Background
Mycovirus, a kind of virus that replicates only inside the cell of fungi or oomycetes [
1,
2], was firstly found in a diseased mushroom [
3]. Since biological control of chestnut blight with hypovirus (Cryphonectria hypovirus 1) was successfully applied under natural conditions in Europe, an increasing number of mycoviruses have been detected from various phytopathogenic fungi belonging to four phyla of
Chytridiomycota,
Zygomycota,
Ascomycota, and
Basidiomycota [
1,
2]. The phytopathologists are showing more interest in mycoviruses that could reduce the pathogenicity of phytopathogenic fungus and enhance plant resistance to abiotic stress [
1,
4‐
6]. Those debilitation-related mycoviruses have great potential to develop biological agents for virocontrol of plant fungal diseases [
1,
2]. So far, more than 300 mycoviruses have been fully or partially sequenced according to the NCBI (National Center for Biotechnology Information) GenBank database, but mycovirus research still significantly lags behind animal or plant virus research.
Mycoviruses usually have double-stranded RNA (dsRNA), single-stranded (ssRNA), and rare ssDNA genomes [
4]. Mycoviruses with dsRNA genome are classified into six families, i.e.
Chysoviridae,
Partitiviridae,
Reoviridae,
Totiviridae,
Megabirnaviridae, and
Quadriviridae. Likewise, mycoviruses with ssRNA genome are also classified into six families:
Alphaflexiviridae,
Barnaviride,
Endornaviridae,
Gammaflexiviridae,
Hypoviridae, and
Narnaviridae. With the development of new sequencing methods, more and more mycoviruses have been identified. However, some of the newly identified mycoviruses showed unique molecular and biological properties different from all known mycoviruses, and could not be classified into any of the established virus families. The proposed family Mycomononegaviridae accommodates Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV-1) that was a newly reported negative-stranded virus in fungi [
4,
7]. A DNA mycovirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), is the prototype of the proposed family Mycodnaviridae [
8]. Two recent reports proposed the establishment of new family Fusariviridae to encompass three previously reported positive-sense RNA mycoviruses: Fusarium graminearum virus 1 (FgV1), Rosellinia necatrix fusarivirus 1 (RnFV1), and Sclerotinia sclerotiorum fusarivirus 1 (SsFV1) [
9,
10]. An EC-approved ICTV proposed family Botybirnaviridae (
https://talk.ictvonline.org/files/ictv_official_taxonomy_updates_since_the_8th_report/m/fungal-official/5871) comprised three potential members of Botrytis porri botybirnavirus 1 (BpRV1), Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1), and soybean leaf-associated botybirnavirus 1 (SlaBRV1) [
11‐
13]. These studies have expanded our knowledge of virus genome, taxonomy, evolution and the interaction between viruses and their hosts.
Sclerotinia sclerotiorum (Lib.) de Bary is a severe world-wide-spread plant pathogenic fungus and attacks more than 400 plant species [
14]. Rapeseed (
Brassica napus) is a major plant oil crop in China and 85 % of rapeseed is planted in the Yangtze River Basin. However, stem rot disease caused by
S. sclerotiorum, the most important disease on rapeseed in China, results in a huge loss of production annually. Currently, no resistant cultivar of
B. napus is available for stem rot disease. While spraying fungicides is an effective method to control stem rot disease at the flowering stage of
B. napus, the application of fungicides is becoming difficult during flowering time due to the change of the cropping system from transplant to broadcast sowing. Furthermore, chemical fungicide-resistant strains (such as carbendazim resistant strains) were isolated frequently from the field [
15]. Screening hypovirulence-associated mycoviruses from
S. sclerotiorum population and probing their potential as bio-control agents to combat diseases is another potential control strategy for rapeseed stem rot.
S. sclerotiorum strains are increasingly recognized to harbor great diverse mycoviruses including the newly reported negative-sense RNA mycovirus and DNA mycovirus in fungi [
2,
7]. Field experiment supplied multiple lines of evidence that SsHADV-1 as a natural fungicide has a great potential to control rapeseed stem rot [
16]. The successful application of SsHADV-1 to the agricultural system has encouraged researchers to screen more strong infective and hypovirulence-associated mycoviruses from
S. sclerotiorum. Characterization of newly discovered mycoviruses will facilitate our understanding of virus ecology, evolution, and establish mycovirus-
S. sclerotiorum interaction system at the molecular level.
In the present research, the strain AH16 of S. sclerotiorum was identified to have the features of hypovirulence. We isolated and sequenced two unrelated mycoviruses of a mitovirus (Sclerotinia sclerotiorum mitovirus 4, SsMV4) and a botybirnavirus (Sclerotinia sclerotiorum botybirnavirus 2, SsBRV2) in strain AH16. Virion transfection experiment directly indicated that SsBRV2 could be responsible for the hypovirulence of S. sclerotiorum.
Conclusions
In the current study, a bisegmented dsRNA virus (SsBRV2/AH16) and a nonsegmented +ssRNA virus (SsMV4/AH16), which co-infects a single strain AH16 of S. sclerotiorum, were characterized. Based on biological characteristics (genome size and organization, etc.) and phylogenetic analysis, SsMV4/AH16 is a new strain of the proposed genus Sclerotinia sclerotiorum mitovirus 4, whereas SsBRV2 is closely related to two previously reported botybirnaviruses, SsBRV1 and BpRV1, which belong to the recently proposed family Botybirnaviridae. The successful introduction of purified VLPs of SsBRV2 into a virus-free isolate of S. sclerotiorum directly confirmed that SsBRV2 is the causal agent of hypovirulence. Our findings also supplied a first evidence that a single S. sclerotiorum strain is co-infected by dsRNA and +ssRNA mycoviruses.
Acknowledgements
This research was financially supported by China National Funds for Distinguished Young Scientists (311250230), the Key Project of the Chinese Ministry of Education (313024), and the Special Fund for Agro-Scientific Research in the Public Interest (201103016). We thank Dr. Said A. Ghabrial for helpful suggestions on the presence research. We would like to thank Professor Hanchang Zhu (college of foreign languages, Huazhong Agricultural University) for helpful suggestions in editing the manuscript.