Dehydration is a major form of osmotic stress in cells. Physiological and molecular basis of dehy... more Dehydration is a major form of osmotic stress in cells. Physiological and molecular basis of dehydration stress responses in cells and organisms has been intensively researched over past years. Almost all of the patented dehydration stress tolerance genes from different organisms were used in engineering drought tolerance in crop plants. In spite of the moral, religious and ethical controversies surrounding use of foreign DNA sequences in crop plants, the numbers of such patents has grown tremendously in recent years. In future, we might witness another rise in patents on use of dehydration stress related gene sequences in creating environmental stress tolerant biological control agents for plant disease and insect pest management in agriculture. This review summarizes some of the recent published patents related to drought tolerance genes and their use.
Entomopathogenic nematodes used as biological control agents encounter various stress conditions ... more Entomopathogenic nematodes used as biological control agents encounter various stress conditions during extended periods in the soil. We investigated gene expression in nematodes that were tolerant or susceptible to desiccation stress to determine whether enhanced tolerance in these populations results from a 'gene-expression response' to desiccation or if, for enhanced tolerance, no such response is needed, perhaps due to a state of constant 'readiness'. The expressions of four genes, aldehyde dehydrogenase, nucleosome assembly protein 1, glutathione peroxidase and heat-shock protein 40, were characterized during desiccation stress in five entomopathogenic nematode species with differing stress tolerance: Steinernema feltiae strain IS-6, S. feltiae Carmiel strain, Steinernema carpocapsae Mexican strain, Steinernema riobrave, and Heterorhabditis bacteriophora strain TTO1. After 24h of desiccation, we observed an inverse relationship between expression of the studied genes and phenotypic desiccation-tolerance capability in the nematodes. H. bacteriophora TTO1 was most susceptible to desiccation but showed the highest expression of all studied genes under desiccation. S. carpocapsae Mexican strain and S. riobrave showed the lowest expression of these genes but were most tolerant to desiccation. Our study showed no induction of gene expression in stress-tolerant nematodes, whereas the stress-susceptible nematodes responded to stress by induced expression of these genes. Since the different levels of gene expression were found to be related to the different stress-tolerance capabilities of the nematodes, these gene-expression ratios can potentially be used as markers of desiccation tolerance in entomopathogenic nematodes.
A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of ... more A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of paper and pulp mill showed high β-1, 4-endoglucanase (2.5 IU/ml) but low activity of β-1, 4-exoglucanase (0.8 IU/ml) and β-glucosidase (0.084 IU/ml). The β-1, 4-endoglucanase gene of IARI-SP-1 was amplified using degenerate primers designed based on sequences already available in NCBI GenBank. A full length gene of β-1, 4-endonuclease consisting of 1499 nucleotides was identified through sequence analysis of the amplified product. The ORF encoded for a protein of 500 amino acids with a predicted molecular weight of 55 kDa. The gene was cloned in pET-28a and over expressed in Escherichia coli BL21 (DE3). In comparison to wild strain (B. subtilis), the transformed E. coli exhibited four times increase in cellulase production. Higher enzyme activity was observed in supernatant (8.2 IU/ml) than cell pellet (2.8 IU/ml) suggesting more extracellular production of β-1, 4-endoglucanase. SDS-PAGE and CMC plate assay also confirmed the overproduction by the transformed E. coli. The pH and temperature optima of expressed β-1, 4-endoglucanase enzyme was identical to that of wild strain and was 8 and 50-60 °C, respectively.
Intestinal nematodes or roundworms (aka soil-transmitted helminths or STHs) cause great disease. ... more Intestinal nematodes or roundworms (aka soil-transmitted helminths or STHs) cause great disease. They infect upwards of two billion people, leading to high morbidity and a range of health problems, especially in infected children and pregnant women. Development of resistance to the two main classes of drugs used to treat intestinal nematode infections of humans has been reported. To fight STH infections , we need new and more effective drugs and ways to improve the efficacy of the old drugs. One promising alternative drug is nitazoxanide (NTZ). NTZ, approved for treating human protozoan infections , was serendipitously shown to have therapeutic activity against STHs. However, its mechanism of action against nematodes is not known. Using the laboratory nematode Caenorhabditis elegans, we show that NTZ acts on the nematodes through avr-14, an alpha-type subunit of a glutamate-gated chloride ion channel known for its role in ivermectin susceptibility. In addition, a forward genetic screen to select C. elegans mutants resistant to NTZ resulted in isolation of two NTZ resistant mutants that are not in avr-14, suggesting that additional mechanisms are involved in resistance to NTZ. We found that NTZ combines synergistically with other classes of anthelmintic drugs, i.e. albendazole and pyrantel, making it a good candidate for further studies on its use in drug combination therapy of STH infections. Given NTZ acts against a wide range of nematode parasites, our findings also validate avr-14 as an excellent target for pan-STH therapy.
In the search for novel Xenorhabdus strains in a recently described nematode species, Steinernema... more In the search for novel Xenorhabdus strains in a recently described nematode species, Steinernema thermophilum, three strains (strain 28 T ¼ DSM 17382 T , strain 42 ¼ DSM 17383 and strain 43 ¼ DSM 17384) were isolated from three independent isolation approaches from crushed mixture of infective juveniles. 16S rRNA gene sequence comparison of strains 28 T and DSM 17383 indicated identity and the phylogenetic position pointed towards an individual taxon within the phylogenetic dendrogram of Xenorhabdus type strains. The nearest phylogenetic relatives of strain 28 T were Xenorhabdus poinarii and Xenorhabdus szentirmaii (97.7% each). The three isolates were almost identical in reaction towards the API and BIOLOG substrate panels but differed in their reactions from those of the established type strains of the genus Xenorhabdus. These clear genomic and metabolic differences let us propose a new species, Xenorhabdus indica sp. nov. for the three clones. The type strain is strain 28 T , DSM 17382 T , CIP 108830 T .
Background: Nematodes are the most numerous animals in the soil. Insect parasitic nematodes of th... more Background: Nematodes are the most numerous animals in the soil. Insect parasitic nematodes of the genus Heterorhabditis are capable of selectively seeking, infecting and killing their insect-hosts in the soil. The infective juvenile (IJ) stage of the Heterorhabditis nematodes is analogous to Caenorhabditis elegans dauer juvenile stage, which remains in 'arrested development' till it finds and infects a new insect-host in the soil. H. indica is the most prevalent species of Heterorhabditis in India. To understand the genes and molecular processes that govern the biology of the IJ stage, and to create a resource to facilitate functional genomics and genetic exploration, we sequenced the transcriptome of H. indica IJs.
Entomopathogenic nematodes (EPNs) have been successfully used in the USA and Europe for the biolo... more Entomopathogenic nematodes (EPNs) have been successfully used in the USA and Europe for the biological management of insect pests, for example, invasive mole crickets, white grubs and other scarab larvae in lawns and turf, fungus gnats in mushroom, black vine weevil and Diaprepes root weevil in nursery plants and citrus, respectively. The use of EPNs in India is still in the preliminary stages. To find the best EPN isolates for use in local insect pest management programs, we previously collected 35 strains of EPNs from different parts of India, and screened them for insect infectivity at broad temperature range. Here we quantitated the virulence of seven EPN strains showing infectivity at broad temperature range against Galleria mellonella and Spodoptera litura. Three isolates of Steinernema and four isolates of Heterorhabditis were tested. The Heterorhabditis sp. Hmg3 was the most virulent EPN isolate among all the tested nematodes (LC 50 D 16.1 infective juveniles (IJs)/insect larvae at 24 h), whereas the isolate Steinernema thermophilum Kr was the most virulent steinernematid (LC 50 D 11.8 IJ/insect larvae at 36 h). These strains would be taken up for mass multiplication to facilitate their actual use in insect pest management programs at farmer's fields. Our results suggest that G. mellonella could be used as a primary model for assessing and predicting the virulence of EPN isolates on other insects such as S. litura.
Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect inf... more Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect infested and another from noninfested, healthy tree. Based on the biological, morphological, and molecular evidences, the nematodes are described as Dela-denus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina). Deladenus albizicus n. sp., isolated from insect-infested tree, multiplied on the fungus Nigrospora oryzae. Myceliophagous females of this nematode reproduced by parthenogenesis and sper-mathecae were indistinct. Infective females, readily produced in the cultures, are dorsally curved. Only one type of males containing small-sized sperms in their genital tracts were produced in the culture. Myceliophagous females: L = 0.75 to 1.71 mm, a = 32.3 to 50.8, b = 9.3 to 11.2, b' = 5.2 to 7.3, c = 27.2 to 35.6, V = 91.0 to 93.3, c' = 2.0 to 2.9, stylet = 11 to 12 mm, excretory pore in the region of median pharyngeal bulb, 43 to 47 mm anterior to hemizonid. Deladenus processus n. sp., isolated from bark of healthy A. lebbeck tree, was cultured on Alternaria alternata. Myceliophagous females reproduced by amphimixis and their spermathecae contained rounded sperms. Infective females were never produced, even in old cultures. Myceliophagous females: L = 0.76 to 0.99 mm, a = 34 to 49, b = 13.3 to 17.7, b' = 3.8 to 5.8, c = 19.6 to 22.8, V = 92.2 to 93.5, c' = 2.7 to 3.5, stylet = 6 to 7 mm, excretory pore in the proximity of hemizonid, tail conoid, tapering from both sides to a long pointed central process. It is proposed to classify Deladenus species in three groups: durus, siricidicola, and laricis groups based on female and spermatogonia dimorphism, mode of reproduction, and insect parasitism.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2007
A novel Gram-positive bacterium, strain 40(T), was isolated in the course of identifying bacteria... more A novel Gram-positive bacterium, strain 40(T), was isolated in the course of identifying bacteria from infective juveniles of the entomopathogenic nematode Steinernema thermophilum. Based on 16S rRNA gene analysis, strain 40(T) was found to be related to the type strains of recognized species of the genus Leucobacter, family Microbacteriaceae. The 16S rRNA gene sequence similarity values of strain 40(T) and Leucobacter albus IAM 14851(T), Leucobacter luti LMG 23118(T), Leucobacter alluvii LMG 23117(T), Leucobacter komagatae DSM 8803(T), Leucobacter chromiireducens CIP 108389(T) and Leucobacter aridicollis CIP 108388(T), respectively, were 97.3, 97.5, 97.6, 97.6, 97.6 and 98.5 %. Chemotaxonomic analysis also supported the affiliation of strain 40(T) to the genus Leucobacter: the major menaquinone was MK-11, the peptidoglycan cross-linkage was of the B-type, the cell wall diamino acid was L-diaminobutyric acid and the major fatty acids were anteiso-C(15 : 0) (42 %), anteiso-C(17 : 0) (34 %) and iso-C(16 : 0) (16 %). Based upon the biochemical and genomic analyses, strain 40(T) is sufficiently distinct from the type strains of recognized Leucobacter species to warrant the description of a novel species, for which the name Leucobacter iarius sp. nov. is proposed. The type strain is strain 40(T) (=DSM 17402(T)=CIP 108831(T)).
Dehydration is a major form of osmotic stress in cells. Physiological and molecular basis of dehy... more Dehydration is a major form of osmotic stress in cells. Physiological and molecular basis of dehydration stress responses in cells and organisms has been intensively researched over past years. Almost all of the patented dehydration stress tolerance genes from different organisms were used in engineering drought tolerance in crop plants. In spite of the moral, religious and ethical controversies surrounding use of foreign DNA sequences in crop plants, the numbers of such patents has grown tremendously in recent years. In future, we might witness another rise in patents on use of dehydration stress related gene sequences in creating environmental stress tolerant biological control agents for plant disease and insect pest management in agriculture. This review summarizes some of the recent published patents related to drought tolerance genes and their use.
Entomopathogenic nematodes used as biological control agents encounter various stress conditions ... more Entomopathogenic nematodes used as biological control agents encounter various stress conditions during extended periods in the soil. We investigated gene expression in nematodes that were tolerant or susceptible to desiccation stress to determine whether enhanced tolerance in these populations results from a 'gene-expression response' to desiccation or if, for enhanced tolerance, no such response is needed, perhaps due to a state of constant 'readiness'. The expressions of four genes, aldehyde dehydrogenase, nucleosome assembly protein 1, glutathione peroxidase and heat-shock protein 40, were characterized during desiccation stress in five entomopathogenic nematode species with differing stress tolerance: Steinernema feltiae strain IS-6, S. feltiae Carmiel strain, Steinernema carpocapsae Mexican strain, Steinernema riobrave, and Heterorhabditis bacteriophora strain TTO1. After 24h of desiccation, we observed an inverse relationship between expression of the studied genes and phenotypic desiccation-tolerance capability in the nematodes. H. bacteriophora TTO1 was most susceptible to desiccation but showed the highest expression of all studied genes under desiccation. S. carpocapsae Mexican strain and S. riobrave showed the lowest expression of these genes but were most tolerant to desiccation. Our study showed no induction of gene expression in stress-tolerant nematodes, whereas the stress-susceptible nematodes responded to stress by induced expression of these genes. Since the different levels of gene expression were found to be related to the different stress-tolerance capabilities of the nematodes, these gene-expression ratios can potentially be used as markers of desiccation tolerance in entomopathogenic nematodes.
A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of ... more A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of paper and pulp mill showed high β-1, 4-endoglucanase (2.5 IU/ml) but low activity of β-1, 4-exoglucanase (0.8 IU/ml) and β-glucosidase (0.084 IU/ml). The β-1, 4-endoglucanase gene of IARI-SP-1 was amplified using degenerate primers designed based on sequences already available in NCBI GenBank. A full length gene of β-1, 4-endonuclease consisting of 1499 nucleotides was identified through sequence analysis of the amplified product. The ORF encoded for a protein of 500 amino acids with a predicted molecular weight of 55 kDa. The gene was cloned in pET-28a and over expressed in Escherichia coli BL21 (DE3). In comparison to wild strain (B. subtilis), the transformed E. coli exhibited four times increase in cellulase production. Higher enzyme activity was observed in supernatant (8.2 IU/ml) than cell pellet (2.8 IU/ml) suggesting more extracellular production of β-1, 4-endoglucanase. SDS-PAGE and CMC plate assay also confirmed the overproduction by the transformed E. coli. The pH and temperature optima of expressed β-1, 4-endoglucanase enzyme was identical to that of wild strain and was 8 and 50-60 °C, respectively.
Intestinal nematodes or roundworms (aka soil-transmitted helminths or STHs) cause great disease. ... more Intestinal nematodes or roundworms (aka soil-transmitted helminths or STHs) cause great disease. They infect upwards of two billion people, leading to high morbidity and a range of health problems, especially in infected children and pregnant women. Development of resistance to the two main classes of drugs used to treat intestinal nematode infections of humans has been reported. To fight STH infections , we need new and more effective drugs and ways to improve the efficacy of the old drugs. One promising alternative drug is nitazoxanide (NTZ). NTZ, approved for treating human protozoan infections , was serendipitously shown to have therapeutic activity against STHs. However, its mechanism of action against nematodes is not known. Using the laboratory nematode Caenorhabditis elegans, we show that NTZ acts on the nematodes through avr-14, an alpha-type subunit of a glutamate-gated chloride ion channel known for its role in ivermectin susceptibility. In addition, a forward genetic screen to select C. elegans mutants resistant to NTZ resulted in isolation of two NTZ resistant mutants that are not in avr-14, suggesting that additional mechanisms are involved in resistance to NTZ. We found that NTZ combines synergistically with other classes of anthelmintic drugs, i.e. albendazole and pyrantel, making it a good candidate for further studies on its use in drug combination therapy of STH infections. Given NTZ acts against a wide range of nematode parasites, our findings also validate avr-14 as an excellent target for pan-STH therapy.
In the search for novel Xenorhabdus strains in a recently described nematode species, Steinernema... more In the search for novel Xenorhabdus strains in a recently described nematode species, Steinernema thermophilum, three strains (strain 28 T ¼ DSM 17382 T , strain 42 ¼ DSM 17383 and strain 43 ¼ DSM 17384) were isolated from three independent isolation approaches from crushed mixture of infective juveniles. 16S rRNA gene sequence comparison of strains 28 T and DSM 17383 indicated identity and the phylogenetic position pointed towards an individual taxon within the phylogenetic dendrogram of Xenorhabdus type strains. The nearest phylogenetic relatives of strain 28 T were Xenorhabdus poinarii and Xenorhabdus szentirmaii (97.7% each). The three isolates were almost identical in reaction towards the API and BIOLOG substrate panels but differed in their reactions from those of the established type strains of the genus Xenorhabdus. These clear genomic and metabolic differences let us propose a new species, Xenorhabdus indica sp. nov. for the three clones. The type strain is strain 28 T , DSM 17382 T , CIP 108830 T .
Background: Nematodes are the most numerous animals in the soil. Insect parasitic nematodes of th... more Background: Nematodes are the most numerous animals in the soil. Insect parasitic nematodes of the genus Heterorhabditis are capable of selectively seeking, infecting and killing their insect-hosts in the soil. The infective juvenile (IJ) stage of the Heterorhabditis nematodes is analogous to Caenorhabditis elegans dauer juvenile stage, which remains in 'arrested development' till it finds and infects a new insect-host in the soil. H. indica is the most prevalent species of Heterorhabditis in India. To understand the genes and molecular processes that govern the biology of the IJ stage, and to create a resource to facilitate functional genomics and genetic exploration, we sequenced the transcriptome of H. indica IJs.
Entomopathogenic nematodes (EPNs) have been successfully used in the USA and Europe for the biolo... more Entomopathogenic nematodes (EPNs) have been successfully used in the USA and Europe for the biological management of insect pests, for example, invasive mole crickets, white grubs and other scarab larvae in lawns and turf, fungus gnats in mushroom, black vine weevil and Diaprepes root weevil in nursery plants and citrus, respectively. The use of EPNs in India is still in the preliminary stages. To find the best EPN isolates for use in local insect pest management programs, we previously collected 35 strains of EPNs from different parts of India, and screened them for insect infectivity at broad temperature range. Here we quantitated the virulence of seven EPN strains showing infectivity at broad temperature range against Galleria mellonella and Spodoptera litura. Three isolates of Steinernema and four isolates of Heterorhabditis were tested. The Heterorhabditis sp. Hmg3 was the most virulent EPN isolate among all the tested nematodes (LC 50 D 16.1 infective juveniles (IJs)/insect larvae at 24 h), whereas the isolate Steinernema thermophilum Kr was the most virulent steinernematid (LC 50 D 11.8 IJ/insect larvae at 36 h). These strains would be taken up for mass multiplication to facilitate their actual use in insect pest management programs at farmer's fields. Our results suggest that G. mellonella could be used as a primary model for assessing and predicting the virulence of EPN isolates on other insects such as S. litura.
Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect inf... more Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect infested and another from noninfested, healthy tree. Based on the biological, morphological, and molecular evidences, the nematodes are described as Dela-denus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina). Deladenus albizicus n. sp., isolated from insect-infested tree, multiplied on the fungus Nigrospora oryzae. Myceliophagous females of this nematode reproduced by parthenogenesis and sper-mathecae were indistinct. Infective females, readily produced in the cultures, are dorsally curved. Only one type of males containing small-sized sperms in their genital tracts were produced in the culture. Myceliophagous females: L = 0.75 to 1.71 mm, a = 32.3 to 50.8, b = 9.3 to 11.2, b' = 5.2 to 7.3, c = 27.2 to 35.6, V = 91.0 to 93.3, c' = 2.0 to 2.9, stylet = 11 to 12 mm, excretory pore in the region of median pharyngeal bulb, 43 to 47 mm anterior to hemizonid. Deladenus processus n. sp., isolated from bark of healthy A. lebbeck tree, was cultured on Alternaria alternata. Myceliophagous females reproduced by amphimixis and their spermathecae contained rounded sperms. Infective females were never produced, even in old cultures. Myceliophagous females: L = 0.76 to 0.99 mm, a = 34 to 49, b = 13.3 to 17.7, b' = 3.8 to 5.8, c = 19.6 to 22.8, V = 92.2 to 93.5, c' = 2.7 to 3.5, stylet = 6 to 7 mm, excretory pore in the proximity of hemizonid, tail conoid, tapering from both sides to a long pointed central process. It is proposed to classify Deladenus species in three groups: durus, siricidicola, and laricis groups based on female and spermatogonia dimorphism, mode of reproduction, and insect parasitism.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2007
A novel Gram-positive bacterium, strain 40(T), was isolated in the course of identifying bacteria... more A novel Gram-positive bacterium, strain 40(T), was isolated in the course of identifying bacteria from infective juveniles of the entomopathogenic nematode Steinernema thermophilum. Based on 16S rRNA gene analysis, strain 40(T) was found to be related to the type strains of recognized species of the genus Leucobacter, family Microbacteriaceae. The 16S rRNA gene sequence similarity values of strain 40(T) and Leucobacter albus IAM 14851(T), Leucobacter luti LMG 23118(T), Leucobacter alluvii LMG 23117(T), Leucobacter komagatae DSM 8803(T), Leucobacter chromiireducens CIP 108389(T) and Leucobacter aridicollis CIP 108388(T), respectively, were 97.3, 97.5, 97.6, 97.6, 97.6 and 98.5 %. Chemotaxonomic analysis also supported the affiliation of strain 40(T) to the genus Leucobacter: the major menaquinone was MK-11, the peptidoglycan cross-linkage was of the B-type, the cell wall diamino acid was L-diaminobutyric acid and the major fatty acids were anteiso-C(15 : 0) (42 %), anteiso-C(17 : 0) (34 %) and iso-C(16 : 0) (16 %). Based upon the biochemical and genomic analyses, strain 40(T) is sufficiently distinct from the type strains of recognized Leucobacter species to warrant the description of a novel species, for which the name Leucobacter iarius sp. nov. is proposed. The type strain is strain 40(T) (=DSM 17402(T)=CIP 108831(T)).
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Papers by Vishal Somvanshi