The ascomycete Botrytis porri causes clove rot and leaf blight of garlic worldwide. We report here the biological and molecular features of a novel bipartite double-stranded RNA (dsRNA) mycovirus named B otrytis p orri R NA v irus 1 (BpRV1) from the hypovirulent strain GarlicBc-72 of B. porri . The BpRV1 genome comprises two dsRNAs, dsRNA-1 (6,215 bp) and dsRNA-2 (5,879 bp), which share sequence identities of 62 and 95% at the 3′- and 5′-terminal regions, respectively. Two open reading frames (ORFs), ORF I (dsRNA-1) and ORF II (dsRNA-2), were detected. The protein encoded by the 3′-proximal coding region of ORF I shows sequence identities of 19 to 23% with RNA-dependent RNA polymerases encoded by viruses in the families Totiviridae , Chrysoviridae , and Megabirnaviridae . However, the proteins encoded by the 5′-proximal coding region of ORF I and by the entire ORF II lack sequence similarities to any reported virus proteins. Phylogenetic analysis showed that BpRV1 belongs to a separate clade distinct from those of other known RNA mycoviruses. Purified virions of ∼35 nm in diameter encompass dsRNA-1 and dsRNA-2, and three structural proteins (SPs) of 70, 80, and 85 kDa, respectively. Peptide mass fingerprinting analysis revealed that the 80- and 85-kDa SPs are encoded by ORF I, while the 70-kDa SP is encoded by ORF II. Introducing BpRV1 purified virions into the virulent strain GarlicBc-38 of B. porri caused derivative 38T reduced mycelial growth and hypovirulence. These combined results suggest that BpRV1 is a novel bipartite dsRNA virus that possibly belongs to a new virus family.

The full-length sequences of Botrytiscinereamitovirus 1 (BcMV1) and an associated RNA (BcMV1-S) in strain CanBc-1c-78 of Botrytis cinerea were determined. Sequence analysis showed that BcMV1 is 2804 nt long and AU-rich (66.8%). BcMV1 shares 95% nucleotide sequence identity with Ophiostomanovo-ulmimitovirus 3b (OnuMV3b). However, it is 472 nt longer than OnuMV3b. Mitochondrial codon usage revealed that BcMV1 contains one open reading frame encoding RdRp, which is 96% identical to the RdRp of OnuMV3b. These findings confirm that BcMV1 belongs to the genus Mitovirus and is a strain of OnuMV3b. BcMV1-S is 2171 nt long and derived from BcMV1 through a single internal in-frame deletion of 633 nt, suggesting that it is a defective RNA of BcMV1. BcMV1-S was found to suppress the replication of BcMV1 and to be co-transmissible with BcMV1 through hyphal anastomosis. Its presence, however, did not alleviate the BcMV1-associated debilitation phenotypes of B. cinerea.

Coniothyrium minitans (Cm) is a mycoparasite of the phytopathogenic fungus Sclerotinia sclerotiorum (Ss). Ss produces a virulence factor oxalic acid (OA) which is toxic to plants and also to Cm, and Cm detoxifies OA by degradation. In this study, two oxalate decarboxylase genes, Cmoxdc1 and Cmoxdc2, were cloned from Cm strain Chy-1. OA and low pH induced expression of Cmoxdc1, but not Cmoxdc2. Cmoxdc1 was partially responsible for OA degradation, whereas Cmoxdc2 had no effect on OA degradation. Disruption of Cmoxdc1 in Cm reduced its ability to infect Ss in dual cultures where OA accumulated. Compared with Chy-1, the Cmoxdc1-disrupted mutants had reduced expression levels of two mycoparasitism-related genes chitinase (Cmch1) and β-1,3-glucanase (Cmg1), and had no detectable activity of extracellular proteases in the presence of OA. On the other hand, the cultural filtrates of the Cmoxdc1-disrupted mutants in OA-amended media showed enhanced antifungal activity, possibly because of increased production of antifungal substances under acidic pH condition resulted from reduced Cmoxdc1-mediated OA degradation. This study provides direct genetic evidence of OA degradation regulating mycoparasitism and antibiosis of Cm against Ss, and sheds light on the sophisticated strategies of Cm in interacting with metabolically active mycelia and dormant sclerotia of Ss.

The current study was conducted to identify Botrytis spp. isolated from symptomatic broad bean plants grown in Hubei Province, China. Among 184 Botrytis strains, three distinct species, B. cinerea, B. fabae and a previously undescribed Botrytis sp., were identified based on morphology of colonies, sclerotia and conidia. The novel Botrytis sp. is described herein as a new species, Botrytis fabiopsis sp. nov. At 20 C B. fabiopsis grew on potato dextrose agar (PDA) at 12-13 mm d(-1), similar to B. fabae (13 mm d(-1)), but slower than B. cinerea (17-19 mm d(-1)). It formed pale gray colonies with short aerial mycelia and produced gray to black sclerotia in concentric rings on PDA. B. fabiopsis produced greater numbers of sclerotia than B. cinerea but fewer than B. fabae. Conidia produced by B. fabiopsis on broad bean leaves are hyaline to pale brown, elliptical to ovoid, wrinkled on the surface and are larger than conidia of B. fabae and B. cinerea. Phylogenetic analysis based on combined DNA sequence data of three nuclear genes (G3PDH, HSP60 and RPB2) showed that B. fabiopsis is closely related to B. galanthina, the causal agent of gray mold disease of Galanthus sp., but distantly related to B. fabae and B. cinerea. Sequence analysis of genes encoding necrosis and ethylene-inducing proteins (NEPs) indicated that B. fabiopsis is distinct from B. galanthina. Inoculation of broad bean leaves with conidia of B. fabiopsis caused typical chocolate spot symptoms with a similar disease severity to that caused by B. fabae but significantly greater than that caused by B. cinerea. This study suggests that B. fabiopsis is a new causal agent for chocolate spot of broad bean.

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Botrytis cinerea is a necrotrophic fungus causing disease on many important agricultural crops. Two novel mycoviruses, namely Botrytis cinerea hypovirus 1 (BcHV1) and Botrytis cinerea fusarivirus 1 (BcFV1), were fully sequenced. The genome of BcHV1 is 10,214 nt long excluding a poly-A tail and possesses one large open reading frame (ORF) encoding a polyprotein possessing several conserved domains including RNA-dependent RNA polymerase (RdRp), showing homology to hypovirus-encoded polyproteins. Phylogenetic analysis indicated that BcHV1 may belong to the proposed genus Betahypovirus in the viral family Hypoviridae. The genome of BcFV1 is 8411 nt in length excluding the poly A tail and theoretically processes two major ORFs, namely ORF1 and ORF2. The larger ORF1 encoded polypeptide contains protein domains of an RdRp and a viral helicase, whereas the function of smaller ORF2 remains unknown. The BcFV1 was phylogenetically clustered with other fusariviruses forming an independent branch, indicating BcFV1 was a member in Fusariviridae. Both BcHV1 and BcFV1 were capable of being transmitted horizontally through hyphal anastomosis. Infection by BcHV1 alone caused attenuated virulence without affecting mycelial growth, significantly inhibited infection cushion (IC) formation, and altered expression of several IC-formation-associated genes. However, wound inoculation could fully rescue the virulence phenotype of the BcHV1 infected isolate. These results indicate the BcHV1-associated hypovirulence is caused by the viral influence on IC-formation-associated pathways.

The PacC/Rim101 pH-responsive transcription factor is an important pathogenicity element for many plant-pathogenic fungi. In this study, we investigated the roles of a PacC homologue, CmpacC, in the mycoparasitic fungus Coniothyrium minitans. CmpacC was confirmed to have the transcriptional activation activity by the transcriptional activation test in Saccharomyces cerevisiae. Disruption of CmpacC resulted in impaired fungal responses to ambient pH. Compared to the wild-type, the CmpacC-disruption mutant ΔCmpacC-29 was significantly suppressed for activities of chitinase and β-1,3-glucanase at pH 5 and 7, consistent with reduced expression levels of Cmch1 and Cmg1 coding for the two enzymes respectively. However, the mutant displayed acidity-mimicking phenotypes such as improved oxalate degradation and increased antifungal activity at pH 6 or higher. Improved efficacy in oxalate degradation by ΔCmpacC-29 was consistent with the enhanced expression level of Cmoxdc1 coding for oxalate decarboxylase. CmpacC transcriptional activation of Cmch1 and Cmg1 and repression of Cmoxdc1 were verified by the presence of the PacC/Rim101 consensus binding-motifs in gene promoter regions and by the promoter DNA-binding assays. This study suggests that CmpacC plays an activator role in regulation of C. minitans mycoparasitism, whereas plays a repressor role in regulation of oxalate degradation and possibly antifungal activity of C. minitans.

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