Perception of the Biocontrol Potential and Palmitic Acid Biosynthesis Pathway of Bacillus subtilis H2 through Merging Genome Mining with Chemical Analysis.

Bacillus has been widely studied for its potential to protect plants from pathogens. Here, we report the whole genome sequence of Bacillus subtilis H2, which was isolated from the tea garden soil of Guiyang Forest Park. Strain H2 showed a broad spectrum of antagonistic activities against many plant fungal pathogens and bacteria pathogens, including the rice blast fungus Magnaporthe oryzae, and showed a good field control effect against rice blast. The complete genome of B. subtilis H2 contained a 4,160,635-bp circular chromosome, with an average G + C content of 43.78%. Through the genome mining of strain H2, we identified 7 known antimicrobial compound biosynthetic gene clusters (BGCs) including sporulation killing factor, surfactin, bacillaene, fengycin, bacillibactin, subtilosin A, and bacilysin. Palmitic acid (PA), a secondary metabolite, was detected and identified in the H2 strain through genome mining analysis and gas chromatography-mass spectrometry (GC-MS). Additionally, we propose, for the first time, that the type II fatty acid synthesis (FAS) pathway in Bacillus is responsible for PA biosynthesis. This finding was confirmed by studying the antimicrobial activity of PA and conducting reverse transcription-quantitative polymerase chain reaction (RT-qPCR) experiments. We also identified numerous genes associated with plant-bacteria interactions in the H2 genome, including more than 94 colonization-related genes, more than 34 antimicrobial genes, and more than 13 plant growth-promoting genes. These findings contribute to our understanding of the biocontrol mechanisms of B. subtilis H2 and have potential applications in crop disease control.

Biocontrol potential of endophytic bacterium Bacillus altitudinis GS-16 against tea anthracnose caused by Colletotrichum gloeosporioides.

Background: As one of the main pathogens causing tea anthracnose disease, Colletotrichum gloeosporioides has brought immeasurable impact on the sustainable development of agriculture. Given the adverse effects of chemical pesticides to the environment and human health, biological control has been a focus of the research on this pathogen. Bacillus altitudinis GS-16, which was isolated from healthy tea leaves, had exhibited strong antagonistic activity against tea anthracnose disease. Methods: The antifungal mechanism of the endophytic bacterium GS-16 against C. gloeosporioides 1-F was determined by dual-culture assays, pot experiments, cell membrane permeability, cellular contents, cell metabolism, and the activities of the key defense enzymes. Results: We investigated the possible mechanism of strain GS-16 inhibiting 1-F. In vitro, the dual-culture assays revealed that strain GS-16 had significant antagonistic activity (92.03%) against 1-F and broad-spectrum antifungal activity in all tested plant pathogens. In pot experiments, the disease index decreased to 6.12 after treatment with GS-16, indicating that strain GS-16 had a good biocontrol effect against tea anthracnose disease (89.06%). When the PE extract of GS-16 treated mycelial of 1-F, the mycelial appeared deformities, distortions, and swelling by SEM observations. Besides that, compared with the negative control, the contents of nucleic acids, protein, and total soluble sugar of GS-16 group were increased significantly, indicating that the PE extract of GS-16 could cause damage to integrity of 1-F. We also found that GS-16 obviously destroyed cellular metabolism and the normal synthesis of cellular contents. Additionally, treatment with GS-16 induced plant resistance by increasing the activities of the key defense enzymes PPO, SOD, CAT, PAL, and POD. Conclusions: We concluded that GS-16 could damage cell permeability and integrity, destroy the normal synthesis of cellular contents, and induce plant resistance, which contributed to its antagonistic activity. These findings indicated that strain GS-16 could be used as an efficient microorganism for tea anthracnose disease caused by C. gloeosporioides.

Chemical Composition, Antibacterial Activity and Mechanism of Action of Fermentation Products from Aspergillus Niger xj.

Six compounds were isolated and purified from the crude acetone extract of Aspergillus niger xj. Characterization of all compounds was done by NMR and MS. On the basis of chemical and spectral analysis structure, six compounds were elucidated as metazachlor (1), nonacosane (2), palmitic acid (3), 5,5'-oxybis(5-methylene-2-furaldehyde) (4), dimethyl 5-nitroisophthalate (5) and cholesta-3,5-dien-7-one (6), respectively, and compounds 1, 4, 5 and 6 were isolated for the first time from A. niger. To evaluate the antibacterial activity of compounds 1-6 against three plant pathogenic bacteria (Agrobacterium tumefaciens T-37, Erwinia carotovora EC-1 and Ralstonia solanacearum RS-2), and the minimum inhibitory concentrations (MICs) were determined by broth microdilution method in 96-well microtiter plates. Results of the evaluation of the antibacterial activity showed that T-37 strain was more susceptible to metazachlor with the lowest MIC of 31.25 µg/mL. The antibacterial activity of metazachlor has rarely been reported, thus the antibacterial mechanism of metazachlor against T-37 strain were investigated. The permeability of cell membrane demonstrated that cells membranes were broken by metazachlor, which caused leakage of ions in cells. SDS-PAGE of T-37 proteins indicated that metazachlor could damage bacterial cells through the destruction of cellular proteins. Scanning electron microscopy results showed obvious morphological and ultrastructural changes in the T-37 cells, further confirming the cell membrane damages caused by metazachlor. Overall, our findings demonstrated that the ability of metazachlor to suppress the growth of T-37 pathogenic bacteria makes it potential biocontrol agents.

First Report of Leaf Spot Caused by Clonostachys rosea on Tea (Camellia sinensis) in China.

Tea (Camellia sinensis), which originated in southwest of China 60 - 70 million years ago, is widely consumed as a beverage for its potential enhancing effect on human health with rich polyphenol content (Pan et al. 2022). From October to December in 2021, a disease with symptoms similar to leaf spot affected the quality and yield of tea Puer (102°73 'E, 25°07' N), Yunnan province, China. Based on the survey, leaf spot symptoms were observed on approximately 60% of tea plants in a 5,700 m2 field. The symptoms initially appeared as shrinking, yellowing, and later became circular or irregular brown spots. To isolate the pathogen, 10 symptomatic leaves were collected from 10 trees, and portions of the diseased tissue (0.5×0.5 cm) were cut at the junction of infected and healthy tissues. After surface sterilization (0.5 min with 75% ethanol and 2 min with 3% NaOCl, washed three times with sterilized distilled water), the disinfected pieces were dried and plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. Four single-spore isolates, FH-1, FH-5, FH-6 and FH-7, were obtained, these isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS] and translation elongation factor 1-alpha [TEF] genes. Therefore, the representative isolate FH-5 was used for further study. Fungal colonies were white or light yellow on PDA after 7 days incubated at 28ºC. Conidia were hyaline, round or oval, aseptate, occur singly or in clusters on hyphae or conidia stalks, and measured as 2.94 ± 1.79 × 1.82 ± 0.2 µm (n = 50). Primary conidiophores is Verticillium-like (Fig1.K,L), which generally formed first, 1-3-level verticillate, mostly with divergent branches and phialides, and measured as 16.67 ± 4.39 µm (n = 50). Secondary conidiophores is penicillate (Fig1.I,J), which generally appearing after one week, sometimes even more often branched, and with a length of 16.02 ± 3.83 µm (n = 50). The morphological features were consistent with the descriptions of Clonostachys rosea Schroers H.J. (Schroers et al. 1999). The pathogen was confirmed to be C. rosea by amplification and sequencing of the internal transcribed spacer region (ITS) and translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4 and EF1-728F/EF1-986R, respectively (Fu Rongtao 2019). The sequences of PCR products were deposited in GenBank with accession numbers ON332533 (ITS) and OP080234 (TEF). BLAST searches of the obtained sequences revealed 99.22% (510/514 nucleotides) and 98.37% (241/245 nucleotides) homology with those of C. rosea HQ-9-1 form GenBank (MZ433177 and MZ451399, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate FH-5 in a well-supported cluster with C. rosea. The pathogenicity of FH-5 was tested through a pot assay. Ten healthy tea plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (105 spores·mL-1) of FH-5 onto leaves until runoff, and the control leaves sprayed with sterile water. Inoculated plants were put in an artificial climate box at 25℃, 70% relative humidity. The pathogenicity test was replicated three times. Symptoms developed on all inoculated leaves but not on the control leaves. Lesions around the wound edge became pale yellow, and brown spots were first observed at 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after two weeks. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses (ITS and TEF) from the infected leaves but not from the noninoculated leaves. In addition, C. rosea has also been reported to cause diseases to broad bean (N. Afshari et al. 2017 ), garlic (Diaz et al. 2022), beet (Haque M.E et al. 2020) and other plants. To our knowledge, this is the first report of leaf spot on tea caused by C. rosea in China. This study provides valuable information for the identification and control of the leaf spot on tea.

Single-cell microliter-droplet screening system (MISS Cell): An integrated platform for automated high-throughput microbial monoclonal cultivation and picking.

Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor- and resource-intensive for high-throughput requirements. Currently, several instruments have been integrated for automated and high-throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single-cell microliter-droplet screening system (MISS Cell), for automated, high-throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high-throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate-producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high-throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.

Insights of microbial community evolution under benzisothiazolinone exposure in different soil environments.

Benzisothiazolinones (1, 2-benzisothiazoline-3-one; BIT) is widely used to control bacterial and fungal diseases of various crops, and their residues in soil may play an important role in the interaction between soil microorganisms. We studied microbial remediation in five representative soils under different soil conditions (unsterilized, sterilized and flooded) using 16S rRNA gene sequencing to investigate the effect of microorganisms on the degradation of BIT residues in soils to minimize the potentially toxic effects of BIT. High-throughput sequencing data showed that the structure and abundance of bacterial communities in BIT soils changed greatly, which might affect their degradation pathways, while Principal Coordinates Analysis (PCoA) results showed that there was no significant difference in the fungal community in different treatments of the five soils, but the degradation rate of BIT was more influenced by anaerobic microorganisms. Furthermore, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Acidobacteria were the predominant bacterial phyla, accounting for 93.74% of all OTUs, which played an important role in the degradation of BIT. Lysinibacillus had a high relative abundance (21.10%) under flooded treatment conditions in Jilin soil, and its bioremediation may be a reason for the rapid degradation of BIT in flooded treatment. Besides, only soil organic matter (SOM) and pH among the soil properties had significant effects on the microbial community. Based on the further analysis of bacterial phenotype, some microorganisms related to the biodegradation of BIT were found, mainly belonging to Proteobacteria, Bacteroidetes and Firmicutes. This study provides a useful theoretical basis for the biodegradation of BIT using isolated microorganisms.

Isolation of Bioactive Compounds, Antibacterial Activity, and Action Mechanism of Spore Powder From Aspergillus niger xj.

Aspergillus fungi can produce a wide range of secondary metabolites, and they have represented a potential resource of novel bioactive compounds. Bacterial plant diseases have a serious impact on the sustainable development of agriculture worldwide, so it is necessary to use natural antibacterial compounds in microorganisms to control plant pathogens. This study was conducted to investigate the bioactive compounds of Aspergillus niger xj, three plant pathogens (Agrobacterium tumefaciens T-37, Erwinia carotovora EC-1, and Ralstonia solanacearum RS-2) were used as indicator bacteria, according to the biological activity tracking, five compounds were isolated from A. niger xj spore powder, and characterization of compounds was done by NMR (1H-NMR and 13C-NMR) and EI-MS and was identified as ergosterol (1), ß-sitosterol (2), 5-pentadecylresorcinol (3), 5-hydroxymethyl-2-furancarboxylic acid (4), and succinimide (5). Compounds 3 and 5 were isolated from A. niger xj for the first time. The minimum inhibitory concentration (MIC) of five compounds against three plant pathogens was evaluated, the results showed that compound 4 exhibited the strongest antibacterial activity against tested bacteria, and RS-2 was the most sensitive to compound 4, showing the lowest MIC of 15.56 µg/ml. We concluded that the mechanism of action of the compound 4 against RS-2 might be described as compound 4 acting on bacterial protein synthesis and intracellular metabolism according to the results of the scanning electron microscopy observation, permeability of cell membrane and SDS-PAGE. These results indicated that compound 4 has good potential to be as a biocontrol agent. In conclusion, the results from this study demonstrated that the compounds with antibacterial activity are of great significance of the prevention and control of plant phytopathogenic bacteria, and they may be applicable to exploring alternative approaches to integrated control of phytopathogens.