Microbiome of Pukzing Cave in India shows high antimicrobial activity against plant and animal pathogens.

Pukzing cave, the largest cave of Mizoram, India was explored for bacterial diversity. Culture dependent method revealed 235 bacterial isolates using three different treatments. Identity of the microbial species was confirmed by 16S rDNA sequencing. The highest bacterial population was recovered from heat treatment (n = 97;41.2%) followed by normal (n = 79;33.6%) and cold treatment (n = 59;25.1%) indicating dominance of moderate thermophiles. Antimicrobial potential of isolates showed 20.4% isolates having antimicrobial ability against tested pathogens. Amplicon sequencing of PKSI, PKSII and NRP specific genes revealed presence of AMP genes in the microbial population. Six microbial pathogens were selected for screening as they are well known for different disease cause organism in various fields such as agriculture and human health. Cave environment harbors unique microbial flora and hypervariable region V4 is more informative. Higher activity of AMP assay against these microbes indicates that cave microbial communities could be potential source of future genomic resources.

Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review.

Plant viral diseases are the foremost threat to sustainable agriculture, leading to several billion dollars in losses every year. Many viruses infecting several crops have been described in the literature; however, new infectious viruses are emerging frequently through outbreaks. For the effective treatment and prevention of viral diseases, there is great demand for new techniques that can provide accurate identification on the causative agents. With the advancements in biochemical and molecular biology techniques, several diagnostic methods with improved sensitivity and specificity for the detection of prevalent and/or unknown plant viruses are being continuously developed. Currently, serological and nucleic acid methods are the most widely used for plant viral diagnosis. Nucleic acid-based techniques that amplify target DNA/RNA have been evolved with many variants. However, there is growing interest in developing techniques that can be based in real-time and thus facilitate in-field diagnosis. Next-generation sequencing (NGS)-based innovative methods have shown great potential to detect multiple viruses simultaneously; however, such techniques are in the preliminary stages in plant viral disease diagnostics. This review discusses the recent progress in the use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases. New portable devices and technologies that could provide real-time analyses in a relatively short period of time are prime important for in-field diagnostics. Current development and application of such tools and techniques along with their potential limitations in plant virology are likewise discussed in detail.

Antimicrobial sensitivity profiling of bacterial communities recovered from effluents of municipal solid waste dumping site.

The diversity of antibiotic-resistance bacteria (ARB) from the effluents of Aizawl city municipal waste dumping site was studied using a culture-dependent method. The present study molecularly identified 73 isolates that were differentiated into three phyla (Proteobacteria, Actinobacteria, and Firmicutes) belonging to 17 family and 22 genera. Bacillus constitutes the most dominant genus comprising 16% of the total isolates. The topology of the phylogenetic tree differentiates them into five major clades. Corynebacterium and Rhodococcus which are morphologically alike were clustered together and the Gram-positive bacteria such as Staphylococcus, Bacillus, and Lysinibacillus formed a separate cluster. Antibiotic resistance of the identified bacterial isolates was performed using 19 different antibiotics. Among the isolates, 70 of them found resistant to polymixin B and nalidixic acid and 10 isolates exhibited resistance to 15 tested antibiotics. The present study revealed that bacteria with antibiotic resistance are extensively distributed in the effluents of the dumping site and may serve as a significant reservoir for the spreading of antibiotic resistance to opportunistic pathogens.

In Vivo Studies of Inoculated Plants and In Vitro Studies Utilizing Methanolic Extracts of Endophytic Streptomyces sp. Strain DBT34 Obtained from Mirabilis jalapa L. Exhibit ROS-Scavenging and Other Bioactive Properties.

Reactive oxygen species (ROS) and other free radicals cause oxidative damage in cells under biotic and abiotic stress. Endophytic microorganisms reside in the internal tissues of plants and contribute to the mitigation of such stresses by the production of antioxidant enzymes and compounds. We hypothesized that the endophytic actinobacterium Streptomyces sp. strain DBT34, which was previously demonstrated to have plant growth-promoting (PGP) and antimicrobial properties, may also have a role in protecting plants against several stresses through the production of antioxidants. The present study was designed to characterize catalase and superoxide dismutase (SOD), two enzymes involved in the detoxification of ROS, in methanolic extracts derived from six endophytic actinobacterial isolates obtained from the traditional medicinal plant Mirabilis jalapa. The results of a preliminary screen indicated that Streptomyces sp. strain DBT34 was the best overall strain and was therefore used in subsequent detailed analyses. A methanolic extract of DBT34 exhibited significant antioxidant potential in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assays. The cytotoxicity of DBT34 against liver hepatocellular cells (HepG2) was also determined. Results indicated that methanolic extract of Streptomyces sp. strain DBT34 exhibited significant catalase and SOD-like activity with 158.21 U resulting in a 55.15% reduction in ROS. The IC50 values of a crude methanolic extract of strain DBT34 on DPPH radical scavenging and ABTS radical cation decolorization were 41.5 µg/mL and 47.8 µg/mL, respectively. Volatile compounds (VOC) were also detected in the methanolic extract of Streptomyces sp. strain DBT34 using GC-MS analysis to correlate their presence with bioactive potential. Treatments of rats with DBT34 extract and sitagliptin resulted in a significant (p ≤ 0.001) reduction in total cholesterol, LDL-cholesterol, and VLDL-cholesterol, relative to the vehicle control and a standard diabetic medicine. The pancreatic histoarchitecture of vehicle control rats exhibited a compact volume of isolated clusters of Langerhans cells surrounded by acinies with proper vaculation. An in-vivo study of Streptomyces sp. strain DBT34 on chickpea seedlings revealed an enhancement in its antioxidant potential as denoted by lower IC50 values for DPPH and ABTS radical scavenging activity under greenhouse conditions in relative comparison to control plants. Results of the study indicate that strain DBT34 provides a defense mechanism to its host through the production of antioxidant therapeutic agents that mitigate ROS in hosts subjected to biotic and abiotic stresses.

Elevated levels of laccase synthesis by Pleurotus pulmonarius BPSM10 and its potential as a dye decolorizing agent.

Laccases (EC 1.10.3.2) are a class of multi-copper oxidases that have industrial value. In the present study, forty-five isolates of wild mushrooms were screened for laccase production. Eight of the isolates exhibited exploitable levels of substrate oxidation capacity. Isolate BPSM10 exhibited the highest laccase activity of 103.50 U/ml. Internal Transcribed Spacer (ITS) rRNA gene sequencing was used to identify BPSM10 as Pleurotus pulmonarius. The response of BPSM10 to two nutritional media supplemented with various inducers was characterized and the results indicated that Malt Extract Broth (MEB) supplemented with Xylidine increased laccase production by 2.8× (349.5 U/ml) relative to the control (122 U/ml), while Potato Dextrose Broth (PDB) supplemented with xylidine increased laccase production by 1.9× (286 U/ml). BPSM10 has the ability to decolorize seven synthetic dyes in a liquid medium. Maximum decolorization was observed of malachite green (MG); exhibiting 68.6% decolorization at 100 mg/L. Fourier-transform infrared spectroscopy (FTIR) was employed to confirm the decolorization capacity. The present study indicates that P. pulmonarius BPSM10 has the potential to be used as a potent alternative biosorbent for the removal of synthetic dyes from aqueous solutions, especially in the detoxification of polluted water.

Correction to: Bioprospection of actinobacteria derived from freshwater sediments for their potential to produce antimicrobial compounds.

Upon publication of this article [1], it was brought to our attention that Figs. 3, 4 and 5 are incorrectly presented in the original version of the article. The figures were inadvertently swapped in the original submission and published. Figure 3 should be treated as Fig. 5; Fig. 4 should be 3 and Fig. 5 should be Fig. 4.

Bioprospection of actinobacteria derived from freshwater sediments for their potential to produce antimicrobial compounds.

BACKGROUND: Actinobacteria from freshwater habitats have been explored less than from other habitats in the search for compounds of pharmaceutical value. This study highlighted the abundance of actinobacteria from freshwater sediments of two rivers and one lake, and the isolates were studied for their ability to produce antimicrobial bioactive compounds. RESULTS: 16S rRNA gene sequencing led to the identification of 84 actinobacterial isolates separated into a common genus (Streptomyces) and eight rare genera (Nocardiopsis, Saccharopolyspora, Rhodococcus, Prauserella, Amycolatopsis, Promicromonospora, Kocuria and Micrococcus). All strains that showed significant inhibition potentials were found against Gram-positive, Gram-negative and yeast pathogens. Further, three biosynthetic genes, polyketide synthases type II (PKS II), nonribosomal peptide synthetases (NRPS) and aminodeoxyisochorismate synthase (phzE), were detected in 38, 71 and 29% of the strains, respectively. Six isolates based on their antimicrobial potentials were selected for the detection and quantification of standard antibiotics using ultra performance liquid chromatography (UPLC-ESI-MS/MS) and volatile organic compounds (VOCs) using gas chromatography mass spectrometry (GC/MS). Four antibiotics (fluconazole, trimethoprim, ketoconazole and rifampicin) and 35 VOCs were quantified and determined from the methanolic crude extract of six selected Streptomyces strains. CONCLUSION: Infectious diseases still remain one of the leading causes of death globally and bacterial infections caused millions of deaths annually. Culturable actinobacteria associated with freshwater lake and river sediments has the prospects for the production of bioactive secondary metabolites.

Pharmacological potential of Bidens pilosa L. and determination of bioactive compounds using UHPLC-QqQLIT-MS/MS and GC/MS.

BACKGROUND: Research of natural products from traditionally used medicinal plants to fight against the human ailments is fetching attention of researchers worldwide. Bidens pilosa Linn. var. Radiata (Asteraceae) is well known for its folkloric medicinal use against various diseases from many decades. Mizoram, North East India, has high plant diversity and the use of this plant as herbal medicine is deep rooted in the local tribes. The present study was executed to understand the pharmacological potential of B. pilosa leaves extract. METHODS: The antimicrobial potential was determined using agar well diffusion and broth microdilution method against bacterial and yeast pathogens. Cytotoxicity was evaluated using MTT and apoptotic DNA fragmentation assays. Further, the antioxidant ability of the extract was analysed using DPPH and ABTS free radical scavenging assay. Mosquitocidal activity was evaluated against third in-star larvae of C. quinquefasciatus using dose response and time response larvicidal bioassay. Additionally, the major phenolic and volatile compounds were determined using UHPLC-QqQLIT-MS/MS and GC/MS respectively. RESULTS: We found that the extract showed highest antimicrobial activity against E. coli (MIC 80 µg/mL and IC50 110.04 µg/mL) and showed significant cytotoxicity against human epidermoid carcinoma (KB-3-1) cells with IC50 values of 99.56 µg/mL among the tested cancer cell lines. The IC50 values for scavenging DPPH and ABTS was 80.45 µg/mL and 171.6 µg/mL respectively. The extract also showed the high phenolics (72 µg GAE/mg extract) and flavonoids (123.3 µg Quercetin /mg extract). Lastly, five bioactive and six volatile compounds were detected using UHPLC-QqQLIT-MS/MS and GC-MS respectively which may be responsible for the plant's bioactivities. An anticancerous compound, Paclitaxel was detected and quantified for the first time from B. pilosa leaves extract, which further showed the anticancerous potential of the tested extract. CONCLUSION: On the basis of the present investigation, we propose that the leaf extract of B. pilosa might be a good candidate for the search of efficient environment friendly natural bioactive agent and pharmaceutically important compounds.

Evaluation of gastrointestinal bacterial population for the production of holocellulose enzymes for biomass deconstruction.

The gastrointestinal (GI) habitat of ruminant and non-ruminant animals sustains a vast ensemble of microbes that are capable of utilizing lignocellulosic plant biomass. In this study, an indigenous swine (Zovawk) and a domesticated goat (Black Bengal) were investigated to isolate bacteria having plant biomass degrading enzymes. After screening and enzymatic quantification of eighty-one obtained bacterial isolates, Serratia rubidaea strain DBT4 and Aneurinibacillus aneurinilyticus strain DBT87 were revealed as the most potent strains, showing both cellulase and xylanase production. A biomass utilization study showed that submerged fermentation (SmF) of D2 (alkaline pretreated pulpy biomass) using strain DBT4 resulted in the most efficient biomass deconstruction with maximum xylanase (11.98 U/mL) and FPase (0.5 U/mL) activities (55°C, pH 8). The present study demonstrated that bacterial strains residing in the gastrointestinal region of non-ruminant swine are a promising source for lignocellulose degrading microorganisms that could be used for biomass conversion.

Determination and production of antimicrobial compounds by Aspergillus clavatonanicus strain MJ31, an endophytic fungus from Mirabilis jalapa L. using UPLC-ESI-MS/MS and TD-GC-MS analysis.

Endophytic fungi associated with medicinal plants are reported as potent producers of diverse classes of secondary metabolites. In the present study, an endophytic fungi, Aspergillus clavatonanicus strain MJ31, exhibiting significant antimicrobial activity was isolated from roots of Mirabilis jalapa L., was identified by sequencing three nuclear genes i.e. internal transcribed spacers ribosomal RNA (ITS rRNA), 28S ribosomal RNA (28S rRNA) and translation elongation factor 1- alpha (EF 1α). Ethyl acetate extract of strain MJ31displayed significant antimicrobial potential against Bacillus subtilis, followed by Micrococccus luteus and Staphylococcus aureus with minimum inhibitory concentrations (MIC) of 0.078, 0.156 and 0.312 mg/ml respectively. In addition, the strain was evaluated for its ability to synthesize bioactive compounds by the amplification of polyketide synthase (PKS) and non ribosomal peptide synthetase (NRPS) genes. Further, seven antibiotics (miconazole, ketoconazole, fluconazole, ampicillin, streptomycin, chloramphenicol, and rifampicin) were detected and quantified using UPLC-ESI-MS/MS. Additionally, thermal desorption-gas chromatography mass spectrometry (TD-GC-MS) analysis of strain MJ31 showed the presence of 28 volatile compounds. This is the first report on A. clavatonanicus as an endophyte obtained from M. jalapa. We conclude that A. clavatonanicus strain MJ31 has prolific antimicrobial potential against both plant and human pathogens and can be exploited for the discovery of new antimicrobial compounds and could be an alternate source for the production of secondary metabolites.

Antimicrobial Potential, Identification and Phylogenetic Affiliation of Wild Mushrooms from Two Sub-Tropical Semi-Evergreen Indian Forest Ecosystems.

The diversity of wild mushrooms was investigated from two protected forest areas in India and 231 mushroom specimens were morphologically identified. Among them, 76 isolates were screened for their antimicrobial potential against seven bacterial and fungal pathogens. Out of 76 isolates, 45 isolates which displayed significant antimicrobial activities were identified using ITS rRNA gene amplification and subsequently phylogenetically characterized using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Sequencing of the ITS rRNA region classified the isolates into 16 genera belonging to 11 families. In total, 11 RAPD and 10 ISSR primers were selected to evaluate genetic diversity based on their banding profile produced. In total 337 RAPD and 312 ISSR bands were detected, among which percentage of polymorphism ranges from 34.2% to 78.8% and 38.6% to 92.4% by using RAPD and ISSR primers respectively. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of selected two methods were structured similarly, grouping the 46 isolates into two clusters which clearly showed a significant genetic distance among the different strains of wild mushroom, with an similarity coefficient ranges from 0.58 to 1.00 and 0.59 to 1.00 with RAPD and ISSR analysis respectively. This reporthas highlighted both DTR and MNP forests provide a habitat for diverse macrofungal species, therefore having the potential to be used for the discovery of antimicrobials. The report has also demonstrated that both RAPD and ISSR could efficiently differentiate wild mushrooms and could thus be considered as efficient markers for surveying genetic diversity. Additionally, selected six wild edible mushroom strains (Schizophyllum commune BPSM01, Panusgiganteus BPSM27, Pleurotussp. BPSM34, Lentinussp. BPSM37, Pleurotusdjamor BPSM41 and Lentinula sp. BPSM45) were analysed for their nutritional (proteins, carbohydrates, fat and ash content), antioxidant potential. The present findings also suggested that the wild edible mushroom strains do not have only nutritional values but also can be used as an accessible source of natural antioxidants.

Phytohormone production endowed with antagonistic potential and plant growth promoting abilities of culturable endophytic bacteria isolated from Clerodendrum colebrookianum Walp.

In this study, culturable endophytic bacterial isolates obtained from an ethnomedicinal plant Clerodendrum colebrookianum Walp., were assessed for their diversity, in vitro screening for their plant growth promoting (PGP) activities and to use them as inoculant for in vivo PGP activities with biocontrol potential. Totally, 73 isolates were recovered from different tissues of C. colebrookianum were identified by 16S rRNA gene sequencing and phylogenetically analyzed by using BOX-PCR fingerprinting. Out of 73 isolates, 52 exhibited varying extents of antagonistic potential were selected for screening for various PGP traits. Concerning the PGP activities, the percentage of isolates positive for P-solubilisation, indolic compounds production, siderophore and ammonia production were 84.6, 92.3, 78.8 and 98.0 respectively. All isolates were positive for the production of hydrocyanic acid (HCN) and 86.5%, 84.6% and 90.3% of isolates showed significant cellulase, amylase and protease production respectively. Further, the top 10 bacterial isolates based on a bonitur scale with multiple PGP activities were screened for root surface colonization and biofilm formation ability. Out of selected 10 isolates, 9 showed significant potential for root surface colonization on tomato roots. Isolate BPSAC6 identified as Bacillus sp. was most efficient in biofilm formation as assessed with respect to the intensity of crystal violet, which further showed their potential to withstand various biotic and abiotic stresses. Furthermore, Bacillus sp. strain BPSAC6 showed a significant increase in shoot and root height as well as fresh weight after 45 and 60 d of inoculation with tomato seedlings. Additionally, biosynthetic potential of antagonistic isolate was detection by using PKSI, PKSII and NRPS biosynthetic genes. Two isolates Pseudomonas psychrotolerans and Labrys wisconsinensis were reported first time as an endophyte. At last, first time an endophytic bacterial strain Bacillus sp. BPSAC6 was reported to produce altogether three phytohormones (IAA, Kinetin and 6-Benzyladenine). This study is the first report that bacteria isolated from C. colebrookianum has biocontrol as well as PGP abilities endowed with phytohormones production and can be used for the preparation of bioinoculant for plant growth promotion.

Detection of biosynthetic gene and phytohormone production by endophytic actinobacteria associated with Solanum lycopersicum and their plant-growth-promoting effect.

In the present study, fifteen endophytic actinobacterial isolates recovered from Solanum lycopersicum were studied for their antagonistic potential and plant-growth-promoting (PGP) traits. Among them, eight isolates showed significant antagonistic and PGP traits, identified by amplification of the 16S rRNA gene. Isolate number DBT204, identified as Streptomyces sp., showed multiple PGP traits tested in planta and improved a range of growth parameters in seedlings of chili (Capsicum annuum L.) and tomato (S. lycopersicum L.). Further, genes of indole acetic acid (iaaM) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) were successively amplified from five strains. Six antibiotics (trimethoprim, fluconazole, chloramphenicol, nalidixic acid, rifampicin and streptomycin) and two phytohormones [indole acetic acid (IAA) and kinetin (KI)] were detected and quantified in Streptomyces sp. strain DBT204 using UPLC-ESI-MS/MS. The study indicates the potential of these PGP strains for production of phytohormones and shows the presence of biosynthetic genes responsible for production of secondary metabolites. It is the first report showing production of phytohormones (IAA and KI) by endophytic actinobacteria having PGP and biosynthetic potential. We propose Streptomyces sp. strain DBT204 for inoculums production and development of biofertilizers for enhancing growth of chili and tomato seedlings.

Thermal properties of tannin extracted from Anacardium occidentale L. using TGA and FT-IR spectroscopy.

The chemical nature of the polyphenols of cashew kernel testa has been determined. Testa contains tannins, which present large molecular complexity and has an ancient use as tanning agents. The use of tannins extracted from cashew testa, considered in many places as a waste, grants an extra value to the cashew. In this work we have analysed through high performance liquid chromatography, infrared spectroscopy (FT-IR) and thermo gravimetric analysis the average molecular weight, main functional groups and thermal properties of tannins extracted from Anacardium occidentale L. The results of these analyses are compared with the commercial grade tannic acid. The FT-IR spectra showed bands characteristic of C = C, C-C and OH bonds. This important bioactive compound present in the cashew nut kernel testa was suggested as an interesting economical source of antioxidants for use in the food and nutraceutical industry.