A Novel Strain of Fusarium oxysporum Alternavirus 1 Isolated from Fusarium oxysporum f. sp. melonis Strain T-BJ17 Confers Hypovirulence and Increases the Sensitivity of Its Host Fungus to Difenoconazole and Pydiflumetofen.

In the current study, a novel strain of Fusarium oxysporum alternavirus 1 (FoAV1) was identified from the Fusarium oxysporum f. sp. melonis (FOM) strain T-BJ17 and was designated as Fusarium oxysporum alternavirus 1-FOM (FoAV1-FOM). Its genome consists of four dsRNA segments of 3515 bp (dsRNA1), 2663 bp (dsRNA2), 2368 bp (dsRNA3), and 1776 bp (dsRNA4) in length. Open reading frame 1 (ORF1) in dsRNA1 was found to encode a putative RNA-dependent RNA polymerase (RdRp), whose amino acid sequence was 99.02% identical to that of its counterpart in FoAV1; while ORF2 in dsRNA2, ORF3 in dsRNA3, and ORF4 in dsRNA4 were all found to encode hypothetical proteins. Strain T-BJ17-VF, which was verified to FoAV1-FOM-free, was obtained using single-hyphal-tip culture combined with high-temperature treatment to eliminate FoAV1-FOM from strain T-BJ17. The colony growth rate, ability to produce spores, and virulence of strain T-BJ17 were significantly lower than those of T-BJ17-VF, while the dry weight of the mycelial biomass and the sensitivity to difenoconazole and pydiflumetofen of strain T-BJ17 were greater than those of T-BJ17-VF. FoAV1-FOM was capable of 100% vertical transmission via spores. To our knowledge, this is the first time that an alternavirus has infected FOM, and this is the first report of hypovirulence and increased sensitivity to difenoconazole and pydiflumetofen induced by FoAV1-FOM infection in FOM.

Complete genome sequence of a novel mitovirus identified in the phytopathogenic fungus Fusarium oxysporum f. sp. melonis strain T-SD3.

A novel mitovirus was identified in Fusarium oxysporum f. sp. melonis strain T-SD3 and designated as "Fusarium oxysporum mitovirus 3" (FoMV3). The virus was isolated from diseased muskmelon plants with the typical symptom of fusarium wilt. The complete genome of FoMV3 is 2269 nt in length with a predicted AU content of 61.40% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a polypeptide of 679 amino acids (aa) containing a conserved RNA-dependent RNA polymerase (RdRp) domain with a molecular mass of 77.39 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5'-untranslated region (UTR) and 3'-UTR of FoMV3 were predicted to fold into stem-loop structures. BLASTp analysis revealed that the RdRp of FoMV3 shared the highest aa sequence identity (83.85%) with that of Fusarium asiaticum mitovirus 5 (FaMV5, a member of the family Mitoviridae) infecting F. asiaticum, the causal agent of wheat fusarium head blight. Phylogenetic analysis further suggested that FoMV3 is a new member of the genus Unuamitovirus within the family Mitoviridae. This is the first report of a new mitovirus associated with F. oxysporum f. sp. melonis.

A Novel Strain of Fusarium oxysporum Virus 1 Isolated from Fusarium oxysporum f. sp. niveum Strain X-GS16 Influences Phenotypes of F. oxysporum Strain HB-TS-YT-1hyg.

A novel strain of Fusarium oxysporum virus 1 (FoV1) was identified from the Fusarium oxysporum f. sp. niveum strain X-GS16 and designated as Fusarium oxysporum virus 1-FON (FoV1-FON). The full genome of FoV1-FON is 2902 bp in length and contains two non-overlapping open reading frames (ORFs), ORF1 and ORF2, encoding a protein with an unknown function (containing a typical -1 slippery motif G_GAU_UUU at the 3'-end) and a putative RNA-dependent RNA polymerase (RdRp), respectively. BLASTx search against the National Center for the Biotechnology Information (NCBI) non-redundant database showed that FoV1-FON had the highest identity (97.46%) with FoV1. Phylogenetic analysis further confirmed that FoV1-FON clustered with FoV1 in the proposed genus Unirnavirus. FoV1-FON could vertically transmit via spores. Moreover, FoV1-FON was transmitted horizontally from the F. oxysporum f. sp. niveum strain X-GS16 to the F. oxysporum strain HB-TS-YT-1hyg. This resulted in the acquisition of the F. oxysporum strain HB-TS-YT-1hyg-V carrying FoV1-FON. No significant differences were observed in the sporulation and dry weight of mycelial biomass between HB-TS-YT-1hyg and HB-TS-YT-1hyg-V. FoV1-FON infection significantly increased the mycelial growth of HB-TS-YT-1hyg, but decreased its virulence to potato tubers and sensitivity to difenoconazole, prochloraz, and pydiflumetofen. To our knowledge, this is the first report of hypovirulence and reduced sensitivity to difenoconazole, prochloraz, and pydiflumetofen in F. oxysporum due to FoV1-FON infection.

Characterization of the First Alternavirus Identified in Fusarium avenaceum, the Causal Agent of Potato Dry Rot.

A novel virus with a double-stranded RNA (dsRNA) genome was isolated from Fusarium avenaceum strain GS-WW-224, the causal agent of potato dry rot. The virus has been designated as Fusarium avenaceum alternavirus 1 (FaAV1). Its genome consists of two dsRNA segments, 3538 bp (dsRNA1) and 2477 bp (dsRNA2) in length, encoding RNA-dependent RNA polymerase (RdRp) and a hypothetical protein (HP), respectively. The virions of FaAV1 are isometric spherical and approximately 30 nm in diameter. Multiple sequence alignments and phylogenetic analyses based on the amino acid sequences of RdRp and HP indicated that FaAV1 appears to be a new member of the proposed family Alternaviridae. No significant differences in colony morphology and spore production were observed between strains GS-WW-224 and GS-WW-224-VF, the latter strain being one in which FaAV1 was eliminated from strain GS-WW-224. Notably, however, the dry weight of mycelial biomass of GS-WW-224 was higher than that of mycelial biomass of GS-WW-224-VF. The depth and the width of lesions on potato tubers caused by GS-WW-224 were significantly greater, relative to GS-WW-224-VF, suggesting that FaAV1 confers hypervirulence to its host, F. avenaceum. Moreover, FaAV1 was successfully transmitted horizontally from GS-WW-224 to ten other species of Fusarium, and purified virions of FaAV1 were capable of transfecting wounded hyphae of the ten species of Fusarium. This is the first report of an alternavirus infecting F. avenaceum and conferring hypervirulence.

A Botybirnavirus Isolated from Alternaria tenuissima Confers Hypervirulence and Decreased Sensitivity of Its Host Fungus to Difenoconazole.

Alternaria alternata botybirnavirus 1 (AaBRV1) was isolated from a strain of Alternaria alternata, causing watermelon leaf blight in our previous research. The effect of AaBRV1 on the phenotype of its host fungus, however, was not determined. In the present study, a novel strain of AaBRV1 was identified in A. tenuissima strain TJ-NH-51S-4, the causal agent of cotton Alternaria leaf spot, and designated as AaBRV1-AT1. A mycovirus AaBRV1-AT1-free strain TJ-NH-51S-4-VF was obtained by protoplast regeneration, which eliminated AaBRV1-AT1 from the mycovirus AaBRV1-AT1-infected strain TJ-NH-51S-4. Colony growth rate, spore production, and virulence of strain TJ-NH-51S-4 were greater than they were in TJ-NH-51S-4-VF, while the sensitivity of strain TJ-NH-51S-4 to difenoconazole, as measured by the EC50, was lower. AaBRV1-AT1 was capable of vertical transmission via asexual spores and horizontal transmission from strain TJ-NH-51S-4 to strain XJ-BZ-5-1hyg (another strain of A. tenuissima) through hyphal contact in pairing cultures. A total of 613 differentially expressed genes (DEGs) were identified in a comparative transcriptome analysis between TJ-NH-51S-4 and TJ-NH-51S-4-VF. Relative to strain TJ-NH-51S-4-VF, the number of up-regulated and down-regulated DEGs in strain TJ-NH-51S-4 was 286 and 327, respectively. Notably, the expression level of one DEG-encoding cytochrome P450 sterol 14α-demethylase and four DEGs encoding siderophore iron transporters were significantly up-regulated. To our knowledge, this is the first documentation of hypervirulence and reduced sensitivity to difenoconazole induced by AaBRV1-AT1 infection in A. tenuissima.

Full genome sequence of a new three-segment gammapartitivirus from the phytopathogenic fungus Alternaria tenuissima on cotton in China.

In this study, a new double-stranded RNA (dsRNA) virus, Alternaria tenuissima partitivirus 1 (AttPV1), was isolated from Alternaria tenuissima strain XJ-BZ-2-6, a phytopathogenic fungus infecting cotton in China. The genome of AttPV1 comprised three dsRNAs of 1,785 nt (dsRNA1), 1,545 nt (dsRNA2), and 1,537 nt (dsRNA3) in length, the nucleotide sequence of which was determined using reverse transcription polymerase chain reaction, random-primed clones, and RNA-ligase-mediated rapid amplification of cDNA ends. dsRNA1 had a single open reading frame encoding a putative 61.54-kDa RNA-dependent RNA polymerase (RdRp). dsRNA2 and dsRNA3 were predicted to encode putative coat proteins (CPs) of 47.90 kDa and 46.25 kDa, respectively. The RdRp domain shared 63.54-73.17% amino acid sequence identity with members of the genus Gammapartitivirus. Phylogenetic trees based on RdRp or CP sequences showed that AttPV1 clustered with members of the genus Gammapartitivirus. Hence, these results indicate that AttPV1 is a new gammapartitivirus from A. tenuissima.

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.

Complete genome sequence of a new botybirnavirus isolated from a phytopathogenic Alternaria alternata in China.

A new double-stranded RNA (dsRNA) virus named Alternaria alternata botybirnavirus 1 (AaBRV1) was isolated from Alternaria alternata strain SD-BZF-19, a phytopathogenic fungus infecting watermelon in China. The genome of AaBRV1 consists of two dsRNA segments (dsRNAs 1 and 2), 6,130 and 5,862 bp in size, respectively. The sequence contains two putative open reading frames (ORFs) which encode two polyproteins of 1,874 and 1,784 amino acids, respectively. Nucleotide sequence comparisons revealed that the two ORFs of AaBRV1 have the highest similarity 60.3% and 56.7%, respectively, with dsRNAs 1 and 2 of Botrytis porri RNA virus 1 (BpRV1). The two polyproteins encoded by dsRNA1 and dsRNA2 shared the highest amino acid identities with the cap-pol fusion protein (60.2%) and hypothetical protein (53.7%) of BpRV1, respectively. AaBRV1 is composed of isometric particles, approximately 35 nm in diameter. Phylogenetic analysis of the RNA dependent RNA polymerase (RdRp) domain of the polyprotein revealed that AaBRV1 clusters together with members of the genus Botybirnavirus. These findings support the discovery of a new botybirnavirus in A. alternata.