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Six insecticides were evaluated for their toxicity against nymphs and adults of assassin bug, Sycanus collaris (Fab.) through contact and stomach mode. The studies revealed that emamectin benzoate (0.4g/L), chlorantraniliprole (0.25ml/L), flubendiamide (0.25 ml/L) and thiamethoxam (0.25 g/L) were considered as relatively safer insecticides for all the nymphal instars and adults of S. collaris. Fenazaquin (1.25ml/L) and quinalphos (2ml/L) caused higher mortality (85-100%) in all the stages of S. collaris. The higher mortality observed in nymphs and relatively lesser mortality rate of adults indicate that the application of the chemical should not be carried out immediately after the release of nymphal instars of S. collaris and adult releases would be ideal in Integrated Pest Management (IPM).

Background Thirty-six entomopathogenic fungi (EPF) were isolated from soil and insect cadaver samples, collected from different forest types, viz., wet evergreen, moist deciduous, dry deciduous and scrub type in South India. Partial sequences of two parsimony informative genes ITS and RPB1 were determined under a phylogenetic approach for assessing the genetic diversity. Results Twenty-seven RPB1 gene sequences and 34 sequences of ITS1, 5.8S and ITS2 regions belonging to 36 EPF were analysed for identification and characterization. Four species of Metarhizium viz., M. anisopliae, M. roberstii, M. majus and M. guizhouense were differentiated. The isolates could be grouped into four main clades of 1–5. Most of the fungi appeared to be closely related to M. anisopliae. Based on the colony characters, colour, conidial size and shape, 27 isolates were morphologically identified as M. anisopliae. Seven strains were apparently related to M. robertsii, three isolates were similar to M. majus and the remaining one was identified as M. guizhouense. Morphological studies in congruence with phylogenetic analysis resolved the species diversity. Bioassay studies showed that M. quizhouense, M. majus and M. robertsii were effective against the banana stem weevil Odoiporus longicollis. Conclusions This is the first attempt to study the diversity and occurrence of Metarhizium species in forests of South India. Wet evergreen forest of Aralam in South India was rich in EPF diversity particularly for three species namely, M. quizhouense, M. robertsii and M. anisopliae.

Spodoptera frugiperda (J E Smith) (fall armyworm) (Lepidoptera: Noctuidae), is a key pest of maize that has recently entered in India causing damage and yield loss. A biocontrol-based integrated pest management (IPM) strategy was designed and evaluated in farmer’s field during rabi and kharif season (2018–2019). IPM strategy comprising installation of controlled release FAW pheromone traps, four releases of Trichogramma pretiosum Riley, two sprays of neem oil, one spray of each Bacillus thuringiensis (NBAIR-BT25) and Metarizium anisopliae (NBAIR Ma-35) resulted in 76 and 71.64% egg mass; 80 and 74.44% larval population reduction at 60 days after treatment during rabi and kharif season, respectively. Cob yield per acre in biocontrol-based IPM field was higher than the farmer’s practice (6–7 sprays of emamectin benzoate 5% SG) during both the seasons, and it resulted in 38.3 and 42.29% gain in yield per acre during rabi and kharif, respectively. Therefore, this module forms a base to manage the fall armyworm in an eco-friendly and farmer friendly manner. Future research with other alternatives has also been discussed.

The search for new Bacillus thuringiensis ( Bt ) strains is a continuous process and researchers are now focusing on finding toxin proteins that are toxic to pests of insect orders that are not reported. In the present study soil and insect cadaver samples were collected from North East India comprising the states of Assam, Tripura and Mehhalaya and native Bt were isolated using standard protocols. At total of 30 Bt isolates were purified and characterized. Various types of crystal morphology were encountered that included bipyramidal, cuboidal, square, rhomboid, spherical and irregular. PCR analysis showed that diverse cry genes were expressed. The cry genes identified were Lepidoptera, Coleoptera and Diptera specific. Detected genes included cry1Ac , cry2A , cry4A , cry10A , cry16A , cry17A , cry19A , cry30Aa , cry44Aa , cry11A , cry4B , cry12A , cry8A and cry7A . Many of them were positive for Vip3A protein. The coleopteran specific Bt were evaluated against Sitophilus oryzae and Callosobruchus chinensis and NBAIR-AgBt6 was found to be toxic. The isolates are being further evaluated for use as biopesticides.

Water stress in one of the limiting factors which influences the plant growth. Microbes being as a partner are an integral part of the ecosystem which influences the plant growth under stress. In the present study, Pseudomonas fluorescens (NBAII- PFDWD) subjected to osmotic stress by altering osmotic potential (-10.28 MPa and -26.82 MPa) using Polyethylene Glycol (PEG) 6000 in its growth media revealed expression of proteins which modulates its cell processes. MALDI TOF studies of selected spots from 2D gel analysis of P. fluorescens (NBAII- PFDWD) grown under different osmotic stresses revealed that the stress kindled genes which were involved in production of osmoprotectants, genes encoding DNA damage repair and increased the translational accuracy. The studies also showed that P. fluorescens possesses unique mechanisms for survival under osmotic stress. The studies indicate the diverse expression of proteins in P. fluorescens (NBAII- PFDWD) under different osmotic potentials which helped them to mitigate impact of osmotic stress. The present method unravelled the mechanisms adopted by P. fluorescens (NBAII- PFDWD) to thrive under osmotic stress. The bacterium is potential stress tolerant isolate which can be exploited as a plant growth promoting rhizobacteria for agricultural crops grown in stressed soils.

Studies were conducted to systematically isolate Metarhizium isolates from the insect cadavers and soils of South India. Morphological and PCR amplified sequences of 5.8S ITS regions and RNA polymerase II largest subunit (RPB1) gene regions were used to identify the isolates at species level. Eight Metarhizium isolates were isolated and initially identified by morphological and microscopic studies. Further identification was confirmed through 5.8SrRNA ITS and RPB1 analysis. They were identified as three isolates of M. robertsii J.F. Bisch., Rehner & Humber sp. nov. (ArMz3R, ArMz3S and ArMz6W), one isolate of M. majus (J.R. Johnst.) J.F. Bisch., Rehner & Humber (VjMz1W) and four isolates of M. anisopliae (WnMz1S, NlMz2S, BgMz2S and DhMz4R). Topical conidial suspensions (TCS) and powder based formulations (PBF) of the eight indigenous isolates of Metarhizium spp. that were isolated from insect cadavers and soils of South India were tested against coleopteran pests Holotricha serrata L. and Oryctes rhinoceros L. that cause serious damage to sugarcane and palm trees respectively. Against H. serrata TCS of M. robertsii (ArMz6W) was the most effective with an LC 50 of 6.893×10 5 cfu/ml and caused 100% mortality against the 3 rd instar larvae in 5 days; PBF elicited an LC 50 of 7.502×10 5 cfu/ml with 96% mortality in 10 days. Against O. rhinoceros TCS (LC 50 of 9.75×10 5 cfu/ml) of M. majus (VjMz1W) caused 90% mortality in 7 days and the PBF (LC 50 of 9.57×10 5 cfu/ml) caused 86% mortality in 14 days. The results establish that M. robertsii is highly effective against H. serrata and against O. rhinoceros, M. majus was the most effective. The TCS formulations of these two strains can be readily deployed for field applications.

Bacillus spp. have long been used as biological control organisms against plant bacterial diseases but the mechanisms by which the bacteria confer protection against the pathogens are not properly understood. Among nine strains of Bacillus spp. three of them, viz. NBAII 63 (B. megaterium), NBAII 71 (B. cereus) and NBAII 65 (B. megaterium) were found highly inhibitory against R. solanacearum. These strains of Bacillus spp. produced indole acetic acid (IAA) and siderophore and solubilized the phosphorous. High amount of IAA (174.2 μg/ml) and siderophore (1.32μg/ml) production followed by the highest phosphorous solubilization (53.3 μg/ml) by the strain NBAII 63 were found. These three potential Bacillus strains showed the increased activity of defense related enzymes, viz. peroxidase (PO), polyphenoloxidase (PPO), phenylalanine ammonia-lyase (PAL) and total phenols in pre treated tomato plants challenged with R. solanacearum. Significant activities of PO, PPO, PAL were observed at 8 days after the treatment of antagonist and declined gradually afterwards. The maximum phenol content (185 μg/g of plant tissue) was observed in the tomato plants whose roots were treated with Bacillus strain 63 at 8 days after inoculation treatment. These Bacillus strains could be used as potential biocontrol agent for the management of bacterial wilt disease of tomato.

The present study was carried out to assess the field efficacy of endophytic isolates of Beauveria bassiana (Bb-5a, Bb-23 and Bb-45) and Metarhizium anisopliae (Ma-35) against maize stem borer (Chilo partellus Swinoe) during kharif seasons of 2015 and 2016 at ICAR-National Bureau of Agricultural Insect Resources (NBAIR), Attur Farm, Bengaluru, Karnataka. All the tested isolates showed suppression of maize stem borer damage during both seasons. Pooled data indicated that lesser dead hearts (9.5-12.71%), stem tunnelling (3.98-4.36 cm/plant) and exit holes (2.59-2.79 /plant) in the treated plants compared to untreated control which showed 21.38% dead hearts, 8.01 cm/plant of stem tunnelling and 6.99 exit holes/plant. Among the four isolates tested, Bb-5a isolate of B. bassiana and Ma-35 isolate of M. anisopliae showed significantly superior effect in lowering the incidence of dead hearts (9.50 and 12.71%), stem tunnelling (3.98 and 4.36 cm/plant) and exit holes (2.59 and 2.79/plant) with higher yields of 72 and 70.0 tonnes/ha respectively.

Five culturable gut bacteria associated with single leafhopper [A. biguttula biguttula (Ishida)] (Hemiptera: Cicadellidae) of Guntur field population were characterized through morphological and 16S rDNA sequences. The characterized gut bacteria were Staphylococcus pasteuri, Enterococcus silesiacus, Bacillus amyloliquefaciens, Enterobacter asburiae and Bacillus pumilus. Among these, Bacillus pumilus was chosen for studying its role on host nutrition and defense. The role of the gut bacterium B. pumilus on insect defense against entomofungal pathogens and nutrition was studied under in vitro condition by dual culture technique and other bioassays. The gut bacterium B. pumilus inhibited the mycelial growth of all four major entomofungal pathogens. Maximum growth inhibition (2.0cm) was recorded on Beauveria bassiana followed by Metarhizium anisopliae (1.7cm), Paecilomyces fumosoroseus (1.6cm) and Lecanicillium lecanii (1.5cm) as compared to control. In vitro experiments proved the production of digestive enzymes such as cellulase, pectinase, amylase and protease from B. pumilus. The present study characterized the gut bacteria associated with A.biguttula biguttula and studied the role of the gut bacterium B.pumilus on host nutrition and defense against entomofungal pathogens for the first time in the world.

Biological control of charcoal root rot disease caused by Macrophomina phaseolina in chickpea was studied by using Streptomyces sp. S160. This biocontrol agent (BCA) inhibited the mycelial growth of M. phaseolina by 50 % in vitro and significantly reduced charcoal rot incidence in the greenhouse by 33.3 %. The greenhouse experiment revealed that seed treatment along with soil application supported the highest germination (88.6 %), vigor index (7326.91) and reduced root rot incidence (12.5 %) in comparison to seed treatment and soil application alone. BCA enhanced the growth and helped in inducing resistance against charcoal rot disease of chickpea caused by M. phaseolina by increasing activity of defense-related enzymes in chickpea plants, leading to the synthesis of defense chemicals in plants. BCA (Streptomyces sp. S160) was also characterized and identified by using polyphasic approaches including 16S rDNA sequencing.

Pseudomonas putida (NBAII-RPF9) was identified as an abiotic stress tolerant bacterium capable of growing at 45 °C as well as in 1 M NaCl. The proteins expressed by this bacterium when subjected to these two stresses were analyzed by 2D gel and MALDI-TOF/MS. Two parameters viz., heat/saline shock (20 min at 45 °C/1 M solid NaCl added at mid log phase and incubated for 1 h) and heat/saline tolerance (24 h growth at 45 °C/in 1 M NaCl) were studied. Under heat shock 13 upregulated proteins and 1 downregulated protein were identified and under tolerance 6 upregulated proteins were identified. GroES and GroEL proteins were expressed under both tolerance and shock. Under saline shock 11 upregulated proteins were identified whereas under saline tolerance 6 upregulated proteins were identified and all these proteins had pI between 3 and 10 with molecular weights ranging from 14.3 to 97 kDa. Aspartate carbamoyltransferase was common under both the saline conditions studied. The analysis revealed involvement of heat stress responsive molecular chaperones and membrane proteins during heat stress. During salt stress, proteins involved in metabolic processes were found to be upregulated to favor growth and adaptation of the bacterium. Heat shock chaperones viz., DnaK and DnaJ were expressed under both saline and heat stress. This is the first report of protein profile obtained from a single bacterium under saline and heat stress and the studies reveal the complex mechanisms adapted by the organism to survive under high temperature or saline conditions.

Thirty isolates of fluorescent pseudomonads were obtained from rhizosphere of different crops in Raichur, India. The fluorescent pseudomonad strains were characterized in vitro for biochemical traits, antimicrobial traits, and pyoluteorin antibiotic production. All the isolates that showed fluorescent pigment production under UV light were rod shaped, Gram negative, positive for oxidase, catalase and citrate utilization tests, and negative for indole test. Out of 30 isolates, 07 isolates were positive for HCN production, 15 isolates were positive for H2S production, and all the isolates were positive for siderophore production. Among all the isolates, RFP-22 showed the maximum percent inhibition of mycelium (46.66 %) of Rhizoctonia solani, the pathogen, and the remaining isolates showed the moderate to least inhibition of mycelium growth of R. solani. The 16S rRNA analysis confirmed that the antibiotic positive isolates belonged to genus Pseudomonas. The amplification of 779 bp region in isolates RFP- 4 and RFP-19 corresponded to pyoluteorin antibiotic-coding pltB gene. Further characterization of pyoluteorin antibiotic through TLC and TOF–MS analysis confirmed the presence of pyoluteorin at 274.26 (g/mol) peak and 2.10 min retention time. Biochemical and molecular analyses confirmed the antagonism of Pseudomonas and isolate through pyoluteorin production.

The efficacy of the biological control agent Trichoderma harzianum was tested on various levels of Fusarium udum, the pigeonpea wilt pathogen. Field plots infested with the pathogen at three inocula levels (log 3.04, log 4.98 and log 5.34 colony-forming units (cfu)/g of soil) were identified. Trichoderma harzianum was applied as a seed treatment (10 and 20 g/kg seed) and as a soil amendment (10 and 20 g/9 m2). The Trichoderma population increased to more than 108 cfu/g soil by 60 days in treated plots, whereas for seed treatments, fungal population reached a maximum of 104.62 cfu/g soil within 45 days, and thereafter started to decline. At log 3.04 pathogen concentration, soil amendment, with T. harzianum at 10 and 20 g gave 42.9% and 61.5% disease control, respectively. Seed treatments resulted in less than 30% disease control when compared to the check. The disease control achieved with fungicide seed treatment was insignificant. At log 4.98 and log 5.34 pathogen concentrations, disease control ranged between 22% and 35.3% for soil treatments. Even at the highest pathogen density (log 5.34), soil amendment with T. harzianum at 10 g gave about 30% disease reduction. In seed treatment plots, disease control ranged between 4.36% and 13.7%. In general, soil application of T. harzianum was found to be more effective than seed treatment for disease suppression. These results suggest the need to augment soil applications of T. harzianum for obtaining effective control of pigeonpea wilt.

Management of Insect Pests by Microorganisms

In the present study, fungal and bacterial antagonists were evaluated against early blight pathogen of tomato, Alternaria solani under in vitro, glasshouse and field conditions. Among the isolates tested, T. harzianum (Th-7) showed significant inhibition of A. solani under in vitro (72.78%) and glasshouse conditions (62.60%). Seedling dip and foliar applications of T. harzianum (Th-7), T. viride (Tv-14) and P. fluorescens (Pf-1) decreased the early blight incidence up to 62% and increased tomato yield up to 37% over control in field trials.

Insects, like other organisms, are susceptible to a variety of diseases caused by bacteria, viruses, fungi and protozoans, and these pathogens are exploited for biological control of insect pests through introductory or inundative applications. Microbial pathogens of insects are intensively investigated to develop environmental friendly pest management strategies in agriculture and forestry. In this paper, the scope for utilization of insect pathogens in pest management in the world and India is reviewed. The most successfully utilized insect pathogen is the bacterium, Bacillus thuringiensis (Bt) which is used extensively for management of certain lepidopteran pests. In India, mostly imported products of Bt kurstaki have been used, which are expensive and there is an urgent need to develop aggressive indigenous Bt strains against various pests. Baculoviruses comprising nuclear polyhedrosis virus (NPV) and granulosis virus (GV) have been successfully used as insect pathogens because of their high virulence and specificity. NPV and GV formulations are used for lepidopteran pests like Helicoverpa armigera (HaNPV) and Spodoptera litura (SlNPV) in India, besides Anticarsia gemmatalis NPV in Brazil, Lymanttria disper NPV, Orgyia pseudotsugata NPV in USA and GV of Pieris rapae in China. Lack of easy mass multiplication methods for the commercial production of baculoviruses calls for R&D to develop production in insect tissue cultures. Entomopathogenic fungi like Beauveria bassiana, B. brongniartii, Metarhizium anisopliae, M. anisopliae var. acridium, Lecanicillium spp., Hirsutella thompsonii, Nomuraea rileyi and Isaria fumosorosea are gaining importance in the crop pest control in recent years due to the simpler, easier and cheaper mass production techniques. Environmental humidity and temperature play an important role in the infection and sporulation of these fungi and as such they are highly suitable during cool and humid cropping seasons. Successful uses include M. anisopliae var. acridium for locust control in Africa, Australia and China, M. anisopliae in sugarcane spittle bug management in Brazil and pine moth (Dendrolimus spp.) control in China using B. bassiana. Since talc based formulations of these fungi have limited shelf life of 3-4 months, alternative formulations with longer shelf life (12-18 months) have to be developed besides suitable oil based formulations for dry land agriculture. There is a scope to utilize the biodiversity of Entomophthorale group of fungi like Entomophthora, Zoophthora, Neozygites etc., which have potential for management of aphids, thrips and lepidopteran pests.