A novel alternavirus with three dsRNA segments from Fusarium pseudograminearum, the pathogen of Fusarium crown rot in wheat.

Three dsRNA segments were detected in Fusarium pseudograminearum strain CF14029, a pathogen causing Fusarium crown rot in China. Characterization and sequence analysis confirmed that these dsRNA sequences originated from the same virus. The viral genome consists of three dsRNA segments: dsRNA1 (3,560 nt in length), encoding an RNA-dependent RNA polymerase (RdRp), dsRNA2 (2,544 nt in length), encoding a hypothetical protein, and dsRNA3 (2,478 nt in length), encoding a putative coat protein (CP). Phylogenetic analysis based on the RdRp and CP amino acid sequences revealed a high degree of similarity of this virus to members of the genus Alternavirus, family Alternaviridae, isolated from other Fusarium fungi. As a novel member of the genus Alternavirus, this virus was provisionally named "Fusarium pseudograminearum alternavirus 1" (FpgAV1). Like other alternaviruses found in Fusarium species, the positive-sense strand of each genomic dsRNA of FpgAV1 possesses a poly(A) tail and a distinctive 5'-terminal octamer sequence (5'-GCT GTG TG-3'). This is the first report of the genomic sequence of an alternavirus identified in F. pseudograminearum.

Efficacy of cyclobutrifluram in controlling Fusarium crown rot of wheat and resistance risk of three Fusarium species to cyclobutrifluram.

Cyclobutrifluram (TYMIRIUM® technology), a new succinate dehydrogenase inhibitor (SDHI) fungicide, is currently being registered by SYNGENTA for controlling Fusarium crown rot (FCR) of wheat in China. The application of 15 or 30 g of active ingredient/100 kg seed of cyclobutrifluram significantly reduced pre-emergence damping-off, discoloration on the stem base and formation of whiteheads caused by FCR. The EC50 values of cyclobutrifluram for 60 isolates of F. pseudograminearum, 30 isolates of F. asiaticum and 30 isolates of F. graminearum ranged from 0.016 to 0.142 mg L-1, 0.010 to 0.041 mg L-1 and 0.012 to 0.059 mg L-1, respectively. One hundred and seven cyclobutrifluram-resistant (CR) mutants were obtained from three Fusarium species isolates, with ten types of mutations identified in Sdh genes. Three Fusarium species isolates exhibited similar resistance mechanisms, with the most prevalent mutations, SdhC1A83V and SdhC1R86K, accounting for 61.68% of mutants. The CR mutants possessed comparable or slightly impaired fitness compared to the corresponding parental isolates. The CR mutants carrying FpSdhBH248Y/Q/D exhibited increased sensitivity to fluopyram. An overall moderate risk of resistance development in three Fusarium species was recommended for cyclobutrifluram.

Genome Analyses Reveal the Secondary Metabolites that Potentially Influence the Geographical Distribution of Fusarium pseudograminearum Populations.

Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, significantly impacts wheat yield and quality in China's Huanghuai region. The rapid F. pseudograminearum epidemic and FCR outbreak within a decade remain unexplained. In this study, two high-quality, chromosome-level genomes of F. pseudograminearum strains producing 3-acetyl-deoxynivalenol (3AcDON) and 15-acetyl-deoxynivalenol (15AcDON) toxins were assembled. Additionally, 38 related strains were resequenced. Genomic differences such as single nucleotide polymorphisms (SNPs), insertions/deletions (indels), and structural variations (SVs) among F. pseudograminearum strains were analyzed. The whole-genome SNP locus-based population classification mirrored the toxin chemotype (3AcDON and 15AcDON)-based classification, indicating the presence of genes associated with the trichothecene toxin gene cluster. Further analysis of differential SNP, indel, and SV loci between the 3AcDON and 15AcDON populations revealed a predominant connection to secondary metabolite synthesis genes. Notably, the majority of the secondary metabolite biosynthesis gene cluster loci were located in SNP-dense genomic regions, suggesting high mutability and a possible contribution to F. pseudograminearum population structure and environmental adaptability. This study provides insightful perspectives on the distribution and evolution of F. pseudograminearum and for forecasting the spread of wheat FCR, thereby aiding in the development of preventive measures and control strategies.

UDP-Galactopyranose Mutase Mediates Cell Wall Integrity, Polarity Growth, and Virulence in Fusarium graminearum.

CHY1 is a zinc finger protein unique to microorganisms that was found to regulate polarized tip growth in Fusarium graminearum, an important pathogen of wheat and barley. To further characterize its functions, in this study we identified CHY1-interacting proteins by affinity purification and selected UDP-galactofuranose (Galf) mutase (UGMA) for detailed characterization, because UGMA and UDP-Galf are unique to fungi and bacteria and absent in plants and animals. The interaction between CHY1 and UGMA was confirmed by yeast two-hybrid assays. Deletion of UGMA in F. graminearum resulted in significant defects in vegetative growth, reproduction, cell wall integrity, and pathogenicity. Infection with the ΔugmA mutant was restricted to the inoculated floret, and no vomitoxin was detected in kernels inoculated with the ΔugmA strain. Compared to the wild type, the ΔugmA mutant produced wide, highly branched hyphae with thick walls, as visualized by transmission electron microscopy. UGMA tagged with green fluorescent protein (GFP) mainly localized to the cytoplasm, consistent with the synthesis of Galf in the cytoplasm. The Δchy1 mutant was more sensitive, while the ΔugmA mutant was more tolerant, to cell wall-degrading enzymes. The growth of the ΔugmA mutant nearly ceased upon caspofungin treatment. More interestingly, nocodazole treatment of the ΔugmA strain attenuated its highly branched morphology, while caspofungin inhibited the degree of the twisted Δchy1 mycelia, indicating that CHY1 and UGMA probably have opposite effects on cell wall architecture. In conclusion, UGMA is an important pathogenic factor that is specific to fungi and bacteria and required for cell wall architecture, radial growth, and caspofungin tolerance, and it appears to be a promising target for antifungal agent development. IMPORTANCE The long-term use of chemical pesticides has had increasingly negative impacts on the ecological environment and human health. Low-toxicity, high-efficiency and environmentally friendly alternative pesticides are of great significance for maintaining the sustainable development of agriculture and human and environmental health. Using fungus- or microbe-specific genes as candidate targets provides a good foundation for the development of low-toxicity, environmentally friendly pesticides. In this study, we characterized a fungus- and bacterium-specific UDP-galactopyranose mutase gene, ugmA, that contributes to the synthesis of the cell wall component Galf and is required for vegetative growth, cell wall integrity, deoxynivalenol (DON) production, and pathogenicity in F. graminearum. The ugmA deletion mutant exhibited increased sensitivity to caspofungin. These results demonstrate the functional importance of UGMA in F. graminearum, and its absence from mammals and higher plants constitutes a considerable advantage as a low-toxicity target for the development of new anti-Fusarium agents.

The CHY-Type Zinc Finger Protein FgChy1 Regulates Polarized Growth, Pathogenicity, and Microtubule Assembly in Fusarium graminearum.

Microtubules (MTs), as transport tracks, play important roles in hyphal-tip growth in filamentous fungi, but MT-associated proteins involved in polarized growth remain unknown. Here, we found that one novel zinc finger protein, FgChy1, is required for MT morphology and polarized growth in Fusarium graminearum. The Fgchy1 mutant presented curved and directionless growth of hyphae. Importantly, the conidia and germ tubes of the Fgchy1 mutant exhibited badly damaged and less-organized beta-tubulin cytoskeletons. Compared with the wild type, the Fgchy1 mutant lost the ability to maintain polarity and was also more sensitive to the anti-MT drugs carbendazim and nocodazole, likely due to the impaired MT cytoskeleton. Indeed, the hyphae of the wild type treated with nocodazole exhibited a morphology consistent with that of the Fgchy1 mutant. Interestingly, the disruption of FgChy1 resulted in the off-center localization of actin patches and the polarity-related polarisome protein FgSpa2 from the hyphal-tip axis. A similar defect in FgSpa2 localization was also observed in the nocodazole-treated wild-type strain. In addition, FgChy1 is also required for conidiogenesis, septation, sexual reproduction, pathogenicity, and deoxynivalenol production. Overall, this study provides the first demonstrations of the functions of the novel zinc finger protein FgChy1 in polarized growth, development, and virulence in filamentous fungi.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Novel positive-sense single-stranded RNA virus related to alphavirus-like viruses from Fusarium graminearum.

A putative novel positive-sense (+) RNA virus was detected in isolate CF16158 of the fungus Fusarium graminearum, the causal agent of Fusarium head blight and crown rot in wheat in China. The full genome of this virus was sequenced and characterized. The complete cDNA sequence is 7,051 nt long and contains four open reading frames (ORFs). ORF2 is predicted to encode helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains that are conserved among the alphavirus-like viruses. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of Hel and RdRp indicated that this (+) RNA mycovirus is a novel member of a new, yet to be established family of alphavirus-like viruses. Therefore, we named this virus "Fusarium graminearum alphavirus-like virus 1" (FgALV1). This is the first report of a full-length genomic sequence of a putative alphavirus-like virus in F. graminearum.

Genome-wide identification, characterization analysis and expression profiling of auxin-responsive GH3 family genes in wheat (Triticum aestivum L.).

Auxin affects many aspects of plant growth and development by regulating the expression of auxin-responsive genes. As one of the three major auxin-responsive families the Gretchen Hagen3 (GH3) gene family maintains hormonal homeostasis by conjugating excess indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acid (JA) to amino acids during hormone and stress-related signaling. Although some work has been carried out the functions of wheat GH3 (TaGH3) family genes in response to abiotic stresses (including salt stress and osmotic stress) are largely unknown. Access to the complete wheat genome sequence permits genome-wide studies on TaGH3s. We performed a systematic identification of the TaGH3 gene family at the genome level and detected 36 members on 14 wheat chromosomes. Many of the genes were segmentally duplicated and Ka/Ks and inter-species synthetic analyses indicated that polyploidization was the contributor to the increased number of TaGH3 members. Phylogenetic analyses revealed that TaGH3 proteins could divided into three major categories (TaGH3-I, TaGH3-II, and TaGH3-III). Diversified cis-elements in the promoters of TaGH3 genes were predicted as essential players in regulating TaGH3 expression patterns. Gene structure and motif analyses indicated that most TaGH3 genes have relatively conserved exon/intron arrangements and motif compositions. Analysis of multiple transcriptome data sets indicated that many TaGH3 genes are responsive to biological and abiotic stresses and possibly have important functions in stress response. qRT-PCR analysis revealed that TaGH3s were induced by salt and osmotic stresses. Customized annotation results revealed that TaGH3s were widely involved in phytohormone response, defense, growth and development, and metabolism. Overall, our work provides a comprehensive insight into the TaGH3 family members, and a basis for the further study of their biological functions in wheat.

A Victorivirus from Fusarium asiaticum, the pathogen of Fusarium head blight in China.

A Victorivirus was detected in isolate F16176 of the fungus Fusarium asiaticum, the causal agent of Fusarium head blight in China. The full genome sequence of the virus was sequenced and characterized. The complete cDNA sequence is 5,281 nucleotides long with 64.2% G + C content and contains two open reading frames (ORFs) that overlap at the pentanucleotide UAAUG. The two ORFs are predicted to encode coat protein (CP) and RNA-dependent RNA polymerase (RdRp), which are conserved among the dsRNA mycoviruses of the genus Victorivirus. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of RdRp indicated that this dsRNA mycovirus is a new virus belonging to the species Rosellinia necatrix victorivirus 1 in the family Totiviridae. This study is the first to report a full-length genomic sequence of a putative member of the genus Victorivirus in F. asiaticum.

A subunit of the HOPS endocytic tethering complex, FgVps41, is important for fungal development and plant infection in Fusarium graminearum.

The signals by which eukaryotic cells communicate with the environment are usually mediated by vesicle trafficking to be attenuated or terminated. However, vesicle trafficking-mediated signal transmission during interactions between pathogens and host plants is poorly understood. Here, we identified and characterized the vacuole sorting protein FgVps41, which is the yeast HOPS tethering complex subunit Vps41 homolog in Fusarium graminearum. Targeted gene deletion demonstrated that FgVps41 is important for vegetative growth, asexual/sexual development, conidial morphology, plant infection and deoxynivalenol production. Cellular localization and cytological examinations revealed that FgVps41 localizes to early/late endosomes and vacuole membrane, and is recruited to prevacuolar compartments and vacuole membrane by interacting with FgRab7 in F. graminearum. Furthermore, we found FgVps41 mediates vacuole membrane fusion and sorting of FgApeI, a cargo protein involving in the cytosol-to-vacuole targeting pathway. In addition, we found that FgVps41 interacts with FgYck3, a vacuolar type I casein kinase, which regulates vesicle fusion in the AP-3 pathway. Deletion of FgYck3 showed similar phenotypes to the ΔFgvps41 mutant, and both FgRab7 and FgYck3 regulate the normal localization of FgVps41. Collectively, our results demonstrate that FgVps41 acts as a HOPS tethering complex subunit and is important for the development of infection-related morphogenesis in F. graminearum.

The FgVps39-FgVam7-FgSso1 Complex Mediates Vesicle Trafficking and Is Important for the Development and Virulence of Fusarium graminearum.

Vesicle trafficking is an important event in eukaryotic organisms. Many proteins and lipids transported between different organelles or compartments are essential for survival. These processes are mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Rab-GTPases, and multisubunit tethering complexes such as class C core vacuole or endosome tethering and homotypic fusion or vacuole protein sorting (HOPS). Our previous study has demonstrated that FgVam7, which encodes a SNARE protein involving in vesicle trafficking, plays crucial roles in growth, asexual or sexual development, deoxynivalenol production, and pathogenicity in Fusarium graminearum. Here, the affinity purification approach was used to identify FgVam7-interacting proteins to explore its regulatory mechanisms during vesicle trafficking. The orthologs of yeast Vps39, a HOPS tethering complex subunit, and Sso1, a SNARE protein localized to the vacuole or endosome, were identified and selected for further characterization. In yeast two-hybrid and glutathione-S-transferase pull-down assays, FgVam7, FgVps39, and FgSso1 interacted with each other as a complex. The ∆Fgvps39 mutant generated by targeted deletion was significantly reduced in vegetative growth and asexual development. It failed to produce sexual spores and was defective in plant infection and deoxynivalenol production. Further cellular localization and cytological examinations suggested that FgVps39 is involved in vesicle trafficking from early or late endosomes to vacuoles in F. graminearum. Additionally, the ∆Fgvps39 mutant was defective in vacuole morphology and autophagy, and it was delayed in endocytosis. Our results demonstrate that FgVam7 interacts with FgVps39 and FgSso1 to form a unique complex, which is involved in vesicle trafficking and modulating the proper development of infection-related morphogenesis in F. graminearum.

Genetic Structure of Populations of the Wheat Sharp Eyespot Pathogen Rhizoctonia cerealis Anastomosis Group D Subgroup I in China.

Sharp eyespot on wheat is caused by Rhizoctonia cerealis anastomosis group D subgroup I (AG-DI) and is an economically important stem-base disease of wheat in temperate regions worldwide. However, the understanding about the field population structure of R. cerealis is limited. In this study, the genetic structure of four wheat-infecting populations in China was investigated using six microsatellite markers characterized from the transcriptome data of R. cerealis AG-DI. A total of 173 unique genotypes were identified among 235 fungal isolates. Departure from Hardy-Weinberg equilibrium, a significant degree of inbreeding, and a significant deficit in heterozygotes indicated a nonrandom mating pattern. Combining the low to intermediate degrees of gametic disequilibrium, although with high genotypic diversity and low to moderate clonal fractions, sexual reproduction probably existed, but the asexual reproduction should be the predominant reproductive mode. Structural analysis showed three gene pools among the four populations, which indicated the existence of three evolutionary origins of R. cerealis AG-DI. The long-distance movement of contaminated material, especially the infected seed, might have caused the moderate gene flow among these populations, which was consistent with the high differentiation among these populations. Overall, the genetic characteristics of the populations suggested a moderate evolutionary potential for R. cerealis AG-DI in China.

Full genome sequence of a putative novel mitovirus isolated from Rhizoctonia cerealis.

A putative novel mitovirus was found in isolate R1084 of the fungus Rhizoctonia cerealis, the causal agent of sharp eyespot of wheat in China. The full genome sequence of the virus was determined and analyzed. The complete cDNA sequence is 3149 nucleotides long with 59.7% A+T content. Using either the fungal mitochondrial or universal genetic code, the viral genome was found to contain a single large open reading frame that is predicted to encode a protein of 812 amino acids with an RNA-dependent RNA polymerase (RdRp) domain that is conserved in the mitovirus RdRp superfamily. The amino acid sequence of the RdRp domain is only 50% identical to the corresponding domain in Sclerotinia sclerotiorum mitovirus 11, and therefore, this virus is proposed to be a novel mitovirus, designated as Rhizoctonia cerealis mitovirus 1-R1084 (RcMV1-R1084). The distinct codon usage of RcMV1-R1084 hints that this virus is potentially able to replicate not only in mitochondria but also in the cytoplasm. This is the first report of a full-length genomic sequence of a putative mitovirus in R. cerealis.

Calreticulin is required for responding to stress, foraging, and fertility in the white-tip nematode, Aphelenchoides besseyi.

Calreticulin (CRT) regulates a wide array of cellular responses in physiological and pathological processes. A full-length cDNA-encoding CRT protein, namely AbCRT-1, was isolated from Aphelenchoides besseyi, an ectoparasitic plant nematode and the agent of white tip disease of rice. The deduced amino acid sequence of AbCRT-1 was highly homologous with other nematode CRTs, and showed the closest evolutionary relationship with BxCRT-1. In-situ hybridization showed that AbCRT-1 is specifically located in the oesophageal gland and gonads of A. besseyi, suggesting its potential role in parasitism and reproduction. Quantity real-time PCR analysis showed that AbCRT-1 is highly expressed in female nematodes but poorly expressed in eggs, juveniles, and male nematodes. Exposing the nematode to relatively low osmotic stress promotes the transcription of AbCRT-1 whereas extreme desiccation suppresses the transcription significantly. Nematodes in which AbCRT-1 mRNA level had been knocked down by soaking them in AbCRT-1 dsRNA solution distributed randomly and did not aggregate temporally, with a decreased capacity of food discernment. Thus the affected nematodes were markedly less fecund. These results demonstrate that AbCRT-1 is required in A. besseyi for responding to stress, foraging, and fertility.

Survey of Fusarium spp. Causing Wheat Crown Rot in Major Winter Wheat Growing Regions of China.

Fusarium crown rot of wheat has become more prevalent in China. To investigate the phylogenetic structure of Fusarium causing wheat crown rot in China, wheat basal stems with symptoms of the disease were collected from 2009 to 2013 in Jiangsu, Anhui, Henan, Hebei, and Shandong provinces. In total, 175 Fusarium isolates were collected and their mycotoxin chemotypes and distribution were identified. Among the 175 isolates, 123 were Fusarium asiaticum; 95 of these were the chemotype 3-acetyl-deoxynivalenol (3-AcDON) and 28 were nivalenol (NIV). Thirty-seven isolates belonged to F. graminearum, which were all 15-AcDON. Smaller numbers of isolates consisted of F. acuminatum, F. pseudograminearum, and F. avenaceum. The virulence of F. asiaticum and F. graminearum isolates on wheat crowns and heads was comparable. The virulence of isolates of the DON and NIV chemotype were statistically similar, but DON tended to be more aggressive. The DON concentrations in grains from wheat heads inoculated with isolates causing either Fusarium head blight or crown rot were similar. In the five provinces, F. asiaticum of the 3-AcDON chemotype was the predominant pathogen causing crown rot, followed by F. graminearum. Recent changes in causal Fusarium species, chemotypes, and distribution in China are discussed.

The heterogeneity of the rDNA-ITS sequence and its phylogeny in Rhizoctonia cerealis, the cause of sharp eyespot in wheat.

The sequence heterogeneity of the ribosomal internal transcribed spacer (ITS) region was investigated for Rhizoctonia cerealis isolates from the anastomosis group AG-DI. Although sequence variability of the ITS has been reported in a few multinucleate R. solani isolates, it has very rarely been reported in binucleate Rhizoctonia spp. isolates and has never been described in R. cerealis, the pathogen of wheat sharp eyespot. In this study, the ITS regions of 15 R. cerealis isolates were cloned and sequenced. The results revealed more than one different ITS sequence within each isolate. This is the first evidence of ITS sequence heterogeneity in R. cerealis. Based on these ITS sequences, different sequences of one isolate did not cluster in one clade, but all of the sequences of the 15 isolates were clustered in the anastomosis subgroup AG-DI, suggesting that the heterogeneity of the ITS did not affect the molecular identification of their anastomosis group. Haplotype analyses indicated that there might be three evolutionary origins of R. cerealis, or a recombination event could be the cause of different ITS sequences in one genome. This study demonstrates the variability and the evolution of Rhizoctonia, especially binucleate R. cerealis. These findings will help design disease control strategies.

Analysis of simple sequence repeats in the Gaeumannomyces graminis var. tritici genome and the development of microsatellite markers.

Understanding the genetic structure of Gaeumannomyces graminis var. tritici is essential for the establishment of efficient disease control strategies. It is becoming clear that microsatellites, or simple sequence repeats (SSRs), play an important role in genome organization and phenotypic diversity, and are a large source of genetic markers for population genetics and meiotic maps. In this study, we examined the G. graminis var. tritici genome (1) to analyze its pattern of SSRs, (2) to compare it with other plant pathogenic filamentous fungi, such as Magnaporthe oryzae and M. poae, and (3) to identify new polymorphic SSR markers for genetic diversity. The G. graminis var. tritici genome was rich in SSRs; a total 13,650 SSRs have been identified with mononucleotides being the most common motifs. In coding regions, the densities of tri- and hexanucleotides were significantly higher than in noncoding regions. The di-, tri-, tetra, penta, and hexanucleotide repeats in the G. graminis var. tritici genome were more abundant than the same repeats in M. oryzae and M. poae. From 115 devised primers, 39 SSRs are polymorphic with G. graminis var. tritici isolates, and 8 primers were randomly selected to analyze 116 isolates from China. The number of alleles varied from 2 to 7 and the expected heterozygosity (He) from 0.499 to 0.837. In conclusion, SSRs developed in this study were highly polymorphic, and our analysis indicated that G. graminis var. tritici is a species with high genetic diversity. The results provide a pioneering report for several applications, such as the assessment of population structure and genetic diversity of G. graminis var. tritici.

Complete genome sequence of a novel endornavirus in the wheat sharp eyespot pathogen Rhizoctonia cerealis.

We report here the presence of a novel double-stranded RNA (dsRNA) virus in an isolate (R0959) of the fungus Rhizoctonia cerealis, the causal agent of sharp eyespot of wheat in China. Sequence analysis showed that the dsRNA segment is 17,486 bp long and contains a single open reading frame (ORF) with the potential to encode a protein of 5,747 amino acids. The predicted protein contains conserved motifs of putative viral methyltransferase, helicase 1, and RNA-dependent RNA polymerase. Sequence similarity and phylogenetic analysis clearly place it in a distinct species within the genus Endornavirus, family Endornaviridae, and therefore we propose its name to Rhizoctonia cerealis endornavirus 1 (RcEV1). This is the first report of the full-length genomic sequence of a dsRNA mycovirus in R. cerealis.

The critical roles of the Zn2Cys6 transcription factor Fp487 in the development and virulence of Fusarium pseudograminearum: A potential target for Fusarium crown rot control.

Fusarium crown rot (FCR) caused by Fusarium pseudograminearum poses a significant threat to wheat production in the Huang-Huai-Hai region of China. However, the pathogenic mechanism of F. pseudograminearum is still poorly understood. Zn2Cys6 transcription factors, which are exclusive to fungi, play pivotal roles in regulating fungal development, drug resistance, pathogenicity, and secondary metabolism. In this study, we present the functional characterization of a Zn2Cys6 transcription factor F. pseudograminearum, designated Fp487. In F. pseudograminearum, Fp487 is shown to be required for mycelial growth through gene knockout and phenotypic analyses. Compared with wild-type CF14047, the ∆Fp487 mutant displayed a slight reduction in growth rate but a significant decrease in conidiogenesis, pathogenicity and 3-acetyl-deoxynivalenol (3AcDON) production. Moreover, the mutant exhibited heightened sensitivity to oxidative and cytomembrane stress. Furthermore, we synthesized dsRNA from the Fp487 gene in vitro, resulting in a reduction in the growth rate of F. pseudograminearum and its virulence on barley leaves through spray-induced gene silencing (SIGS). Notably, this study makes the first instance of inducing the expression of abundant dsRNA from F. pseudograminearum by engineering the Escherichia coli strain HT115 (DE3) and utilizing the SIGS technique to evaluate the virulence effect of dsRNA on F. pseudograminearum. In conclusion, our findings revealed the crucial role of Fp487 in regulating pathogenicity, stress responses, DON production, and conidiogenesis in F. pseudograminearum. Furthermore, Fp487 is a potential RNAi-based target for FCR control.

Extreme Diversity of Mycoviruses Present in Single Strains of Rhizoctonia cerealis, the Pathogen of Wheat Sharp Eyespot.

Rhizoctonia cerealis is the pathogen of wheat sharp eyespot, which occurs throughout temperate wheat-growing regions of the world. In this project, the genomes of viruses from four strains of R. cerealis were analyzed based on Illumina high-throughput transcriptome sequencing (RNA-Seq) data. After filtering out reads that mapped to the fungal genome, viral genomes were assembled. In total, 131 virus-like sequences containing complete open reading frames (ORFs), belonging to 117 viruses, were obtained. Based on phylogenetic analysis, some of them were identified as novel members of the families Curvulaviridae, Endornaviridae, Hypoviridae, Mitoviridae, Mymonaviridae, and Phenuiviridae, while others were unclassified viruses. Most of these viruses from R. cerealis were significantly different from the viruses already reported. We propose the establishment of a new family, Rhizoctobunyaviridae, and two new genera, Rhizoctobunyavirus and Iotahypovirus. We further clarified the distribution and coinfection of these viruses in the four strains. Surprisingly, 39 viral genomes of up to 12 genera were found in strain R1084. Strain R0942, containing the fewest viruses, also contained 21 viral genomes belonging to 10 genera. Based on the RNA-Seq data, we estimated the accumulation level of some viruses in host cells and found that the mitoviruses in R. cerealis generally have very high accumulation. In conclusion, in the culturable phytopathogenic fungus R. cerealis, we discovered a considerable diversity of mycoviruses and a series of novel viruses. This study expands our understanding of the mycoviral diversity in R. cerealis and provides a rich resource for the further use of mycoviruses to control wheat sharp eyespot. IMPORTANCE Rhizoctonia cerealis is a binucleate fungus that is widely distributed worldwide and can cause sharp eyespot disease in cereal crops. In this study, 131 virus-like sequences belonging to 117 viruses were obtained based on analysis of high-throughput RNA-Seq data from four strains of R. cerealis. Many of these viruses were novel members of various virus families, while others were unclassified viruses. As a result, a new family named Rhizoctobunyaviridae and two new genera, Rhizoctobunyavirus and Iotahypovirus, were proposed. Moreover, the discovery of multiple viruses coinfecting a single host and the high accumulation levels of mitoviruses have shed light on the complex interactions between different viruses in a single host. In conclusion, a significant diversity of mycoviruses was discovered in the culturable phytopathogenic fungus R. cerealis. This study expands our understanding of mycoviral diversity, and provides a valuable resource for the further utilization of mycoviruses to control wheat diseases.

Trehalase Inhibitor Validamycin May Have Additional Mechanisms of Toxicology against Rhizoctonia cerealis.

Sharp eyespot is a crucial disease affecting cereal plants, such as bread wheat (Triticum aestivum) and barley (Hordeum vulgare), and is primarily caused by the pathogenic fungus Rhizoctonia cerealis. As disease severity has increased, it has become imperative to find an effective and reasonable control strategy. One such strategy is the use of the trehalose analog, validamycin, which has been shown to have a potent inhibitory effect on several trehalases found in both insects and fungi, and is widely used as a fungicide in agriculture. In this study, we demonstrated that 0.5 µg/mL validamycin on PDA plates had an inhibitory effect on R. cerealis strain R0301, but had no significant impact on Fusarium graminearum strain PH-1. Except for its inhibiting the trehalase activity of pathogenic fungi, little is known about its mechanism of action. Six trehalase genes were identified in the genome of R. cerealis, including one neutral trehalase and five acidic trehalase genes. Enzyme activity assays indicated that treatment with 5 µg/mL validamycin significantly reduces trehalase activity, providing evidence that validamycin treatment does indeed affect trehalase, even though the expression levels of most trehalase genes, except Rc17406, were not obviously affected. Transcriptome analysis revealed that treatment with validamycin downregulated genes involved in metabolic processes, ribosome biogenesis, and pathogenicity in the R. cerealis. KEGG pathway analysis further showed that validamycin affected genes related to the MAPK signaling pathway, with a significant decrease in ribosome synthesis and assembly. In conclusion, our results indicated that validamycin not only inhibits trehalose activity, but also affects the ribosome synthesis and MAPK pathways of R. cerealis, leading to the suppression of fungal growth and pesticidal effects. This study provides novel insights into the mechanism of action of validamycin.