Evaluation of Antifungal Activity of Endophytic Bacillus spp. and Identification of Secondary Metabolites Produced Against the Phytopathogenic Fungi.

Endophytic bacteria serve as a rich source of diverse antimicrobial compounds. Recently, there has been a growing interest in utilizing endophytic Bacillus spp. as biological agents against phytogenic fungi, owing to their potential to produce a wide range of antimicrobial substances. The objective of this research was to investigate the protective abilities of 15 endophytic Bacillus spp. isolated from previous study from wheat plant, against the phytopathogenic fungi, Fusarium graminearum and Macrophomina phaseolina. A dual culture plate assay was conducted as a preliminary analysis, revealing that 7 out of 15 endophytic Bacillus spp. demonstrated inhibition against one or both of the phytopathogenic fungi used in this study. All seven endophytes were further assessed for the presence of diffusible antifungal metabolites. The cultures were grown in potato dextrose broth for 120 h, and the cell-free supernatant was extracted and analyzed using the cup plate method. The methanolic extract yielded similar results to the dual culture plate analysis, except for WL2-15. Additionally, deformities in the mycelial structure were examined under the light microscope upon exposure to methanolic extract. Furthermore, the analysis and identification of metabolites were carried out via gas chromatography-mass spectrometry of methanolic extract from selected seven endophytic Bacillus spp. The chromatogram revealed the presence of some major peaks such as tridecanoic acid, methyl ester, hydroperoxide, 1-methylbutyl, 9-octadecenamide, (z)-, hexane-1,3,4-triol, 3,5-dimethyl- tetradecanoic acid. To the best of our knowledge, this is the first report of these biocontrol agents in endophytic Bacillus spp. Interestingly, volatile organic compound production was also seen in all the isolates against the phytopathogenic fungi.

Wheat Growth Dependent Succession of Culturable Endophytic Bacteria and Their Plant Growth Promoting Traits.

Endophytic bacteria present ubiquitously in all plant parts. Their community structure may vary depending on plant tissue and growth condition. This work mainly focused on exploring the diversity of culturable nitrogen-fixing endophytic bacteria in above-ground plant parts of wheat by harvesting it during various growth points (Seed stage, 1st, 2nd, and 3rd month old plants, respectively). Distinct endophytic bacterial colonies were selected on Jensen's agar plate. Based on the 16S rRNA sequencing, 43 putative nitrogen-fixing endophytic bacteria were identified. Most of the isolates were found unique to the plant growth phase except for Pseudomonas sp., Bacillus sp., Paenibacillus sp., Microbacterium sp., Exiguobacterium sp. Further, endophytic bacteria were scrutinized for their plant growth promoting traits. They were found positive for IAA production (100%), P-solubilization (21%), Zn-solubilization (63%), ammonia production (93%), and nifH gene (33%). Extracellular enzyme production was found positive for cellulase (98%), pectinase (98%), and protease (100%). Their endophytic colonization ability was assessed using reactive oxygen species (ROS) induction assay, upon their entry inside the host plant.

Comparative negation of amphiphile production using nutrition factors: Amyloids versus biosurfactants.

Microbial amphiphiles play an important role in environmental activities such as microbial signaling, bioremediation, and biofilm formation. Microorganisms rely on their unique characteristics of interfaces to carry out critical biological functions, which are helped by amphipathic biomolecules known as amphiphiles. Bacillus amyloids aid in cell adhesion and biofilm formation. Pseudomonas sp. are essential in biofilm development and are a vital survival strategy for many bacteria. Furthermore, Pseudomonas and Bacillus are well-known for their ability to produce biosurfactants with a range of applications, including bioremediation and removing biological pollutants from different environments. The study employed 31 different media types and a range of analytical techniques to assess the presence of amyloid proteins and the absence of biosurfactants in Bacillus licheniformis K125 (GQ850525.1) and Pseudomonas fluorescens CHA0. The presence of amyloid proteins was confirmed through Congo red and thioflavin T staining. The carefully constructed medium also efficiently inhibited the synthesis of biosurfactants by these bacteria. Additionally, surface tension measurements, emulsification index, thin-layer chromatography, and high-performance thin-layer chromatography analyses indicated the absence of biosurfactants in the tested media.

Structural and biochemical analysis of phosphoethanolamine methyltransferase from the pine wilt nematode Bursaphelenchus xylophilus.

In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the ß1d-α6 loop (W123F, M126I, and Y127F) and ß1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.

Antibiotic producing endophytic Streptomyces spp. colonize above-ground plant parts and promote shoot growth in multiple healthy and pathogen-challenged cereal crops.

The Streptomyces spp. used in this work were previously isolated as diazotrophic endophytes from sorghum stems. Here, we characterized the Streptomyces spp. for their colonization ability, plant growth promotion and protection against fungal disease in three cereals. In vitro analysis by dual culture study showed inhibitory effect on the rice pathogen Magnaporthe oryzae B157 along with inhibition of the ubiquitous phytopathogen Rhizoctonia solani by the Streptomyces spp. used in this study. The active compounds responsible for phytopathogen inhibition were extracted with ethyl acetate and tested positive against the fungal pathogens. GC-MS based identification of the active compounds responsible for fungal pathogen inhibition showed them to be 2-(chloromethyl)-2-cyclopropyloxirane, 2, 4- ditert-butylphenol and 1-ethylthio-3-methyl-1, 3-butadiene in extracts of culture supernatants from the three different strains respectively. EGFP tagged Streptomyces strains showed profuse colonization in roots as well as aerial parts of cereal plants. Direct inhibitory action against M. oryzae B157 and R. solani correlated with the observation that upon fungal pathogen challenge, the bacterized rice, sorghum and wheat plants showed significantly good plant growth, particularly in aerial parts as compared to unbacterized controls. In addition, benefit was seen in inoculated healthy plants in terms of increase in wet weight of roots and shoots as compared to the uninoculated controls. The mechanism of biocontrol also involved induction of plant defense response as evidenced by the upregulation of PR10a, NPR1, PAL and LOX2 in Streptomyces colonized plants.