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 the Fusarium pathogens associated with crown rot in the Huanghuai wheat-growing region of China.

To investigate the distribution and diversity of the pathogens associated with Fusarium crown rot in the Huanghuai wheat-growing region (HHWGR) of China, we collected wheat samples with symptomatic stem bases from seven provinces in the HHWGR between 2013 and 2016. A total of 1196 isolates obtained from 222 locations were identified as 9 Fusarium species based on morphological and molecular identification. Of these pathogen species, F. pseudograminearum was the dominant species. Furthermore, F. sinensis was isolated from the disease specimens and tested for virulence to wheat. The result of the pathogenicity revealed that an intraspecific differentiation existed in F. pseudograminearum; sequence analysis of the EF-1α gene showed that 194 F. pseudograminearum isolates were differentiated into two distinct clades which closed to the strains from Australia and China respectively, but neither pathogenicity nor EF-1α sequence was related to the geographic origins of these isolates. However, universal rice primers-polymerase chain reaction showed a correlation with the geographical origins of the 194 isolates, which were divided into eight subclusters, the level of genetic diversity was higher within a geographical population than among the different populations. The results of these analyses can be directly used to facilitate disease monitoring and development of control strategies.

The complete mitochondrial genomes of five critical phytopathogenic Bipolaris species: features, evolution, and phylogeny.

In the present study, three mitogenomes from the Bipolaris genus (Bipolaris maydis, B. zeicola, and B. oryzae) were assembled and compared with the other two reported Bipolaris mitogenomes (B. oryzae and B. sorokiniana). The five mitogenomes were all circular DNA molecules, with lengths ranging from 106,403 bp to 135,790 bp. The mitogenomes of the five Bipolaris species 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). The PCG length, AT skew and GC skew showed large variability among the 13 PCGs in the five mitogenomes. Across the 13 core PCGs tested, nad6 had the least genetic distance among the 16 Pleosporales species we investigated, indicating that this gene was highly conserved. In addition, the Ka/Ks values for all 12 core PCGs (excluding rps3) were < 1, suggesting that these genes were subject to purifying selection. Comparative mitogenomic analyses indicate that introns were the main factor contributing to the size variation of Bipolaris mitogenomes. The introns of the cox1 gene experienced frequent gain/loss events in Pleosporales species. The gene arrangement and collinearity in the mitogenomes of the five Bipolaris species were almost highly conserved within the genus. Phylogenetic analysis based on combined mitochondrial gene datasets showed that the five Bipolaris species formed well-supported topologies. This study is the first report on the mitogenomes of B. maydis and B. zeicola, as well as the first comparison of mitogenomes among Bipolaris species. The findings of this study will further advance investigations into the population genetics, evolution, and genomics of Bipolaris species.

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.

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.

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.