Halomonas icarae sp. nov., a moderately halophilic bacterium isolated from beach soil in India.

A moderately halophilic, Gram-stain-negative, aerobic bacterium, strain D1-1T, belonging to the genus Halomonas, was isolated from soil sampled at Pentha beach, Odisha, India. Phylogenetic trees reconstructed based on 16S rRNA genes and multilocus sequence analysis of gyrB and rpoD genes revealed that strain D1-1T belonged to the genus Halomonas and was most closely related to Halomonas alimentaria YKJ-16T (98.1 %) followed by Halomonas ventosae Al12T (97.5 %), Halomonas sediminicola CPS11T (97.5 %), Halomonas fontilapidosi 5CRT (97.4 %) and Halomonas halodenitrificans DSM 735T (97.2 %) on the basis of 16S rRNA gene sequence similarity. Sequence identities with other species within the genus were lower than 97.0 %. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of 22.4-30 % and 79.5-85.4 % with close relatives of H. halodenitrificans DSM 735T, H. alimentaria YKJ-16T, H. ventosae Al12T and H. fontilapidosi 5CRT were lower than the threshold recommended for species delineation (70 % and 95-96 % for dDDH and ANI, respectively). Further, strain D1-1T formed yellow-coloured colonies; cells were rod-shaped, motile with optimum growth at 30 °C (range, 4-45 °C) and 2-8 % NaCl (w/v; grew up to 24 % NaCl). The major fatty acids were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c) and C16 : 0 and the main respiratory quinone was ubiquinone Q-9 in line with description of the genus. Based on its chemotaxonomic and phylogenetic characteristics and genome uniqueness, strain D1-1T represents a novel species in the genus Halomonas, for which we propose the name Halomonas icarae sp. nov., within the family Halomonadaceae. The type strain is D1-1T (=JCM 33602T=KACC 21317T=NAIMCC-B-2254T).

Sustainable use of the spent mushroom substrate of Pleurotus florida for production of lignocellulolytic enzymes.

Spent mushroom substrate (SMS), a major byproduct of the mushroom industry, is a lignocellulosic biomass, which contains approximately 57-74.3% of holocellulose fraction. This study was aimed at utilizing SMS of Pleurotus florida for recovery of lignocellulolytic enzymes and sugars and also as a substrate for production of cellulolytic enzymes using different isolates of Trichoderma and Aspergillus under solid-state fermentation (SSF). SMS of P. florida extracts contained significant amounts of laccase (3,015.8 ± 29.5 U/g SMS) and xylanase (1,187.9 ± 12 U/g SMS) activity. Crystallinity pattern and chemical changes in SMS revealed that SMS had a lower crystallinity index (34.2%) as compared with the raw biomass (37.8%), which, in turn, helps in enhancing the accessibility of cellulolytic enzymes to holocellulose. Among the isolates, Trichoderma longibrachiatum A-01 showed maximum activity of endoglucanase (220.4 ± 5.9 U/mg), exoglucanase (78.5 ± 3.2 U/mg) and xylanase (1,550.4 ± 11.6 U/mg) while Aspergillus aculeatus C-08 showed maximum activity of cellobiase (113.9 ± 3.9 U/mg). Extraction with sodium citrate buffer (pH 4.8) showed maximum cellulolytic enzyme activity as compared with other solvents tested. Partial purification of endoglucanase, exoglucanase, xylanase, and cellobiase resulted in 56.3% (1,112.5 U/mg), 48.4% (212.5 U/mg), 44% (4,492.3 U/mg), and 62% (705.0 U/mg) yield with an increase by 5.2-, 4.5-, 4.1-, and 5.0-fold as compared with crude extract. The results reveal that SMS from P. florida could be a potential and cost-effective substrate for production of cellulolytic enzymes from T. longibrachiatum A-01 and A. aculeatus C-08.

Endophytic Bacillus subtilis antagonize soil-borne fungal pathogens and suppress wilt complex disease in chickpea plants (Cicer arietinum L.).

The present study aimed to identify potential endophytic bacteria antagonistic against three soil-borne fungal pathogens, Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum f.sp. ciceri causing root rot, collar rot, and fungal wilt diseases in chickpea plants, respectively. A total of 255 bacterial endophytes were isolated from the leaves, stems, and roots of seven different crop plants (chickpea, tomato, wheat, berseem, mustard, potato, and green pea). The dual culture-based screening for antifungal properties indicated that three endophytic isolates had strong inhibition (>50%) against all three pathogens tested. Based on morphological, biochemical, and molecular characterization, the selected isolates (TRO4, CLO5, and PLO3) were identified as different strains of Bacillus subtilis. The bacterial endophytes (TRO4 and CLO5) were positive for plant growth promoting (PGP) traits viz., ammonia, siderophore, and indole-3-acetic acid (IAA) production. The bio-efficacy of the endophytes (TRO4, CLO5, and PLO3) was tested by an in planta trial in chickpea pre-challenged with R. solani, S. rolfsii, and F. oxysporum f.sp. ciceri. The B. subtilis strains TRO4 and CLO5 were found to be effective in reducing percent disease incidence (p ≤ 0.05) and enhancing plant growth parameters. The different root parameters viz. root length (mm), surface area (cm2), root diameter (mm), and root volume (cm3) were significantly (p ≤ 0.05) increased in TRO4 and CLO5 inoculated chickpea plants. Confocal Scanning Laser Microscopy showed heavy colonization of bacteria in the roots of endophyte-inoculated chickpea plants. The inoculation of endophytic Bacillus subtilis strains TRO4 and CLO5 in chickpea plants through seed biopriming reduced the accumulation of superoxide, enhanced the plant defense enzymes, and induced the expression of Pathogenesis-Related (PR) genes. Semi-quantitative analysis of defense-related genes showed differential activation of PR genes (60srp and IFR) by endophyte inoculation. The results of the present study reveal the antagonistic potential of B. subtilis strains TRO4 and CLO5 against three major soil-borne fungal pathogens and their ability to suppress wilt complex disease in chickpea plants. This is the first report on the simultaneous suppression of three major soil-borne fungal pathogens causing wilt complex in chickpea plants by endophytic B. subtilis strains.