Phytopathogenic fungi decrease crop yield and quality and cause huge losses in agricultural production. To prevent the occurrence of crop diseases and insect pests, farmers have to use many synthetic chemical pesticides. The extensive use of these pesticides has resulted in a series of environmental and ecological problems, such as the increase in resistant weed populations, soil compaction, and water pollution, which seriously affect the sustainable development of agriculture. This review discusses the main advances in research on plant-pathogenic fungi in terms of their pathogenic factors such as cell wall-degrading enzymes, toxins, growth regulators, effector proteins, and fungal viruses, as well as their application as biocontrol agents for plant pests, diseases, and weeds. Finally, further studies on plant-pathogenic fungal resources with better biocontrol effects can help find new beneficial microbial resources that can control diseases.

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Research Progress on Phytopathogenic Fungi and Their Role as Biocontrol Agents.

Members of the family Chrysoviridae are isometric, non-enveloped viruses with segmented, linear, dsRNA genomes. There are 3-7 genomic segments, each of which is individually encapsidated. Chrysoviruses infect fungi, plants and possibly insects, and may cause hypovirulence in their fungal hosts. Chrysoviruses have no known vectors and lack an extracellular phase to their replication cycle; they are transmitted via intracellular routes within an individual during hyphal growth, in asexual or sexual spores, or between individuals via hyphal anastomosis. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the family Chrysoviridae, which is available at ictv.global/report/chrysoviridae.

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ICTV Virus Taxonomy Profile: Chrysoviridae.

The cosmopolitan fungus Rhizoctonia solani has a wide host range and is the causal agent of numerous crop diseases, leading to significant economic losses. To date, no cultivars showing complete resistance to R. solani have been identified and it is imperative to develop a strategy to control the spread of the disease. Fungal viruses, or mycoviruses, are widespread in all major groups of fungi and next-generation sequencing (NGS) is currently the most efficient approach for their identification. An increasing number of novel mycoviruses are being reported, including double-stranded (ds) RNA, circular single-stranded (ss) DNA, negative sense (-)ssRNA, and positive sense (+)ssRNA viruses. The majority of mycovirus infections are cryptic with no obvious symptoms on the hosts; however, some mycoviruses may alter fungal host pathogenicity resulting in hypervirulence or hypovirulence and are therefore potential biological control agents that could be used to combat fungal diseases. R. solani harbors a range of dsRNA and ssRNA viruses, either belonging to established families, such as Endornaviridae, Tymoviridae, Partitiviridae, and Narnaviridae, or unclassified, and some of them have been associated with hypervirulence or hypovirulence. Here we discuss in depth the molecular features of known viruses infecting R. solani and their potential as biological control agents.

Viruses Infecting the Plant Pathogenic Fungus Rhizoctonia solani

Mycoviruses are wide spread throughout almost all groups of fungi but only a small number of mycoviruses can attenuate the growth and virulence of their fungal hosts. Alternaria alternata is an ascomycete fungus that causes leaf spot diseases on various crop plants. In this study, we identified a novel ssRNA mycovirus infecting an A. alternata f. sp. mali strain isolated from an apple orchard in China. Sequence analyses revealed that this virus is related to hypoviruses, in particular to Wuhan insect virus 14, an unclassified hypovirus identified from insect meta-transcriptomics, as well as other hypoviruses belonging to the genus Hypovirus, and therefore this virus is designed as Alternaria alternata hypovirus 1 (AaHV1). The genome of AaHV1 contains a single large open-reading frame encoding a putative polyprotein (∼479 kDa) with a cysteine proteinase-like and replication-associated domains. Curing AaHV1 from the fungal host strain indicated that the virus is responsible for the slow growth and reduced virulence of the host. AaHV1 defective RNA (D-RNA) with internal deletions emerging during fungal subcultures but the presence of D-RNA does not affect AaHV1 accumulation and pathogenicities. Moreover, AaHV1 could replicate and confer hypovirulence in Botryosphaeria dothidea, a fungal pathogen of apple white rot disease. This finding could facilitate better understanding of A. alternata pathogenicity and is relevant for development of biocontrol methods of fungal diseases.

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Identification of a Novel Hypovirulence-Inducing Hypovirus From Alternaria alternata.

High-throughput virome analyses with various fungi, from cultured or uncultured sources, have led to the discovery of diverse viruses with unique genome structures and even neo-lifestyles. Examples in the former category include splipalmiviruses and ambiviruses. Splipalmiviruses, related to yeast narnaviruses, have multiple positive-sense (+) single-stranded (ss) RNA genomic segments that separately encode the RNA-dependent RNA polymerase motifs, the hallmark of RNA viruses (members of the kingdom Orthornavirae). Ambiviruses appear to have an undivided ssRNA genome of 3∼5 kb with two large open reading frames (ORFs) separated by intergenic regions. Another narna-like virus group has two fully overlapping ORFs on both strands of a genomic segment that span more than 90% of the genome size. New virus lifestyles exhibited by mycoviruses include the yado-kari/yado-nushi nature characterized by the partnership between the (+)ssRNA yadokarivirus and an unrelated dsRNA virus (donor of the capsid for the former) and the hadaka nature of capsidless 10-11 segmented (+)ssRNA accessible by RNase in infected mycelial homogenates. Furthermore, dsRNA polymycoviruses with phylogenetic affinity to (+)ssRNA animal caliciviruses have been shown to be infectious as dsRNA-protein complexes or deproteinized naked dsRNA. Many previous phylogenetic gaps have been filled by recently discovered fungal and other viruses, which have provided interesting evolutionary insights. Phylogenetic analyses and the discovery of natural and experimental cross-kingdom infections suggest that horizontal virus transfer may have occurred and continue to occur between fungi and other kingdoms. Expected final online publication date for the Annual Review of Phytopathology, Volume 60 is August 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Mycovirus Diversity and Evolution Revealed/Inferred from Recent Studies.

A novel hepta-segmented double-stranded RNA (dsRNA) virus was isolated and characterized from the strain FJ-4 of the phytopathogenic fungus Colletotrichum fructicola, and was named Colletotrichum fructicola chrysovirus 1 (CfCV1). The full-length cDNAs of dsRNA1-7 were 3620, 2801, 2687, 2437, 1750, 1536, and 1211 bp, respectively. The 5'- and 3'-untranslated regions of the seven dsRNAs share highly similar internal sequence and contain conserved sequence stretches, indicating that they have a common virus origin. The 5'-and 3'-UTRs of the seven dsRNAs were predicted to fold into stable stem-loop structures. CfCV1 contains spherical virions that are 35 nm in diameter consisting of seven segments. The largest dsRNA of CfCV1 encodes an RNA-dependent RNA polymerase (RdRp), and the second dsRNA encodes a viral capsid protein (CP). The dsRNA5 encodes a C2H2-type zinc finger protein containing an R-rich region and a G-rich region. The smallest dsRNA is a satellite-like RNA. The functions of the other proteins encoded by dsRNA3, dsRNA4, dsRNA6 are unknown. Phylogenetic analysis, based on RdRp and CP, indicated that CfCV1 is phylogenetically related to Botryosphaeria dothidea chrysovirus 1 (BdCV1), and Penicillium janczewskii chrysovirus 2 (PjCV2), a cluster of an independent cluster II group in the family Chrysoviridae. Importantly, all the seven segments of CfCV1 were transmitted successfully to other virus-free strains with an all-or-none fashion. CfCV1 exerts minor influence on the growth of C. fructicola but can confer hypovirulence to the fungal host. To our knowledge, this is the first report of a hepta-segmented tentative chrysovirus in C. fructicola.

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Identification and Characterization of a Novel Hepta-Segmented dsRNA Virus From the Phytopathogenic Fungus Colletotrichum fructicola.

Alternaria fungi are important pathogens infecting a wide variety of organisms. Here, we report a novel double-stranded RNA (dsRNA) mycovirus named Alternaria botybirnavirus 1 (ABRV1) isolated from a phytopathogenic Alternaria sp. strain (SCFS-3) infecting a pear tree in China. ABRV1 has two dsRNA components (dsRNAs 1 and 2) with the sizes of 6,188 and 5,903 bp, containing two putative open reading frames encoding two polyproteins (202 and 192 kDa, respectively). The polyprotein encoded by ABRV1 dsRNA1 shares 41% amino acid (aa) sequence identity with the one encoded by dsRNA2 (instead of dsRNA1) of Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1). Conversely, the polyprotein encoded by ABRV1 dsRNA2 shares 46% aa sequence identity with the one (i.e., cap-pol fusion protein) encoded by SsBRV1 dsRNA1. ABRV1 has isometric spherical virus particles (~40 nm in diameter), putatively composed of the 60-, 70- and 80-kDa structural proteins. The genomic organization and phylogenetic analyses revealed that ABRV1 belongs to a newly proposed family “Botybirnaviridae”, and to our knowledge, this is the first report of a botybirnavirus infecting an Alternaria sp. strain.

Characterization of a novel botybirnavirus isolated from a phytopathogenic Alternaria fungus

Botryosphaeria dothidea is an important pathogenic fungus that causes serious diseases in fruits and trunks of many wood plant species worldwide. In this study, 28 B. dothidea strains isolated from pear trunk samples showing stem wart or canker symptoms were used to detect double-stranded RNA (dsRNA) viruses. The dsRNA bands with the size of ~ 1.0 to ~ 6.0 kbp were examined from ten strains. Here, we reported a novel dsRNA mycovirus, tentatively named as Botryosphaeria dothidea victorivirus 2 (BdVV2), isolated from the B. dothidea strain MSD53. BdVV2 contained spherical virions that were ~ 38 nm in diameter consisting of a single linear dsRNA genome of 5,090 bp in length. The BdVV2 genome contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp), which shared significant amino acid identities of 68% and 60% with the corresponding proteins of Sphaeropsis sapinea RNA viruses 1 (SsRV1). Phylogenetic analyses based on the aa sequences of CP and RdRp both showed that BdVV2 was phylogenetically related to the members of the genus Victorivirus in the family Totiviridae. BdVV2 is thus a novel victorivirus isolated from the phytopathogenic fungus B. dothidea.

Characterization of a novel victorivirus isolated from the phytopathogenic fungus Botryosphaeria dothidea

In this study, we report the molecular characterization of a novel positive-sense single-stranded RNA (+ssRNA) mycovirus from Botryosphaeria dothidea strain G91, which we name "Botryosphaeria dothidea botourmiavirus 1" (BdBOV-1). The complete genome of BdBOV-1 is 2547 nucleotides (nt) long, contains one open reading frame (ORF) potentially encoding an RNA-dependent RNA polymerase (RdRp), and shows a close relationship to mycoviruses of the family Botourmiaviridae. Phylogenetic analysis based on the RdRp sequences confirmed that BdBOV-1 clustered together with the members of the family Botourmiaviridae. To the best of our knowledge, this is the first report of a +ssRNA mycovirus infecting B. dothidea.

Molecular characterization of a novel mycovirus infecting the phytopathogenic fungus Botryosphaeria dothidea

In this study, we describe a novel positive, single-stranded (+ss) RNA mycovirus, named Botryosphaeria dothidea botrexvirus 1 (BdBV1), from a phytopathogenic fungus Botryosphaeria dothidea showing abnormal morphology and attenuated virulence. BdBV1 is phylogenetically related to Botrytis virus X (BotVX) and is the second potential member of the proposed genus Botrexvirus in the family Alphaflexiviridae. However, it differs from the monopartite BotVX in that BdBV1 possesses a bipartite genome comprised of two ssRNA segments (RNA1 and RNA2 with lengths of 5,035 and 1,063 nt, respectively). BdBV1 RNA1 and RNA2 encode putative RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes, which share significant identity with corresponding genes in both fungal and plant viruses. Moreover, open reading frames (ORFs) 2-4 of BdBV1 RNA1 shared no detectable identity with any known viral proteins. Immunosorbent electron microscopy (ISEM) analysis using an antibody against the virus CP generated in vitro revealed that BdBV1 is encapsidated in filamentous particles. A comparison of the biological effects of BdBV1 infection on symptoms and growth in isogenic lines of virus-free and virus-infected B. dothidea revealed that BdBV1 is probably involved in reduced growth and virulence of the host fungus. This study describes and characterizes a novel bipartite botrexvirus, which is closely related to uni- and multi-partite fungal and plant viruses and contributes useful information to a better understanding of virus evolution.

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Feng Xiao

Papers

Identification and Characterization of a Novel Hepta-Segmented dsRNA Virus From the Phytopathogenic Fungus Colletotrichum fructicola.

Characterization of a novel botybirnavirus isolated from a phytopathogenic Alternaria fungus

Characterization of a novel victorivirus isolated from the phytopathogenic fungus Botryosphaeria dothidea

Molecular characterization of a novel mycovirus infecting the phytopathogenic fungus Botryosphaeria dothidea

Discovery and Characterization of a Novel Bipartite Botrexvirus From the Phytopathogenic Fungus Botryosphaeria dothidea