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. 2023 Oct 7;15(10):2059.
doi: 10.3390/v15102059.

Identification and Characterization of a Novel Hypovirus from the Phytopathogenic Fungus Botryosphaeria dothidea

Yongqi Wen  1 , Jinyue Qu  1 , Honglin Zhang  1 , Yi Yang  1 , Rui Huang  1 , Jili Deng  1 , Jiayu Zhang  1 , Yanping Xiao  1 , Jiali Li  1 , Meixin Zhang  1 , Guoping Wang  2 , Lifeng Zhai  1
Affiliations

Identification and Characterization of a Novel Hypovirus from the Phytopathogenic Fungus Botryosphaeria dothidea

Yongqi Wen et al. Viruses. .

Abstract

Many mycoviruses have been accurately and successfully identified in plant pathogenic fungus Botryosphaeria dothidea. This study discovered three mycoviruses from a B. dothidea strain SXD111 using high-throughput sequencing technology. A novel hypovirus was tentatively named Botryosphaeria dothidea hypovirus 1 (BdHV1/SXD111). The other two were known viruses, which we named Botryosphaeria dothidea polymycovirus 1 strain SXD111 (BdPmV1/SXD111) and Botryosphaeria dothidea partitivirus 1 strain SXD111 (BdPV1/SXD111). The genome of BdHV1/SXD111 is 11,128 nucleotides long, excluding the poly (A) tail. A papain-like cysteine protease (Pro), a UDP-glucose/sterol glucosyltransferase (UGT), an RNA-dependent RNA polyprotein (RdRp), and a helicase (Hel) were detected in the polyprotein of BdHV1/SXD111. Phylogenetic analysis showed that BdHV1/SXD111 was clustered with betahypovirus and separated from members of the other genera in the family Hypoviridae. The BdPmV1/SXD111 genome comprised five dsRNA segments with 2396, 2232, 1967, 1131, and 1060 bp lengths. Additionally, BdPV1/SXD111 harbored three dsRNA segments with 1823, 1623, and 557 bp lengths. Furthermore, the smallest dsRNA was a novel satellite component of BdPV1/SXD111. BdHV1/SXD111 could be transmitted through conidia and hyphae contact, whereas it likely has no apparent impact on the morphologies and virulence of the host fungus. Thus, this study is the first report of a betahypovirus isolated from the fungus B. dothidea. Importantly, our results significantly enhance the diversity of the B. dothidea viruses.

Keywords: Betahypovirus; Botryosphaeria dothidea; Botryosphaeria dothidea hypovirus 1; Hypoviridae; genome.

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Conflict of interest statement

The authors declare no conflict of interes in this paper.

Figures

Figure 1
Figure 1
DsRNAs extracted from strain SXD111. (A) 1.0% agarose gel electrophoretic profiles of dsRNA preparations extracted from strain SXD111 without treatment (lane 2) and treated with S1 nuclease (lane 3) and DNase I (lane 4), respectively. (B) The dsRNA nature of Botryosphaeria dothidea hypovirus 1 (BdHV1), Botryosphaeria dothidea polymycovirus 1 (BdPmV1), Botryosphaeria dothidea partitivirus 1 (BdPV1), Botryosphaeria dothidea chrysovirus 1 (BdCV1), and Botryosphaeria dothidea victorivirus 3 (BdVV3) determined by enzymatic treatment with S1 nuclease and DNase I. (C) Detection of BdHV1, BdPmV1, BdPV1, BdCV1, and BdVV3 in the tested strains via RT-PCR using specific primers (Supplementary Table S1). The virus-free strain JNT1111 was used as a negative control. Marker, DNA marker.
Figure 2
Figure 2
BdHV1/SXD111 genomic organization. (A) Genomic organization of the ORF1-encoding polyprotein. The conserved motifs for protease (Pro), UDP-glucose/sterol glucosyltransferase (UGT), RNA-dependent RNA polyprotein (RdRp), and helicase (Hel) domain blocks on the polyprotein are marked in blue, orange, green, and yellow, respectively, with the lengths corresponding to their amino acid (aa) sizes. The numbers under the line indicate the start and end positions of the genome, 5’- and 3’-untranslated regions (UTRs), and the conserved domains. (B–E) Multiple alignments of the RdRp, Hel, UGT, and Pro of BdHV1/SXD111 amino acid sequences with members of the family Hypoviridae. Black shading indicates the conserved sequence level, and the darkest color indicates the most conserved sequence. For abbreviations of viruses and viral protein accession numbers used in alignment analysis, see Supplementary Table S2. *, conserved amino acid residues.
Figure 3
Figure 3
Phylogenetic analysis of Botryosphaeria dothidea hypovirus 1 (BdHV1/SXD111). A maximum-likelihood tree (model LG+G+I+F) was constructed based on the polyprotein sequences encoded by BdHV1/SXD111 and those of known hypoviruses using MEGA X. The putative hypovirus BdHV1/SXD111 is indicated in red. The values in the upper left corner of the branches indicate the bootstrap probability based on 1000 replicates. Bootstrap values < 50% are hidden.
Figure 4
Figure 4
BdPV1/SXD111 genomic organization. (A) Schematic representation of the genomic organization of BdPV1/LW-1 and BdPV1/SXD111. The length and sequences of their satellite-like RNA were different. (B) Multiple alignment of the dsRNAs of BdPV1/SXD111. Asterisks show identical nucleotides in dsRNAs. (C) Secondary structures proposed for 5′- and 3′-terminus of BdPV1/SXD111 dsRNA3 with the lowest energies. *, conserved nucleotides.
Figure 5
Figure 5
Colony morphology on the PDA plate and virulence on the pear fruit of strains SXD111, JNT1111, JST1, JST2, and JST3. (A) Detection of BdHV1/SXD111, BdPmV1/SXD111, and BdPV1//SXD111 in different strains of B. dothidea via RT-PCR with specific primers (see Supplementary Table S1). (B) Colony morphology on PDA medium (28 °C, 4 days). (C) Statistical analysis of the growth rate of BdHV1/SXD111-infected and virus-free strains on a PDA medium. (D) Statistical analysis of the lesion size of BdHV1/SXD111-infected and virus-free strains on wound-inoculated pear (P. bretschneideri cv. ‘Huangguan’) at four dpi. (E) Fruits of pear wound-inoculated with colonized plugs of strains SXD111, JNT1111, JST1, JST2, and JST3 at four dpi. Strains JST1, JST2, and JST3 were derived from JNT1111 in the pairing cultures of strains JNT1111 and SXD111. The error bars indicate the standard deviations from different sample means. Based on a multiple-range test, there was no significant difference at the p < 0.05 confidence level for growth rates and lesion sizes.
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Grants and funding

This work was financially supported by the National Natural Science Foundation of China (32072476); the Science and Technology Research Program of Chongqing Municipal Education Commission (KJQN202001411, KJQN202201401); the Innovation Research and Entrepreneurship Training Program for Undergraduate of Chongqing (S202210647031); and the Research Funding Project of Yangtze Normal University (2017KYQD54).