Mingde Wu

Hypovirulence and Double-Stranded RNA in Botrytis cinerea.

ABSTRACT Twenty-one strains of Botrytis cinerea isolated from 13 species of plants grown in China were compared for pathogenicity on Brassica napus, mycelial growth on potato dextrose agar, and presence of double-stranded (ds)RNA. The results showed that the strain CanBc-1 was severely debilitated in pathogenicity and mycelial growth, compared with the 20 virulent strains. A dsRNA of approximately 3.0 kb in length was detected in CanBc-1 and 4 hypovirulent single-conidium (SC) isolates of CanBc-1, but was not detected in the 20 virulent strains of B. cinerea and 4 virulent SC isolates of CanBc-1. Results of the horizontal transmission experiment showed that the hypovirulent trait of CanBc-1 was transmissible and the 3.0-kb dsRNA was involved in the transmission of hypovirulence. Analysis of a 920-bp cDNA sequence generated from the 3.0-kb dsRNA of CanBc-1 indicated that the dsRNA element was a mycovirus, designated as B. cinerea debilitation-related virus (BcDRV). Further analyses showed that BcDRV is closely related to Ophiostoma mitovirus 3b infecting O. novo-ulmi, the causal agent of Dutch elm disease. Mitochondria and cytoplasm in hyphal cells of CanBc-1 became degenerated, compared with the virulent isolate CanBc-1c-66 of B cinerea. This is the first report on the occurrence of Mitovirus-associated hypovirulence in B. cinerea.

Genome characterization of a debilitation-associated mitovirus infecting the phytopathogenic fungus Botrytis cinerea

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.

Characterization of a Novel Bipartite Double-Stranded RNA Mycovirus Conferring Hypovirulence in the Phytopathogenic Fungus Botrytis porri

ABSTRACT 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 RNA virus 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.

Botrytis fabiopsis, a new species causing chocolate spot of broad bean in central China

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.

Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum.

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.

Novel Hypovirulence-Associated RNA Mycovirus in the Plant-Pathogenic Fungus Botrytis cinerea: Molecular and Biological Characterization

ABSTRACT Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A −1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The −1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.

CmpacC Regulates Mycoparasitism, Oxalate Degradation and Antifungal Activity in the Mycoparasitic Fungus Coniothyrium minitans

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.

Production of anti-fungal volatiles by non-pathogenic Fusarium oxysporum and its efficacy in suppression of Verticillium wilt of cotton

AimsThe study aimed at identifying volatile organic compounds (VOCs) produced by the non-pathogenic Fusarium oxysporum (Fo) strain CanR-46, and to determine the anti-fungal spectrum and the control efficacy of the Fo-VOCs.MethodsThe Fo-VOCs were identified by GC-MS. The antifungal activity of the Fo-VOCs was tested using the double-dish method. Soil contaminated with Verticillium dahliae was fumigated with the Fo-VOCs to test the control efficacy. The GFP-tagged derivative strain CanR-46GFP was tested to colonize cotton roots for prevention of infection by V. dahliae.ResultsNineteen VOCs were identified with eremophila-1 (10),11-diene being the most abundant. The Fo-VOCs inhibited mycelial growth of 14 fungal species including V. dahliae, delayed V. dahliae conidial germination, suppressed V. dahliae germ-tube elongation, and caused V. dahliae hyphal shriveling and collapse. Four synthetic chemicals, 5-hexenoic acid, limonene, octanoic acid and 3,4-2H-dihydropyran, in the Fo-VOCs profile showed antifungal activity against V. dahliae. The Fo-VOCs significantly (P < 0.05) reduced severity of Verticillium wilt of cotton compared to the control. CanR-46GFP extensively colonized the cotton root tissues, thus effectively prevented V. dahliae infection.ConclusionsThe VOC-producing fungus F. oxysporum CanR-46 is a promising biocontrol agent against V. dahliae.

Development of PCR-Based Assays for Detecting and Differentiating Three Species of Botrytis Infecting Broad Bean

Botrytis cinerea, B. fabae, and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop polymerase chain reaction (PCR)-based assays to detect and differentiate these three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, were designed based on two sequence-characterized amplified region markers derived from two random amplified polymorphic DNA assays. The other primer set, Bfa-f/Bfa-r for B. fabae, was designed based on the necrosis and ethylene-inducing protein 1 gene sequence. The three primer sets were highly specific for the corresponding species of Botrytis in both single and multiplex PCR assays. The PCR detection limit was 40, 40, and 400 pg of DNA per 25-μl reaction mixture for B. fabae, B. fabiopsis, and B. cinerea, respectively. Presence of the broad bean DNA in the PCR reactions at 1:1000 (Botrytis DNA/broad bean DNA [wt/wt]) had negligible effects on detection of the targeted Botrytis spp. The multiplex PCR assay was able to detect three Botrytis spp. in artificially infected and naturally infected broad bean leaves. These results suggest that the multiplex PCR assay developed in this study could be used to monitor the epidemics of chocolate spot of broad bean in the field.

Characterization of three mycoviruses co-infecting the plant pathogenic fungus Sclerotinia nivalis

Two dsRNAs of approximately 6.0-kb and 3.0-kb in length were detected in strain SsSn-1 of Sclerotinia nivalis. Genomic analysis showed that the 6.0-kb dsRNA was a victorivirus, named as Sclerotinia nivalis victorivirus 1 (SnVV1). The genome of SnVV1 is 5162bp in length containing two large open reading frames (ORFs), ORF1 and ORF2. ORF1 was deduced to encode a coat protein (CP) showing homology to CPs of viruses belonging to the family Totiviridae. The stop codon of ORF1 overlaps the start codon of ORF2 in the tetranucleotide sequence AUGA. ORF2 was predicted to encode for a RNA-dependent RNA polymerase (RdRp) that was very similar to the RdRps of victoriviruses. The 3.0-kb dsRNA was consisted of two species of mitoviruses, named as Sclerotinia nivalis mitovirus 1/SsSn-1 (SnMV1/SsSn-1) and Sclerotinia sclerotiorum mitovirus 3/SsSn-1 (SsMV3/SsSn-1). The genomes of SnMV1/SsSn-1 and SsMV3/SsSn-1 were 2720nt and 2583nt in length, respectively. Both mitoviruses were AU-rich and deduced to contain a major large ORF encoding a mitoviral RdRp with the fungal mitochondrial codon usages. SnMV1/SsSn-1 was most closely related to Sclerotinia sclerotiorum mitovirus 4 (SsMV4/NZ1) and shared 76.5% and 80.1% identity with SsMV4/NZ1 for nucleotide and RdRp sequences, respectively. In addition, the nucleotide and RdRp sequences of SsMV3/SsSn-1 were 90.6% and 95.9% identical to the nucleotide and RdRp sequences of SsMV3/NZ1, respectively. Considering their nucleotide and RdRp sequence identities with other mitoviruses, SnMV1/SsSn-1 may belong to the genus Mitovirus, whereas SsMV3/SsSn-1 is possibly a strain of SsMV3. Both SnMV1/SsSn-1 and SsMV3/SsSn-1 were transmitted to a recipient virus-free colony faster than was SnVV1 through hyphal anastomosis. Co-infection by these mycoviruses had no apparent effects on growth and pathogenicity of S. nivalis.