A Circular Single-Stranded DNA Mycovirus Infects Plants and Confers Broad-Spectrum Resistance against Fungal Diseases.

Circular single-stranded (ss) DNA viruses have been rarely found in fungi, and the evolutionary and ecological relationships among ssDNA viruses infecting fungi and other organisms remain unclear. Here, a novel circular ssDNA virus, tentatively named Diaporthe sojae circular DNA virus 1 (DsCDV1), was identified in the phytopathogenic fungus Diaporthe sojae isolated from pear trees. DsCDV1 has a monopartite genome (3,185 nt in size) encapsidated in isometric virions (21-26 nm in diameter). The genome comprises seven putative open reading frames encoding a discrete replicase (Rep) split by an intergenic region, a putative capsid protein (CP), several proteins of unknown function (P1 to P4), and a long intergenic region. Notably, the two split parts of DsCDV1 Rep share high identities with the Reps of Geminiviridae and Genomoviridae, respectively, indicating an evolutionary linkage with both families. Phylogenetic analysis based on Rep or CP sequences placed DsCDV1 in a unique cluster, supporting the establishment of a new family, tentatively named Gegemycoviridae, intermediate to both families. DsCDV1 significantly attenuates fungal growth and nearly erases virulence when transfected into the host fungus. Remarkably, DsCDV1 can systematically infect tobacco and pear seedlings, providing broad-spectrum resistance to fungal diseases. Subcellular localization analysis revealed that P3 systematically localizes in plasmodesmata, while and its expression in trans-complementation experiments restores the wild-type phenotype of a movement-deficient plant virus; thus P3 is identified as a movement protein. DsCDV1 exhibits unique molecular and biological traits not observed in other ssDNA viruses, serving as a link between fungal and plant ssDNA viruses and presenting an evolutionary connection between ssDNA viruses and fungi. These findings contribute to expanding our understanding of ssDNA virus diversity and evolution, offering potential biocontrol applications for managing crucial plant diseases.

A novel botybirnavirus with a unique satellite dsRNA causes latent infection in Didymella theifolia isolated from tea plants.

IMPORTANCE: A novel botybirnavirus, infecting the tea plant pathogen Didymella theifolia and tentatively named Didymella theifolia botybirnavirus 1 (DtBRV1), together with an additional double-stranded RNA (dsRNA), was characterized. DtBRV1 comprises two dsRNAs (1 and 2) encapsidated in isometric virions, while dsRNA3 is a satellite. The satellite represents a unique specimen since it contains a duplicated region and has high similarity to the two botybirnavirus dsRNAs, supporting the notion that it most likely originated from a deficient genomic component. The biological characteristics of DtBRV1 were further determined. With their unique molecular traits, DtBRV1 and its related dsRNA expand our understanding of virus diversity, taxonomy, and evolution.

Genomic analysis of Colletotrichum camelliae responsible for tea brown blight disease.

BACKGROUND: Colletotrichum camelliae, one of the most important phytopathogenic fungi infecting tea plants (Camellia sinensis), causes brown blight disease resulting in significant economic losses in yield of some sensitive cultivated tea varieties. To better understand its phytopathogenic mechanism, the genetic information is worth being resolved. RESULTS: Here, a high-quality genomic sequence of C. camelliae (strain LT-3-1) was sequenced using PacBio RSII sequencing platform, one of the most advanced Three-generation sequencing platforms and assembled. The result showed that the fungal genomic sequence is 67.74 Mb in size (with the N50 contig 5.6 Mb in size) containing 14,849 putative genes, of which about 95.27% were annotated. The data revealed a large class of genomic clusters potentially related to fungal pathogenicity. Based on the Pathogen Host Interactions database, a total of 1698 genes (11.44% of the total ones) were annotated, containing 541 genes related to plant cell wall hydrolases which is remarkably higher than those of most species of Colletotrichum and others considered to be hemibiotrophic and necrotrophic fungi. It's likely that the increase in cell wall-degrading enzymes reflects a crucial adaptive characteristic for infecting tea plants. CONCLUSION: Considering that C. camelliae has a specific host range and unique morphological and biological traits that distinguish it from other species of the genus Colletotrichum, characterization of the fungal genome will improve our understanding of the fungus and its phytopathogenic mechanism as well.

Amplicon Sequencing Reveals Novel Fungal Species Responsible for a Controversial Tea Disease.

Amplicon sequencing is a powerful tool for analyzing the fungal composition inside plants, whereas its application for the identification of etiology for plant diseases remains undetermined. Here, we utilize this strategy to clarify the etiology responsible for tea leaf brown-black spot disease (LBSD), a noticeable disease infecting tea plants etiology that remains controversial. Based on the ITS-based amplicon sequencing analysis, Didymella species were identified as separate from Pestalotiopsis spp. and Cercospora sp., which are concluded as the etiological agents. This was further confirmed by the fungal isolation and their specific pathogenicity on diverse tea varieties. Based on the morphologies and phylogenetic analysis constructed with multi-loci (ITS, LSU, tub2, and rpb2), two novel Didymella species-tentatively named D. theae and D. theifolia as reference to their host plants-were proposed and characterized. Here, we present an integrated approach of ITS-based amplicon sequencing in combination with fungal isolation and fulfillment of Koch's postulates for etiological identification of tea plant disease, revealing new etiology for LBSD. This contributes useful information for further etiological identification of plant disease based on amplicon sequencing, as well as understanding, prevention, and management of this economically important disease.

A Novel Heptasegmented Positive-Sense Single-Stranded RNA Virus from the Phytopathogenic Fungus Colletotrichum fructicola.

In this study, a novel positive-sense single-stranded RNA (+ssRNA) mycovirus, tentatively named Colletotrichum fructicola RNA virus 1 (CfRV1), was identified in the phytopathogenic fungus Colletotrichum fructicola. CfRV1 has seven genomic components, encoding seven proteins from open reading frames (ORFs) flanked by highly conserved untranslated regions (UTRs). Proteins encoded by ORFs 1, 2, 3, 5, and 6 are more similar to the putative RNA-dependent RNA polymerase (RdRp), hypothetical protein (P2), methyltransferase, and two hypothetical proteins of Hadaka virus 1 (HadV1), a capsidless 10- or 11-segmented +ssRNA virus, while proteins encoded by ORFs 4 and 7 showed no detectable similarity to any known proteins. Notably, proteins encoded by ORFs 1 to 3 also share considerably high similarity with the corresponding proteins of polymycoviruses. Phylogenetic analysis conducted based on the amino acid sequence of CfRV1 RdRp and related viruses placed CfRV1 and HadV1 together in the same clade, close to polymycoviruses and astroviruses. CfRV1-infected C. fructicola strains demonstrate a moderately attenuated growth rate and virulence compared to uninfected isolates. CfRV1 is capsidless and potentially encapsulated in vesicles inside fungal cells, as revealed by transmission electron microscopy. CfRV1 and HadV1 are +ssRNA mycoviruses closely related to polymycoviruses and astroviruses, represent a new linkage between +ssRNA viruses and the intermediate double-stranded RNA (dsRNA) polymycoviruses, and expand our understanding of virus diversity, taxonomy, evolution, and biological traits. IMPORTANCE A scenario proposing that dsRNA viruses evolved from +ssRNA viruses is still considered controversial due to intergroup knowledge gaps in virus diversity. Recently, polymycoviruses and hadakaviruses were found as intermediate dsRNA and +ssRNA stages, respectively, between +ssRNA and dsRNA viruses. Here, we identified a novel +ssRNA mycovirus, Colletotrichum fructicola RNA virus 1 (CfRV1), isolated from Colletotrichum fructicola in China. CfRV1 is phylogenetically related to the 10- or 11-segmented Hadaka virus 1 (HadV1) but consists of only seven genomic segments encoding two novel proteins. CfRV1 is naked and may be encapsulated in vesicles inside fungal cells, representing a potential novel lifestyle for multisegmented RNA viruses. CfRV1 and HadV1 are intermediate +ssRNA mycoviruses in the linkage between +ssRNA viruses and the intermediate dsRNA polymycoviruses and expand our understanding of virus diversity, taxonomy, and evolution.

Characterization of a Novel Mitovirus Infecting Melanconiella theae Isolated From Tea Plants.

A dsRNA segment was identified in the fungus Melanconiella theae isolated from tea plants. The complete dsRNA sequence, determined by random cloning together with RACE protocol, is 2,461 bp in length with an AU-rich content (62.37%) and comprises a single ORF of 2,265-nucleotides encoding an RNA-dependent RNA-polymerase (RdRp, 754 amino acids in size). The terminus sequences can fold into predicted stable stem-loop structures. A BLASTX and phylogenetic analysis revealed the dsRNA genome shows similarities with the RdRp sequences of mitoviruses, with the highest identity of 48% with those of grapevine-associated mitovirus 20 and Colletotrichum fructicola mitovirus 1. Our results reveal a novel member, tentatively named Melanconiella theae mitovirus 1 (MtMV1), belongs to the family Mitoviridae. MtMV1 is capsidless as examined by transmission electron microscope, efficiently transmitted through conidia as 100 conidium-generated colonies were analyzed, and easily eliminated by hyphal tipping method combined with green-leaf tea powder. MtMV1 has a genomic sequence obviously divergent from those of most members in the family Mitoviridae and some unique characteristics unreported in known members. This is the first report of a mycovirus infecting Melanconiella fungi to date.