RESUMO
Integrated pest management (IPM) is a comprehensive approach to managing diseases, focusing on combining various strategies to reduce pathogen populations effectively and in an environmentally conscious way. We investigated the effects of IPM on beneficial microbial populations and its relationship with pathogen populations in both direct-seeded rice (DSR) and transplanted rice (TR) systems. This study demonstrates that IPM practices have significantly higher populations of beneficial microbes, such as Trichoderma harzianum and Pseudomonas fluorescens, and lower level of the pathogen Fusarium verticillioides compared to non-IPM (farmer practices). The average mean population of T. harzianum was 6.38 × 103 CFU/g in IPM compared to 3.22 × 103 CFU/g in non-IPM during 2019 in TR at Bambawad. P. fluorescens mean population in 2019 was significantly higher in IPM (4.67 × 103 CFU/g) than in non-IPM (3.82 × 103 CFU/g) at the Karnal location in DSR. The F. verticillioides populations were significantly lower in IPM fields (9.46 × 103 CFU/g) compared to non-IPM fields (11.48 × 103 CFU/g) during 2017 at Haridwar in TR. Over three years, a significant increase in the populations of beneficial microbes in IPM plots was observed in all three locations of both TR and DSR, highlighting the sustainable impact of IPM practices. Disease dynamics analysis revealed that IPM effectively managed key diseases in both DSR and TR systems, with significant correlations between microbial density and disease severity. A significant positive correlation was recorded between F. verticillioides population and bakanae incidence at all three locations. Sheath blight incidence was negatively correlated with P. fluorescens population in both TR and DSR. In DSR, bacterial blight and brown spot diseases are reduced with the increased population of T. harzianum. Bioagents T. harzianum and P. fluorescens reduced disease incidence, underscoring the role of beneficial microbes in disease suppression and their importance for sustainable production using IPM practices.
RESUMO
Bacillus thuringiensis (Bt) strains were isolated from soil samples of Great Nicobar Islands, one of the "hottest biodiversity hotspots," where no collection has been characterized previously. The 36 new Bt isolates were obtained from 153 samples analyzed by crystal protein production with light/phase-contrast microscopy, determination of cry gene profile by SDS-PAGE, evaluation of toxicity against Coleopteran, and Lepidopteran insect pests, finally cloning and sequencing. Majority of the isolates showed the presence of 66-35 kDa protein bands on SDS-PAGE while the rest showed >130, 130, 73, and 18 kDa bands. The variations in crystal morphology and mass of crystal protein(s) purified from the isolates of Bt revealed genetic and molecular diversity. Based on the toxicity test, 50 % of isolates were toxic to Ash weevils, 16 % isolates were toxic to cotton bollworm, 38 % isolates were toxic both to ash weevil as well as cotton bollworm, while 11 % of the isolates did not exhibit any toxicity. PCR analysis unveiled prepotency of cry1B- and cry8b-like genes in these isolates. This study appoints the first isolation and characterization of local B. thuringiensis isolates in Great Nicobar Islands. Some of these isolates display toxic potential and, therefore, could be adopted for future applications to control some agriculturally important insect pests in the area of integrated pest management for sustainable agriculture.
Assuntos
Bacillus thuringiensis/genética , Bacillus thuringiensis/isolamento & purificação , Besouros/microbiologia , Endotoxinas/toxicidade , Lepidópteros/microbiologia , Controle Biológico de Vetores , Microbiologia do Solo , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/análise , DNA Bacteriano/genética , Endotoxinas/química , Endotoxinas/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Ilhas do Oceano Índico , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Miraculin-like proteins, belonging to the Kunitz superfamily, are natural plant defense agents against pests and predators, and therefore are potential biopesticides for incorporation into pest-resistant crops. Here, a miraculin-like protein from Murraya koenigii was assessed for its in vitro and in vivo effects against two polyphagous lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura. M. koenigii miraculin-like protein (MKMLP) inhibited the trypsin-like activity and total protease activity of H. armigera gut proteinases (HGP) by 78.5 and 40%, respectively, and S.litura gut proteinases (SGP) by 81 and 48%, respectively. The inhibitor was stable and actively inhibited the proteolysis of both HGP and SGP enzymes for up to 72 h. Incorporation of MKMLP into artificial diet adversely affected the growth and development of pests in a dose-dependent manner. After 10 days of feeding on diets containing 200 µM MKMLP, larval weight was reduced to 69 and 44.8% and larval mortality was increased to 40 and 43.3% for H. armigera and S litura, respectively. The LC(50) of MKMLP was 0.34 and 0.22% of the diet for H.armigera and S. litura, respectively. These results demonstrate the efficacy of MKMLP as a potential plant defense agent against H. armigera and S. litura.
Assuntos
Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Murraya/química , Proteínas de Plantas/farmacologia , Inibidores de Proteases/farmacologia , Spodoptera/efeitos dos fármacos , Animais , Sistema Digestório/enzimologia , Ativação Enzimática/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/enzimologia , Mariposas/enzimologia , Peptídeo Hidrolases/metabolismo , Controle Biológico de Vetores , Estabilidade Proteica , Spodoptera/enzimologiaRESUMO
Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.
Assuntos
Chaperonina 60/química , Chaperonina 60/fisiologia , Quitina/química , Xenorhabdus/metabolismo , Sequência de Aminoácidos , Animais , Genoma , Inseticidas/química , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Frações Subcelulares/metabolismoRESUMO
Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.
Assuntos
Proteínas de Fímbrias/isolamento & purificação , Inseticidas/isolamento & purificação , Aglutinação , Sequência de Aminoácidos , Animais , Sequência de Bases , Dicroísmo Circular , Clonagem Molecular , Proteínas de Fímbrias/química , Proteínas de Fímbrias/farmacologia , Hemócitos/metabolismo , Larva/efeitos dos fármacos , Dados de Sequência Molecular , Subunidades Proteicas , Proteínas Recombinantes/isolamento & purificação , XenorhabdusRESUMO
Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.