First detection of mycoviruses in Gnomoniopsis castaneae suggests a putative horizontal gene transfer event between negative-sense and double-strand RNA viruses.

Gnomoniopsis castaneae is an ascomycetous fungus mainly known as a major pathogen of chestnut causing nut rots, although it is often found as an endophyte in chestnut tissues. To date, no virus has been reported as associated with to this fungus. Here, a collection of G. castaneae isolates from several European countries was screened to detect mycoviruses infecting the fungus: for the first time we report the identification and prevalence of mitovirus Gnomoniopsis castaneae mitovirus 1 (GcMV1) and the chrysovirus Gnomoniopsis castaneae chrysovirus 1 (GcCV1). Interestingly, we provide evidence supporting a putative horizontal gene transfer between members of the phyla Negarnaviricota and Duplornaviricota: a small putative protein of unknown function encoded on the RNA3 of GcCV1 (Chrysoviridae) has homologs in the genome of viruses of the family Mymonaviridae.

The Interaction between Hypovirulence-Associated Chrysoviruses and Their Host Fusarium Species.

Chrysoviruses are isometric virus particles (35-50 nm in diameter) with a genome composed of double-stranded RNAs (dsRNA). These viruses belonged to the Chrysoviridae family, named after the first member isolated from Penicillium chrysogenum. Phylogenetic classification has divided the chrysoviruses into Alphachrysovirus and Betachrysovirus genera. Currently, these chrysoviruses have been found to infect many fungi, including Fusarium species, and cause changes in the phenotype and decline in the pathogenicity of the host. Thus, it is a microbial resource with great biocontrol potential against Fusarium species, causing destructive plant diseases and substantial economic losses. This review provides a comprehensive overview of three chrysovirus isolates (Fusarium graminearum virus 2 (FgV2), Fusarium graminearum virus-ch9 (FgV-ch9), and Fusarium oxysporum f. sp. dianthi mycovirus 1 (FodV1)) reported to decline the pathogenicity of Fusarium hosts. It also summarizes the recent studies on host response regulation, host RNA interference, and chrysovirus transmission. The information provided in the review will be a reference for analyzing the interaction of Fusarium species with chrysovirus and proposing opportunities for research on the biocontrol of Fusarium diseases. Finally, we present reasons for conducting further studies on exploring the interaction between chrysoviruses and Fusarium and improving the accumulation and transmission efficiency of these chrysoviruses.

Co-infection of Aspergillus ochraceopetaliformis strain RCEF7483 by a novel chrysovirus and a known partitivirus.

An airborne microflora isolate, Aspergillus ochraceopetaliformis RCEF7483, was found to harbor seven dsRNA elements, indicating co-infection with a novel chrysovirus and a known partitivirus. Sequence analysis and RT-PCR confirmed dsRNA5-7 as components of Aspergillus ochraceous virus (AOV), a member of the Partitiviridae family. In light of its distinct host, we have designated it Aspergillus ochraceopetaliformis partitivirus 1 (AoPV1). The dsRNA segments, named dsRNA1-4, with lengths of 3706 bp, 3410 bp, 3190 bp, and 3158 bp, respectively, constitute the genome of a novel chrysovirus designated Aspergillus ochraceopetaliformis chrysovirus 1 (AoCV1). The dsRNA1-4 segments contain five open-reading frames (ORF1-5). Specifically, ORF1 encodes a putative RNA-dependent RNA polymerase (RdRp) with a length of 1112 amino acids, and ORF2 encodes a putative coat protein (CP) spanning 976 amino acids. Additionally, ORF3-5 encode hypothetical proteins (HP1, HP2, and HP3) with lengths of 108, 843, and 914 amino acids, respectively. Comparative analysis revealed the highest similarity of dsRNA1-4 with corresponding proteins in Aspergillus terreus chrysovirus 1 (AtCV1) (RdRp, 66.58%; CP, 51.02%; HP2, 61.80%; and HP3, 41.30%). Due to falling below the threshold for a new species in the Chrysoviridae, we propose that dsRNA1-4 in A. ochraceopetaliformis strain RCEF7483 constitute the novel chrysovirus AoCV1. Moreover, phylogenetic analysis using RdRp amino acid sequences placed AoCV1 within the Alphachrysovirus genus of the Chrysoviridae family, clustering with AtCV1 and other alphachrysoviruses. Our study contributes to the understanding of mycoviruses in A. ochraceopetaliformis and expands our knowledge of the diversity and evolution of chrysoviruses in fungal hosts.

A GFP-expressing minigenome of a chrysovirus replicating in fungi.

The Fusarium graminearum virus China 9 (FgV-ch9) is a member of the genus Betachrysovirus in the Chrysoviridae family and causes hypovirulence in its host, Fusarium graminearum, the causal agent of Fusarium head blight. Although insights into viral biology of FgV-ch9 have expanded in recent years, questions regarding the function of virus-encoded proteins, cis-acting elements, and virus transmission are yet to be answered. Therefore, we developed a tool for the establishment of an artificial 6th segment of FgV-ch9, which encodes a GFP gene flanked by the non-translated regions of FgV-ch9 segment 1. Subsequently, we have proved successful encapsidation of this artificial segment into virus particles as well as its horizontal transmission. Expression of GFP was further verified via immunoassay and life cell imaging. Thus far, we were able to establish for the first time a mini-replicon system for segmented dsRNA viruses replicating in fungi.

A virus from Aspergillus cibarius with features of alpha- and betachrysoviruses.

From the ascomycete Aspergillus cibarius strain NW-FVA 2590, which was originally isolated from a root, associated with stem collar necrosis of Fraxinus excelsior L., a novel virus was isolated and characterized. Its genome is encoded on three monocistronic dsRNA segments ranging from 3683 bp (dsRNA 1) over 3093 (dsRNA 2) to 2902 bp (dsRNA 3), which are packed in isometric particles of around 35 nm. While the viral RdRp (P1) is encoded on segment 1, protein sequencing showed that two more structural proteins are present which are translated from dsRNA 2 (P2) and dsRNA 3 (P3) and possibly form the viral capsid. Additionally, P2 and P3 may undergo posttranslational modifications since the detected proteins bands deviated from the calculated sizes. Due to its phylogenetic position, the novel virus was grouped in the family of Chrysoviridae and was tentatively denominated as Aspergillus cibarius chrysovirus 1 (AcCV1). Due to its composition, biological properties and phylogenetic position, distant from the genera Alphachrysovirus and Betachrysovirus, we suggest to position AcCV1 in a proposed genus "Gammachrysovirus".

Detection of the Xanthi Chryso-like Virus in New Geographical Area and a Novel Arthropod Carrier.

Here, we report on a serendipitous finding of a chryso-like virus associated with Culex pipiens mosquitos in the course of study aimed to detect and characterize West Nile virus (WNV) circulating in mosquitos in Serbia, Southern Europe. Upon initial detection of unexpected product in a PCR protocol for partial WNV NS5 gene amplification, further confirmation and identification was obtained through additional PCR and Sanger sequencing experiments. Bioinformatic and phylogenetic analysis identified the obtained sequences as Xanthi chryso-like virus (XCLV). The finding is particular for the fact that it associates XCLV with a new potential vector species and documents a novel geographical area of its distribution.

Molecular and biological characteristics of a novel chrysovirus infecting the fungus phytopathogenic Setosphaeria turcica f.sp. sorghi.

A new double-stranded RNA (dsRNA) virus has been identified in the filamentous fungus Setosphaeria turcica f.sp. sorghi, whose genome consists of four segments (dsRNA1-4). Each dsRNA carries single open reading frame (ORF) flanked by 5' and 3' untranslated regions (UTRs) containing strictly conserved termini. The putative protein encoded by dsRNA1 showed 80.50% identity to the RNA-dependent RNA polymerase (RdRp) of the most closely related virus, Alternaria alternata chrysovirus 1 (AaCV1), belonging to the Chrysoviridae. dsRNA2 encodes the putative coat protein, while dsRNA3 and dsRNA4 respectively encode the hypothetical proteins of unknown functions. Phylogenetic analysis based on the RdRp protein indicated the virus clustered with members of the genus Betachrysovirus in the family Chrysoviridae. Based on the dsRNA profile, amino acid sequence comparisons, and phylogenetic analyses, the mycovirus is thought to be a new member of the family Chrysoviridae and designated as Setosphaeria turcica chrysovirus 1 (StCV1). Moreover, obvious differences were observed in the colony, mycelial and spore morphology between StCV1-infected and virus-cured strains of S. turcica f.sp. sorghi. StCV1 infection strongly reduced colony growth rate, spore production ability and virulence on host fungus. To our knowledge, this is the first report about mycovirus infecting S. turcica f.sp. sorghi and also the first chrysovirus infecting S. turcica.

Cryphonectria nitschkei chrysovirus 1 with unique molecular features and a very narrow host range.

Cryphonectria nitschkei chrysovirus 1 (CnCV1), was described earlier from an ascomycetous fungus, Cryphonectria nitschkei strain OB5/11, collected in Japan; its partial sequence was reported a decade ago. Complete sequencing of the four genomic dsRNA segments revealed molecular features similar to but distinct from previously reported members of the family Chrysoviridae. Unique features include the presence of a mini-cistron preceding the major large open reading frame in each genomic segment. Common features include the presence of CAA repeats in the 5'-untranslated regions and conserved terminal sequences. CnCV1-OB5/11 could be laterally transferred to C. nitschkei and its relatives C. radicalis and C. naterciae via coculturing, virion transfection and protoplast fusion, but not to fungal species other than the three species mentioned above, even within the genus Cryphonectria, suggesting a very narrow host range. Phenotypic comparison of a few sets of CnCV1-infected and -free isogenic strains showed symptomless infection in new hosts.

Population Structure of Double-Stranded RNA Mycoviruses That Infect the Rice Blast Fungus Magnaporthe oryzae in Japan.

Various viruses infect Magnaporthe oryzae (syn. Pyricularia oryzae), which is a well-studied fungus that causes rice blast disease. Most research has focused on the discovery of new viruses and the hypovirulence-associated traits conferred by them. Therefore, the diversity and prevalence of viruses in wild fungal populations have not been explored. We conducted a comprehensive screening of M. oryzae mycoviruses from various regions in Japan using double-stranded RNA (dsRNA) electrophoresis and RT-PCR assays. We detected three mycoviruses, Magnaporthe oryzae virus 2 (MoV2), Magnaporthe oryzae chrysovirus 1 (MoCV1), and Magnaporthe oryzae partitivirus 1 (MoPV1), among 127 of the 194 M. oryzae strains screened. The most prevalent virus was MoPV1 (58.8%), which often co-infected in a single fungal strain together with MoV2 or MoCV1. MoV2 and MoCV1 were found in 22.7 and 10.8% of strains, respectively, and they were usually distributed in different regions so that mixed-infection with these two mycoviruses was extremely rare. The predominance of MoPV1 in M. oryzae is supported by significant negative values from neutrality tests, which indicate that the population size of MoPV1 tends to increase. Population genetic analyses revealed high nucleotide diversity and the presence of phylogenetically diverse subpopulations among the MoV2 isolates. This was not the case for MoPV1. Furthermore, studies of a virus-cured M. oryzae strain revealed that MoV2 does not cause any abnormalities or symptoms in its host. However, a leaf sheath inoculation assay showed that its presence slightly increased the speed of mycelial growth, compared with virus-free mycelia. These results demonstrate that M. oryzae in Japan harbors diverse dsRNA mycovirus communities with wide variations in their population structures among different viruses.

Molecular and biological characterization of a novel strain of Alternaria alternata chrysovirus 1 identified from the pathogen Alternaria tenuissima causing watermelon leaf blight.

The leaf blight caused by the genus Alternaria is one of the most epidemic diseases on watermelon, and A. tenuissima is the dominant pathogenic species in China. Mycoviruses are found ubiquitously in filamentous fungi, and an increasing number of novel mycoviruses infecting the genus Alternaria have been reported. In this study, a mycovirus from A. tenuissima strain SD-BZF-12 was identified and characterized, whose genome size was very similar with Alternaria alternata chrysovirus 1-N18 (AaCV1-N18). The dsRNA1- and dsRNA2-encoded proteins of the virus had 99 % identities with counterparts of AaCV1-N18; and the dsRNA3- and dsRNA4-encoded proteins of the virus showed the 80 % and 94 % sequence identities with proteins deduced from dsRNA4 and dsRNA3 of AaCV1-N18, respectively. Intriguingly, dsRNA5 of the virus encoded a truncated protein with 68 amino acids (aa) by comparing with 115 aa of AaCV1-N18 dsRNA5. Phylogenetic analysis of RNA-dependent RNA polymerase domain suggested that the virus clustered together with AaCV1-N18. Based on these characteristics, the mycovirus was identified to be a novel strain of AaCV1 and designated as AaCV1-AT1. In addition, no obvious differences were observed on colony morphology between AaCV1-AT1-infected and virus-cured strains of A. tenuissima; however, AaCV1-AT1 infection reduced colony growth rate and spore production ability on host fungus, and increased the median effective concentration of difenoconazole or tebuconazole on its host. This is the first report of AaCV1-AT1 associated with A. tenuissima.

Chrysoviruses Inhabited Symbiotic Fungi of Lichens.

A lichen body is formed most often from green alga cells trapped in a net of ascomycetous fungi and accompanied by endolichenic or parasitic fungi, other algae, and symbiotic or free-living bacteria. The lichen's microcosmos is inhabited by mites, insects, and other animals for which the lichen is a source of food or a place to live. Novel, four-segmented dsRNA viruses were detected in saxicolous Chrysothrixchlorina and Leprariaincana lichens. Comparison of encoded genome proteins revealed classification of the viruses to the genus Alphachrysovirus and a relationship to chrysoviruses from filamentous ascomycetous fungi. We propose the names Chrysothrix chrysovirus 1 (CcCV1) and Lepraria chrysovirus 1 (LiCV1) as acronyms for these viruses. Surprisingly, observation of Chrysothrixchlorina hybridization with fluorescent-labelled virus probe by confocal microscope revealed that the CcCV1 virus is not present in the lichen body-forming fungus but in accompanying endolichenic Penicilliumcitreosulfuratum fungus. These are the first descriptions of mycoviruses from a lichen environment.

Blue-White Colony Selection of Virus-Infected Isogenic Recipients Based on a Chrysovirus Isolated from Penicillium italicum.

Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum (P. italicum). In this study, we isolated and characterized a new double-stranded RNA (dsRNA) virus, designated Penicillium italicum chrysovirus 1 (PiCV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that PiCV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1ΔpksP defective in the polyketide synthase gene (pksP), which is involved in pigment biosynthesis, and these mutants formed albino (white) colonies. Then we applied hyphal anastomosis method to horizontally transmit PiCV1 from the white virus-donors (i.e., HSPi-YN1 mutants) to wild-type recipients (i.e., P. italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable PiCV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic dsRNA profile analysis. This blue-white colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized PiCV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen PiCV1-infected isogenic recipients.

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.

Molecular Characterization of a Chrysovirus Isolated From the Citrus Pathogen Penicillium crustosum and Related Fungicide Resistance Analysis.

Penicillium sp. are damaging to a range of foods and fruits including citrus. To date, double-stranded (ds)RNA viruses have been reported in most Penicillium species but not in citrus pathogen P. crustosum. Here we report a novel dsRNA virus, designated as Penicillium crustosum chrysovirus 1 (PcCV1) and isolated from P. crustosum strain HS-CQ15. PcCV1 genome comprises four dsRNA segments, referred to as dsRNA1, dsRNA2, dsRNA3, and dsRNA4, which are 3600, 3177, 3078, and 2808 bp in length, respectively. Sequence analysis revealed the presence of four open reading frames (ORFs) in the PcCV1 genome. ORF1 in dsRNA1 encodes a putative RNA-dependent RNA polymerase (RdRp) and ORF2 in dsRNA2 encodes a putative coat protein (CP). The two remaining ORFs, ORF3 in dsRNA3 and ORF4 in dsRNA4, encode proteins of unknown function. Phylogenetic analysis based on RdRp sequences showed that PcCV1 clusters with other members of the genus Chrysovirus, family Chrysoviridae. Transmission electron microscope (TEM) analysis revealed that the PcCV1 visions are approximately 40 nm in diameter. Regarding biological effects of PcCV1, HS-CQ15 harboring the chrysovirus exhibited no obvious difference in colony morphology under fungicide-free conditions but decreased resistance to demethylation inhibitor (DMI)-fungicide prochloraz, as compared to PcCV1-cured strain. Here we provide the first evidence of a virus present in citrus pathogenic fungus P. crustosum and the chrysovirus-induced change in fungicide-resistance of its host fungus.

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.

Comprehensive analysis of full genome sequence and Bd-milRNA/target mRNAs to discover the mechanism of hypovirulence in Botryosphaeria dothidea strains on pear infection with BdCV1 and BdPV1.

Pear ring rot disease, mainly caused by Botryosphaeria dothidea, is widespread in most pear and apple-growing regions. Mycoviruses are used for biocontrol, especially in fruit tree disease. BdCV1 (Botryosphaeria dothidea chrysovirus 1) and BdPV1 (Botryosphaeria dothidea partitivirus 1) influence the biological characteristics of B. dothidea strains. BdCV1 is a potential candidate for the control of fungal disease. Therefore, it is vital to explore interactions between B. dothidea and mycovirus to clarify the pathogenic mechanisms of B. dothidea and hypovirulence of B. dothidea in pear. A high-quality full-length genome sequence of the B. dothidea LW-Hubei isolate was obtained using Single Molecule Real-Time sequencing. It has high repeat sequence with 9.3% and DNA methylation existence in the genome. The 46.34 Mb genomes contained 14,091 predicted genes, which of 13,135 were annotated. B. dothidea was predicted to express 3833 secreted proteins. In bioinformatics analysis, 351 CAZy members, 552 transporters, 128 kinases, and 1096 proteins associated with plant-host interaction (PHI) were identified. RNA-silencing components including two endoribonuclease Dicer, four argonaute (Ago) and three RNA-dependent RNA polymerase (RdRp) molecules were identified and expressed in response to mycovirus infection. Horizontal transfer of the LW-C and LW-P strains indicated that BdCV1 induced host gene silencing in LW-C to suppress BdPV1 transmission. To investigate the role of RNA-silencing in B. dothidea defense, we constructed four small RNA libraries and sequenced B. dothidea micro-like RNAs (Bd-milRNAs) produced in response to BdCV1 and BdPV1 infection. Among these, 167 conserved and 68 candidate novel Bd-milRNAs were identified, of which 161 conserved and 20 novel Bd-milRNA were differentially expressed. WEGO analysis revealed involvement of the differentially expressed Bd-milRNA-targeted genes in metabolic process, catalytic activity, cell process and response to stress or stimulus. BdCV1 had a greater effect on the phenotype, virulence, conidiomata, vertical and horizontal transmission ability, and mycelia cellular structure biological characteristics of B. dothidea strains than BdPV1 and virus-free strains. The results obtained in this study indicate that mycovirus regulates biological processes in B. dothidea through the combined interaction of antiviral defense mediated by RNA-silencing and milRNA-mediated regulation of target gene mRNA expression.

Chrysoviruses in Magnaporthe oryzae.

Magnaporthe oryzae, the fungus that causes rice blast, is the most destructive pathogen of rice worldwide. A number of M. oryzae mycoviruses have been identified. These include Magnaporthe oryzae. viruses 1, 2, and 3 (MoV1, MoV2, and MoV3) belonging to the genus, Victorivirus, in the family, Totiviridae; Magnaporthe oryzae. partitivirus 1 (MoPV1) in the family, Partitiviridae; Magnaporthe oryzae. chrysovirus 1 strains A and B (MoCV1-A and MoCV1-B) belonging to cluster II of the family, Chrysoviridae; a mycovirus related to plant viruses of the family, Tombusviridae (Magnaporthe oryzae. virus A); and a (+)ssRNA mycovirus closely related to the ourmia-like viruses (Magnaporthe oryzae. ourmia-like virus 1). Among these, MoCV1-A and MoCV1-B were the first reported mycoviruses that cause hypovirulence traits in their host fungus, such as impaired growth, altered colony morphology, and reduced pigmentation. Recently we reported that, although MoCV1-A infection generally confers hypovirulence to fungi, it is also a driving force behind the development of physiological diversity, including pathogenic races. Another example of modulated pathogenicity caused by mycovirus infection is that of Alternaria alternata chrysovirus 1 (AaCV1), which is closely related to MoCV1-A. AaCV1 exhibits two contrasting effects: Impaired growth of the host fungus while rendering the host hypervirulent to the plant, through increased production of the host-specific AK-toxin. It is inferred that these mycoviruses might be epigenetic factors that cause changes in the pathogenicity of phytopathogenic fungi.

The Effect of Aspergillus Thermomutatus Chrysovirus 1 on the Biology of Three Aspergillus Species.

This study determined the effects of Aspergillus thermomutatus chrysovirus 1 (AthCV1), isolated from Aspergillus thermomutatus, on A. fumigatus, A. nidulans and A. niger. Protoplasts of virus-free isolates of A. fumigatus, A. nidulans and A. niger were transfected with purified AthCV1 particles and the phenotype, growth and sporulation of the isogenic AthCV1-free and AthCV1-infected lines assessed at 20 °C and 37 °C and gene expression data collected at 37 °C. AthCV1-free and AthCV1-infected A. fumigatus produced only conidia at both temperatures but more than ten-fold reduced compared to the AthCV1-infected line. Conidiation was also significantly reduced in infected lines of A. nidulans and A. niger at 37 °C. AthCV1-infected lines of A. thermomutatus and A. nidulans produced large numbers of ascospores at both temperatures, whereas the AthCV1-free line of the former did not produce ascospores. AthCV1-infected lines of all species developed sectoring phenotypes with sclerotia produced in aconidial sectors of A. niger at 37 °C. AthCV1 was detected in 18% of sclerotia produced by AthCV1-infected A. niger and 31% of ascospores from AthCV1-infected A. nidulans. Transcriptome analysis of the naturally AthCV1-infected A. thermomutatus and the three AthCV1-transfected Aspergillus species showed altered gene expression as a result of AthCV1-infection. The results demonstrate that AthCV1 can infect a range of Aspergillus species resulting in reduced sporulation, a potentially useful attribute for a biological control agent.

De novo transcriptomic assembly and mRNA expression patterns of Botryosphaeria dothidea infection with mycoviruses chrysovirus 1 (BdCV1) and partitivirus 1 (BdPV1).

BACKGROUND: Pear ring rot, caused by Botryosphaeria species, is responsible for substantial economic losses by causing severe recession of pear tree growth in China. Mycovirus-mediated hypovirulence in plant pathogenic fungi is a crucial biological method to control fungal diseases. METHODS: We conducted a large-scale and comprehensive transcriptome analysis to identify mRNA in B. dothidea in response to mycovirus. De novo sequencing technology from four constructed libraries of LW-C (Botryosphaeria dothidea chrysovirus 1, BdCV1), LW-P (Botryosphaeria dothidea partitivirus 1, BdPV1), LW-CP (LW-1 strain infection with BdCV1 and BdPV1), and Mock (free virus) was used to investigate and compare gene expression changes in B.dothidea strains infected with mycovirus. RESULTS: In total, 30,058 Unigenes with an average length of 2128 bp were obtained from 4 libraries of B. dothidea strains. These were annotated to specify their classified function. We demonstrate that mRNAs of B. dothidea strains in response to mycovirus are differentially expressed. In total, 5598 genes were up-regulated and 3298 were down-regulated in the LW-CP group, 4468 were up-regulated and 4291 down-regulated in the LW-C group, and 2590 were up-regulated and 2325 down-regulated in the LW-P group. RT-qPCR was used to validate the expression of 9 selected genes. The B. dothidea transcriptome was more affected by BdCV1 infection than BdPV1. We conducted GO enrichment analysis to characterize gene functions regulated by B. dothidea with mycovirus infection. These involved metabolic process, cellular process, catalytic activity, transporter activity, signaling, and other biological pathways. KEGG function analysis demonstrated that the enriched differentially expressed genes are involved in metabolism, transcription, signal transduction, and ABC transport. mRNA is therefore involved in the interaction between fungi and mycovirus. In addition, changes in differential accumulation levels of cp and RdRp of BdCV1 and BdPV1 in B. dothidea strains were evaluated, revealing that the accumulation of BdCV1 and BdPV1 is related to the phenotype and virulence of B. dothidea strain LW-1. CONCLUSIONS: The identification and analysis of mRNAs from B. dothidea was first reported at the transcriptome level. Our analysis provides further insight into the interaction of B. dothidea strains infection with chrysovirus 1 (BdCV1) and partitivirus 1 (BdPV1) at the transcriptome level.

Identification and Characterization of a Novel Hepta-Segmented dsRNA Virus From the Phytopathogenic Fungus Colletotrichum fructicola.

A novel hepta-segmented double-stranded RNA (dsRNA) virus was isolated and characterized from the strain FJ-4 of the phytopathogenic fungus Colletotrichum fructicola, and was named Colletotrichum fructicola chrysovirus 1 (CfCV1). The full-length cDNAs of dsRNA1-7 were 3620, 2801, 2687, 2437, 1750, 1536, and 1211 bp, respectively. The 5'- and 3'-untranslated regions of the seven dsRNAs share highly similar internal sequence and contain conserved sequence stretches, indicating that they have a common virus origin. The 5'-and 3'-UTRs of the seven dsRNAs were predicted to fold into stable stem-loop structures. CfCV1 contains spherical virions that are 35 nm in diameter consisting of seven segments. The largest dsRNA of CfCV1 encodes an RNA-dependent RNA polymerase (RdRp), and the second dsRNA encodes a viral capsid protein (CP). The dsRNA5 encodes a C2H2-type zinc finger protein containing an R-rich region and a G-rich region. The smallest dsRNA is a satellite-like RNA. The functions of the other proteins encoded by dsRNA3, dsRNA4, dsRNA6 are unknown. Phylogenetic analysis, based on RdRp and CP, indicated that CfCV1 is phylogenetically related to Botryosphaeria dothidea chrysovirus 1 (BdCV1), and Penicillium janczewskii chrysovirus 2 (PjCV2), a cluster of an independent cluster II group in the family Chrysoviridae. Importantly, all the seven segments of CfCV1 were transmitted successfully to other virus-free strains with an all-or-none fashion. CfCV1 exerts minor influence on the growth of C. fructicola but can confer hypovirulence to the fungal host. To our knowledge, this is the first report of a hepta-segmented tentative chrysovirus in C. fructicola.