ICRISAT Archival Report 2006 - The seedlings of success in the ...

Serological and nucleic acid-based diagnostics tests for HaNPV, SliNPV, and AmblNPV:
Nucleopolyhedroviruses (NPVs) are commonly used as biological pesticides to control insect pests over a wide
array of agricultural crops on commercial basis under integrated pest management (IPM) programs. Many viral
products are failing to meet acceptable standards because of poor inability to accurately assess the product quality.
Therefore, it is necessary to have an efficient strategy for virus production and diagnostic tools for ensuring quality
control of the NPVs: Helicoverpa armigera nucleopolyhedrovirus (HaNPV), Spodoptera litura
nucleopolyhedrovirus (SliNPV), and Amsacta albistriga nucleopolyhedrovirus (AmblNPV).
To compliment our ongoing NPV production activities, we produced polyclonal antibodies against polyhedra
protein of HaNPV, SliNPV, and AmblNPV infecting H. armigera, S. litura, and A. albistriga, respectively, to
develop an enzyme-linked immunosorbent assay (ELISA) for diagnosis and quality control of the NPVs. Poly
occlusion bodies (POBs) were purified from NPV infected insect cadavers and used for the purification of
polyhedrin protein by alkali treatment, followed by differential centrifugation and isoelectric focusing at 5.6 pH.
The purified polyhedrin protein was used as antigen. Three New Zealand White inbred rabbits were used for
immunization with 0.5 mg ml -1 purified polyhedrin protein of HaNPV, SliNPV, and AmblNPV emulsified in
Freund’s complete adjuvant through intramuscular route. Four injections were given at weekly intervals. A week
after 4 th injection, the rabbit was bled for polyclonal antiserum at weekly intervals for 4 weeks, and then the animals
were boosted with polyhedrin protein in Freund’s incomplete adjuvant. The purity and titer of the antiserum was
assessed by direct-antigen coating ELISA and western immunoblotting. The polyclonal antibodies did not react with
the insect protein extracts reveling high specificity of the antibodies, and the antibody titer in various bleeds ranged
between 1: 2,000 to 1: 40,000 for detecting respective polyhedrin proteins. In addition, the antibodies of each virus
were cross-reacted with polyhedrins of other viruses in the study, indicating high conservation in polyhedrin protein
among the three species of NPVs. The detection limits of antiserum in indirect ELISA was 20 ng of polyhedrin in
case of HaNPV and SliNPV, whereas 8 ng in case of AmblNPV. Further work is continuing towards quantification
of polyhedrin for the early diagnosis of infection progress in the field, as well as quality control of NPV to
accurately quantify the virus titer in the formulations.
In addition, three degenerate oligonucleotide primers (PgC, PgN, and RedPgC) were developed for the amplification
of polyhedrin gene from by polymerase chain reaction (PCR). Using these primers, HaNPV polyhedrin gene (~750
bp) was amplified by PCR and cloned into pUC18 vector. This sequence is being characterized, and the sequence
information will be used to assess the diversity of HaNPV isolates occurring in India. Attempts are also being made
to develop oligomers for the amplification of hyper variable regions in HaNPV genome for studying virus diversity.
Lava Kumar, GV Ranga Rao and Farid Waliyar
Milestone: Tools for the integrated pest management in different crops developed (HCS/GVRR) 2010
Development of insect pest diagnostic and management tools: Crop losses caused by biotic stresses are estimated
to be in tens of billions of dollars across the world in spite of the huge investments in plant protection. Pest
epidemics continue to occur causing severe hardships to poor farmers. We developed computer based insect pest
diagnostic and management tools for all ICRISAT mandate crops to minimize crop losses and increase crop
productivity without jeopardizing environmental safety. In this process, the E-learning systems developed in
collaboration with Knowledge Management and Sharing unit on groundnut and chickpea pest diagnosis and
management have been shared with NARS and updated. This is an effective extension tool for NARS extension staff
and scientists.
GV Ranga Rao
Synergism of host plant resistance to Helicoverpa with insecticides its implications for ETLs in pigeonpea: We
evaluated the effect of different protection regimes on H. armigera-resistant (ICPL 332) and susceptible (ICPL
87119) genotypes of pigeonpea to quantify the contribution of host plant resistance in Helicoverpa management.
The plots were sprayed at the 10% flowering, 75% flowering, 50% podding, and dough stages in different
combinations. There were three replications in a RCBD for each variety. Untreated plots served as a control.
Percentage pod damage was 13.2 and 47.4% in the completely protected and unprotected plots of ICPL 87119,
respectively. The protected plots of ICPL 332 had a pod damage of 7.0% compared to 37.6% in unprotected plots.
The grain yield was 2491 kg ha -1 in completely protected plots of ICPL 87119 compared to 1950kg in the
unprotected plots. The protected plots of ICPL 332 yielded 2539 kg ha -1 compared to 1811 kg ha -1 in the unprotected
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