Isolation and Genome-Based Characterization of Biocontrol Potential of Bacillus siamensis YB-1631 against Wheat Crown Rot Caused by Fusarium pseudograminearum.

Fusarium crown rot (FCR) caused by Fusarium pseudograminearum is one of the most serious soil-borne diseases of wheat. Among 58 bacterial isolates from the rhizosphere soil of winter wheat seedlings, strain YB-1631 was found to have the highest in vitro antagonism to F. pseudograminearum growth. LB cell-free culture filtrates inhibited mycelial growth and conidia germination of F. pseudograminearum by 84.14% and 92.23%, respectively. The culture filtrate caused distortion and disruption of the cells. Using a face-to-face plate assay, volatile substances produced by YB-1631 inhibited F. pseudograminearum growth by 68.16%. In the greenhouse, YB-1631 reduced the incidence of FCR on wheat seedlings by 84.02% and increased root and shoot fresh weights by 20.94% and 9.63%, respectively. YB-1631 was identified as Bacillus siamensis based on the gyrB sequence and average nucleotide identity of the complete genome. The complete genome was 4,090,312 bp with 4357 genes and 45.92% GC content. In the genome, genes were identified for root colonization, including those for chemotaxis and biofilm production, genes for plant growth promotion, including those for phytohormones and nutrient assimilation, and genes for biocontrol activity, including those for siderophores, extracellular hydrolase, volatiles, nonribosomal peptides, polyketide antibiotics, and elicitors of induced systemic resistance. In vitro production of siderophore, ß-1, 3-glucanase, amylase, protease, cellulase, phosphorus solubilization, and indole acetic acid were detected. Bacillus siamensis YB-1631 appears to have significant potential in promoting wheat growth and controlling wheat FCR caused by F. pseudograminearum.

Response of Fusarium pseudograminearum to Biocontrol Agent Bacillus velezensis YB-185 by Phenotypic and Transcriptome Analysis.

The use of biological control agents (BCAs) is a promising alternative control measure for Fusarium crown rot (FCR) of wheat caused by Fusarium pseudograminearum. A bacterial strain, YB-185, was isolated from the soil of wheat plants with FCR and identified as Bacillus velezensis. YB-185 exhibited strong inhibition of F. pseudograminearum mycelial growth and conidial germination in culture. Seed treatment with YB-185 in greenhouse and field resulted in reductions in disease by 66.1% and 57.6%, respectively, along with increased grain yield. Microscopy of infected root tissues confirmed that YB-185 reduced root invasion by F. pseudograminearum. RNA-seq of F. pseudograminearum during co-cultivation with B. velezensis YB-185 revealed 5086 differentially expressed genes (DEGs) compared to the control. Down-regulated DEGs included genes for glucan synthesis, fatty acid synthesis, mechanosensitive ion channels, superoxide dismutase, peroxiredoxin, thioredoxin, and plant-cell-wall-degrading enzymes, whereas up-regulated DEGs included genes for chitin synthesis, ergosterol synthesis, glutathione S-transferase, catalase, and ABC transporters. In addition, fungal cell apoptosis increased significantly, as indicated by TUNEL staining, and the scavenging rate of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS·+) in the fungus significantly decreased. Thus, F. pseudograminearum may be trying to maintain normal cell functions by increasing cell wall and membrane synthesis, antioxidant and anti-stress responses, detoxification of bacterial antimicrobial compounds, and transportation of damaging compounds from its cells. However, cell death and free radical accumulation still occurred, indicating that the responses were insufficient to prevent cell damage. Bacillus velezensis YB-185 is a promising BCA against FCR that acts by directly damaging F. pseudograminearum, thus reducing its ability to colonize roots and produce symptoms.

Genomic and Phenotypic Insights into the Potential of Bacillus subtilis YB-15 Isolated from Rhizosphere to Biocontrol against Crown Rot and Promote Growth of Wheat.

Fusarium crown rot caused by Fusarium pseudograminearum is one of the most devastating diseases of wheat worldwide causing major yield and economic losses. In this study, strain YB-15 was isolated from soil of wheat rhizosphere and classified as Bacillus subtilis by average nucleotide identity analysis. It significantly reduced Fusarium crown rot with a control efficacy of 81.50% and significantly improved the growth of wheat seedlings by increasing root and shoot fresh weight by 11.4% and 4.2%, respectively. Reduced Fusarium crown rot may have been due to direct antagonism by the production of ß-1, 3-glucanase, amylase, protease and cellulase, or by the ability of B. subtilis YB-15 to induce defense-related enzyme activities of wheat seedlings, both alone and in seedlings infected with F. pseudograminearum. Improved plant growth may be related to the ability of B. subtilis YB-15 to secrete indole acetic acid and siderophores, as well as to solubilize phosphorus. In addition, the genome of strain YB-15 was determined, resulting in a complete assembled circular genome of 4,233,040 bp with GC content of 43.52% consisting of 4207 protein-encoding genes. Sequencing the B. subtilis YB-15 genome further revealed genes for encoding carbohydrate-active enzymes, biosynthesis of various secondary metabolites, nutrient acquisition, phytohormone production, chemotaxis and motility, which could explain the potential of strain YB-15 to be plant growth-promoting bacteria and biological control agent. B. subtilis YB-15 appears to be a promising biocontrol agent against Fusarium crown rot as well as for wheat growth promotion.

Biological Control of Take-All and Growth Promotion in Wheat by Pseudomonas chlororaphis YB-10.

Wheat is a worldwide staple food crop, and take-all caused by Gaeumannomyces graminis var. tritici can lead to a tremendous decrease in wheat yield and quality. In this study, strain YB-10 was isolated from wheat rhizospheric soil and identified as Pseudomonas chlororaphis by morphology and 16S rRNA gene sequencing. Pseudomonas chlororaphis YB-10 had extracellular protease and cellulase activities and strongly inhibited the mycelium growth of Gaeumannomyces graminis var. tritici in dual cultures. Up to 87% efficacy of Pseudomonas chlororaphis YB-10 in controlling the take-all of seedlings was observed in pot experiments when wheat seed was coated with the bacterium. Pseudomonas chlororaphis YB-10 was also positive for indole acetic acid (IAA) and siderophore production, and coating wheat seed with the bacterium significantly promoted the growth of seedlings at 107 and 108 CFU/mL. Furthermore, treatment with Pseudomonas chlororaphis YB-10 increased activities of the wheat defense-related enzymes POD, SOD, CAT, PAL and PPO in seedlings, indicating induced resistance against pathogens. Overall, Pseudomonas chlororaphis YB-10 is a promising new seed-coating agent to both promote wheat growth and suppress take-all.

Isolation, Identification, and Complete Genome Assembly of an Endophytic Bacillus velezensis YB-130, Potential Biocontrol Agent Against Fusarium graminearum.

Wheat scab caused by F. graminearum is a highly destructive disease that leads to yield reduction and mycotoxin contamination of grains. In this study, an endophytic bacterium of strain YB-130 was isolated from surface sterilized wheat spikes with scab symptoms and identified as Bacillus velezensis by whole genome annotation, 16S rRNA gene and average nucleotide identities analysis. The whole-genome sequence of strain YB-130 was obtained by PacBio sequencing. 88 putative Carbohydrate-Active Enzymes and 12 gene clusters encoding for secondary metabolites were identified in the YB-130 genome, including one gene cluster for the synthesis of lanthipeptide only found in strain YB-130 genome. In dual cultures, strain YB-130 significantly inhibited the growth of F. graminearum PH-1 and other eight fungal plant pathogens, indicating a broad antifungal activity. Furthermore, strain YB-130 was able to significantly inhibit spore morphology and hyphal development of F. graminearum PH-1. Strain YB-130 also reduced deoxynivalenol production by F. graminearum PH-1 in dual cultures, possibly due to its ability to suppress the expression of tri5, tri3, and tri8 that are required for deoxynivalenol production in F. graminearum. Overall, B. velezensis YB-130 is a promising biological control agent of both F. graminearum infection and mycotoxin production.

Selection of Reference Genes for Real-time Quantitative PCR Normalization in the Process of Gaeumannomyces graminis var. tritici Infecting Wheat.

Gaeumannomyces graminis var. tritici is a soil borne pathogenic fungus associated with wheat roots. The accurate quantification of gene expression during the process of infection might be helpful to understand the pathogenic molecular mechanism. However, this method requires suitable reference genes for transcript normalization. In this study, nine candidate reference genes were chosen, and the specificity of the primers were investigated by melting curves of PCR products. The expression stability of these nine candidates was determined with three programs-geNorm, Norm Finder, and Best Keeper. TUBß was identified as the most stable reference gene. Furthermore, the exopolygalacturonase gene (ExoPG) was selected to verify the reliability of TUBß expression. The expression profile of ExoPG assessed using TUBß agreed with the results of digital gene expression analysis by RNA-Seq. This study is the first systematic exploration of the optimal reference genes in the infection process of Gaeumannomyces graminis var. tritici.