Genetic polymorphism and plant growth promotion traits of potent fungal entomopathogens of rice leaf folder.

Entomopathogenic fungal biocides are preferred for environment friendly sustainable management of insect pests due to their host specificity and harmlessness to non-target insects. Plant growth promotion (PGP) functions of the entomofungi are also important attributes but hitherto insignificantly explored. Therefore, virulence of 17 natural fungal entomocides (Cordyceps, Beauveria, Metarhizium, Nomuraea, Fusarium, Verticillium, Trichoderma and Paecilomyces spp.) were evaluated for pathogenicity against five rice pests (brown plant hopper (Nilaparvata lugens) and green leaf hopper (Nephotettix virescens) nymphs, leaf folder (Cnaphalocrosis medinalis) and yellow stem borer (Scirpophaga incertulas) larvae and swarming caterpillar (Spodoptera mauritia), respectively), and PGP traits of the potent leaf folder pathogens. Among the fungi, only the leaf folder pathogens (3 isolates each of Beauveria and Metarhizium spp.) infected > 50% (80-90%) larvae but other fungi were ineffective as infected < 50% (0-47%) insects. Besides, the leaf folder pathogens exhibited diverse PGP traits such as organic/inorganic phosphate solubilization (104.7-236.4 µg/ml), and siderophore, ammonia, hydrogen cyanide (HCN), indole production etc. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequences repeat (SSR) and internal transcribed spacers (ITS) analysis ascertained strain identity and genetic (inter and intra-specific) diversity among the potent biocides Beauveria and Metarhizium spp. The virulent natural fungal pathogens of rice pests with polyvalent PGP traits may be prospected for rice growth promotion and biocontrol of leaf folder.

Neem-based products as potential eco-friendly mosquito control agents over conventional eco-toxic chemical pesticides-A review.

Mosquitoes cause serious health hazards for millions of people across the globe by acting as vectors of deadly communicable diseases like malaria, filariasis, dengue and yellow fever. Use of conventional chemical insecticides to control mosquito vectors has led to the development of biological resistance in them along with adverse environmental consequences. In this light, the recent years have witnessed enormous efforts of researchers to develop eco-friendly and cost-effective alternatives with special emphasis on plant-derived mosquitocidal compounds. Neem oil, derived from neem seeds (Azadirachta indica A. Juss, Meliaceae), has been proved to be an excellent candidate against a wide range of vectors of medical and veterinary importance including mosquitoes. It is environment-friendly, and target-specific at the same time. The active ingredients of neem oil include limonoids like azadirachtin A, nimbin, salannin and numerous other substances that are still waiting to be discovered. Of these, azadirachtin has been shown to be very effective and is mainly responsible for its toxic effects. The quality of the neem oil depends on its azadirachtin content which, in turn, depends on its manufacturing process. Neem oil can be used directly or as nanoemulsions or nanoparticles or even in the form of effervescent tablets. When added to natural breeding habitat waters they exert their mosquitocidal effects by acting as ovicides, larvicides, pupicides and/or oviposition repellents. The effects are generated by impairing the physiological pathways of the immature stages of mosquitoes or directly by causing physical deformities that impede their development. Neem oil when used directly has certain disadvantages mainly related to its disintegration under atmospheric conditions rendering it ineffective. However, many of its formulations have been reported to remain stable under environmental conditions retaining its efficiency for a long time. Similarly, neem seed cake has also been found to be effective against the mosquito vectors. The greatest advantage is that the target species do not develop resistance against neem-based products mainly because of the innumerable number of chemicals present in neem and their combinations. This makes neem-based products highly potential yet unexplored candidates of mosquito control agents. The current review helps to elucidate the roles of neem oil and its various derivatives on mosquito vectors of public health concern.

Characterization and mosquitocidal potency of a Bacillus thuringiensis strain of rice field soil of Burdwan, West Bengal, India.

Bacillus thuringiensis is the most popular mosquitocidal bacteria, strains of which are effective against almost all mosquito larvae. It has host specificity and thus, has no adverse effect on non-target species of the ecosystem. Culex tritaeniorhynchus, a vector of Japanese encephalitis (JE), breeds in vast area of rice fields in Burdwan district of West Bengal, India, which has already confronted JE epidemic. Entomological investigation and ecological studies on this vector mosquito showed that JE epidemic may reoccur anytime in the area. A strain of Bt (BU55) was isolated from rice field soil, efficacy was tested against Cx. tritaeniorhynchus and mosquitocidal role was confirmed against Cx. quinquefascistus also. The LC50 of Bacillus thuringiensis BU55 against Cx. tritaeniorhynchus and Cx. quinquefascistus after 72 h was 8.59 ml (final dose 2.49 x107 CFU/ml) and 7.52 ml (final dose 2.20 x 107 CFU/ml), respectively. Insecticidal crystal protein profile of BU55 produced 136.89, 64.80, 43.45, 33.65 and 26.98 kDa bands. Among them 136.89, 64.29, 26.98 kDa proteins are comparable to actual toxins viz. Cry1Ac (138.3 kDa, Lepidoptera specific), Cry4D (68.0 kDa, Diptera specific) and Cyt (27.4 kDa, Diptera specific). The results clearly showed that the Bt strain is a potent dipteran larvicide and can be used against the JE vectors to control the disease.

Isolation and characterization of marine bacteria from East Coast of India: functional screening for salt stress tolerance.

Soil salinization has become a severe constraint for crop production world-wide which necessitated development or induced enhancement of salt stress tolerance in plant life to sustain production in saline lands. Recognition and prospecting of valuable stress tolerant genes from natural microbial resources of saline habitat is obscure to date. Therefore, the investigation was towards isolation and characterization of marine salt stress tolerant microbes along the East coast of India for revelation of effective salt stress tolerant genes. Salt stress tolerance was assessed from 98 bacterial isolates obtained from 28 water and soil samples. Among them, 35 isolates which failed to grow beyond 4% salt were discarded and remainder 63 isolates were selected for further functional analysis and only seven isolates recorded ≥8% NaCl stress tolerance. Phylogeny revealed that four isolates belong to Firmicutes and three isolates were members of Proteobacteria. Ribosomal Database Project Release-11 and SILVA SSU database based genotyping and taxonomic identity analysis confirmed that the higher (20%) salt stress tolerant bacteria were Staphylococcus sp., Enterococcus sp., Enterobacter sp. and Proteus sp. To investigate candidate, as well as, novel salt stress tolerant genes, the seven bacterial isolates would provide new horizon to focus on the recent developments of salinity stress tolerance. In addition, the findings evidently point out the diversity of salt stress tolerant marine bacteria in coastal Odisha and West Bengal, India.

Application of rice (Oryza sativa L.) root endophytic diazotrophic Azotobacter sp. strain Avi2 (MCC 3432) can increase rice yield under green house and field condition.

Use of plant-associated beneficial microbes, especially endophytes are getting popular day by day as they occupy a relatively privileged niche inside different plant tissues with lesser competition for food and shelter than rhizosphere. The effects of different physical factors like temperature, rainfall, and seasonal variation and UV radiation on plant growth promoting endophytic communities are less pronounced than those on the rhizospheric and phylloplane microbes. This present work has been compromised with further utilization of an indigenous rice (Oryza sativa L.) root endophytic Azotobacter sp. strain Avi2 (MCC 3432) (AzA) as a bio-formulation for sustainable rice production based on several physiological parameters (plant height, root length/weight, leaf area, yield, chlorophyll contain), in-vitro comparative plant growth promoting assays, greenhouse and field experiments (dry and wet season). Treatments with AzA exhibited higher yield as well as maximal chlorophyll fluorescence (Fm) of flag leaves in flowering and grain filling stages indicating higher photosynthetic rates. Scanning electron microscopic image of rice roots demonstrated accumulation of bacterial biofilm at the junction of primary and lateral root confirming the root-colonizing ability of the bacterium. The results of the study were quite encouraging as AzA exhibited better vegetative and reproductive growth of rice in pot and field experiment compared to formulated rhizospheric Azotobacter sp. (commercial product). Apart from that plants treated with AzA (supplemented 50% nitrogenous fertilizer of recommended dose) exhibited similar yield parameters when it was compared with the recommended dose of fertilizer (RDF; 120:60:60 mg N:P:K kg-1 soil/ without any bacterial). Therefore, it can be concluded that application of this plant growth promoting endophyte can reduce a substantial amount of N-fertilizer for field application.

Larvicidal potential of Skermanella sp. against rice leaf folder (Cnaphalocrosis medinalis Guenee) and pink stem borer (Sesamia inferens Walker).

Insect pests in the rice agroecosystem, particularly the leaf folder, Cnaphalocrosis medinalis (Guenee) and stem borer, Sesamia inferens (Walker), cause significant yield losses. These pests are generally managed by farmers by application of insecticides and a few biocontrol agents. As a component of integrated pest management, biocontrol agents play a dynamic role in pest control. Although diverse microbial communities are available in the rice ecosystem, bacterial genera such as Bacillus and Pseudomonas spp. are broadly used as biocontrol agents. Therefore, an attempt was made to identify other effective entomopathogenic bacteria to manage the above mentioned pests. In this study, the two entomopathogenic bacteria isolated from diseased pink stem borer (S. inferens Walker) larvae collected from rice fields were identified as Skermanella sp. (KX611462) and Serratia sp. (KX761232). The larvicidal activity of these two bacteria was evaluated against third instar larvae of C. medinalis and S. inferens in in vitro assays and on potted rice plants (Oryza sativa var. TN1). The results of this study demonstrated 50% (LC50) larval mortality of C. medinalis at 2.95 × 103 and 5.88 × 103 colony forming units (CFU) ml-1 for Skermanella sp. and Serratia sp., respectively, under in vitro conditions, 2.57 × 104 and 3.38 × 104 CFU ml-1, respectively, in whole plant assays. Similarly, the LC50 value for Skermanella sp. was 3.80 × 104 CFU ml-1 and Serratia sp. was 2.29 × 105 CFU ml-1 for S. inferens larvae. Our study reports the larvicidal activity of Skermanella sp. against C. medinalis and S. inferens.

Virulence assay and role of Bacillus thuringiensis TS110 as biocontrol agent against the larval stages of rice leaffolder Cnaphalocrocis medinalis.

The bacterial isolate Bacillus thuringiensis TS110 was isolated from the rice field soil of Burdwan district, West Bengal, India. Bioassay test of the bacteria TS110 against 3rd, 4th and 5th instar larvae of Cnaphalocrocis medinalis was carried out. Cut leaf assay, potted plant assay and field assay were done. During filed assay, it has been observed that the LC50 (×107) values of TS110 against 3rd, 4th and 5th instar larvae of C. medinalis were 3.77, 5.29, 4.83 and 4.93, 4.42, 4.72 in dry and wet season, respectively. The morphological, biochemical and phylogenetic analysis of the isolate TS110 were done. TS110 was positive for catalase, nitrate reductase, methyl red, voges-proskauer, oxidase, urease, indole, citrate utilization, arginine dihydrolase test, starch, lipid, gelatin, casein, and lecithin hydrolysis test. TS110 showed fermentation test positive for glucose, fructose, mannose, arabinose and trehalose in nutrient broth medium. The antibiotic sensitivity test showed that the bacterial isolate was sensitive to kanamycin (30 µg/disc), nalidixic acid (30 µg/disc), rifampicin (5 µg/disc), doxycycline (30 µg/disc), gatifloxacin (10 µg/disc), vancomycin (30 µg/disc), gentamycin (10 µg/disc), ampicillin (10 µg/disc), ofloxacin (5 µg/disc), levofloxacin (5 µg/disc), streptomycin (10 µg/disc), gentamycin (10 µg/disc). The phylogenetic analysis revealed that TS110 was closely related to different species of B. thuringiensis submitted to the GenBank. On the basis of morpho-physiological and molecular characterization, the bacterial isolate was identified as B. thuringiensis.

Tuning of azine derivatives for selective recognition of Ag+ with the in vitro tracking of endophytic bacteria in rice root tissue.

Several azine derivatives have been prepared and structurally characterized by spectroscopic and single-crystal X-ray diffraction analysis. Two of them, viz. naphthalene based (A10) and anthracene based (A11) show fluorescence enhancement in the presence of Ag+ in aqueous-methanol. Moreover, A11 efficiently tracks Ag+in vitro endophytic bacteria infected rice root tissue. Experimental results have been substantiated by theoretical DFT calculations.

Dual mode ratiometric recognition of zinc acetate: nanomolar detection with in vitro tracking of endophytic bacteria in rice root tissue.

Several naphthalene-based aldazine derivatives were developed as efficient colorimetric and fluorescence probes for selective ratiometric recognition of traces of zinc acetate. The derivative structures were characterized by single-crystal X-ray diffraction. The probes were used for in vitro tracking of zinc acetate in endophytic bacteria within rice root tissue and to image zinc acetate in human breast cancer cells (MCF7) by normal and fluorescence microscopy. Density functional theoretical studies were in close agreement with the experimental findings.

Characterization of N2-fixing plant growth promoting endophytic and epiphytic bacterial community of Indian cultivated and wild rice (Oryza spp.) genotypes.

MAIN CONCLUSION: The diversity of endophytic and epiphytic diazotrophs in different parts of rice plants has specificity to the niche (i.e. leaf, stem and root) of different genotypes and nutrient availability of the organ. Inoculation of the indigenous, polyvalent diazotrophs can facilitate and sustain production of non-leguminous crops like rice. Therefore, N2-fixing plant growth promoting bacteria (PGPB) were isolated from different parts of three Indian cultivated [Oryza sativa L. var. Sabita (semi deep/deep water)/Swarna (rain fed shallow lowland)/Swarna-Sub1(submergence tolerant)] and a wild (O. eichingeri) rice genotypes which respond differentially to nitrogenous fertilizers. Thirty-five isolates from four rice genotypes were categorized based on acetylene reduction assay on nitrogenase activity, biochemical tests, BIOLOG and 16S rRNA gene sequencing. The bacteria produced 9.36-155.83 nmole C2H4 mg(-1) dry bacteria h(-1) and among them nitrogenase activity of 11 potent isolates was complemented by nifH-sequence analysis. Phylogenetic analysis based on 16S rDNA sequencing divided them into five groups (shared 95-100 % sequence homology with type strains) belonging to five classes-alpha (Ancylobacter, Azorhizobium, Azospirillum, Rhizobium, Bradyrhizobium, Sinorhizobium, Novosphingobium, spp.), beta (Burkholderia sp.), gamma (Acinetobacter, Aeromonas, Azotobacter, Enterobacter, Klebsiella, Pantoea, Pseudomonas, Stenotrophomonas spp.) Proteobacteria, Bacilli (Bacillus, Paenibacillus spp.) and Actinobacteria (Microbacterium sp.). Besides, all bacterial strains possessed the intrinsic PGP traits of like indole (0.44-7.4 µg ml(-1)), ammonia (0.18-6 mmol ml(-1)), nitrite (0.01-3.4 mol ml(-1)), and siderophore (from 0.16-0.57 µmol ml(-1)) production. Inoculation of rice (cv. Swarna) seedlings with selected isolates had a positive impact on plant growth parameters like shoot and root elongation which was correlated with in vitro PGP attributes. The results indicated that the diverse polyvalent phytonic PGP bacteria, which may be exploited as bio-inoculants to improve rice production.

Diversity and seasonal fluctuation of predominant microbial communities in Bhitarkanika, a tropical mangrove ecosystem in India.

Different groups of microorganisms are present in mangrove areas, and they perform complex interactions for nutrient and ecological balances. Since little is known about microbial populations in mangroves, this study analyzed the microbial community structure and function in relation to soil physico-chemical properties in Bhitarkanika, a tropical mangrove ecosystem in India. Spatial and seasonal fluctuations of thirteen important groups of microorganisms were evaluated from the mangrove forest sediments during different seasons, along with soil physico-chemical parameters. The overall microbial load (x10(5)cfu/g soil) in soil declined in the order of heterotrophic, free living N2 fixing, Gram-negative nitrifying, sulphur oxidizing, Gram-positive, spore forming, denitrifying, anaerobic, phosphate solubilizing, cellulose degrading bacteria, fungi and actinomycetes. Populations of the heterotrophic, phosphate solubilizing, sulphur oxidizing bacteria and fungi were more represented in the rainy season, while, Gram-negative, Gram-positive, nitrifying, denitrifying, cellulose decomposing bacteria and actinomycetes in the winter season. The pool size of most of other microbes either declined or maintained throughout the season. Soil nutrients such as N, P, K (Kg/ha) and total C (%) contents were higher in the rainy season and they did not follow any common trend of changes throughout the study period. Soil pH and salinity (mS/cm) varied from 6-8 and 6.4-19.5, respectively, and they normally affected the microbial population dynamics. Determination of bacterial diversity in Bhitarkanika mangrove soil by culture method showed the predominance of bacterial genera such as Bacillus, Pseudomonas, Desulfotomaculum, Desulfovibrio, Desulfomonas, Methylococcus, Vibrio, Micrococcus, Klebsiella and Azotobacter. Principal component analysis (PCA) revealed a correlation among local environmental variables with the sampling locations on the microbial community in the mangrove soil.

Reduction of selenite to red elemental selenium by moderately halotolerant Bacillus megaterium strains isolated from Bhitarkanika mangrove soil and characterization of reduced product.

Two Gram (+) bacterial strains, BSB6 and BSB12, showing resistance and potential for Se(IV) reduction among 26 moderately halotolerant isolates from the Bhitarkanika mangrove soil were characterized by biochemical and 16S rDNA sequence analyses. Both of them were strictly aerobic and able to grow in a wide range of pH (4-11), temperature (4-40°C) and salt concentration (4-12%) having an optimum growth at 37°C, pH ∼7.5 and 7% salt (NaCl). The biochemical characteristics and 16S rDNA sequence analysis of BSB6 and BSB12 showed the closest phylogenetic similarity with the species Bacillus megaterium. Both the strains effectively reduced Se(IV) and complete reduction of selenite (up to 0.25 mM) was achieved within 40 h. SEM with energy dispersive X-ray and TEM analyses revealed the formation of nano size spherical selenium particles in and around the bacterial cells which were also supported by the confocal micrograph study. The UV-Vis diffuse reflectance spectra and XRD of selenium precipitates revealed that the selenium particles are in the nanometric range and crystalline in nature. These bacterial strains may be exploited further for bioremediation process of Se(IV) at relatively high salt concentrations and green synthesis of selenium nanoparticles.