Fungal disease suppression and growth promotion potential of endophytic bacteria from ethnomedicinal plants.

103 bacterial isolates obtained from 8 ethnomedicinal plants in Manipur, India were studied for antifungal and plant growth promoting (PGP) activities. Forty-six (46), out of 62 antifungal isolates, showed potent activities against R. solani. Since R. solani (RS), a sheath blight pathogen, threatens rice yields worldwide, the present study was aimed at discovering promising bioinoculants with anti-RS and PGP potential on rice. Twenty-nine (29) endophytic isolates exhibiting promising anti-RS and PGP activities were subjected to seed vigor assays on rice (var. Jatra) and 16 were found to enhance rice seedling vigour by 70% or more over the control. Four (4) strains, Streptomyces sp. (AcRz21), Alkalihalobacillus sp. (PtL11), Bacillus sp. (TgIb5), and Priestia sp. (TgIb12) with the highest vigor indices were studied for growth promotion of rice in field conditions under pathogen-challenged and pathogen-free conditions. These bioactive strains were able to significantly enhance root and shoot biomass and reduce lesion heights caused by R. solani.

In vitro degradation of ß-amyloid fibrils by microbial keratinase.

INTRODUCTION: Amyloid fibrils are misfolded, protease-resistant forms of normal proteins. They are infectious such as prions or noninfectious such as ß-amyloid (Aß) fibrils causing Alzheimer's disease (AD). Prions and amyloids are structurally similar, possessing cross ß-pleated sheet-like structures. As microbial keratinase could degrade prions, we tested keratinase activity on Aß fibrils. METHODS: Lysozyme treated with urea generates Aß fibrils demonstrated by immunoblotting with anti-Aß antibody, high-performance liquid chromatography, and Congo red absorption spectroscopy. Two keratinases, Ker1 and Ker2, were purified from an actinomycete Amycolatopsis sp. MBRL 40 and incubated with Aß fibrils. RESULTS: Soluble Ker1 and Ker1 reconstituted on neutral/cationic liposomes degraded Aß fibrils efficiently. Ker 2 was less potent. DISCUSSION: Drugs that target AD inhibit acetylcholinesterase or formation of Aß fibrils and downstream effects. These drugs have side effects and do not benefit globally in cognition. Keratinases are novel molecules for drug development against AD.

Feather degradation by keratinolytic bacteria and biofertilizing potential for sustainable agricultural production.

Feathers account for 5-7% of the total weight of chicken have become one of the major pollutants due to their recalcitrant nature. Feather which is constituted of 90% keratin can be a good source of peptides, amino acids, and minerals for use as organic fertilizer. Traditional feather degradation methods consume large amount of energy and reduces the overall quality of the proteins. However, degradation of keratin by keratinolytic bacteria may represent as an alternative for the development of cheap, cost effective, eco-friendly, and easily available nitrogen (N) and minerals rich source as potential organic fertilizers. Keratinase enzymes from bacteria are serine-type proteases showing optimal activity at pH 6 to 9 and 30 to 50 °C. Mechanism of degradation includes, sulfitolysis, proteolysis, followed by deamination. Keratinolytic bacteria showing antagonism against important plant pathogens may act as biocontrol agent. Feather hydrolyzate can also be employed as nitrogenous fertilizers for plant growth. Tryptophan release from the feather degradation can act as precursor for plant phytohormone, indole-3-acetic acid (IAA). Solubilization of inorganic phosphate (P) by keratinolytic bacteria may further elevate the growth of plant. Application of hydrolyzate increases the water holding capacity, N, carbon (C) and mineral content of the soil. It elevates protein, amino acids, and chlorophyll content of plant. Feather hydrolyzate enhances seed germination and growth of plant. Soil application further increases the population of beneficial bacteria. The use of keratinolytic bacteria having antagonistic and plant growth promoting activities, and feather hydrolyzate can emerge as sustainable and alternative tools to promote and improve organic farming, agro-ecosystem, environment, human health, and soil biological activities.

Biocontrol and plant growth promoting activities of a Streptomyces corchorusii strain UCR3-16 and preparation of powder formulation for application as biofertilizer agents for rice plant.

Streptomyces corchorusii strain UCR3-16, obtained from rice rhizospheric soils showed antifungal activities against 6 major rice fungal pathogens by diffusible and volatile compounds production. The strain was found positive for production of fungal cell wall degrading enzymes such as chitinase, ß-1,3-glucanase, ß-1,4-glucanase, lipase and protease. The strain was also positive for plant growth promoting traits. It produced up to 30.5µg/ml of IAA and solubilized a significant amount of inorganic phosphate (up to 102µg/ml). It also produced 69% siderophore units. The strain also produced ammonia and gave positive result for ACC deaminase activity. Highest vigor index of inoculated seedlings was observed when rice seeds were treated with cell suspension of UCR3-16 corresponding to 4.5×10(8)cfu/ml. Bioinoculant-treated seeds also showed similar results under pathogen challenged conditions. In pot trial experiments, UCR3-16-treated rice plants showed significantly increased growth and grain yield production. Powder formulation of the strain was developed using talcum and corn starch as carriers and the shelf-lives were monitored. Talcum formulation showed higher cell-count than corn starch even after 6 months of storage, and optimum condition for storage of the powder formulation were found to be at 4°C. Pot trial experiments using talcum powder formulation also showed significant positive effects on growth of rice plants. Field trial using talcum powder formulation also exhibited significant enhancement in shoot length and weight of shoot and root, and total grain yield and weight of grains in rice plants. Talcum formulation also significantly reduced the sheath blight disease in rice leaves.

Keratinolytic activities of alkaliphilic Bacillus sp. MBRL 575 from a novel habitat, limestone deposit site in Manipur, India.

Microbial degradation of keratinous wastes is preferred over physicochemical methods as the latter is costlier and not eco-friendly. Novel habitats are promising for discovery of new microbial strains. Towards discovery of novel keratinolytic bacteria, screening of bacterial strains from a novel limestone habitat in Hundung, Manipur, India was done and a promising isolate, MBRL 575, was found to degrade native chicken feather efficiently. It could grow over a broad pH range (Langeveld et al. in J Infect Dis 188:1782-1789, 2003; Park and Son in Microbiol Res 164:478-485, 2009; Zaghloul et al. in Biodegradation 22:111-128, 2011; Takami et al. in Biosci Biotechnol Biochem 56:1667-1669, 1992; Riffel et al. in J Biotechnol 128:693-703, 2007; Wang et al. in Bioresour Technol 99:5679-5686, 2008) and in presence of 0-15 % NaCl. Based on phenotypic characterization and 16S rRNA gene sequence analysis, the new keratinolytic limestone isolate was identified as Bacillus sp. MBRL 575. It produced 305 ± 12 U/ml keratinase and liberated 120 ± 5.5 mg of soluble peptides and 158 ± 4 mg of amino acids per gram of feather after 48 h of incubation at 30 °C in chicken feather medium. The strain could also degrade feathers of other species besides chicken. The cell-free enzyme was also able to degrade feather. Citrate and soybean meal were found to be the best carbon and nitrogen supplements for enhanced enzyme, soluble peptide and amino acid production. In addition to keratinolytic activity, MBRL 575 also exhibited antagonistic activity against two major rice fungal pathogens, Rhizoctonia oryzae-sativae (65 %) and Rhizoctonia solani (58 %).

Actinobacterial diversity in limestone deposit sites in Hundung, Manipur (India) and their antimicrobial activities.

Studies on actinobacterial diversity in limestone habitats are scarce. This paper reports profiling of actinobacteria isolated from Hundung limestone samples in Manipur, India using ARDRA as the molecular tool for preliminary classification. A total of 137 actinobacteria were clustered into 31 phylotypic groups based on the ARDRA pattern generated and representative of each group was subjected to 16S rRNA gene sequencing. Generic diversity of the limestone isolates consisted of Streptomyces (15 phylotypic groups), Micromonospora (4), Amycolatopsis (3), Arthrobacter (3), Kitasatospora (2), Janibacter (1), Nocardia (1), Pseudonocardia (1) and Rhodococcus (1). Considering the antimicrobial potential of these actinobacteria, 19 showed antimicrobial activities against at least one of the bacterial and candidal test pathogens, while 45 exhibit biocontrol activities against at least one of the rice fungal pathogens. Out of the 137 actinobacterial isolates, 118 were found to have at least one of the three biosynthetic gene clusters (PKS-I, PKS-II, NRPS). The results indicate that 86% of the strains isolated from Hundung limestone deposit sites possessed biosynthetic gene clusters of which 40% exhibited antimicrobial activities. It can, therefore, be concluded that limestone habitat is a promising source for search of novel secondary metabolites.

Streptomyces canchipurensis sp. nov., isolated from a limestone habitat.

Hundung Limestone habitat, Manipur, India is an unexplored site for microbial diversity studies. Using polyphasic taxonomy, a Streptomyces strain, MBRL 172(T), has been characterized. The strain was found to show highest 16S rRNA gene sequence similarity with Streptomyces coeruleofuscus NBRC 12757(T) (99.2 %). The DNA relatedness between MBRL 172(T) and S. coeruleofuscus NBRC 12757(T), and between MBRL 172(T) and Streptomyces nogalater NBRC 13445(T), were 36.8 ± 4.4 and 52.5 ± 2.7 %, respectively. Strain MBRL 172(T) was found to contain LL-diaminopimelic acid as the diagnostic diamino acid and glucose, mannose and xylose as the major sugars in whole cell hydrolysates. The polar lipids in the cell membrane were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositolmannoside. The predominant menaquinones detected were MK-9(H6) and MK-9(H8). The cellular fatty acids identified were mainly saturated fatty acids: anteiso-C15:0, iso-C16:0 and iso-C15:0. Based on differences in the biochemical and molecular characteristics from its closest relatives, the strain can be proposed to represent a novel taxon in the genus Streptomyces, for which the name Streptomyces canchipurensis is proposed, with the type strain MBRL 172(T) (=JCM 17575(T) = KCTC 29105(T)).

Streptomyces muensis sp. nov.

A strain of Streptomyces, MBRL 179(T), isolated from a sample from a Limestone quarry located at Hundung, Manipur, India, was characterized by polyphasic taxonomy. The strain formed a monophyletic clade with Streptomyces spinoverrucosus NBRC 14228(T) (16S rRNA gene sequence similarity of 99.3 %) in the Neighbour-joining tree. DNA-DNA hybridization experiment gave a DNA-DNA relatedness value of 34.7 % between MBRL 179(T) and S. spinoverrucosus NBRC 14228(T). Strain MBRL 179(T) contained LL-diaminopimelic acid, xylose, glucose, and mannose in the whole cell-wall hydrolysates along with small amount of ribose. The major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside, with other unknown phospholipids and aminophospholipid. MK-9(H6), MK-9(H8) and MK-9(H4) were the predominant menaquinones detected. The major fatty acids were anteiso-C16:0 (28.1 %), iso-C16:0 (20.3 %), C16:0 (9.4 %) and anteiso-C17:0 (8.3 %). The G+C content of the genomic DNA was 71.1 %. Based on the polyphasic experiment results, the strain MBRL 179(T) merits recognition as a representative of a novel species of the genus Streptomyces for which the name Streptomyces muensis sp. nov. is proposed; the type strain is MBRL 179(T) (=JCM 17576(T) = KCTC 29124(T)).

Micromonospora kangleipakensis sp. nov., isolated from a sample of limestone quarry.

Strain MBRL 34(T), isolated from a sample of limestone quarry located at Hundung, Manipur, India, was characterized by polyphasic taxonomy. The strain showed the highest 16S rRNA gene sequence similarity with Micromonospora echinaurantica DSM 43904(T) (98.4 %), but formed a monophyletic clade with Micromonospora coerulea DSM 43143(T) (98.3 %) in the neighbour-joining tree. DNA-DNA hybridization experiments gave a DNA-DNA relatedness value of 53.1 % between MBRL 34(T) and M. coerulea DSM 43143(T). Strain MBRL 34(T) contained meso-diaminopimelic acid, galactose and glucose in the whole-cell hydrolysates along with small amounts of mannose, xylose, rhamnose and ribose. The major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside, along with an unknown lipid. MK-10(H6), MK-10(H2) MK-11(H4) and MK-10(H4) were the predominant menaquinones detected. The major fatty acids were iso-C16 : 0 and iso-C15 : 0. The G+C content of the genomic DNA was 73.5 %. Based on the taxonomic characteristics from a polyphasic study, strain MBRL 34(T) merits recognition as a representative of a novel species of the genus Micromonospora for which the name Micromonospora kangleipakensis sp. nov. is proposed; the type strain is MBRL 34(T) ( = DSM 45612(T) = JCM 17696(T)).

Streptomyces hundungensis sp. nov., a novel actinomycete with antifungal activity and plant growth promoting traits.

A novel actinobacterium MBRL 251(T), isolated from a sample collected from a limestone quarry at Hundung, Manipur, India, was characterized using a polyphasic approach. The 16S ribosomal RNA gene sequence of strain MBRL 251(T) showed closest similarities with Streptomyces xanthochromogenes NRRL B-5410(T) (99.6%) and Streptomyces michiganensis NBRC 12797(T) (99.6%). The DNA relatedness between MBRL 251(T) and S. xanthochromogenes NBRC 12828(T), and S. michiganensis NBRC 12797(T) was 46.6% and 40.7%, respectively. Strain MBRL 251(T) contained LL-diaminopimelic acid as the diagnostic diamino acid, with glucose and xylose as the main cell wall sugars, whereas small amounts of galactose, mannose, rhamnose and ribose were also detected in the whole-cell wall hydrolysates. The major fatty acids identified were anteiso-C15:0 (35.1%), iso-C16:0 (21.1%) and anteiso-C17:1 (13.2%). The predominant menaquinones detected were MK-9(H6) and MK-9(H8), whereas the polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositolmannosides. The G+C content of the genomic DNA was 72.3%. The phenotypic and genotypic data showed that strain MBRL 251(T) merits the recognition as a representative of a novel species of the genus Streptomyces. It is proposed that the isolate should be classified in the genus Streptomyces as a novel species, Streptomyces hundungensis sp. nov. The type strain is MBRL 251(T) (=JCM 17577(T)=KCTC 29124(T)).

Rhodococcus canchipurensis sp. nov., an actinomycete isolated from a limestone deposit site.

A novel actinobacterial strain, MBRL 353(T), was isolated from a sample collected from a limestone quarry at Hundung, Manipur, India. Comparison of 16S rRNA gene sequences of strain MBRL 353(T) and other members of the genus Rhodococcus showed sequence similarities ranging from 95.5 to 98.2 %, with strain MBRL 353(T) showing closest sequence similarity to Rhodococcus triatomae IMMIB RIV-085(T) (98.2 %) and Rhodococcus equi DSM 20307(T) (97.2 %). DNA-DNA hybridization results, however, revealed that DNA-DNA relatedness values between strain MBRL 353(T) and R. triatomae DSM 44892(T) (43.4 %) and R. equi DSM 20307(T) (33.4 %) were well below the 70 % limit for species identification. Strain MBRL 353(T) contained meso-diaminopimelic acid as the diagnostic diamino acid and galactose and arabinose in the cell wall. Mycolic acids were present. The major fatty acids were C(16 : 0) (45.7 %), C(18 : 1)ω9c (18.2 %) and 10-methyl C(18 : 0) (11.3 %). The only menaquinone detected was MK-8(H(2)), while the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and one unknown phospholipid. The G+C content of the genomic DNA was 69.2 mol%. The phenotypic and genotypic data showed that strain MBRL 353(T) merits recognition as a representative of a novel species of the genus Rhodococcus for which the name Rhodococcus canchipurensis sp. nov. is proposed; the type strain is MBRL 353(T) (= KCTC 19851(T) = JCM 17578(T)).

Streptomyces manipurensis sp. nov., a novel actinomycete isolated from a limestone deposit site in Manipur, India.

A novel actinobacterium, designated MBRL 201(T), was isolated from a sample collected from a limestone quarry at Hundung, Manipur, India. The strain was characterized using polyphasic taxonomy. Comparison of the 16S rRNA gene sequence of strain MBRL 201(T) and other Streptomyces species showed sequence similarities ranging from 93.0 to 99.6 % and strain MBRL 201(T) showed closest similarities to Streptomyces virginiae NBRC 12827(T) (99.6 %) and Streptomyces cinnamonensis NBRC 15873(T) (99.6 %). The DNA relatedness between MBRL 201(T) and the type strains of S. virginiae NBRC 12827(T) and S. cinnamonensis NBRC 15873(T) were 44.5 and 35.6 % respectively. Strain MBRL 201(T) contained LL: -diaminopimelic acid (A(2)pm) as the diagnostic diamino acid, with glucose as the main sugar, while small amounts of galactose, glucose, mannose, rhamnose, ribose and xylose were also present in cell-wall hydrolysates. The major fatty acids identified were anteiso-C(15:0) (38.9 %), iso-C(15:0) (19.9 %) and anteiso-C(17:1) (14.7 %). The predominant menaquinones detected were MK-9(H(6)) and MK-9(H(8)), while the polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositolmannosides, with other unknown phospholipids and lipids. The G+C content of the genomic DNA was 72.9 %. The phenotypic and genotypic data showed that strain MBRL 201(T) merits recognition as a representative of a novel species of the genus Streptomyces. It is proposed that the isolate should be classified in the genus Streptomyces as a novel species, Streptomyces manipurensis sp. nov. The type strain is MBRL 201(T) (=DSM 42029(T) = JCM 17351(T)).