Abstract
The whole genome of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) from India, HearNPV-L1, was sequenced and analyzed, with a view to look for genes and/or nucleotide sequences that might be involved in the differences and virulence among other HearNPVs sequenced from other countries like SP1A (Spain), NNg1 (Kenya) and G4 (China). The entire nucleotide sequence of the HearNPV-L1 genome was 136,740 bp in length having GC content of 39.19% and contained 113 ORFs that could encode polypeptides with more than 50 amino acids (GenBank accession number KT013224). Two ORFs, viz., ORF 18 (300 bp) and ORF 19 (401 bp) identified were unique in HearNPV-L1 genome. Most of the HearNPV-L1 ORFs showed high similarity to NNg1, SP1A and G4 genomes. HearNPV-L1 genome contains 5 h (hr1-hr5), these regions were found 84–100% similar to hr region of NNg1, SP1A and G4 genomes. A total of four bro genes were observed in HearNPV-L1 genome, of which bro-a gene was 12 and 351 bp bigger than SP1A and G4 bro-a, respectively, while bro-b was 15 bp bigger SP1A and NNg1 bro-b, whereas 593 bp shorter than G4 bro-b, while bro-c was 12 bp shorter than NNg1, however bro-c was absent in G4 genome. HearNPV-L1 bro-d was 100% homologous to bro-d of SP1A, NNg1 and G4 genomes, respectively. The comparative analysis of HearNPV-L1 genome indicated that there are several other putative genes and nucleotide sequences that may be responsible for insecticidal activity in HearNPV-L1 isolate, however, further functional analysis of the hypothetical (putative) genes may help identifying the genes that are crucial for the virulence and insecticidal activity.
Electronic supplementary material
The online version of this article (doi:10.1007/s13337-016-0352-6) contains supplementary material, which is available to authorized users.
Keywords: Helicoverpa armigera NPV, Whole genome sequencing, Lepidoptera, Comparative genome analysis
Introduction
Helicoverpa armigera nucleopolyhedrovirus HearNPV (Baculoviridae), is a rod shaped, single nucleocapsid alphabaculovirus with circular closed, double stranded DNA genomes that ranges in size from 80 to 180 kb [27]. It has proved to have excellent insecticidal propriety for control of H. armigera (Lepidoptera: Noctuidae) larvae on number of crops [14, 25]. The family Baculoviridae comprises of four genera, viz., Alphabaculovirus (Lepidopteran specific nucleopolyhedroviruses), Betabaculovirus (Lepidopteran specific granuloviruses), Gammabaculovirus (Hymenopteran specific nucleopolyhedroviruses) and Deltabaculovirus (Dipteran specific nucleopolyhedroviruses). The NPVs produce large, polyhedron-shaped occlusion bodies containing many virions, are further designated as single nucleopcapsid or multinucleocapsid. Depending on the number of nucleocapsids packaged in an envelope of the virion, NPVs are called as SNPV (single) and MNPV (multi). NPVs that are isolated from different insects in the field populations differ from one another with regard to virulence against respective insects. [2, 8, 10, 17, 23]. These viruses are known to play role for gene delivery vectors for human gene therapy [13] and for display system for complex eukaryotic proteins [6, 20].
The gram pod borer H. armigera is an important pest that feeds on a number of agricultural and other economically important plants and is recorded on more than 120 host-plants causing severe crop losses to over 80 cultivated crops throughout tropical and sub-tropical region of our planet [30, 31]. H. armigera has developed a resistance to all groups of chemical insecticides [29], In order to mitigate the resistance developed, several options have been explored for the control of this devastating pest. Among them, NPVs have come as one of the most promising larvicidal agents against such pests [4]. To date, more than 60 baculoviruses have been isolated and sequenced completely, a number of NPVs have also been isolated from H. armigera so far and each one has very good biological activity [9, 12].
HearNPV to control H. armigera was one of the first commercially available baculoviral pesticide produced in China, and has been successfully used to control the pest in agricultural crops [32]. Whole genome sequencing and comparative studies with HearNPV-NNg1 (Kenya) and HearNPV-G4 (China) exhibit the most and least virulence respectively against H. arimgera larvae [24]. Recently five naturally occurring HearNPV isolates, viz., SP1A, SP1B, LB1, LB3 and LB6 from Spain have been fully sequenced. The insecticidal properties of these five genotypes showed significant differences among them, the principal variation among them was located in homologous regions (hrs) and baculovirus repeat ORFs (bro) genes [1]. The current study is the first report on the complete genome of HearNPV of the Indian isolate.
In this study, we sequenced and analyzed HearNPV-L1 isolate from Ludhiana, Punjab, India. The HearNPV-L1 isolate showed high insecticidal activity against H. armigera larvae in the laboratory conditions, having lowest LC50 values compared to several isolates, viz., Hingoli, Nanded, Nagpur, Bengaluru, Faridkot and Manasa obtained from different parts of India. We also compared the complete genome of HearNPV-L1 isolate with those of previously reported NNg1 from Kenya, G4 from China and a recently sequenced SP1A isolate from Spain. Our data showed that two ORFs were unique to HearNPV-L1 genome and showed highest similarity to NNg1 isolate of Kenya, apart from these there are marked similarities were also found between HearNPV-L1 and NNg1 in homologous region and bro genes.
Materials and methods
Virus and insects
HearNPV-L1 was originally isolated from a naturally infected H. armigera from Ludhiana (30.9100°N, 75.8500°E), Punjab, India, in the year 2011. The virus was propagated in laboratory stocks of healthy third instar H. armigera larvae. These insects were maintained on artificial diet using BOD (Biological Oxygen Demand) incubator maintained at 27.5 °C and 60% RH.
Virus purification, isolation and production
The original virus isolated was obtained from diseased larvae collected from the pigeonpea (Cajanus cajan) crop in Ludhiana, India. Occlusion bodies (polyhedra) from the infected larvae were purified by centrifugation at 2500 revolutions per minute (rpm) for 1 min at 4 °C. The debris was pelleted, whereas the occlusion bodies (OBs) remained in the supernatant. For the isolation of pure virus, the suspension was again subjected to centrifugation at 10,000 rpm for 5 min at 4 °C. The OBs settled as a pellet at the sides of the walls of the centrifuge tube. This process was repeated twice to isolate only the OBs leaving behind the contaminants. The pellet obtained was then suspended in 1 ml of double distilled water and stored at −20 °C for further use. The virus obtained from the above procedure isolated from the field collected diseased larvae was further confirmed by conducting a series of bioassay tests on H. armigera in order to determine their specificity. The same stock was used for isolation of Genomic DNA for Next generation sequencing through Illumina NextSeq 500 technology.
Isolation of viral DNA
DNA extractions were performed using 0.1 M sodium carbonate solution, the final suspension included 0.5 mgml−1 of proteinase K and 1% SDS and incubated overnight at 37 °C. This was followed by Phenol: Chloroform: Isoamyl Alcohol (25:24:1 v/v) extraction with the final absolute ethyl alcohol precipitation [28]. The DNA was re-suspended in a TrisEDTA buffer (composed of 10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The extracted DNA was visualized in Vilber Lourmat gel documentation system on 1% agarose gel.
Next generation sequencing of HearNPV-L1
The quality of DNA extracted from the NPV was checked on 1% agarose gel for the single intact band. 1 µL of each sample used for determining concentration using Qubit 2.0 fluorometer. The paired end sequencing libraries was prepared using Illumina True seq Nano DNA HT library preparation kit as per the manufacturer’s instructions. The amplified libraries were analyzed in bioanalyser 2100 (M/s Agilient Technologies) using High sensitivity HS DNA chip as per manufacturer’s instructions. The library was further sequenced on Next Seq 500 Illumina 2 × 150 bp chemistry.
Sequence analysis
The genome analysis was performed by reference genome assembly of the Nucleopolyhedrosis virus from National Centre for Biotechnology Information (NCBI). The analysis revealed that high quality data covered more than 95% of reference genome. All mapped reads were de-novo assembled by SPAdes (3.5.0) software on default parameter. A total of 99 scaffolds were generated. A gene prediction was carried out from generated scaffold, which was annotated by using BlastX against NCBI nr database. ORFs encoding more than 50 amino acids (150 bp) were considered to be protein-encoding and named as putative genes. One hundred and thirteen (113) ORFs in the final dataset were checked for maximum alignment with known baculoviruse gene homologues from GenBank. ORFs with an overlaps of hr region were not included from the alignment analysis. Homology searches were done through the NCBI using BLAST. Multiple alignment and percentage identities were performed using the Clustal W 2.0.
Results
Analysis of HearNPV-L1 genome
The HearNPV-L1 genome was assembled into a contiguous sequence of 136,740 bp in length (GenBank accession number; KT013224) with GC content of 39.19% and contained 113 ORFs that could encode polypeptides with more than 50 amino acids. The contiguous sequence of the entire HearNPVL1 genome was analyzed and compared with SP1A [1], NNg1 [21], G4 [34] and Ac [2] genomes. The identified HearNPV-L1 ORFs were plotted on the circular map shown in (Fig. 1). Detailed information on the size, location and percent homology of HearNPV-L1 ORFs is given in electronic supplementary material Table 1 together with those of SP1A, NNg1, G4 and Autographa californica multicapsid NPV (AcMNPV).
Fig. 1.
Circular genome map of HearNPV-L1 isolated from Helicoverpa armigera collected from Ludhiana, India. The position, size and direction for the 113 open reading frames (ORFs) listed in Table 1 are presented as different colour bars, along with ORF numbers and gene names. The Pink coloured bars indicate the location and relative size of the five homologous regions (hrs) and a direct repeat sequence (dr)
Table 1.
Comparative analysis of five core genes of HearNPV-L1 isolated form Helicoverpa armigera collected from Ludhiana, India
| Sl no | L1 | SPA1 | NNg1 | G4 | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Genes | ORF | Bp | Aa | ORF | Bp | Aa | ID% | ORF | Bp | aa | ID% | ORF | Bp | aa | ID% | |
| 1 | Polyhedrin | 1 | 741 | 246 | 1 | 741 | 246 | 100 | 1 | 741 | 246 | 100 | 1 | 741 | 246 | 100 |
| 2 | Dbp | 16 | 972 | 323 | 23 | 972 | 322 | 100 | 24 | 972 | 323 | 100 | 25 | 972 | 323 | 100 |
| 3 | DNAPol | 52 | 3063 | 1020 | 66 | 3063 | 1020 | 99 | 68 | 3063 | 1020 | 99 | 67 | 3063 | 1020 | 99 |
| 4 | Iap2 | 47 | 753 | 250 | 61 | 759 | 252 | 98 | 64 | 753 | 250 | 99 | 62 | 753 | 250 | 99 |
| 5 | Iap3 | 83 | 807 | 267 | 102 | 807 | 268 | 97 | 106 | 807 | 267 | 97 | 103 | 807 | 268 | 97 |
| 6 | Pif3 | 78 | 600 | 199 | 97 | 600 | 199 | 99 | 101 | 600 | 199 | 99 | 98 | 600 | 199 | 99 |
| 7 | Pif1 | 90 | 1587 | 528 | 110 | 1587 | 528 | 100 | 114 | 1587 | 528 | 99 | 111 | 1587 | 528 | 99 |
| 8 | Pif2 | 110 | 1152 | 383 | 133 | 1152 | 383 | 99 | 140 | 1152 | 383 | 99 | 132 | 1152 | 383 | 100 |
| 9 | Lef6 | 15 | 564 | 187 | 22 | 564 | 187 | 100 | 23 | 564 | 187 | 100 | 24 | 564 | 187 | 99 |
| 10 | Lef11 | 24 | 384 | 127 | 30 | 384 | 127 | 100 | 32 | 384 | 122 | 100 | 32 | 384 | 127 | 100 |
| 11 | Lef8 | 28 | 2706 | 901 | 36 | 2706 | 901 | 99 | 38 | 2706 | 901 | 100 | 38 | 2706 | 901 | 98 |
| 12 | Lef9 | 39 | 1560 | 519 | 53 | 1560 | 519 | 99 | 55 | 1560 | 519 | 99 | 56 | 1560 | 359 | 99 |
| 13 | Lef3 | 50 | 1140 | 379 | 64 | 1140 | 379 | 99 | 67 | 1140 | 379 | 99 | 65 | 1140 | 379 | 99 |
| 14 | Lef4 | 62 | 2019 | 461 | 77 | 2019 | 461 | 98 | 81 | 2019 | 461 | 100 | 78 | 2019 | 461 | 100 |
| 15 | Lef5 | 70 | 849 | 282 | 85 | 948 | 315 | 100 | 89 | 948 | 315 | 100 | 87 | 948 | 315 | 99 |
| 16 | Lef2 | 95 | 696 | 231 | 117 | 717 | 238 | 98 | 123 | 681 | 226 | 98 | 117 | 726 | 241 | 93 |
| 17 | Lef1 | 102 | 738 | 245 | 125 | 738 | 245 | 99 | 131 | 738 | 245 | 98 | 124 | 738 | 245 | 96 |
The 113 ORFs identified in the HearNPV-L1 genome were distributed almost evenly along both strands of genomic DNA, with 55 ORFs facing towards clockwise and 58 ORFs towards anti clockwise. The HearNPV-L1 genome is almost collinear with those of SP1A, NNg1 and G4. In addition, a total of 76 L1 ORFs had homologous ORFs on the AcMNPV genome, sharing low amino acid sequence identities ranging from 7 to 64.6%, except of Pol-h, which shared higher amino acid sequence identities of 85% (Electronic supplementary material Table 1).
Presence and absence of ORFs in genomes with higher virulence
The HearNPV-L1 genome contained two putative ORFs, ORF 18 and ORF 19 of 300 bp and 401 bp respectively unique to HearNPV-L1 genome. These ORFs even not found in AcMNPV also. However, the function of these ORFs is unknown as per comparison of L1 with SP1A, NNg1 and G4. It shows that ORF 62 of NNg1 is absent in L1, SP1A and G4 genome. Out of 113 ORFs only one had early promoter motif. Out of 113 ORFs 20 ORFs were 100% identical in SP1A, 21 in NNg1 and 25 in G4 genomes, respectively.
Comparative analysis of five core genes
Five Core genes were chosen for comparison among L1, SP1A, NNg1 and G4 genomes which play an important role in insecticidal activity. Polh (1), DNA binding protein (dbp) (1), Inhibitor of apoptosis (iap) (2), per os infectivity factor (pif) (3) and Late expression factor (lef) (9) were studied. Upon their comparison it was found that there was slight nucleotide level difference was present in lef5of HearNPV-L1 (849 bp in size) which was 99 bp shorter than the SP1A, NNg1 and G4 genome, whereas lef 2 (696 bp in size) and was 21 bp shorter than lef2 of SP1A, 15 bp greater than lef2 of NNg1 and 30 bp shorter that lef2 of G4 genome. All the core protein genes of HearNPV-L1 were 97–100% identical to SP1A, NNg1 and G4 genome (Table 1).
Analysis of homologous regions (hrs)
Most of the baculovirus genome contain homologous repeated region, which also contains a “core” imperfect palindrome and dispersed unevenly among the genome in AT rich intergenic region [15]. The hr repeats initiates the DNA replication in transient assays [22]. The baculovirus hrs acts as enhancers for RNA polymerase mediated transcription of baculovirus early promoter [7, 26]. It functions as origin of the DNA Replication in transient replication assays [5, 6]. They are also site for recombinant and rearrangement in baculovirus genomes [16]. There are five hr regions are identified as hr1, hr2, hr3, hr4 and hr5, whose sizes were 1540, 1395, 133, 1790 and 1011 bp, respectively. A palindrome was found in hr1region of HearNPV-L1 genome, which looks quite similar to those of NNg1, SP1A, and G4 genomes, respectively. The repeat contains an imperfect palindrome sequence of 31 bp, in which two homologous sequences of the IE-1 binding motif (IBM) represented by 5′-ACBYGTAA-3′ in AcMNPV hrs [18] were located in an inverted manner (Fig. 2 5′-ATATGAAA-3′) in HearNPV-L1 genome.
Fig. 2.
The consensus repeat sequence identified in the genome of HearNPV-L1 h. An imperfect palindrome in repeat region indicated in grey shade (a) and boxed sequence represent putative IE-1 binding motif (AcMNPV and IBM) like sequence
The locations of hr2 and hr3 were conserved among SP1A, NNg1 and G4. However, hr2 and hr3 in HearNPV-L1, SP1A and NNg1 were separated by three bro genes (ORF43 (bro-a), ORF44 (bro-b) and ORF45 (bro-c) for HearNPV-L1, ORF57 (bro-a), ORF58(bro-b) and ORF59 (bro-c) for SP1A and ORF59 (bro-a), ORF60(bro-b) and ORF61 (bro-c) for NNg1 whereas there was only two bro genes were found in G4 genome, viz., ORF59 (bro-a) and ORF60 (bro-b) between hr2 and hr3. It has previously been suggested that hr2 and hr3 comprised a single hr before the insertion of bro genes occurred between them [3]. Our comparative analysis of bro gene sequences indicated that the NNg1 bro-b gene, the third bro gene present between hr2 and hr3, was generated through duplication of the bro-c gene, thus suggesting that this region was a hot spot for recombination. These baculovirus variants contain different numbers of repeats, and this suggests that hrs show high structural plasticity. It is also interesting to note that, despite the high degree of structural similarity among hrs, the actual sequences are entirely different among baculovirus species. The arrangement of these hrs on the genome is almost the same as NNg1 and it is possible that variability in the hr sequences affect not only progeny of virus production, but also the insecticidal activity of the Helicoverpa spp. NPVs [17]. The homologous regions are also suggested to be responsible for the difference in the virulence factor of NPVs [11].
Analysis of bro genes
Baculovirus-repeated ORF genes (bro) were first identified in baculovirus genomes as a repetitive gene and constitute a gene family. Most bro genes contain a 41 aa core sequence at the N-terminal half and several different domains throughout the sequence. Based on the similarity of those different domains, bro genes were separated into four groups, I–IV [9].
The HearNPV-L1 genome contained four bro genes, bro-a (ORF44), bro-b (ORF45), bro-c (ORF46) and bro-d (ORF85), encoded 246, 412, 267 and 500 amino acids, respectively (Table 2). The HearNPV-L1 bro-a, which shared relatively high amino acid sequence identities of 75% with bro-a and 57% with bro-b (ORF60) from SP1A and G4 respectively, however, bro-c was found absent in G4 genome. The HearNPV-L1 bro-b had no homologues in any of NNg1, SPA1 and G4. The HearNPV-L1 bro-c, which shared 91% amino acid sequence identity with bro-b (ORF61) of SP1A and (ORF61) of NNg1 (ORF60) in G4. The HearNPV-L1 bro-d, was the most conserved bro gene among the Helicoverpa spp. NPVs, sharing amino acid sequence identities of 99% with bro-c (ORF 59) from SPA1 and ORF 61 of NNg1. NPVs, shared high amino acid sequence identity with other NPV genomes based on the fact that HearNPV-L1 bro-b and bro-c, along with bro-a, were located between hr2 and hr3, which was considered to be a hot spot for baculovirus genome recombination [19], these hrs may be responsible for bro-c duplication.
Table 2.
Comparative analysis of bro genes of HearNPV-L1 isolated form Helicoverpa armigera collected from Ludhiana, India
| Sl no | L1 | SPA1 | NNg1 | G4 | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Genes | ORF | Bp | Aa | ORF | Bp | aa | Identity | ORF | Bp | aa | Identity | ORF | Bp | Aa | Identity | |
| 1 | Bro-a | 44 | 1086 | 246 | 57 | 1074 | 357 | 91 | 59 | 1086 | 361 | 99 | 59 | 735 | 244 | 100 |
| 2 | Bro-b | 45 | 465 | 412 | 58 | 306 | 101 | 84 | 60 | 306 | 101 | 81 | 60 | 1058 | 352 | 99 |
| 3 | Bro-c | 46 | 1047 | 267 | 59 | 1047 | 352 | 75 | 61 | 1059 | 352 | 73 | ||||
| 4 | Bro-d | 85 | 1506 | 500 | 104 | 1506 | 501 | 99 | 108 | 1506 | 501 | 99 | 104 | 1506 | 501 | 99 |
Discussion
Development of an effective biopesticide against H. armigera is the current need in India and rest of the world. In this direction an effort was made to identify the most virulent NPV. HearNPV-L1 isolate was identified after a series of bioassay experiments and its insecticidal activity was determined. The insecticidal activity of HearNPV-L1 isolate expressed in terms of 50% lethal concentration was 1.92 × 107 in third instar of H. armigera larvae compared to the isolates obtained from Hingoli (8.2 × 107), Nanded (6.8 × 107), Nagpur (4.7 × 107), Bengaluru (4.7 × 107), Faridkot (10.6 × 107) and Manasa (7.5 × 107) collected from different parts of India. In the current study we attempted whole genome sequencing through next generation sequencing (Illumina NextSeq 500) technology of HearNPV-L1 isolate.
We compared L1 genome with those of SP1A, NNg1 and G4 genomes and found that HearNPV-L1 showed 97–99% homology to these genomes. Majority of putative genes identified in HearNPV-L1 genome shared a high degree of similarity and amino acid sequence identity with NNg1 genome than SP1A and G4 genomes. Genes involved in insecticidal activity crucial for infectivity and productivity such as p74 and per os infectivity factor (promote preoral infection of insect larvae) were analyzed at nucleotide level to find observed differences in insecticidal activity between Helicoverpa spp NPVs, HearNPV-L1 shared 97–99% amino acid sequence identity to those of SP1A, NNg1 and G4, suggesting that p74 and pifs are not responsible for the insecticidal activity in L1 isolate. Further comparative analysis of five core genes, viz., Polyhedrin (pol-h), DNA binding protein (dbp), DNA polymerase (DNApol), Inhibitor of apoptosis (iap) and Late expression factor (lef) resulted’’ in 97–100% identity among Helicoerpa spp. genomes (SP1A, NNg1 and G4), indicating the role of these genes is not significant in insecticidal activity.
Detailed comparative study of HearNPV-L1 genome identified several putative genes whose functions were unknown, however, might be responsible for insecticidal activity of HearNPV-L1. Two putative genes viz; ORF 18 and ORF 19 of 299 and 399 bp respectively found unique to HearNPV-L1 genome. These two genes had no homologue in any of the genome (SP1A, NNg1 and G4) compared in this study. Functional characterization of these putative ORFs of HearNPV-L1 genome may help to elucidate whether these genes are associated with the differences in insecticidal activity among Helicoverpa spp. NPVs.
HearNPV-L1 genome contains 5 h (hr1-hr5), these regions were found 84–100% similar to hr region of NNg1, SP1A and G4 genomes. However, size of hr significantly varied among HearNPV-L1, NNg1, SP1A and G4 genomes. Among all the 5 h present in L1 genome hr2 was found to be most identical to those of SP1A, NNg1 and G4 genomes. It may be possible that observed differences among hrs or variations in overall hr sequence length affects not only insecticidal activity but progeny virus production also in Helicoverpa spp. NPVs [24]. A direct repeat (dr) was found of 152 bp in HearNPV-L1 genome which was 99% identical to the direct repeat of NNg1 (162 bp). The presence of a homologue of HearNPV-L1 direct repeat in NNg1 shows sharing of unique traits among strains of different geographical regions.
A total of four bro genes were observed in L1 genome, viz., bro-a, bro-b, bro-c and bro-d, differences were observed between HearNPV-L1 and other three Helicoverpa spp. NPVs in bro gene diversity. The early promoter present in bro-b gene of L1 has a homologue in SP1A and NNg1 genome but lacked in G4 genome [33]. bro-c and bro-d of L1 genome was homologous to bro-c and bro-d of SP1A and NNg1 genomes respectively. Based on this comparative analysis of bro genes, it is possible that observed bro gene diversity may be responsible for observed insecticidal activity between HearNPV-L1, SP1A, NNg1 and G4 genomes.
Overall comparative analysis of HearNPV-L1 genome with three other Helicoverpa spp. NPVs SP1A, NNg1 and G4 genomes indicates that apart from hr region and bro genes there are several other putative genes and nucleotide sequences may be responsible for insecticidal activity in HearNPV-L1 isolate of India and other virulent genomes, viz., SP1A from Spain and NNg1 isolate of Kenya. However, further functional analysis of the hypothetical (putative) genes of HearNPV-L1 isolate may identify the genes which are crucial for the virulence and insecticidal activity.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
This study was funded by “National Agricultural Science Fund” (NASF) of Indian Council of Agricultural Research. New Delhi, India. Authors are thankful to the Director, Dr. Abraham Verghese, ICAR-National Bureau of Agricultural Insect Resources (NBAIR), Hebbal, Bengaluru, for his support to conduct the work. First author is thankful to Jain University, Bengaluru, as this work is part of her Ph.D. Thesis.
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