Showkat Ahmad Lone

Molecular and insecticidal characterization of Vip3A protein producing Bacillus thuringiensis strains toxic against Helicoverpa armigera (Lepidoptera: Noctuidae)

Vegetative insecticidal proteins (Vip) represent the second generation of insecticidal proteins produced by Bacillus thuringiensis (Bt) during the vegetative growth stage of growth. Bt-based biopesticides are recognized as viable alternatives to chemical insecticides; the latter cause environmental pollution and lead to the emergence of pest resistance. To perform a systematic study of vip genes encoding toxic proteins, a total of 30 soil samples were collected from diverse locations of Kashmir valley, India, and characterized by molecular and analytical methods. Eighty-six colonies showing Bacillus-like morphology were selected. Scanning electron microscopy observations confirmed the presence of different crystal shapes, and PCR analysis of insecticidal genes revealed a predominance of the lepidopteran-specific vip3 (43.18%) gene followed by coleopteran-specific vip1 (22.72%) and vip2 (15.90%) genes in the isolates tested. Multi-alignment of the deduced amino acid sequences revealed that vip3 sequences were highly conserved, whereas vip1 and vip2 showed adequate differences in amino acid sequences compared with already reported sequences. Screening for toxicity against Helicoverpa armigera larvae was performed using partially purified soluble fractions containing Vip3A protein. The mortality levels observed ranged between 70% and 96.6% in the isolates. The LC50 values of 2 of the native isolates, JK37 and JK88, against H. armigera were found to be on par with that of Bt subsp. kurstaki HD1, suggesting that these isolates could be developed as effective biopesticides against H. armigera.

Identification of phenazine-1-carboxylic acid gene (phc CD) from Bacillus pumilus MTCC7615 and its role in antagonism against Rhizoctonia solani.

Bacillus pumilus MTCC7615, a biocontrol agent isolated from rice rhizosphere was characterized to be antagonistic to Rhizoctonia solani, the pathogen causing sheath blight disease of rice. The phenazine-1-carboxylic acid gene (phc CD) of this bacterium was PCR amplified (1400 bp), cloned, and sequenced. The sequence analysis revealed the presence of two ORFs of phc CD gene commonly found in Pseudomonas species. The sequence showed 98% similarity to phc CD gene of the Pseudomonas isolate LBUM223 (DQ788993). The crude antibiotic extract from B. pumilus MTCC7615 was observed to inhibit mycelial growth of R. solani under in vitro conditions. The HPLC analysis of crude antibiotic extract from B. pumilus MTCC7615 confirmed the presence of phenazine. The study has also reported the presence of phc CD gene which is responsible for the synthesis of phenazine-1-carboxylic acid in B. pumilus. The ability of the bacterial isolate to control sheath blight disease in rice seedlings under in vivo conditions was confirmed by the pot culture experiment. The structural and functional genomics of phc C and phc D genes would lead to a better understanding of phenazine biosynthesis in B. pumilus for its efficient utilization in plant protection strategies.

Applications of Bacillus thuringiensis for Prevention of Environmental Deterioration

The indiscriminate use of chemical insecticides for the control of insect pests over the years has led to serious environmental problems such as persistence of toxicity, which can in turn lead to the acquisition of resistance in target pests. These broad-spectrum insecticides, in addition to target pests, also kill non-target predators and parasites that otherwise check the pest populations. Furthermore, these pesticides keep on accumulating throughout aquatic and terrestrial food webs, creating ecological imbalances, and impairing human health.

Recent Approaches in Risk Assessment of Foods

Food safety is important not only for the people’s general health and daily life, economic development, and social stability but also for the government’s and country’s image. Microbiological and chemical contamination in food is a major cause of illnesses. Food-borne disease remains a real and formidable problem in both developed and developing countries, causing great human suffering and significant economic losses. New science-based approaches to food safety provide an effective way for governments to protect consumers against food-borne disease and plan appropriate response measures when necessary. Risk assessment is a structured science-based process to estimate the likelihood and severity of risk with attendant uncertainty. For risk assessment many organizations recognize four major elements: hazard identification; exposure assessment; dose–response assessment or hazard characterization; and risk characterization. Risk assessment can be descriptive or narrative, qualitative, semi-quantitative, or quantitative. Both qualitative and quantitative risk assessments are important in different circumstances. Food safety risk assessments are undertaken in response to identified chemical or microbial risks to human health. Chemical risk assessments focus on the presence of chemicals such as food additives, food contaminants, or residues of veterinary drugs. A microbial risk assessment evaluates the likelihood of adverse human health effects occurring after exposure to a pathogenic microorganism or to the medium in which the organism occurs. Activities within risk assessment focus on estimating the risk that a hazardous event or factor will negatively affect a population or subpopulation. This chapter deals with the food safety risk assessment process. It introduces a range of techniques that can be used to support risk assessment in practice and outlines the essential characteristics of a good risk assessment.

Cloning and expression of dnaK gene from Bacillus pumilus of hot water spring origin

A set of thermotolerant strains isolated from hot springs of Manikaran and Bakreshwar (India) were selected with an aim to isolate dnak gene which encodes DnaK protein. The gene dnaK along with its flanking region was successfully amplified from 5 different strains (4 from Bakreshwar and one from Manikaran). Restriction fragment length polymorphism (RFLP) revealed that amplicons were almost identical in sequence. The dnak gene from one representative, Bacillus pumilus strain B3 isolated from Bakreshwar hot springs was successfully cloned and sequenced. The dnaK gene was flanked by gene grpE on one side. The dnaK gene was 1842 bp in length encoding a polypeptide of 613 amino acid residues. Calculated molecular weight and pI of the protein were 66,128.36 Da and 4.72 respectively. The deduced amino acid sequence of this gene shared high sequence homology with other DnaK proteins and its homologue Hsp 70 from other microorganisms, but possessed 36 substitutions and two insertions, as compared to DnaK protein of Bacillus subtilis. The dnaK gene of B. pumilus was successfully expressed in Escherichia coli BL 21 (DE3) using pET expression systems. Heterologous expression of dnaK of B. pumilus in E. coli BL 21 (DE3) allowed for the growth of E. coli up to 50 °C and survival up to 60 °C for 16 h, suggesting that dnak from B. pumilus imparts tolerance to host cells under high temperature. This novel gene can be an important component for possible utilization in abiotic stress management of plants.

Transcriptome sequence analysis and mining of SSRs in Jhar Ber (Ziziphus nummularia (Burm.f.) Wight & Arn) under drought stress

Ziziphus nummularia (Burm.f.) Wight & Arn., a perennial shrub that thrives in the arid regions, is naturally tolerant to drought. However, there are limited studies on the genomics of drought tolerance in Ziziphus sp. In this study, RNA-sequencing of one month old seedlings treated with PEG 6000 was performed using Roche GS-FLX454 Titanium pyrosequencing. A total of 367,176 raw sequence reads were generated, and upon adapter trimming and quality filtration 351,872 reads were assembled de novo into 32,739 unigenes. Further characterization of the unigenes indicated that 73.25% had significant hits in the protein database. Kyoto encyclopedia of genes and genomes database (KEGG) identified 113 metabolic pathways from the obtained unigenes. A large number of drought-responsive genes were obtained and among them differential gene expression of 16 highly induced genes was validated by qRT-PCR analysis. To develop genic-markers, 3,425 simple sequence repeats (SSRs) were identified in 2,813 unigene sequences. The data generated shall serve as an important reservoir for the identification and characterization of drought stress responsive genes for development of drought tolerant crops.

Step-Stress Partially Accelerated Life Testing Plan for Rayleigh Distribution Using Adaptive Type-II Progressive Hybrid Censoring

This study deals with estimating data of failure times under step-stress partially accelerated life tests based on adaptive Type-II progressive hybrid censoring. The mathematical model related to the lifetime of the test units is assumed to follow Rayleigh distribution. The point and interval maximum likelihood estimations are obtained for distribution parameters and tampering coefficient. The Monte Carlo simulation algorithm along with R software is used to evaluate the performances of the estimators of the tempering coefficient and model parameters. The performances are carried out in terms of mean square errors and biases.

Selection and characterization of Bacillus thuringiensis strains from northwestern Himalayas toxic against Helicoverpa armigera

In this study, we present the selection and the characterization of Bacillus thuringiensis (Bt) strains with respect to their cry/cyt gene content and toxicity evaluation toward one of the most important polyphagous lepidopteran pest, Helicoverpa armigera. Fifty‐six Bt isolates were obtained from 10 different regions of northwestern Himalayas, recording a total B. thuringiensis index of 0.62. Scanning electron microscopy revealed presence of bipyramidal, spherical, flat and irregular crystal shapes; SDS‐PAGE analysis of spore‐crystal mixtures showed the prominence of 130, 70, and 100 kDa protein bands in majority of the isolates; PCR analysis with primers for eight cry and cyt gene families and 13 cry gene subfamilies resulted in isolates showing different combinations of insecticidal genes. Strains containing cry1 were the most abundant (57.1%) followed by cyt2 (46.42%), cry11 (37.5%), cry2 (28.57%), cry4 (21.42%), cyt1 (19.64%), cry3 (8.9%), and cry7, 8 (7.14%). A total of 30.35% of the strains did not amplify with any of the primers used in this study. Median lethal concentration 50 (LC50) estimates of spore‐crystal mixtures of Bt‐JK12, 17, 22, 48, and 72 against second instar larvae of H. armigera was observed to be 184.62, 275.39, 256.29, 259.93 μg ml−1, respectively. B. thuringiensis presents great diversity with respect to the presence of crystal protein encoding genes and insecticidal activity. Four putative toxic isolates identified in this study have potential application in insect pest control. B. thuringiensis isolate JK12 exhibited higher toxicity against H. armigera than that of B. thuringiensis HD1, hence can be commercially exploited to control insect pest for sustainable crop production. The results of this study confirm the significance of continuous exploration of new Bt stains from different ecological regions of the world.

Characterization of lepidopteran-specific cry1 and cry2 gene harbouring native Bacillus thuringiensis isolates toxic against Helicoverpa armigera

Highlights • The manuscript deals with the characterization of lepidopteran-specific cry1 and cry2 gene harbouring Bt isolates.• We were able to find certain Bt isolates containing both cry1 and cry2 genes.• Both cry1 and cry2 genes were found in isolates containing vip3A genes, hence can result in complementation of toxicity.• 13.63% of the Bt isolates were found to be toxic against Helicoverpa armigera.

Microbial Resource Centers Towards Harnessing Microbial Diversity for Human Welfare

Microbes are known to play an important role in numerous metabolic processes like nutrient cycling, environmental detoxification, production of antibiotics, vitamins, industrial enzymes etc. Therefore it is important to efficiently harness and utilize the biologically important properties of microbes and their products to tackle the ever growing challenges of food security, healthcare and environmental pollution. A complete knowledge of these microbes with respect to the role played by them in ecosystem function is essential to fully exploit them for the benefit of mankind. Unfortunately only 4–5% of the microbes have been explored so far whereas the rest ≈95% is are still un-culturable. A number of uncertainties still exist with respect to the microbial diversity as knowledge regarding the number of species of microorganisms that exist, their distribution, stability in the environment and the important roles played by them are lacking to a greater extent. Microbial diversity is an unseen global resource that deserves to be conserved and utilized judiciously. Microbial resource centers play an important role in this regard as they act as living libraries holding microorganisms. The primary function of these centers is to collect, maintain and distribute microbial strains and/or their products to researchers and industrialists all over the world. The role of microbial Culture Collections with respect to conservation and propagation of microbial resources and the difficulties and uncertainties of conservation faced are discussed.