Enrichment of bacteria involved in the nitrogen cycle and plant growth promotion in soil by sclerotia of rice sheath blight fungus.

Rice sheath blight pathogen, Rhizoctonia solani, produces numerous sclerotia to overwinter. As a rich source of nutrients in the soil, sclerotia may lead to the change of soil microbiota. For this purpose, we amended the sclerotia of R. solani in soil and analyzed the changes in bacterial microbiota within the soil at different time points. At the phyla level, Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chloroflexi and Firmicutes showed varied abundance in the amended soil samples compared to those in the control. An increased abundance of ammonia-oxidizing bacterium (AOB) Nitrosospira and Nitrite oxidizing bacteria (NOB) i.e., Nitrospira was observed, where the latter is reportedly involved in the nitrifier denitrification. Moreover, Thiobacillus, Gemmatimonas, Anaeromyxobacter and Geobacter, the vital players in denitrification, N2O reduction and reductive nitrogen transformation, respectively, depicted enhanced abundance in R. solani sclerotia-amended samples. Furthermore, asymbiotic nitrogen-fixing bacteria, notably, Azotobacter as well as Microvirga and Phenylobacterium with nitrogen-fixing potential also enriched in the amended samples compared to the control. Plant growth promoting bacteria, such as Kribbella, Chitinophaga and Flavisolibacter also enriched in the sclerotia-amended soil. As per our knowledge, this study is of its kind where pathogenic fungal sclerotia activated microbes with a potential role in N transformation and provided clues about the ecological functions of R. solani sclerotia on the stimulation of bacterial genera involved in different processes of N-cycle within the soil in the absence of host plants.

Exploring the Symbiotic Mechanism of a Virus-Mediated Endophytic Fungus in Its Host by Dual Unique Molecular Identifier-RNA Sequencing.

The symbiosis of endophytes and plants is universal in nature. However, how endophytes grow in plants is not entirely clear. Previously, we reported that a virus-infected fungal pathogen could grow in plants as an endophyte. In this study, we utilized Sclerotinia sclerotiorum strain DT-8, a virus-mediated endophyte, to investigate the mechanism of symbiosis with rapeseed by dual unique molecular identifier-RNA sequencing (dual-UMI RNA-seq). We found that the expressions of genes encoding S. sclerotiorum amylase/glucoamylase, glucose transporters, and rapeseed sugars will eventually be exported transporter 11 (SWEET11) were upregulated. It suggested that strain DT-8 might utilize plant starch as a nutrient. The defense systems of rapeseed were also activated, such as production of reactive oxygen species, phenylpropanoids, and brassinin, to control the growth of strain DT-8, while strain DT-8 counteracted host suppression by producing effector-like proteins, detoxification enzymes, and antioxidant components. Moreover, rapeseed also upregulated pectate lyase and pectinesterase genes to facilitate the colonization by strain DT-8. Our findings provide novel insights into the interaction of virus-mediated endophytes and their hosts that warrant further study. IMPORTANCE Although endophytes are widespread in nature, the interactions between endophytes and their hosts are still not fully understood. Members of a unique class of endophytes, the virus-mediated endophytic fungi, are continuously being discovered and have received wide attention. In this study, we investigated the interaction between a mycovirus-mediated endophytic fungus and its host rapeseed by using dual-UMI RNA-seq. According to the dual-UMI RNA-seq results, an aerial view of symbiotic mechanism under balanced regulation was suggested. This research expands our understanding of the symbiotic mechanisms of virus-fungus-plant interactions and could establish a foundation for the further development of practical application with virus-mediated hypovirulent fungi.

Isolation and evaluation of the biocontrol potential of Talaromyces spp. against rice sheath blight guided by soil microbiome.

Rice sheath blight caused by Rhizoctonia solani is the major disease of rice that seriously threatens food security worldwide. Efficient and eco-friendly biological approaches are urgently needed since no resistant cultivars are available. In this study, fallow and paddy soils were initially subjected to microbiome analyses, and the results showed that Talaromyces spp. were significantly more abundant in the paddy soil, while Trichoderma spp. were more abundant in the fallow soil, suggesting that Talaromyces spp. could live and survive better in the paddy soil. Five Talaromyces isolates, namely, TF-04, TF-03, TF-02, TF-01 and TA-02, were isolated from the paddy soil using sclerotia of R. solani as baits and were further evaluated for their activity against rice sheath blight. These isolates efficiently parasitized the hyphae and rotted the sclerotia even at higher water contents in the sterilized sand and the soil. Isolate TF-04 significantly promoted rice growth, reduced the severity of rice sheath blight and increased the rice yield under outdoor conditions. Defence-related genes were upregulated and enzyme activities were enhanced in rice treated with isolate TF-04. Our research supplies a microbiome-guided approach to screen biological control agents and provides Talaromyces isolates to biologically control rice sheath blight.

Bio-priming with a hypovirulent phytopathogenic fungus enhances the connection and strength of microbial interaction network in rapeseed.

Plant disease is one of the most important causes of crop losses worldwide. The effective control of plant disease is related to food security. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum leads to serious yield losses in rapeseed (Brassica napus) production. Hypovirulent strain DT-8 of S. sclerotiorum, infected with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), has the potential to control SSR. In this study, we found rapeseed bio-priming with strain DT-8 could significantly decrease the disease severity of SSR and increase yield in the field. After bio-priming, strain DT-8 could be detected on the aerial part of the rapeseed plant. By 16S rRNA gene and internal transcribed spacer (ITS) sequencing technique, the microbiome on different parts of the SSR lesion on bioprimed and non-bioprimed rapeseed stem was determined. The results indicated that SSR and bio-priming treatment could influence the structure and composition of fungal and bacterial communities. Bio-priming treatment could reduce the total abundance of possible plant pathogens and enhance the connectivity and robustness of the interaction network at the genus level. This might be one of the mechanisms that rapeseed bioprimed with strain DT-8 had excellent tolerance on SSR. It might be another possible mechanism of biocontrol and will provide a theoretical guide for agricultural practical production.

Genome-Wide Identification and Expression Analysis of the bZIP Transcription Factors in the Mycoparasite Coniothyrium minitans.

The basic leucine zipper (bZIP) proteins family is one of the largest and most diverse transcription factors, widely distributed in eukaryotes. However, no information is available regarding the bZIP gene family in Coniothyrium minitans, an important biocontrol agent of the plant pathogen Sclerotinia sclerotiorum. In this study, we identified 34 bZIP genes from the C. minitans genome, which were classified into 8 groups based on their phylogenetic relationships. Intron analysis showed that 28 CmbZIP genes harbored a variable number of introns, and 15 of them shared a feature that intron inserted into the bZIP domain. The intron position in bZIP domain was highly conserved, which was related to recognize the arginine (R) and could be treated as a genomic imprinting. Expression analysis of the CmbZIP genes in response to abiotic stresses indicated that they might play distinct roles in abiotic stress responses. Results showed that 22 CmbZIP genes were upregulated during the later stage of conidial development. Furthermore, transcriptome analysis indicated that CmbZIP genes are involved in different stages of mycoparasitism. Among deletion mutants of four CmbZIPs (CmbZIP07, -09, -13, and -16), only ΔCmbZIP16 mutants significantly reduced its tolerance to the oxidative stress. The other mutants exhibited no significant effects on colony morphology, mycelial growth, conidiation, and mycoparasitism. Taken together, our results suggested that CmbZIP genes play important roles in the abiotic stress responses, conidial development, and mycoparasitism. These results provide comprehensive information of the CmbZIP gene family and lay the foundation for further research on the bZIP gene family regarding their biological functions and evolutionary history.

Mycoparasitism illuminated by genome and transcriptome sequencing of Coniothyrium minitans, an important biocontrol fungus of the plant pathogen Sclerotinia sclerotiorum.

Coniothyrium minitans is a mycoparasite of the notorious plant pathogen Sclerotinia sclerotiorum. To further understand the parasitism of C. minitans, we assembled and analysed its genome and performed transcriptome analyses. The genome of C. minitans strain ZS-1 was assembled into 350 scaffolds and had a size of 39.8 Mb. A total of 11 437 predicted genes and proteins were annotated, and 30.8 % of the blast hits matched proteins encoded by another member of the Pleosporales, Paraphaeosphaeria sporulosa, a worldwide soilborne fungus with biocontrol ability. The transcriptome of strain ZS-1 during the early interaction with S. sclerotiorum at 0, 4 and 12 h was analysed. The detected expressed genes were involved in responses to host defenses, including cell-wall-degrading enzymes, transporters, secretory proteins and secondary metabolite productions. Seventeen differentially expressed genes (DEGs) of fungal cell-wall-degrading enzymes (FCWDs) were up-regulated during parasitism, with only one down-regulated. Most of the monocarboxylate transporter genes of the major facilitator superfamily and all the detected ABC transporters, especially the heavy metal transporters, were significantly up-regulated. Approximately 8 % of the 11 437 proteins in C. minitans were predicted to be secretory proteins with catalytic activity. In the molecular function category, hydrolase activity, peptidase activity and serine hydrolase activity were enriched. Most genes involved in serine hydrolase activity were significantly up-regulated. This genomic analysis and genome-wide expression study demonstrates that the mycoparasitism process of C. minitans is complex and a broad range of proteins are deployed by C. minitans to successfully invade its host. Our study provides insights into the mechanisms of the mycoparasitism between C. minitans and S. sclerotiorum and identifies potential secondary metabolites from C. minitans for application as a biocontrol agent.

Host Transcriptional Response of Sclerotinia sclerotiorum Induced by the Mycoparasite Coniothyrium minitans.

Mycoparasite Coniothyrium minitans parasitizes specifically the mycelia or sclerotia of Sclerotinia sclerotiorum, a worldwidely spread plant fungal pathogen causing serious diseases on crops. The interaction of C. minitans with S. sclerotiorum remains reciprocal and complex and little is known, especially on the side of the host (S. sclerotiorum). In this study, the early transcriptional response of S. sclerotiorum to the mycoparasitism by C. minitans was explored and the differentially expressed genes (DEGs) were analyzed. Based on GO ontology, KEGG pathway and fungal categories database, 887 up-regulated DEGs were enriched in the growth related function (i.e., rRNA processing, ribosome biogenesis, binding and transport), while the 546 down-regulated DEGs were enriched in the stress-related functions (i.e., oxidoreductase, response to stress and heat and the chorismate biosynthetic process). The expression of shikimate pathway and the biosynthesis of phenylalanine involving genes was significantly suppressed. Furthermore, 581 unenriched DEGs were explored in the parasitizing process and were mapped on the Pfam domains of redox enzymes, Alpha/Beta hydrolase, haloacid dehalogenase, and other universal conserved domain containing proteins. Thirty-two DEGs encoding candidate effectors, with 16 up-regulated and 16 down-regulated, were observed with diverse function. SS1G_11912 (encoding SsNEP2) was significantly up-regulated and may function in the parasitism. The involving of the shikimate pathway of phenylalanine biosynthesis and effector candidates were discussed. The results provide a basal understand on the interaction of S. sclerotiorum and C. minitans.

Sclerotia of a phytopathogenic fungus restrict microbial diversity and improve soil health by suppressing other pathogens and enriching beneficial microorganisms.

Sclerotinia sclerotiorum, a notorious soil-borne pathogen of various important crops, produces numerous sclerotia to oversummer in the soil. Considering that sclerotia may also be attacked by other microbes in the soil, we hypothesized that sclerotia in soil may affect the community of soil microbes directly and/or indirectly. In this study, we inoculated sclerotia of S. sclerotiorum in soil collected from the field to observe changes in microbial diversity over three months using 16S rRNA and ITS2 sequencing techniques. Alpha diversity indices exhibited a decline in the diversity of microbial communities, while permanova results confirmed a significant difference in the microbial communities of sclerotia-amended and non-amended soil samples. In sclerotia-amended soil, fungal diversity showed enrichment of antagonists such as Clonostachys, Trichoderma, and Talaromyces and a drastic reduction in the plant pathogenic microbes compared to the non-amended soil. Sclerotia not only activated the antagonists but also enhanced the abundance of plant growth-promoting bacteria, such as Chitinophaga, Burkholderia, and Dyella. Moreover, the presence of sclerotia curtailed the growth of several notorious plant pathogenic fungi belonging to various genera such as Fusarium, Colletotrichum, Cladosporium, Athelia, Alternaria, and Macrophomina. Thus, we conclude that S. sclerotiorum when dormant in soil can reduce the diversity of soil microbes, including suppressing plant pathogens and enriching beneficial microbes. To the best of our knowledge, this is the first time a plant pathogen has been found in soil that can significantly suppress other pathogens. Our findings may provide novel cues to understand the ecology of crop pathogens in soil and maintaining soil conditions that could be beneficial for constructing a healthy soil microorganism community required for mitigating soil-borne diseases.

CmAim24 Is Essential for Mitochondrial Morphology, Conidiogenesis, and Mycoparasitism in Coniothyrium minitans.

Coniothyrium minitans is an important mycoparasite of the notorious phytopathogenic fungus Sclerotinia sclerotiorum The mycoparasitism system of C. minitans-S. sclerotiorum is unique and important in probing fungi and fungal interactions. Here, we report a conidiation-deficient mutant, ZS-1TN1961, which was screened from a transfer DNA (T-DNA) insertional library of C. minitans A single-copy gene, encoding a protein with high sequence similarity to Aim24 (altered inheritance of mitochondria protein 24) in Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Gene replacement and complementation experiments confirmed that mutants lacking CmAim24 exhibited significantly reduced conidial production and germination as well as reduced sclerotial mycoparasitic ability. Furthermore, cellular localization assays showed that CmAim24 localized to mitochondria, and abnormal mitochondria were observed in the ΔCmAim24 mutant. The ΔCmAim24 mutant exhibited significant accumulation of reactive oxygen species (ROS) and a reduced ATP content in mycelia. In summary, our results suggest that CmAim24 plays a key role in mitochondrial architecture and function, conidiogenesis, and mycoparasitism in C. minitansIMPORTANCE Aim24 proteins are involved in mitochondrial biogenesis and accumulate between the two membranes of a mitochondrion. Their function in prokaryotes and filamentous fungi is as yet unknown. In the present study, we characterized an Aim24 protein, CmAim24, in the mycoparasite Coniothyrium minitans and proved its critical role in mitochondrial morphology and function, conidiogenesis, conidial germination, and mycoparasitism to S. sclerotiorum.

Functional Analysis of the Melanin-Associated Gene CmMR1 in Coniothyrium minitans.

Coniothyrium minitans is a sclerotial parasite, which has been investigated for commercial control of crop diseases caused by Sclerotinia sclerotiorum. Previously, we obtained a T-DNA insertional mutant, ZS-1TN24363, which did not produce melanin during conidiation. To understand the function of melanin in C. minitans, we cloned the gene that was disrupted by the T-DNA insertion, and found that this gene, called CmMR1, encoded a putative protein of 1,011 amino acids, which is a homolog of the transcription factor MR. Full-length CmMR1 contains 3,167 bp, with three exons and two introns. To confirm that the disrupted gene is responsible for the melanin-deficiency of the mutant, CmMR1 was disrupted and three targeted knockout mutants were obtained. Biological assays showed that the phenotype of the targeted knockout mutants was similar to that of the T-DNA insertional mutant. Furthermore, gene complementation confirmed that CmMR1 is responsible for the mutant phenotype. CmMR1 disruption did not affect hyphal growth, conidiation, and parasitization of C. minitans, however, the ROS accumulation increased and tolerance to UV light decreased significantly in the mutants. Our result may enhance the understanding of melanin in the ecology of C. minitans on molecular level.