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.

Molecular characterization of a novel narnavirus infecting the phytopathogenic fungus Botryosphaeria dothidea.

Here, a novel mycovirus, Botryosphaeria dothidea narnavirus 5 (BdNV5), was discovered in the plant-pathogenic fungus Botryosphaeria dothidea strain ZM210167-1. The BdNV5 genome sequence is 2,397 nucleotides (nt) in length and contains a putative open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) with a molecular mass of 72.77 kDa. A BLASTp search using the RdRp amino acid (aa) sequence showed that it was most similar to the RdRp of Botryosphaeria dothidea narnavirus 4 (42.35%). In a phylogenetic tree based on RdRp aa sequences, BdNV5 clustered with members of the family Narnaviridae. BdNV5 is thus a novel member of the family Narnaviridae infecting the phytopathogenic fungus B. dothidea.

Diversity of Lasiodiplodia Species Associated with Canker and Dieback in Fruit Trees in the Henan and Shandong Provinces of China.

Lasiodiplodia is a widely distributed genus that is associated with a variety of diseases in many plant species, especially fruit trees. In this study, a disease survey of fruit trees growing in 12 orchards located in the Henan and Shandong provinces of China was conducted between 2020 and 2022. The symptoms observed included stem canker, branch dieback, and gummosis. Phylogenetic analyses of internal transcribed spacer, tub2, tef1, and rpb2 sequence data combined with morphological characteristics revealed that the 19 isolates collected during the survey belonged to five documented Lasiodiplodia species, namely, Lasiodiplodia citricola, L. chiangraiensis, L. huangyanensis, L. pseudotheobromae, and L. theobromae, and two previously undescribed species, L. xinyangensis and L. ziziphi. In addition, the survey identified three novel host-pathogen interactions: L. chiangraiensis on loquat, L. citricola on apple, and L. huangyanensis on grapevine. Furthermore, the detailed phylogenic analysis indicated that four previously described Lasiodiplodia species were genetically very closely related that they would be better classified as synonyms rather than distinct species, so L. paraphysoides and L. nanpingensis should be considered synonyms of L. citricola, L. fujianensis should be a synonym of L. iraniensis, and L. henanica should be a synonym of L. huangyanensis. Pathogenicity tests confirmed that representative isolates of the two novel species and three new host-pathogen interactions identified in the current study were pathogenic to their original hosts, thereby fulfilling Koch's postulates. Similarly, all of the isolates were found to be pathogenic on four alternative hosts, although a high degree of variation in virulence was observed.

Molecular characterization of a novel ourmia­like virus from the phytopathogenic fungus Botryosphaeria dothidea.

Here, we describe a novel ourmia-like virus, Botryosphaeria dothidea ourmia-like virus 2 (BdOLV2), derived from the phytopathogenic fungus Botryosphaeria dothidea strain ZM180192-1 infecting maize in Henan province of China. The complete genome sequence of BdOLV2 consists of a positive-sense single-stranded RNA (+ ssRNA) segment with a length of 2,532 nucleotides (nt). The sequence contains a large open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) consisting of 605 amino acids (aa) with a molecular mass of 68.59 kDa. This RdRp protein contains eight typical conserved motifs associated with ourmia-like viruses. BLASTp analysis revealed that the RdRp protein of BdOLV2 had the highest similarity (62.10%, 58.15%, and 55.75% identity, respectively) to a virus previously identified as "Botourmiaviridae sp.", Macrophomina phaseolina ourmia-like virus 2, and Macrophomina phaseolina ourmia-like virus 2-A. Phylogenetic analysis based on the RdRp aa sequence indicated that BdOLV2 is a new member of the genus Magoulivirus in the family Botourmiaviridae.

Lasiodiplodia regiae sp. nov.: A New Species Causing Canker and Dieback of Fruit Trees in China.

Canker and dieback are serious fungal diseases of woody plants that can cause huge economic losses to orchards. The purpose of this study was to classify and assess the pathogenicity of fungal species associated with canker and dieback on fruit trees growing in Henan Province, China. In total, 150 isolates of Botryosphaeriaceae were obtained from six different fruit trees exhibiting typical symptoms of stem canker, branch dieback, and gummosis. Morphological examinations and phylogenetic analysis of ITS, tef1, tub2, and rpb2 revealed two Botryosphaeriaceae species, which are Botryosphaeria dothidea and a novel species, Lasiodiplodia regiae, respectively. Using Koch's postulates, we confirmed that the different isolates of L. regiae can cause disease in their original hosts. The pathogenicity tests showed that L. regiae can cause canker, dieback, and gummosis symptoms in four different hosts, indicating a relatively wider host range. Moreover, 10 L. regiae isolates exhibited similar symptoms but different levels of virulence on shoots of peach trees under field conditions. This study demonstrated that L. regiae was a new causal agent of canker and dieback of six fruit tree species, which could be a serious risk to the orchard industry in China. Furthermore, the findings provide a foundation for further epidemiological studies and the development of management strategies.

BsTup1 is required for growth, conidiogenesis, stress response and pathogenicity of Bipolaris sorokiniana.

Tup1, a conserved transcriptional repressor, plays a critical role in the growth and development of fungi. Here, we identified a BsTup1 gene from the plant pathogenic fungus Bipolaris sorokiniana. The expression of BsTup1 showed a more than three-fold increase during the conidial stage compared with mycelium stage. Deletion of BsTup1 led to decrease hyphal growth and defect in conidia formation. A significant difference was detected in osmotic, oxidative, or cell wall stress responses between the WT and ΔBsTup1 strains. Pathogenicity assays showed that virulence of the ΔBsTup1 mutant was dramatically decreased on wheat and barely leaves. Moreover, it was observed that hyphal tips of the mutants could not form appressorium-like structures on the inner epidermis of onion and barley coleoptile. Yeast two-hybrid assays indicated that BsTup1 could interact with the BsSsn6. RNAseq revealed significant transcriptional changes in the ΔBsTup1 mutant with 2369 genes down-regulated and 2962 genes up-regulated. In these genes, we found that a subset of genes involved in fungal growth, sporulation, cell wall integrity, osmotic stress, oxidation stress, and pathogenicity, which were misregulated in the ΔBsTup1 mutant. These data revealed that BsTup1 has multiple functions in fungal growth, development, stress response and pathogenesis in B. sorokiniana.

Comparative mitochondrial genome analyses reveal conserved gene arrangement but massive expansion/contraction in two closely related Exserohilum pathogens.

Exserohilum turcicum and E. rostratum, two closely related fungal species, are both economically important pathogens but have quite different target hosts (specific to plants and cross-kingdom infection, respectively). In the present study, complete circular mitochondrial genomes of the two Exserohilum species were sequenced and de novo assembled, which mainly comprised the same set of 13 core protein-coding genes (PCGs), two rRNAs, and a certain number of tRNAs and unidentified open reading frames (ORFs). Comparative analyses indicated that these two fungi had significant mitogenomic collinearity and consistent mitochondrial gene arrangement, yet with vastly different mitogenome sizes, 264,948 bp and 64,620 bp, respectively. By contrast with the 17 introns containing 17 intronic ORFs (one-to-one) in the E. rostratum mitogenome, E. turcicum involved far more introns (70) and intronic ORFs (126), which was considered as the main contributing factors of their mitogenome expansion/contraction. Within the generally intron-rich gene cox1, a total of 18 and 10 intron position classes (Pcls) were identified separately in the two mitogenomes. Moreover, 16.16% and 10.85% ratios of intra-mitogenomic repetitive regions were detected in E. turcicum and E. rostratum, respectively. Based on the combined mitochondrial gene dataset, we established a well-supported topology of phylogeny tree of 98 ascomycetes, implying that mitogenomes may act as an effective molecular marker for fungal phylogenetic reconstruction. Our results served as the first report on mitogenomes in the genus Exserohilum, and would have significant implications in understanding the origin, evolution and pathogenic mechanisms of this fungal lineage.

Transcriptome Analysis of the Molecular Patterns of Pear Plants Infected by Two Colletotrichum fructicola Pathogenic Strains Causing Contrasting Sets of Leaf Symptoms.

Colletotrichum fructicola infects pear leaves, resulting in two major symptoms: tiny black spots (TS) followed by severe early defoliation and big necrotic lesions (BnL) without apparent damage depending on the pathotypes. How the same fungal species causes different symptoms remains unclear. To understand the molecular mechanism underlying the resulting diseases and the diverse symptoms, two C. fructicola pathogenetic strains (PAFQ31 and PAFQ32 responsible for TS and BnL symptoms, respectively) were inoculated on Pyrus pyrifolia leaves and subjected to transcriptome sequencing at the quiescent stage (QS) and necrotrophic stage (NS), respectively. In planta, the genes involved in the salicylic acid (SA) signaling pathway were upregulated at the NS caused by the infection of each strain. In contrast, the ethylene (ET), abscisic acid (ABA), and jasmonic acid (JA) signaling pathways were specifically related to the TS symptoms caused by the infection of strain PAFQ31, corresponding to the yellowish and early defoliation symptoms triggered by the strain infection. Correspondingly, SA was accumulated in similar levels in the leaves infected by each strain at NS, but JA was significantly higher in the PAFQ31-infected as measured using high-performance liquid chromatography. Weighted gene co-expression network analysis also reveals specific genes, pathways, phytohormones, and transcription factors (TFs) associated with the PAFQ31-associated early defoliation. Taken together, these data suggest that specific metabolic pathways were regulated in P. pyrifolia in response to the infection of two C. fructicola pathotypes resulting in the diverse symptoms: JA, ET, and ABA accumulated in the PAFQ31-infected leaves, which negatively affected the chlorophyll metabolism and photosynthesis pathways while positively affecting the expression of senescence-associated TFs and genes, resulted in leaf yellowing and defoliation; whereas SA inhibited JA-induced gene expression in the PAFQ32-infected leaves, which led to hypersensitive response-like reaction and BnL symptoms.

Characterization of a Novel Mycovirus from the Phytopathogenic Fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is, globally, one of the most economically important phytopathogenic fungi worldwide, causing the canker and dieback of fruit trees. An increasing number of viruses infecting B. dothidea have lately been reported, several of which could confer hypovirulence. In this study, isolated from strain ZM170285-1 of B. dothidea, a novel double-stranded RNA (dsRNA) mycovirus, tentatively named Botryosphaeria dothidea partitivirus 2 (BdPV2), was identified well. The BdPV2 harbored three dsRNA segments (1-3) with lengths of 1751, 1568, and 1198 bp, which encoded an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), and a hypothetical protein of unknown function, respectively. BLASTp searches revealed that the predicted protein sequences of dsRNA1 and dsRNA2 had the highest identities (74.95% and 61.01%) with the corresponding dsRNAs of Penicillium stoloniferum virus S (PsV-S), whereas dsRNA3 shared the highest identity (32.95%) with the dsRNA3 of Aspergillus ochraceous virus 1 (AoV1). Phylogenetic analysis indicated that BdPV2 belonged to the Gammapartitivirus genus and Partitiviridae family. To our knowledge, this is the first report of a Gammapartitivirus in B. dothidea.

Mitogenome-wide comparison and phylogeny reveal group I intron dynamics and intraspecific diversification within the phytopathogen Corynespora cassiicola.

Corynespora cassiicola, the causal agent of an extensive range of plant diseases worldwide, is a momentous fungus with diverse lifestyles and rich in intraspecies variations. In the present study, a total of 56 mitochondrial genomes of C. cassiicola were assembled (except two available online) and analyzed, of which 16 mitogenomes were newly sequenced here. All these circular mitochondrial DNA (mtDNA) molecules, ranging from 39,223 bp to 45,786 bp in length, comprised the same set of 13 core protein-coding genes (PCGs), two rRNAs and 27 tRNAs arranged in identical order. Across the above conserved genes, nad3 had the largest genetic distance between different isolates and was possibly subjected to positive selection pressure. Comparative mitogenomic analysis indicated that seven group I (IB, IC1, and IC2) introns with a length range of 1013-1876 bp were differentially inserted in three core PCGs (cox1, nad1, and nad5), resulting in the varied mitogenome sizes among C. cassiicola isolates. In combination with dynamic distribution of the introns, a well-supported mitogenome-wide phylogeny of the 56 C. cassiicola isolates revealed eight phylogenetic groups, which only had weak correlations with host range and toxin class. Different groups of isolates exhibited obvious differences in length and GC content of some genes, while a degree of variance in codon usage and tRNA structure was also observed. This research served as the first report on mitogenomic comparisons within C. cassiicola, and could provide new insights into its intraspecific microevolution and genetic diversity.

Discovery and Characterization of a Novel Bipartite Botrexvirus From 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.

Characterization of Diaporthe species associated with peach constriction canker, with two novel species from China.

Species of Diaporthe infect a wide range of plants and live in vivo as endophytes, saprobes or pathogens. However, those in peach plants are poorly characterized. In this study, 52 Diaporthe strains were isolated from peach branches with buds, showing constriction canker symptoms. Phylogenetic analyses were conducted using five gene regions: internal transcribed spacer of the ribosomal DNA (ITS), translation elongation factor 1-α (TEF), ß-tubulin (TUB), histone (HIS), and calmodulin (CAL). These results coupled with morphology revealed seven species of Diaporthe, including five known species (D. caryae, D. cercidis, D. eres, D. hongkongensis, and D. unshiuensis). In addition, two novel species D. jinxiu and D. zaofenghuang are introduced. Except for the previously reported D. eres, this study represents the first characterization of Diaporthe species associated with peach constriction canker in China, and contributes useful data for practicable disease management.

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

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.