Genomewide Transcriptome Profiles Reveal How Bacillus subtilis Lipopeptides Inhibit Microsclerotia Formation in Verticillium dahliae

Verticillium dahliae is a soilborne fungus and the primary causal agent of vascular wilt diseases worldwide. The fungus produces melanized microsclerotia that are crucially important for the survival and spread of V. dahliae. There are no fungicides available that are both effective and environmentally friendly to suppress the fungus. Previously, Bacillus subtilis C232 was isolated from soil and was demonstrated to suppress microsclerotia formation in V. dahliae. In this study, liquid chromatography coupled with mass spectrometry revealed that the antifungal substance is actually a mixture of lipopeptides. Exposure of V. dahliae to these lipopeptides resulted in hyphal swelling, cell lysis, and downregulation of melanin-related genes. RNA sequencing analyses of the lipopeptide-suppressed transcriptome during microsclerotial development revealed that 5,974 genes (2,131 upregulated and 3,843 downregulated) were differentially expressed versus nonsuppressive conditions. Furthermore, gene ontology enrichment analyses revealed that genes involved in response to stress, cellular metabolic processes, and translation were significantly enriched. Additionally, the lipopeptides inhibited expression of genes associated with secondary metabolism, protein catabolism, and the high-osmolarity glycerol response signaling pathway. Together, these findings provide evidence for the mechanism by which B. subtilis lipopeptides suppress microsclerotia formation. The transcriptomic insight garnered here may facilitate the development of biological agents to combat Verticillium wilt.

Bioactive substances produced by Burkholderia sp. with antifungal action in Candida spp

Yeasts of the genus Candida are commensals, colonizing the gastrointestinal and genital tract. Accounting for 15% of hospital acquired infections, they are considered a pathogen of clinical importance. The emergence of fungal infections and the occurrence of intrinsic and acquired resistance have reflected in the increased search for new antimicrobials. The objective of this study was to evaluate the antifungal activity of extracellular substances produced by Burkholderia sp. strain RV7S3, for yeast control of the genus Candida. The substance responsible for the antifungal activity was identified and characterized biochemically, its activity was evaluated by agar diffusion tests, minimum inhibitory concentration (MIC), action effect on biofilm formation, and hemolytic activity. The data suggested that the antifungal substance is a hydrolase that exhibits lipolytic activity. The lowest concentration of this enzyme, capable of inhibiting 90% of fungal growth, was 0.38 µg.mL-1. The agar diffusion test showed inhibition halo formation of fungal growth with a diameter of 10 mm or greater, presenting 17.5 ± 0.5 mm. The substance showed low hemolytic activity and reduced biofilm cell viability, demonstrating its potential as an antifungal agent.

Extraction, purification and characterization of antifungal and antibacterial substances produced from Bacillus velezensis CP 1604

Bacillus velezensis is one of the members of the Bacillus subtilis species complex which is generally considered as safety organism. The bacterium possesses several beneficial properties for crops, especially production of antifungal and antibacterial agents against plant pathogens. In this study, we aimed to investigate the biological properties of antimicrobial subtances produced from B. velezensis CP 1604. From liquid culture, substances with activity against Fusarium oxysporum and Xanthomonas oryzae were extracted by means of adsorption with Amberlite XAD-7, extraction from lyophilized powder using ethanol, precipitation at low pH and extraction with organic solvents of 1-butanol and 2-pentanol. The substances were subsequently purified using High Performance Liquid Chromatography (HPLC). Antifungal substance was eluted at 5.328 min while antibacterial substance was observed at 15.313 min. Mass spectrometry analysis showed that antifungal substance was iturin A with molecular weight of 1042 Da and antibacterial substance was macrolactin A with molecular weight of 402 Da. The antifungal substance was stable at high temperature but antibacterial activity was significantly reduced when treated at 100° C for 2 hours. Both substances reduced the activity at low pH but the activities still maintained at high pH. The antimicrobial activities against the fungal and bacterium were not affected when treated with hydrolytic enzymes such as trypsin, α-chymotrypsin, amylase, lipase and proteinase K. Further safety and efficiency investigations of the antifungal and antibacterial substances from B. velezensis CP 1604 on the plant disease control are required in order to seek for the potential application on sustainable agricultural production.

Biological Characteristics and Antimicrobial Activity of EndophyticStreptomycessp. TQR12-4 Isolated from EliteCitrus nobilisCultivar Ham Yen of Vietnam

Ham Yen orange (Citrus nobilisLour) is the highly valuable commercial fruit of Vietnam. With the blooming of fruit production and farming area, this specialty crop is facing threats from several serious diseases; therefore the search for new effective biocontrollers is required to prevent the existing excessive use of fertilizers and plant protection chemicals. Endophytic actinomycetes are of great scientific interest due to their high potential of application in agriculture and pharmaceutical research. In this work, endophytic actinomycetes were isolated from a native orange species of Northeast mountainous province Tuyen Quang. Among 49 isolates obtained, the isolate TQR12-4 strongly inhibited test pathogensColletotrichum truncatum,Geotrichum candidum,Fusarium oxysporum, andF. udum. This isolate gave comparatively high biomass yields on different substrates, for example, carboxy methyl cellulose, starch, protein, and chitin, within a wide range of temperature from 15 to 45°C and pH from 4 to 10. Sequence analysis of 16S rDNA gene showed that TQR12-4 shared 99% similarity toStreptomyces prasinopilosus; however, it slightly differed from the latter in spore morphology and hence was named asStreptomycessp. TQR12-4. A thermostable antifungal substance of nonpeptide nature produced byStreptomycessp. TQR12-4 had MIC againstFusarium udumof 100 μg/mL and 400 μg/mL respective to extract fractionsX4andX5.