Natural Biopesticide of Insect pest Nuclear Polyhedrosis Virus
Authors: Rajdeep Mundiyara1, Prem Kumar2 and Mamta Bajya3
1Seed Officer, Rajasthan State Seeds Corporation,Mandore, Jodhpure
2 Department of Plant Philology, Jobner
Email of corresponding author: rmundiyara5@gmail.com

Among all microbials, entomopathogenic viruses are of special interest because of their pest specific nature with no hazardous impact on environment. Insects are susceptible to various diseases caused by bacteria, fungi, virus and protozoa and these are being exploited in biocontrol programme of major insect pest in ecofriendly manner. More than 600 viruses from 15 families have been reported as infecting insects, of the15 families, only three baculoviruses, polydnaviruses and ascoviruses are not suspected of having mammalian hosts. Baculoviruses comprising nuclear polyhedrosis virus (NPV) and granulosis virus (GV) have been successfully used as insect pathogens because of their high virulence and specificity.

Introduction

Microbial pesticides occupy around 1.3 per cent of the world’s total pesticide market. Biopesticides mean the use of fungi, bacteria, viruses, protozoa and nematodes through inundative or inoculative release for the biological control of insect pest, diseases, weeds and nematodes in agriculture, medicinal, veterinary, forestry and horticultural eco-systems. At present, the world market for microbial pesticides is in excess of US $ 125 million per annum which is still less than 1 percent of the total global market for agrochemical crop protection of $ 20-25 billion. Among all microbial biopesticides, Bacillus thuringiensis (80%) shares maximum followed by nematodes (13.3%) and others (6.67%). During last decade, government of India spent nearly Rs.14, 926 million for biocontrol programme in different crops. More than 600 viruses from 15 families have been reported as infecting insects. These insect viruses are submicroscopic, obligate, intracellular, pathogenic entities; the viruses belonging to Baculoviridae, Polydnaviridae and Ascoviridaewhich are pathogenic to insects and related invertebrates. Most of the insect viruses belong to the family Baculoviridae and they could be used against nearly all the major pests of food and fibre crops. Baculoviruses (nuclear polyhedrosis virus NPV and granulosisvirus GV) are mainly associated with the orders of Lepidoptera and Hymenoptera but few isolates from Diptera, Neuroptera, Coleoptera, Trichoptera, Crustacea and mites are also known. These viruses are often genus or species specific and highly virulent to their hosts. Members of the Baculoviridae are characterized by the presence of large, double-stranded covalently closed circular DNA genome of 88 to 200kbp. This is associated with arginine rich protein. This protein- DNA complex is contained by rod shaped nucleocapsid. One or more nucleocapsids are packaged within a single lipoprotein envelop to form singly embedded (SNPVs) or multiply embedded virus particle (MNPVs) or virion respectively (The virions are rod-shaped, 40-70 nm X 250-400 nm, comprising a lipoprotein envelope around nucleo-capsid). These structures may be occluded within crystalline matrix, known as polyhedron, composed of polyhedrin protein for NPVs and granules or capsules, composed of granulin protein for GVs. Granulosis virus contains only one virion per virus.

Baculoviruses have been described in over 600 insect species. The family Baculoviridae consisting of four Genera: Alphabaculovirus (Lepidoptean specific NPVs), Betabaculoviruses (Lepidopteran specific GVs), Gammabaculoviruses (Hymenopteran specific NPVs) and Deltabaculoviruses (Dipteran specific NPVs). The NPV from Autographica californica (AcMNPV) is one of the most intensively studied species.

Mode of Action

Virus after ingested by larva, the occlusion body dissolves in alkaline gut juice (pH 9.0-10.5) and virus particles released in gut. The virus particles are attached to the peritrophic membrane lining the midgut. The lipoprotein membrane surrounding the virus fuses with plasma membrane of the gut wall cells and liberates nucleocapsids into the cytoplasm. The nucleotide transports virus DNA into the nucleus of the cell and virus gene expression begins. The virus multiplies rapidly and eventually fills the body of the host with virus particles. These virus particle become occluded late in life cycle. After the death of larvae, they release massive amount of occlusion bodies in environment which further infect other larvae.

Symptoms

After 2-4 days of virus ingestion, the larvae cease to feed and become lethargic. During advanced stage, epidermis becomes very fragile and ruptures easily. The larvae become wilted and body contents become a fluidized mass of decomposed tissue and polyhedral. Just prior to death, infected larvae often climb to the highest point of substrate and attach themselves by their prolegs. Upon death, they hang in a characteristic V shape.

Table 1. List of HaNPV and SlNPV registered under Insecticide act in India for different crops

Table 2. Nuclear polyhedrosis viruses recorded in India



Table 3. Commercial formulations available world wide

Source: Microbial biopesticide by Koul and Dhaliwal


Mass production of NPV:

The NPV of H. armigera is propagated in early fifth instar larvae. For HaNPV and SINPV production, the synthetic diet prepared is poured at 4gm/cell in the multi-cavity trays and the diet surface is uniformly sprayed with virus prepared in distilled sterilised water. The dose of the inoculum used is 5 x 10⁵ polyhedral occlusion bodies (POB) in 10 ml suspension. A blunt end polished glass rod (6 mm) is used to distribute the suspension containing the virus uniformly over the diet surface. Early fifth instar stage of larvae are released singly into the glass vials after inoculation and plugged with cotton and incubated at a constant temperature of 25°C in a laboratory incubator. The larvae are observed for the development of virosis and the cadavers collected carefully from individual bottles starting from fifth day, where the dead caterpillar will have 2-6 billion poly occlusion bodies (POB) which is in terms of larval equivalent (LE). 1 LE of H. armiegera NPV = 6 x 10⁹ POBs; 1 LE of S. litura = 2 x 10 ⁹ POBs. The contents are frozen immediately. Depending upon the need, cadavers are removed from the refrigerator and thawed very rapidly by agitation in water.

Processing of NPV: The cadavers are brought to normal room temperature by repeatedly thawing the container with cadaver under running tap water. The cadavers are homogenized in sterile ice cold distilled water at the ratio 1: 2.5 (w/v) in a blender or precooled all glass pestle and mortar. The homogenate is filtered through double layered muslin and repeatedly washed with distilled water. The ratio of water to be used for this purpose is 1: 7.5-12.5 (w/v) for the original weight of the cadaver processed. The left over mat on the muslin is discarded and the filtrate can be semi-purified by differential centrifugation. The filtrate is centrifuged for 30-60 sec. at 500 rpm to remove debris. The supernatant is next centrifuged for 20 min at 5,000 rpm. Then the pellet containing the polyhedral occlusion bodies (POB) is suspended in sterile distilled water and washed three times by centrifuging the pellet in distilled water at low rpm followed by centrifugation at high rpm. The pellet finally collected is suspended in distilled water and made up to a known volume, which is necessary to calculate the strength of the POB in the purified suspension.

Directions for use of NPV

• The recommended dosage of HaNPV is 250-300LE, 500LE and 250 LE/ha, for chickpea, pigeon pea and cotton, respectively. (One LE = 6 x 10⁹ POBs).

• 100 ml of NPV could be diluted in 200-400 litres of water when high volume sprayer is used and in 50-70 litres of water in case of power sprayers.

• Preferable to spray using high volume knap-sack sprayer. Virus should be sprayed during evening hours and addition of UV blockers are recommended to improve the effectiveness of NPV. Spray should be initiated as soon as some newly hatched larvae are observed or three to five days after a trap catch of 5 moths per pheromone trap. Subsequent sprays should be made at 7-10 days intervals depending upon the pest population.
• The NPV based biopesticides are marketed under different trades names such as Spodo-Cide®, Spodopterin®, Heli-Cide®, Heliokill® etc., in the Indian market.

Conclusion

NPV can be an ideal tool for use in IPM programme because it is having less residual toxicity, compatible with many chemical pesticides, self-perpetuating nature and ecofriendly. To make it more effective there is urgent need of to develop more virulent and potent strains by biotechnological methods. Other areas which require attention are to develop quality control guidelines and methodologies, systematic registration policies, to identify effective stains and to develop UV (Ultraviolet) resistant strains. Finally to develop stronger IPM strategies, it is necessary to study of Interactionof NPV with other control measures thoroughly.

References

Erayya, J.Jagdish, P.K. Sajeesh and UpadhyayVinod. 2013.Nuclear Polyhedrosis Virus (NPV), A Potential Biopesticide: A Review. Research Journal of Agriculture and Forestry Sciences.1 (8): 30-33.

Ramanujam B., Rangeshwaran R.,SivakmarG.,Mohan M. and Yandigeri M.S. 2014. Management of Insect Pests by Microorganisms. Proc Indian NatnSci Acad. 80 (2): 455-471.




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