Fig 1.
Characterization of Metarhizium majus partitivirus 1 (MmPV1).
(A) Electrophoretic profiles of MmPV1 in RCEF0578. M, DNA molecular weight marker; lane 1, dsRNAs of MmPV1. (B) Schematic representation of the genomic organisation of MmPV1. (C) The 5′- and 3′-terminal nucleic acid sequences of MmPV1. The shaded areas represent 100% nucleotide identity. (D) Multiple amino acid sequence alignment of the putative RdRp of MmPV1 and ten other gammapartitiviruses. The shaded areas indicate a position with a single, fully conserved residue.
Fig 2.
Phylogenetic analysis of MmPV1.
The ML phylogenetic tree based on RdRp sequences of 34 partitiviruses including MmPV1 and representatives from seven genera of the family Partitiviridae, (Table S1), with Beauveria bassiana polymycovirus 1 (BbPmV-1) as the outgroup.
Fig 3.
Horizontal transmission of MmPV1.
(A) MmPV1 dsRNA segments in RCEF0578 and RCEF0577. (B) ISSR-PCR with primer P9 of M. majus isolates. (C) Dual-culture of M. majus isolates, the front of the colony (above) and the back of the colony (below). (D) Confirmation of MmPV1 horizontal transmission in RCEF0577 strains with ISSR primer P9. (E) dsRNAs extraction from RCEF0577 strains. (F) RT-PCR of dsRNA with MmPV1 specific primers. (G) RT-PCR of total RNA with specific primers ORF1-F/ORF1-R of MmPV1.
Fig 4.
Effect of MmPV1 on conidiation and growth of M. majus.
(A) Conidiation of Mm and Mm/MmPV1 strains cultured on PDA for 14 d, together with colony morphology. (B) Colony morphology of different strains cultured on PDA plates for 14days. (C) Relative expression levels of conidiation-related genes as shown by qRT-PCR. ANOVA *, P <0.05; **, P <0.01; ****, P <0.0001.
Fig 5.
Effect of MmPV1 on heat shock and UV-B irradiation tolerance.
(A) Relative germination rates of Mm and Mm/MmPV1 strains following heat shock treatment for 24 h. (B) Relative expression levels of genes involved in response to heat shock as shown by RT-qPCR. (C) Relative germination rates of different strains following UV-B irradiation treatment for 24 h. (D) Relative expression levels of genes involved in DNA damage repair genes as shown by qRT-PCR. ANOVA *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001.
Fig 6.
Effect of MmPV1 on fungal virulence.
(A) Survival rates of G. mellonella larvae following injection with conidial suspensions from Mm and Mm/MmPV1 strains. Control insects were treated with sterile water. (B) Mean lethal times (LT50) of different strains after injection. (C) Survival rates of G. mellonella larvae following topical infection with conidial suspensions from different strains. Control insects were treated with sterile water. (D) The mean lethal times (LT50) of different strains after topical application. **, P <0.01.
Fig 7.
Analysis of fungal virulence-related phenotypes.
(A) Appressorium formation rate of Mm and Mm/MmPV1 strains in plastic hydrophobic plate. (B) Colony diameter of different strains following penetration of the wings of C. atrata and subsequent culturing for 4 d, together with colony morphology. (C) Hydrophobicity of different strains. (D) Adherence of different strains in hydrophobic plate. (E) Relative expression levels of virulence-related genes as shown by qRT-PCR. ANOVA *, P <0.05; **, P <0.01; ***, P <0.001.
Fig 8.
Total ion flow chromatograms of extracts from MmPV1-infected and -free M. majus.
(A) The MmPV1-free strain in negative model. (B) The MmPV1-free strain in positive model. (C) The MmPV1-infected strain in negative model. (D) The MmPV1-infected strain in positive model. Number 1 to 22 are the main metabolites which changed more than two times. There are marked on the relative bigger peaks.
Table 1.
The metabolites changed more than two times in virus-free and virus-infected Metarhizium majus strains.