of the northern boreal forests, and are known to host a diverse community of partitiviruses. The transmission of these mycoviruses occurs horizontally among host strains via mycelial anastomoses. We revealed using dual cultures that virus transmission rates are affected by pre-existing virus infections among two strains of H. annosum. The transmission efficacy of mycovirus HetPV15-pa1 to a pre-infected host was elevated from zero to 50% by the presence of HetPV13-an1, and a double infection of these viruses in the donor resulted in an overall transmission rate of 90% to a partitivirus-free recipient. On contrary, pre-existing virus infections of two closely related strains of HetPV11 hindered each other's transmission, but had unexpectedly dissimilar effects on the transmission of more distantly related viruses. The co-infection of HetPV13-an1 and HetPV15-pa1 significantly reduced host growth, whereas double infections including HetPV11 strains had variable effects. Moreover, the results showed that RdRp transcripts are generally more abundant than capsid protein (CP) transcripts and the four different virus strains express unique transcripts ratios of RdRp and CP. Taken together, the results show that the interplay between co-infecting viruses and their host is extremely complex and highly unpredictable.

Mycovirus Fusarium oxysporum f. sp. dianthi Virus 1 Decreases the Colonizing Efficiency of Its Fungal Host.

Mycoviruses that induce hypovirulence in phytopathogenic fungi are interesting because their potential use as biological control agents of the plant diseases caused by their fungal hosts. The recently identified chrysovirus Fusarium oxysporum f. sp. dianthi virus 1 (FodV1) has been associated to the induction of hypovirulence in Fusarium oxysporum f. sp. dianthi, the forma specialis of F. oxysporum that causes vascular wilt in carnation (Dianthus caryophyllus). In this work, we have used confocal laser scanner microscopy and two isogenic GFP-labeled strains of F. oxysporum f. sp. dianthi infected (V+) and not infected (V-) with the Fusarium oxysporum f. sp. dianthi virus 1, respectively, to analyze the effect of mycovirus FodV1 on the plant colonization pattern of its fungal host. Results demonstrate that FodV1-viral infection affects the speed and spatial distribution of fungal colonization into the plant. Initial stages of external root colonization were similar for both strains, but the virus-free strain colonized the internal plant tissues faster than the virus-infected strain. In addition, other differences related to the specific zone colonized and the density of colonization were observed between both F. oxysporum f. sp. dianthi strains. The hyphae of both V- and V+ strains progressed up through the xylem vessels but differences in the number of vessels colonized and of hyphae inside them were found. Moreover, as colonization progressed, V- and V+ hyphae propagated horizontally reaching the central medulla but, while the virus-free strain V- densely colonized the interior of the medulla cells, the virus-infected strain V+ appeared mainly in the intercellular spaces and with a lower density of colonization. Finally, the incidence of FodV1-viral infections in a collection of 221 isolates sampled between 2008 and 2012 in the geographic area where the originally infected isolate was obtained has been also analyzed. The very low (<2%) incidence of viral infections is discussed here. To the best of our knowledge, this work provides the first microscopic evidence about the effect of a hypovirulence-inducing mycovirus on the pattern of plant colonization by its fungal host.

Mycovirus therapy for invasive pulmonary aspergillosis?

With the current revived interest in the use of bacteriophages for the treatment of bacterial infections, the study of mycoviruses as novel therapeutic solutions for invasive aspergillosis is the logical next step. Although ssRNA, dsRNA, and ssDNA mycoviruses have been identified, the majority of characterised mycoviruses have dsRNA genomes. Prevalence of dsRNA mycoviruses in Aspergillus spp. varies, and mycoviruses can have different effects on their fungal hosts: hypovirulence, hypervirulence, or a killer phenotype. Therapeutically, extracellular transmission of the mycovirus is essential. DsRNA mycoviruses lack an extracellular phase; however, a single ssDNA mycovirus with homologues in Aspergillus genomes has been described with an extracellular mode of transmission. Mycoviruses can induce hypovirulence or a killer phenotype, and both can be exploited therapeutically. Mycoviruses inducing hypovirulence have been used to control chestnut blight, however for aspergillosis no such mycovirus has been identified yet. Mycovirus encoded killer toxins or anti-idiotypic antibodies and killer peptides derived from these have been demonstrated to control fungal infections including aspergillosis in animals. This indicates that mycoviruses inducing both phenotypes could be exploited therapeutically as long as the right mycovirus has been identified.

Biology of viral satellites and their role in pathogenesis.

Extraviral components that can influence the accumulation and pathogenesis of their associated helper viruses are known as 'satellites'. The maintenance of satellites requires their ability to associate with their helper viruses. Satellites can be categorized as either satellite viruses or satellite nucleic acids based on their ability to encode capsid proteins. Understanding the biology of satellites is important since they are pathogenic to a wide range of plant, animal, and yeast organisms. Most satellites influence the pathogenesis of their helper viruses by altering the interaction between the host and helper virus. However, the molecular mechanism that governs the trilateral interaction between host, satellites, and helper virus remains largely unexplored. This review comprehensively describes details of the association and interaction of helper viruses with satellite viruses, satellite RNAs, and satellite DNAs, and their implications for pathogenesis.

Production of fumonisins by endophytic strains of Tolypocladium cylindrosporum and its relation to fungal virus infection.

Fumonisins were first discovered in Fusarium verticillioides, a fungus associated to disease and asymptomatic infections in maize. Afterwards, other fungal taxa have been found to produce fumonisins. The entomopathogenic ascomycete Tolypocladium cylindrosporum has been isolated from soil and also as an endophyte from leaves of grasses. The objectives of this work were to determine the in vitro production of fumonisin B (FB) mycotoxins and the immunosuppressive compound cyclosporine A (CyA) in several strains of T. cylindrosporum, and to examine the effect of fungal virus infection and temperature in FB production. FB1 was detected in 30% of the strains, ranging from 0.16 to 5.52 µg cm-2 in solid media, and FB2 was detected in 78% of the strains, ranging from 0.764 to 40.92 µg cm-2. CyA was not detected in any strain. The mean FB2 concentration of the endophytic strain Tc37W was three times greater (p < 0.05) than that of any other strain. Up to 34% more of FB2 was detected in strains infected by the virus TcV3 than in the corresponding virus-free versions. The effect of temperature on FB2 content was interactively significantly dependent on fungal strain and growth medium; in the YES medium, the FB2 of virus-infected strains Tc37-1V and Tc37W increased by 67 and 16%, respectively, at 26 °C as compared to 20 °C. The FB concentration in some fungal strains was similar to that in fungi associated to food and feed intoxications.

A capsidless ssRNA virus hosted by an unrelated dsRNA virus.

Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication(1). Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix(2). YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses(3), while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal totiviruses(4). Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+)ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus.

Generation of a high resolution map of sRNAs from Fusarium graminearum and analysis of responses to viral infection.

Previously, we characterized F. graminearum hypovirus 1 (FgHV1) and F. graminearum hypovirus 2 (FgHV2), which are the only two hypoviruses in F. graminearum that are closely related to Cryphonectria hypovirus 1 (CHV1) and Cryphonectria hypovirus 2 (CHV2) in the Hypoviridae family. In this study, we preliminarily elucidated the RNA silencing mechanism of the F. graminearum/hypovirus system from a small RNA (sRNA) perspective by using HiSeq deep sequencing. The length distributions of F. graminearum sRNA were altered by hypoviral infection. Potential microRNA-like (milRNA) candidates were differentially expressed between the hypovirus-free and hypovirus-infected library types. Extensive virus-derived small interfering RNAs (vsiRNAs) were also principally defined. The 1,831,081 and 3,254,758 total reads generated from the FgHV1 and FgHV2 genomes in F. graminearum yielded the first high-resolution sRNA maps of fungal viruses. In addition, extensive bioinformatics searches identified a large number of transcripts that are potentially targeted by vsiRNAs, several of which were effectively down-regulated. In particular, the RNA silencing-related genes FgDicer1 and FgRdRp5 were predicted targets of FgHV1- and FgHV2-derived siRNAs, possibly revealing a novel anti-RNA silencing strategy employed by mycoviruses.