Insect pests are major biotic constraints limiting soybean production and quality. Soybean growers rely heavily on synthetic chemical insecticides to combat insect pests. Increasing concern for environmental awareness about the use of chemical insecticides has sparked interest in eco-friendly pest management alternatives. An experiment was conducted to assess Bt-127 suspension concentrate (SC) formulation as a component of IPM under real farm situation in Hegdoli village of Kotgiri Mandal, Nizamabad District, Telangana during kharif 2017-18. Incidence of semiloopers (Chrysodeixis acuta and Mocis undata) and tobacco caterpillar (Spodoptera litura) was lowered by 99.5% and 92.9% with the first spray of Bt-127 SC formulation while their incidence was lowered by 99.6% and 97.3%, respectively in farmer's practice with spray of emamectin benzoate. After second spray of Bt-127 SC formulation, incidence of S. litura was lowered by 91.3% and found on par with 91.5% lowering from spray of chlorantraniliprole in farmer's practice. Incidence of natural enemies in IPM field after first and second spray was higher at 0.84 and 0.72 per meter row length in comparison to 0.12 and 0.40, respectively in farmer's practice. The IPM module resulted in higher cost-benefit ratio (1: 2.36) as compared with farmer's practice (1: 1.61).

The tobacco caterpillar, Spodoptera litura is a key insect pest affecting the crop stand and yield of a wide range of crops, mainly castor. Entomopathogens are known to play a vital role in eco-friendly management of insect pests. The present study was undertaken with the objective of developing a freely flowable WDG formulation of Bacillus thuringiensis (Bt) containing a high percentage of active ingredient coupled with biodegradable adjuvants. Water dispersible granule (WDG) formulation of DOR Bt-127 - a native strain of B. thuringiensis var. kurstaki was prepared by a combination of agglomeration and pan drying method using a binder (guar gum), a wetting agent (Tween-80) and a water-soluble carrier (starch). WDG had good wettability and was effective against S. litura. X-ray diffraction studies revealed semi-crystalline nature of WDG formulation. FTIR spectra of Bt WDG formulation revealed change in transmittance peaks to 1631, 1547 and 1147 cm(-1) from 1636, 1534 and 1075 cm(-1) of Bt (technical) indicating binding of Bt with starch and guar gum. The formulation has a high protein content of 134.7 mg/g and potency of 74,200 SU/mg against 9 days old S. litura larvae. Pot culture studies and field trials revealed that efficacy of Bt-127 WDG formulation was on par with the commercial Btk formulation against 7 and 9 days old larvae. Bt-127 WDG formulation was promising against early as well as older instar larvae and therefore has a prospective to be used in Bio-intensive Integrated Pest Management programmes to reduce the usage of synthetic chemical insecticides.

Bacillus thuringiensis (Bt) and entomopathogenic fungi (EPF) are in use for management of insect pests. Continuous use of Bt can lead to problem of resistance development in insect pests. Hence use of combination formulations (CF) of microbials with diverse modes of action has been attempted to slow down the process of resistance development. Suspension concentrate (SC) formulations of a local strain of Bt var. kurstaki DOR Bt-127 were developed singly and in combination with conidia of the EPF Nomuraea rileyi (Nr) and Beauveria bassiana (Bb). Electron microscopy of Bt + Bb CF treated larvae of Helicoverpa armigera revealed simultaneous infection by both microbials indicating their compatibility. Endotoxin contents in Bt-SC, Bt + Bb and Bt + Nr CFs were 5.0, 4.7 and 4.7%, respectively. These formulations were effective against larvae of Spodoptera litura, H. armigera and Achaea janata coupled with a lowering of the effective requirement of Bt and EPF. In multi-location field trials, Bt-SC and Bt + Nr CF were highly effective against S. litura and A. janata on castor as well as H. armigera and Thysanoplusia orichalcea on sunflower. However, Bt + Bb CF was highly effective only on sunflower against H. armigera and T. orichalcea. All formulations had 24 months shelf-life at room temperature. DOR Bt-127 based SC formulations developed singly and in combination with Nr and Bb were effective against major lepidopteran pests of castor and sunflower and did not lose viability under storage at room temperature. The CFs of Bt with EPF could prove promising for mitigating resistance development to Bt.

Insect pest management is important to save the crop from yield losses and increase productivity. In India, about 33% of the crop loss occurs due to insect pests and is worth of about ` 200 thousand crores annually. In additionsubstantial volume of agricultural exports is rejected every year due to pesticide residues. The pesticides are unsafe for environment and human health; their misuse by farmers further aggravates the problem. Therefore, sustainable and eco-friendly pest management practices are needed. In thisarticle, I share my personal experiences during the two-decade sojourn, marked by struggles and breakthroughs, which culminated in a successfully commercialized biocontrol technology that has reached the farmers.

Semilooper, Achaea janata feeds on many different species of plants among which castor is one of the mostpreferred hosts. DOR Bt-127 was multiplied through solid state fermentation and subjected to milling in a planetary ball mill. 559 nm particles of Bt technical gave 100% mortality against A. janata @ 0.6 mg/l. Field evaluation of Bt127 SC (33.3%) formulation lowered A. janata larval population (97.3 to 98.1% reduction over control by 3 days after treatment).

Biopesticides have long been attracting global attention as a safer strategy over chemical pest control, with potentially less risk to humans and the environment. Globally, the use of biopesticides is increasing steadily by 10% every year. Nanotechnology has provided new tools in the form of nanopesticides and nanosensors for pest management in agriculture with the concept of minimal usage for maximum effect. Nanopesticides are formulations of active ingredient of a pesticide in nanoform that have slow degradation, targeted delivery, and controlled release of active ingredient for longer period that make them environmentally safe and less toxic in comparison with conventional chemical pesticides. Several studies have reported an enhancement in the efficacy of certain biological substances on pests and a reduction of losses due to physical degradation through encapsulation of these substances in nanoparticulate systems. Research pertaining to nanobiopesticide development and evaluation till date has been limited to laboratory with approaches like development of nanocomposites, nanoengineered biopesticides, and capping of nanoparticles with biopesticides, etc. Enabling successful utilization of these formulations for pest management at the field level requires drafting of suitable biosafety and registration guidelines that are globally acceptable. In this chapter, we review the progress of research carried out in development of nanobiopesticides for crop protection, viz., management of insect pests, diseases, and nematodes.

Microencapsulation of Beauveria bassiana (Bb) conidia with sodium humate (SH) was undertaken successfully through spray drying at a high inlet air temperature of 175 degrees C with corresponding outlet air temperature of 86.5 +/- 1.3 degrees C using 0.2% SH. The obtained product was a free-flowing, dark-brown powder containing microcapsules of Bb conidia coated with sodium humate (Bb-SH). These microcapsules measured 2.47-3.57 mu m and possessed an uneven, fluffy surface. The colony-forming units (CFU) of Bb-SH microcapsules spray-dried at 175 degrees C were 21.54 LCFUg(-1), on par with 21.59 LCFUg(-1) for Bb conidial powder not subjected to spray drying. Bb-SH microcapsules resulted in a high mortality of 93.0% against six-day-old Helicoverpa armigera larvae within five days after treatment. Bb-SH microcapsules readily dispersed in water, releasing sodium humate from the conidial surface. Germination of conidia was not affected by sodium humate as visualized by scanning electron microscopy of the cuticular surface of treated larvae. Bb-SH microcapsules showed good viability (21.11 LCFUg(-1)) at the end of six months of storage at room temperature (approximate to 30 degrees). Thus, sodium humate is a promising biopolymer for encapsulation of Bb conidia for extended shelf-life at room temperature.

The technical powder of a local isolate of Bacillus thuringiensis (Bt) var. kurstaki namely DOR Bt-1 was subjected to high pressure homogenisation (HPH) to obtain nanoparticles (NPs) ranging 32–1,106 nm as determined through dynamic light scattering. NPs of Bt were also characterised for shape and size through scanning electron microscopy, field emission scanning electron microscopy and transmission electron microscopy that helped in visualising the smallest nanoparticles, i.e., less than even 50 nm. The larger particles comprised mainly Bt spores with a length and width of 1,139 and 813 nm respectively. HPH showed little effect on the spore viability. NPs had a zeta potential above +30 and were stable, resulted in higher mortality of Helicoverpa armigera larvae at a lower dose. Alkali solubilisation of the NPs resulted in higher values of protein and endotoxin quantified through Lowry’s method and ELISA respectively. This is indicative of an increased solubility of the toxin in the midgut alkaline fluid of the insect leading to higher mortality coupled with quicker kill. Thus Bt as NPs has brighter prospects for insect pest management with increased speed of kill at lower doses.

A local strain DOR Bt-1 belonging to Bacillus thuringiensis var. kurstaki (Bt.k) was multiplied through solid state fermentation and the resulting technical powder was milled and sieved to obtain particles of various sizes. Efficacy of Bt.k. against larvae of Helicoverpa armigera was found to increase with the decrease in particle size. Boric acid was found to be synergistic to DOR Bt-1 technical powder. LC50 of the Bt and boric acid mixture (75:25) was lower at 89.63 mg/100 mL in comparison to Bt alone at 116.75 mg/100 mL. A suspension concentrate formulation with DOR Bt-1 technical was developed using boric acid as an adjuvant. The formulation was found to be highly effective against H. armigera in laboratory bioassays with an LC50 of 185 µL (containing 53.36 mg Bt). The formulation gave effective control of H. armigera on sunflower within 3 days after spray even at the lowest dose of 1.0 mL/L under field conditions.

Eight Bacillus thuringiensis (Bt) strains were isolated from the soil samples collected from different fields in Andhra Pradesh and Karnataka, India. The isolates were characterized on the basis of primary bioassay against 2nd instars larvae of Helicoverpa armigera and Spodoptera litura and also screened with REP-PCR (Repetitive Extragenic Palindromic sequence) to distinguish closely related relationship among the strains. Based on the bioassay of eight isolates, Bt-52 and Bt-127 isolates were showed high toxicity against 2nd instars of H. armigera and S. litura. Rep-PCR profiles with these isolates showed Bt-52 and Bt-127 isolates formed separate clusters in phylogenetic tree. Further PCR amplification was carried out with cry gene specific primers used for 8 Bt isolates revealed the presence of amplified fragments characteristic of ten different cry genes. Results showed the expected sizes of PCR products of cry1Aa, cry1Ac, cry1Ad, cry1Ae, cry2Aa, cry2Ab, cry2Ac, cry1C, cry1D and cry1E genes ranging from 1000-1286, 844, 500, 443 and 300 bp, respectively. Out of the eleven cry genes investigated, only cry1Ab gene was not found any of the 8 Bt isolates. One Bt isolate Bt-112 showed only cry2Aa gene with PCR product size of 500bp. While, six of the 8 isolates were harboring a combination of different cry genes, which are Lepidoptera active cry1 genes and Lepidoptera-Diptera active Lepidoptera active cry2 genes. Consequently, combined strains are good candidates in the search for bio-control agents with a wider spectrum of action against many different insects. On the other hand, cry1 genes were the most frequently found among the 8 local Bt isolates and the most common profile of cry2 genes contained cry2Aa and the second abundant genes are cry1Ad, cry2Ab, cry2Ac and cry1C.

Bacillus thuringiensis (Bt) crystal proteins (Cry) bind to aminopeptidase N (APN) receptors on insect midgut membrane leading to pore formation and subsequent death. However, evolution of insect resistance to Bt toxins threatens their long-term application. Therefore, search for new targets which could function as Cry toxin receptors is an immediate mandate. In the present study, two full-length APN cDNAs were cloned from Malpighian tubule and salivary gland tissues of the moth, Achaea janata. Both these APNs showed 99% and 32% sequence homology with fat body and midgut APNs respectively. Tissue distribution analysis revealed the presence of two different APN isoforms, one predominant in non-gut visceral tissues while the other exclusively expressed in the midgut. Immunofluorescence and western blot analyses showed cross-reactivity in Malpighian tubule and salivary gland when probed with anti-fat body APN antiserum. These results clearly indicated the presence of non-gut (AjAPN1) and gut-specific (AjAPN4) isoforms in this moth. The expression of both the isoforms steadily increased during the larval development. Hormonal studies indicated regulation of the APN genes by the morphogenetic hormones, 20-hydroxyecdyone and juvenile hormone. Further, in vitro ligand-blotting studies demonstrated binding of Cry toxins to APNs in Malpighian tubule and salivary gland indicating their potential as alternate targets.

Biological control constitutes an important component of integrated pest management (IPM). However, the non-availability o f efficient biocontrol agents is one of the major constraints in adopting IPM practices. Microbial control, which makes use o f naturally occurring microbes to control insect pests, pathogens, and weeds, is less harmful to nontarget organisms and the environment than the chemical pesticides. Microbials are promising alternatives to chemical pesticides and have opened up new vistas in insect pest management to aid promotion o f safe, eco-friendly pest management. The use o f microbial pesticides in pest management is quite limited because o f lack of appropriate formulations and the availability of quality products to the farmers. Since 2006, the registration o f the microbial pesticides for commercial purposes has been made mandatory in India. It warrants information on toxicological results against mammals and eco-toxicity data on nontargets such as fishes, birds, earthworms, honeybees, and silkworm. The data is to be generated with technical product and the formulation of every strain intended for commercialization. It is also mandatory to generate data on the safety of the formulation to natural enemies along with data on the bioefficacy and phytotoxicity to the crop. Fourteen primary microbial pesticide products and their formulations have been registered in India by 2009. There are 478 products o f the 14 microbial pesticides registered in India. There are 184 products for the management of plant pathogens

A sporulating culture of Bacillus thuringiensis ssp. kurstaki strain DOR Bt-1 was isolated from castor semilooper (Achaea janata L.) cadavers from a castor bean (Ricinus communis L.) field at Kothakota mandal of Mahaboobnagar district, Andhra Pradesh, India. This strain has shown high toxicity against many insect pests and the wettable power formulation of this strain obtained through solid-state fermentation has been registered with the Central Insecticides Board (CIB), India under section 9 (3b). Based on the bioassay results and the efficacy of the formulation on different Lepidopteran insects, it was concluded that the strain must be carrying diverse cry genes. Therefore, to understand the cry gene profile of this strain, a PCR-based assay was carried out. Genomic PCR analysis of the DOR Bt-1 isolate with universal primers specific to cry gene classes indicated the presence of both cry1 and cry2 class-specific genes. Further, PCR analysis with cry gene-specific primers revealed the presence of cry1Aa, cry1Ab, cry1Ac, cry2Aa and cry2Ab genes. Thus, DOR Bt-1 isolate harbors a combination of different cry genes, such as Lepidoptera-active cry1Aa, cry1Ab and cry1Ac, and Lepidoptera-Diptera-active cry2Aa and cry2Ab genes, indicating that this isolate has the potential to be effective against both Lepidopteran and Dipteran insect pests. _____________________________________________________________________________________________________________

A new cry1Ab gene was cloned from the promising local isolate, DOR Bt-1, a Bacillus thuringiensis strain isolated from castor semilooper ( Achaea janata L.) cadavers from castor bean ( Ricinus communis L.) field. The nucleotide sequence of the cloned cry1Ab gene indicated that the open reading frame consisted of 3,465 bases encoding a protein of 1,155 amino acid residues with an estimated molecular weight of 130 kDa. Homology comparisons revealed that the deduced amino acid sequence of cry1Ab had a variation of seven amino acid residues compared to those of the known Cry1Ab proteins in the NCBI database and this gene has been designated as cry1Ab26 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry1Ab26 was cloned into pET 29a(+) vector and expressed in E. coli strain BL21 (DE3) under the control of T7 promoter with IPTG induction. ELISA, SDS-PAGE, and Western blot analysis confirmed the expression of 130-kDa protein. Insect bioassays with neonate larvae of Helicoverpa armigera showed that the partially purified Cry1Ab26 caused 97 % mortality within 5 days of feeding.

Castor is an industrially important non-edible oilseed crop belonging to the family Euphorbiaceae. Development of location-specific varieties and hybrids with appropriate crop production technologies led to increased production and productivity of the crop. However, cultivation of the crop under input-intensive conditions resulted in increased vulnerability of the high yielding cultivars to a multitude of insect pests attacking the crop at all phenological stages and includes seedling pests, foliage feeders and inflorescence pests. The red hairy caterpillar in endemic areas, the defoliators, viz., castor semilooper, Spodoptera and other hairy caterpillars, and sucking pests, such as jassid, whitefly, thrips and mites, cause huge damage to castor crop. Yield loss estimates indicate a reduction of seed yield up to 35-50% depending on the crop growth stage and the pest outbreak. Several insect pest management tactics are being developed for control of the major pests and includes cultural, biological, insecticidal and combinations of two or more methods, which are employed at three stages of prevention, observation, and intervention. In the recent past, attempts are directed towards formulation of potent microbial pesticides besides development of genetically modified castor. This chapter deals with the biology of major insects attacking castor along with the control methods being adopted, and gaps in the knowledge of pest management strategies that need to be addressed are discussed.

A local isolate of the entomopathogenic fungus, Beauveria bassiana (ITCC-4513) was formulated as a Suspension Concentrate (SC) using mineral oil as carrier. In laboratory bioassays against 5-day-old Helicoverpa armigera larvae, the formulation had an LC 50 value of 61.22 mg l -1 at 3 days after treatment. Data on shelf-life of the formulation stored in HDPE bottles at room temperature (29±1°C) was generated for 24 months. The formulation was found to be effective at 200 mg l-1 in field trials against H. armigera on sunflower crop. There was no phytotoxic effect of the formulation.

We have isolated a strain of Bacillus thuringiensis (Bt) from Indian soil samples that was shown to be toxic to Achaea janata larvae. The isolate, named B. thuringiensis DOR4, serotypically identified with the standard subspecies kurstaki (H3a3b3c) and produced bipyramidal inclusions along with an amorphous type. Although the plasmid pattern of DOR4 was different from that of the reference strain, a crystal protein profile showed the presence of two major bands (130 and 65 kDa) similar to those of Bt subsp. kurstaki HD-1. To verify the cry gene content of DOR4, triplex PCR analysis was performed; it showed amplification of the cry1C gene in addition to cry1Aa, cry1Ac, cry2A, and cry2B genes, but not the cry1Ab gene. RT-PCR analysis showed the expression of cry1Aa and cry1Ac genes. In vitro proteolysis of DOR4 protoxin with midgut extract generated products of different sizes. Zymogram analysis of DOR4 protoxin as substrate pointed to a number of distinct proteases that were responsible for activation of protoxins. Furthermore, toxin overlay analysis revealed the presence of multiple toxin-binding proteins in midgut epithelium. Based on all these characterizations, we suggest that the Bt DOR4 strain can be exploited for an A. janata control program.

Formulation of a local strain of Bacillus thuringiensis ( B r ) var. kttrstaki, viz DOR Bt- I . obtained through multiplication on a wheat bran based solid substrate was tested against castor semilooper field. Field trials showed that a single spray of the formulation at the dose of 1.5 g / litre gave ef'kctive control of castor semi looper till 30 days atier spraying. Incedence of the larval parasitoid (micropilitis maculipennis was observed only in Bt sprayed fields. Continuous egg laying and high larval incidence necessitated five sprays in tlie cluinalphos sprayed fields at 6-7 days intervals. Iticremcntal cost:benefit ratlo for thc Br spray was found to be 3.85 while it was 1.62 for quinalphos.

The low temperature adapted strain of Trichogramma chilonis was developed after rearing for 30 generations under laboratory conditions at 18–24°C with 95.0±0.44% parasitism. The development, searching ability and biological parameters of low temperature (18–24°C) adapted strain of T. chilonis was studied in comparison with the strain reared in the laboratory at ambient temperature, with a view to utilize them in cooler months. Non-significant variation among adapted and nonadapted strain was observed with respect to % parasitism, fecundity and longevity when reared in small glass vials. However, in the cage studies for parasitoid's searching ability, 58% eggs were parasitized by the low temperature adapted strain as compared to 17.2% by the non-adapted strain. The host searching test indicated that adaptation at low temperature led to better host searching and such strain could be successfully utilized under a low temperature regime.