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. 2019 Mar 17;11(3):266.
doi: 10.3390/v11030266.

Characterization of a Botybirnavirus Conferring Hypovirulence in the Phytopathogenic Fungus Botryosphaeria dothidea

Lifeng Zhai  1   2 , Mengmeng Yang  3   4 , Meixin Zhang  5 , Ni Hong  6   7 , Guoping Wang  8   9
Affiliations

Characterization of a Botybirnavirus Conferring Hypovirulence in the Phytopathogenic Fungus Botryosphaeria dothidea

Lifeng Zhai et al. Viruses. .

Abstract

A double-stranded RNA (dsRNA) virus was isolated and characterized from strain EW220 of the phytopathogenic fungus Botryosphaeria dothidea. The full-length cDNAs of the dsRNAs were 6434 bp and 5986 bp in size, respectively. The largest dsRNA encodes a cap-pol fusion protein that contains a coat protein gene and an RNA-dependent RNA polymerase (RdRp) domain, and the second dsRNA encodes a hypothetical protein. Genome sequence analysis revealed that the sequences of the dsRNA virus shared 99% identity with Bipolaris maydis botybirnavirus 1(BmBRV1) isolated from the causal agent of corn southern leaf blight, Bipolaris maydis. Hence, the dsRNA virus constitutes a new strain of BmBRV1 and was named Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmBRV1-BdEW220). BmBRV1-BdEW220 contains spherical virions that are 37 nm in diameter and consist of two dsRNA segments. The structural proteins of the BmBRV1-BdEW220 virus particles were 110 kDa, 90 kDa, and 80 kDa and were encoded by dsRNA1 and 2-ORFs. Phylogenetic reconstruction indicated that BmBRV1 and BmBRV1-BdEW220 are phylogenetically related to the genus Botybirnavirus. Importantly, BmBRV1-BdEW220 influences the growth of B. dothidea and confers hypovirulence to the fungal host. To our knowledge, this is the first report of a botybirnavirus in B. dothidea.

Keywords: Botryosphaeria dothidea; Botybirnavirus; double-stranded RNA virus; genome; hypovirulence.

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Conflict of interest statement

The authors declare that they do not have any conflict of interest.

Figures

Figure 1
Figure 1
Colony morphology and virulence of strains EW220 and JNT1111 on pear shoots (P. pyrifolia cv. ‘Hohsui’). (A) Colony morphology in potato dextrose agar (PDA) medium (28 °C, 4 days); (B) pear shoots unwound-inoculated with colonized plugs of the tested strains photographed at 60 dpi.
Figure 2
Figure 2
Double-stranded RNAs extracted from strain EW220 and the genomic organization of Botryosphaeria dothidea botybirnavirus 1 (BmBRV1-BdEW220). (A) One percent agarose gel electrophoretic profiles of dsRNA preparations extracted from strains EW220 and JNT1111 after digestion with DNase I and S1 nuclease; (B) schematic diagrams of the genomic organization of BmBRV1-BdEW220.
Figure 3
Figure 3
Multiple sequence alignments and predicted secondary structures for the terminal regions of the coding strand of dsRNA1 and dsRNA2. (A) Conserved sequences of the 5′-UTRs of dsRNA1 and dsRNA2; (B) Conserved sequences of the 3′-UTRs of dsRNA1 and dsRNA2; the conserved sequences are shown in black; (C) Predicted secondary structures for the 5′- and 3′-terminus of the coding strands of dsRNA-1 and dsRNA-2 of BmBRV1-BdEW220.
Figure 4
Figure 4
Multiple alignments of the amino acid sequences of the RNA-dependent RNA polymerase of BmBRV1-EW220 with other selected members in Botybirnavirus (A) and phylogenetic analysis of BmBRV1-EW220 (B). The phylogenetic trees were constructed based on the best-fit model of protein evolution (LG + G + I + F). The gamma value was 2. Bootstrap values (relative) generated using 1000 replicates are shown on the branches. Only bootstrap values of ≥50% are presented, and the branch lengths are proportional to the genetic distances. The black circle represents BmBRV1-EW220. Abbreviations used in Figure 4A: BmBRV1-EW220, Bipolaris maydis botybirnavirus 1 strain BdEW220; BmBRV1, Bipolaris maydis botybirnavirus 1; SsBRV1, Sclerotinia sclerotiorum botybirnavirus 1; SsBRV2, Sclerotinia sclerotiorum botybirnavirus 2; ABRV1, Alternaria botybirnavirus 1; SlBRV1, Soybean leaf-associated botybirnavirus 1; BpRV1, Botrytis porri RNA virus 1.
Figure 5
Figure 5
Features of the BmBRV1-BdEW220 viral particles (A) Transmission electron microscopy (TEM) images of the BmBRV1-BdEW220 viral particles; (B) agarose gel electrophoresis of the dsRNAs extracted from purified virus particles of BmBRV1-BdEW220 and the mycelia of strain EW220 (line EW220); (C) SDS-PAGE analysis of the purified viral particles shows the three distinct protein bands. The scale bar represents 50 nm.
Figure 6
Figure 6
Detection of BmBRV1-BdEW220 in different strains of B. dothidea by dsRNAs profiling and RT-PCR with specific primers. Strains EW220-64-T1, EW220-64-T2, and EW220-64-T3 were derived from EW220-64 in the pairing cultures of EW220/EW220-64. The primers BmBRV1-BdEW220-S1F (5′-GCGCTGAGTGGATGATCAAAG-3′) and BmBRV1-BdEW220-S1R (5′-CTCTTCGTCTGGCAAAAAGCC-3′) were used for detecting the BmBRV1-BdEW220-dsRNA1 segment, and BmBRV1-BdEW220-S2F (5′-GCACTAAGAGAGACTTTCGAG-3′) and BmBRV1-BdEW220-S2R (5′-CGGTAGGATC ATCCATAGTG-3′) were used for detecting the BmBRV1-BdEW220-dsRNA2 segment.
Figure 7
Figure 7
Colony morphology and virulence of strain EW220, strain JNT1111, and derived sub-strains on pear fruit (P. bretschneideri cv. Huangguan). Strains EW220-64-T1, EW220-64-T2, and EW220-64-T3 were derived from EW220-64 in the pairing cultures of EW220/EW220-64. (A) Colony morphology in PDA medium (28 °C, 3 days); (B) pear fruits wound-inoculated with colonized plugs of tested strains and photographed at 4 dpi; (C) statistical analysis of the growth rate and the lesion size. The error bars indicate the standard deviations from different sample means. The letter indicates a significant difference at the p < 0.05 level of confidence based on a multiple range test.
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