Vishal Somvanshi | INDIAN AGRICULTURAL RESEARCH INSTITUTE, NEW DELHI, INDIA (original) (raw)

Papers by Vishal Somvanshi

Microbial Factories, 2015

Recent Patents on DNA & Gene Sequences, 2009

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.

Molecular Microbiology, 2010

Fimbriae are adhesive organelles known to enable pathogens to colonize animal tissue, but little ... more Fimbriae are adhesive organelles known to enable pathogens to colonize animal tissue, but little is known of their function in mutualistic symbioses. Photorhabdus colonization of Heterorhabditis bacteriophora nematodes is essential for the pair's insect pathogenic lifestyle. Maternal nematodes acquire Photorhabdus symbionts as a persistent intestinal biofilm prior to transmission to infective juvenile (IJ) stage offspring developing inside the maternal body. Screening 8000 Photorhabdus mutants for defects in IJ colonization revealed that a single fimbrial locus, named mad for maternal adhesion defective, is essential. The mad genes encode a novel usher/ chaperone assembled fimbria regulated by an ON/OFF invertible promoter switch. Adherent Photorhabdus cells in maternal nematode intestines had the switch ON opposite to the OFF orientation of most other cells. A DmadA mutant failed to adhere to maternal intestines and be transmitted to the IJs. Mad fimbriae were detected on TT01 phase ON cells but not on DmadA phase ON cells. Also required for transmission is madJ, predicted to encode a transcriptional activator related to GrlA. Expression of madA-K or madIJK restored the ability of madJ mutant to adhere. The Mad fimbriae were not required for insect pathogenesis, indicating the specialized function of Mad fimbriae for symbiosis.

Molecular and Biochemical Parasitology, 2008

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.

Microbiological Research, 2014

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.

Euphytica, 2010

Orobanche and Phelipanche, commonly known as broomrape, are dicotyledonous holoparasitic flowerin... more Orobanche and Phelipanche, commonly known as broomrape, are dicotyledonous holoparasitic flowering plants that cause heavy economic losses in a wide variety of plant species. Breeding for Orobanche resistance is still one of the most effective management strategies for this weed. However, previous efforts to find broomrape-resistant tomato (Solanum lycopersicon) genotypes have been unsuccessful. Here, we report on the isolation and characterization of a fast-neutron-mutagenized M-82 tomato mutant, Sl-ORT1. The Sl-ORT1 mutant showed resistance to Phelipanche aegyptiaca as compared to cultivar M-82; segregation analysis suggested a single recessive ort1 allele. Sl-ORT1 broomrape resistance was reflected in a lower number of broomrapes per plant, reduced P. aegyptiaca fresh weight per plant, and the absence of broomrape's negative effect on plant host growth and yield. Sl-ORT1 was shown to be resistant to high concentrations of P. aegyptiaca seeds, and to another three broomrape species: Phelipanche ramosa, Orobanche cernua, and Orobanche crenata. Grafting experiments demonstrated that roots, rather than shoots, are necessary for Sl-ORT1 broomrape resistance. In addition, Sl-ORT1 was shown to be resistant to broomrape under field conditions. Since yield parameters are slightly affected by the mutation, this resistance gene should be introduced into tomato varieties with different genetic backgrounds; this newly identified Orobanche-resistant mutant may be further utilized in breeding programs for Orobanche resistance.

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.

Applied and Environmental Microbiology, 2009

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed fo... more A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.

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.

Science, 2012

Microbial populations stochastically generate variants with strikingly different properties, such... more Microbial populations stochastically generate variants with strikingly different properties, such as virulence or avirulence and antibiotic tolerance or sensitivity. Photorhabdus luminescens bacteria have a variable life history in which they alternate between pathogens to a wide variety of insects and mutualists to their specific host nematodes. Here, we show that the P. luminescens pathogenic variant (P form) switches to a smaller-cell variant (M form) to initiate mutualism in host nematode intestines. A stochastic promoter inversion causes the switch between the two distinct forms. Mform cells are much smaller (one-seventh the volume), slower growing, and less bioluminescent than P-form cells; they are also avirulent and produce fewer secondary metabolites. Observations of form switching by individual cells in nematodes revealed that the M form persisted in maternal nematode intestines, were the first cells to colonize infective juvenile (IJ) offspring, and then switched to P form in the IJ intestine, which armed these nematodes for the next cycle of insect infection.

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)).

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2006

In the course of isolating bacteria from infective juveniles of the entomopathogenic nematode Ste... more In the course of isolating bacteria from infective juveniles of the entomopathogenic nematode Steinernema thermophilum Ganguly & Singh, 2000, three isolates were obtained (OP1 T , OP29 and VS3). On the basis of 16S rRNA gene sequence analysis and riboprint patterns, these three strains were identical to each other but distinct from the type strains of the five recognized species of the genus Providencia. Based on biochemical and genomic analysis and supported by the low (<35 %) DNA-DNA relatedness between strain OP1 T and the type strain of its phylogenetically closest relative, Providencia rettgeri (99?5 % 16S rRNA gene sequence similarity), strain OP1 T was considered to be sufficiently distinct from recognized Providencia species to warrant the description of a novel species. The name Providencia vermicola sp. nov. is proposed, with OP1 T (=DSM 17385 T =CIP 108829 T ) as the type strain.

Infection and Immunity, 2010

Burkholderia cenocepacia AU1054 is an opportunistic pathogen isolated from the blood of a person ... more Burkholderia cenocepacia AU1054 is an opportunistic pathogen isolated from the blood of a person with cystic fibrosis. AU1054 is a multihost pathogen causing rapid pathogenicity to Caenorhabditis elegans nematodes. Within 24 h, AU1054 causes greater than 50% mortality, reduced growth, emaciated body, distended intestinal lumen, rectal swelling, and prolific infection of the nematode intestine. To determine virulence mechanisms, 3,000 transposon mutants were screened for attenuated virulence in nematodes. Fourteen virulence-attenuated mutants were isolated, and the mutant genes were identified. These genes included paaA, previously identified as being required for full virulence of B. cenocepacia K56-2. Six mutants were restored in virulence by complementation with their respective wild-type gene. One of these contained an insertion in gspJ, predicted to encode a pseudopilin component of the type 2 secretion system (T2SS). Nematodes infected with AU1054 gspJ had fewer bacteria present in the intestine than those infected with the wild type but still showed rectal swelling. The gspJ mutant was also defective in pathogenicity to onion and in degradation of polygalacturonic acid and casein. This result differs from previous studies where no or little role was found for T2SS in Burkholderia virulence, although virulence factors such as zinc metalloproteases and polygalacturonase are known to be secreted by the T2SS. This study highlights strain specific differences in B. cenocepacia virulence mechanisms important for understanding what enables environmental microbes to function as opportunistic pathogens.

Applied and Environmental Microbiology, 2009

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed fo... more A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.

Microbial Factories, 2015

Recent Patents on DNA & Gene Sequences, 2009

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.

Molecular Microbiology, 2010

Fimbriae are adhesive organelles known to enable pathogens to colonize animal tissue, but little ... more Fimbriae are adhesive organelles known to enable pathogens to colonize animal tissue, but little is known of their function in mutualistic symbioses. Photorhabdus colonization of Heterorhabditis bacteriophora nematodes is essential for the pair's insect pathogenic lifestyle. Maternal nematodes acquire Photorhabdus symbionts as a persistent intestinal biofilm prior to transmission to infective juvenile (IJ) stage offspring developing inside the maternal body. Screening 8000 Photorhabdus mutants for defects in IJ colonization revealed that a single fimbrial locus, named mad for maternal adhesion defective, is essential. The mad genes encode a novel usher/ chaperone assembled fimbria regulated by an ON/OFF invertible promoter switch. Adherent Photorhabdus cells in maternal nematode intestines had the switch ON opposite to the OFF orientation of most other cells. A DmadA mutant failed to adhere to maternal intestines and be transmitted to the IJs. Mad fimbriae were detected on TT01 phase ON cells but not on DmadA phase ON cells. Also required for transmission is madJ, predicted to encode a transcriptional activator related to GrlA. Expression of madA-K or madIJK restored the ability of madJ mutant to adhere. The Mad fimbriae were not required for insect pathogenesis, indicating the specialized function of Mad fimbriae for symbiosis.

Molecular and Biochemical Parasitology, 2008

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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;gene-expression response&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; to desiccation or if, for enhanced tolerance, no such response is needed, perhaps due to a state of constant &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;readiness&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;. 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.

Microbiological Research, 2014

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.

Euphytica, 2010

Orobanche and Phelipanche, commonly known as broomrape, are dicotyledonous holoparasitic flowerin... more Orobanche and Phelipanche, commonly known as broomrape, are dicotyledonous holoparasitic flowering plants that cause heavy economic losses in a wide variety of plant species. Breeding for Orobanche resistance is still one of the most effective management strategies for this weed. However, previous efforts to find broomrape-resistant tomato (Solanum lycopersicon) genotypes have been unsuccessful. Here, we report on the isolation and characterization of a fast-neutron-mutagenized M-82 tomato mutant, Sl-ORT1. The Sl-ORT1 mutant showed resistance to Phelipanche aegyptiaca as compared to cultivar M-82; segregation analysis suggested a single recessive ort1 allele. Sl-ORT1 broomrape resistance was reflected in a lower number of broomrapes per plant, reduced P. aegyptiaca fresh weight per plant, and the absence of broomrape's negative effect on plant host growth and yield. Sl-ORT1 was shown to be resistant to high concentrations of P. aegyptiaca seeds, and to another three broomrape species: Phelipanche ramosa, Orobanche cernua, and Orobanche crenata. Grafting experiments demonstrated that roots, rather than shoots, are necessary for Sl-ORT1 broomrape resistance. In addition, Sl-ORT1 was shown to be resistant to broomrape under field conditions. Since yield parameters are slightly affected by the mutation, this resistance gene should be introduced into tomato varieties with different genetic backgrounds; this newly identified Orobanche-resistant mutant may be further utilized in breeding programs for Orobanche resistance.

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.

Applied and Environmental Microbiology, 2009

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed fo... more A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.

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.

Science, 2012

Microbial populations stochastically generate variants with strikingly different properties, such... more Microbial populations stochastically generate variants with strikingly different properties, such as virulence or avirulence and antibiotic tolerance or sensitivity. Photorhabdus luminescens bacteria have a variable life history in which they alternate between pathogens to a wide variety of insects and mutualists to their specific host nematodes. Here, we show that the P. luminescens pathogenic variant (P form) switches to a smaller-cell variant (M form) to initiate mutualism in host nematode intestines. A stochastic promoter inversion causes the switch between the two distinct forms. Mform cells are much smaller (one-seventh the volume), slower growing, and less bioluminescent than P-form cells; they are also avirulent and produce fewer secondary metabolites. Observations of form switching by individual cells in nematodes revealed that the M form persisted in maternal nematode intestines, were the first cells to colonize infective juvenile (IJ) offspring, and then switched to P form in the IJ intestine, which armed these nematodes for the next cycle of insect infection.

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)).

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2006

In the course of isolating bacteria from infective juveniles of the entomopathogenic nematode Ste... more In the course of isolating bacteria from infective juveniles of the entomopathogenic nematode Steinernema thermophilum Ganguly & Singh, 2000, three isolates were obtained (OP1 T , OP29 and VS3). On the basis of 16S rRNA gene sequence analysis and riboprint patterns, these three strains were identical to each other but distinct from the type strains of the five recognized species of the genus Providencia. Based on biochemical and genomic analysis and supported by the low (<35 %) DNA-DNA relatedness between strain OP1 T and the type strain of its phylogenetically closest relative, Providencia rettgeri (99?5 % 16S rRNA gene sequence similarity), strain OP1 T was considered to be sufficiently distinct from recognized Providencia species to warrant the description of a novel species. The name Providencia vermicola sp. nov. is proposed, with OP1 T (=DSM 17385 T =CIP 108829 T ) as the type strain.

Infection and Immunity, 2010

Burkholderia cenocepacia AU1054 is an opportunistic pathogen isolated from the blood of a person ... more Burkholderia cenocepacia AU1054 is an opportunistic pathogen isolated from the blood of a person with cystic fibrosis. AU1054 is a multihost pathogen causing rapid pathogenicity to Caenorhabditis elegans nematodes. Within 24 h, AU1054 causes greater than 50% mortality, reduced growth, emaciated body, distended intestinal lumen, rectal swelling, and prolific infection of the nematode intestine. To determine virulence mechanisms, 3,000 transposon mutants were screened for attenuated virulence in nematodes. Fourteen virulence-attenuated mutants were isolated, and the mutant genes were identified. These genes included paaA, previously identified as being required for full virulence of B. cenocepacia K56-2. Six mutants were restored in virulence by complementation with their respective wild-type gene. One of these contained an insertion in gspJ, predicted to encode a pseudopilin component of the type 2 secretion system (T2SS). Nematodes infected with AU1054 gspJ had fewer bacteria present in the intestine than those infected with the wild type but still showed rectal swelling. The gspJ mutant was also defective in pathogenicity to onion and in degradation of polygalacturonic acid and casein. This result differs from previous studies where no or little role was found for T2SS in Burkholderia virulence, although virulence factors such as zinc metalloproteases and polygalacturonase are known to be secreted by the T2SS. This study highlights strain specific differences in B. cenocepacia virulence mechanisms important for understanding what enables environmental microbes to function as opportunistic pathogens.

Applied and Environmental Microbiology, 2009

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed fo... more A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.