More than 3000 microorganisms have been reported to cause diseases in insects. The honey bees and silkworms were the first arthropods which were observed and recorded to suffer from various diseases, since their products were very important in the man’s economy.
Pasteur’s detailed studies on the ‘Flacherie’, as well as ‘Pebrine’ diseases of the silkworms and Agostino Bassi’s excellent contribution towards the establishment of a relationship between a microorganism, the fungus, Beauveria bassiana (Balsamo) Vuillemin and the infectious white muscardine of silkworm, led to the development of insect pathology as a specific section or discipline in the science of Entomology.
Metchnikoff is generally credited with actually initiating the experimental work to demonstrate that a disease may be intentionally caused in insect pests of economic importance. In 1879 he had published his work on the role of green muscardine, Metarhizium anisopliae (Metchnikoff) Sorokin in the control of the wheat cockchafer, Anisoplia austriaca Herbst.
Krassilstchik in 1888, mass produced spores of the fungus and applied them in field tests against insect pests. Subsequently, contributions on insect diseases by Steinhaus (1963) and his co-workers at the University of California, Berkeley, USA, gave the real impetus to researches on the use of insect pathogens as pesticidal agents.
Steinhaus (1949) coined the term ‘microbial control’ to express the pest population management through disease causing microorganisms. Microbial control includes all aspects of utilization of microorganisms or their by-products in the control of pest species. For the control of insect pest populations, many of the principles governing the role of insect parasitoids and predators also apply to entomogenous microorganisms.
In nature, viruses, bacteria, protozoa, fungi and rickettsia perform important role in the dynamics and natural regulation of insect and mite population. Their effect is most evident in epizootics of diseases which occur at intervals and under some circumstances decimate the host populations. Less obvious is the effect of insect pathogens in the enzootic stage or those which cause chronic or low grade infections.
Other than causing the outright death, pathogens may interfere with insect development, alter reproduction, lower insect resistance to attack by parasitoids, predators and other pathogens, and influence the susceptibility of insects to control by chemical insecticides or other artificial methods.
Insect pathogens may be divided into two groups according to the means by which they enter and infect their hosts. One group which includes bacteria, protozoa and viruses, must be ingested in order to cause infection and mortality, and can be considered similar to chemical insecticides which act as stomach poisons.
Some of these microorganisms, such as the viruses, are quite specific in their sites of development and they multiply only in certain tissues within the body of the host. Others, including bacteria, may cause a spreading septicaemia by growing diffusely in the tissues and body fluids. Some bacteria may kill their hosts purely by the activity of toxins which they produce during growth.
The second group, which includes the pathogenic fungi, enters their hosts through the outer integument of the insect’s body. These invasive organisms can be equated to contact chemical insecticides since they need not be ingested to cause infection. They are more subject to regulation by physical factors in the environment, since their penetrative stages generally are not very resistant to adverse effects of external conditions.
The successful use of diseases for insect control depends upon the biology and characteristics of the host insects and the pathogenic microorganisms as well as the environment. Host insects must occupy the habitats suitable for introduction of a pathogen and they must have habits that enhance the possibilities of infection.
Since disease is generally considered as a density-dependent factor of mortality, the insects that live in aggregations or which form large populations are more susceptible to epizootics as compared with the species which generally maintain low population densities.
The major emphasis in the application of microbes has been to field-collect or artificially mass culture a specific insect pathogen and disseminates it when the host is most susceptible to its effect. One approach is to introduce and colonize pathogens as permanent mortality factor in the host populations. This approach is called the microbial introduction.
The success of this method has been demonstrated for (i) the milky disease bacteria, Bacillus popilliae Dutky and B. lentimorbus Dutky, to control the Japanese beetle, Popillia japonica Newman, in USA, (ii) the accidental introduction and establishment of a polyhedrosis virus which lead to the control of devastating European spruce sawfly, Gilpinia hercyniae (Hartig), in Canada, and (iii) the importation and successful dissemination of a nuclear polyhedrosis virus for the control of the European pine sawfly, Neodiprion sertifer (Geoffroy) in Canada.
Another microbial technique is to make the repeated applications of a pathogen as microbial insecticide for temporary suppression of insect pests. The best illustration of this is the development of a bacterium, Bacillus thuringiensis Berliner. This bacterium is produced by fermentation and is formulated as a dust, wettable powder or emulsion. The material is handled and applied in the same manner as a chemical insecticide.
Its effect is short-lived and many applications are needed. In general, microbial agents meant for introduction must spread from relatively small inocula and should persist in the environment, allowing the nature to take its course. Those unable to spread and persist must be used as microbial insecticides, to be applied repeatedly.
The microbial control has numerous advantages. The relatively high degree of specificity of most pathogens tends to protect the beneficial insects. They are harmless and non-toxic to other forms of life because they do not possess any toxic residues. Many pathogens are compatible with insecticides to the degree that they may be used concurrently and in some cases, synergistically; the infection may cause the insect to be more susceptible to chemical poisons.
Microbial pathogens are highly versatile in so far as the method of their application is concerned. Some of the pathogens may be introduced and then colonized, so that the control brought about may be permanent; while other pathogens may be used as sprays or dusts, just like insecticides.
It is also easier and comparatively inexpensive to produce some of the pathogens. The low dosage required in some instances to get the desired control and the slow development of resistance to a microbial pathogen, further make the insect pathogens as ideal pesticidal agents for their use in pest management programmes.
Besides these advantages, microbial control methods also have certain disadvantages or limitations. These limitations include the correct timing of application with respect to the incubation period of the disease, the specificity of the pathogens, narrowing the spectrum of effectiveness where several pests are involved, the necessity to maintain the pathogens in a viable condition, the difficulty of culturing in large quantities and the required favourable climatic conditions for obtaining best efficiency.
In addition to this, a thorough understanding of the interactions within the ecosystem is necessary for attempting the microbial control efficiently, effectively and economically. The major problems include the short residual effectiveness of pathogens under field conditions and the inadequate methods for obtaining their dissemination.
Bacteria:
Approximately one hundred species of pathogenic bacteria have been recorded from various species of insects which could be either obligate or crystalliferous or facultative. Spores of obligate pathogens like Bacillus popilliae Dutky and B. lentimorbus Dutky, which are the causal agents of ‘milky’ diseases- Types A and B, respectively, in the population of Japanese beetle, P. japonica, are commercially available under different trade names, viz. ‘Doom’, ‘Japidemic,’ etc. Some of the selected soil sites, as for example a patch in a house lawn, can be spot-treated with the spores of these pathogens.
The soil offers an excellent habitat for the pathogen which is mainly carried from one site to another either by the irrigation water or by the movement of diseased grubs. Hence soil once treated ensures the persistence of pathogens and the control of the beetle on a long term basis. These bacteria being obligate pathogens have to be mass-produced on living insect hosts. White grubs, belonging to the genus, Holotrichia, have been found to be susceptible to an infection caused by B.popilliae.
Field trials with the milky disease spore formulations have been attempted in Gujarat State with considerable success and as many as 20-25 per cent of the grubs were found infected with the disease in subsequent years, in areas where the pathogen had been applied earlier. There is a considerable potential for the use of this bacterium against the white grubs of groundnut in India which are not easily controlled by insecticides.
Bacillus thuringiensis Berliner (Bt) is a crystalliferous spore former and unlike milky disease organisms, this bacterium can only be used for short term pest control. This is a unique microbial control agent in the sense that it is free from pollution, residual toxicity, biomagnification in non-target organisms, besides having several other advantages. B. thuringiensis has a wide spectrum of insecticidal activity in the class Insecta. More than 525 insect species belonging to 13 orders have been found to be infected by Bt around the world. It can be easily produced using ordinary fermentation technology.
The bacterium, B. thuringiensis also produces toxins which are poisonous to insects. As many as 25 varieties/serotypes are known with different capacities to produce various toxins and poisons. This is the reason for variable toxicity of this bacterium towards different species of insects. The pH of midgut of an insect is an important factor in determining its susceptibility to the crystalliferous bacteria. In general, susceptible insect species have a high midgut pH value.
Under such circumstances the endotoxin releases the toxic principle which further damages the tissues, thus resulting in an increase in the potassium ions in the haemolymph, the condition responsible for the paralysis of the host insect. These toxins alone can be formulated without the live bacteria and such preparations can be even more useful as control agents in areas wherever silkworm rearing is carried out; the chemicals will be free from the infectivity found in B. thuringiensis.
This bacterium is also reported to be safe to the honey bees wherever it has been used for treating the agricultural crops against various pests. It is non-toxic to man and vertebrates. The reason for this lack of toxicity is that, in mammals, the primary digestion of proteins is at low pH. The stomach enzyme pepsin, which has optimum pH value of 2, degrades the endotoxin/crystal toxin into an atoxic compound. But the exotoxin produced by some of the strains of this bacterium has been found to be toxic to mice when given through injection. It has also been observed that the thermostable exotoxin kills several dipterous species of insects in animal droppings.
The exotoxin is a nucleotide and thus it is quite possible that it may produce mutagenic effects. Fortunately, not all the strains of B. thuringiensis are capable of producing the exotoxin. The presence of endotoxin alone would be sufficient for the consideration of this bacterium as the most powerful microbial control agent yet developed for the control of insect pests.
In India, different commercial preparations of B. thuringiensis have been tested against more than 50 species of crop pests. For example, ‘Thuricide’ has been used in an attempt to control sugarcane Gurdaspur borer in Punjab, reducing its infestation from 11.34 to 4.8-5.8 per cent.
The effective control of Pieris brassicae (Linnaeus) and Plutella xylostella (Linnaeus) was achieved, when cauliflower crop was treated with the bacterium and the control thus achieved was only next to carbaryl. ‘Dipel’ – a commercial preparation when applied on Bengal gram gave effective control of the pod borer, Helicoverpa armigera (Hubner).
While spraying B. thuringiensis it is important to maintain the pH of the spray fluid at neutrality. The performance of this bacterium has been enhanced by spray additives, Plyac, Triton-X, corn oil, mollasses, etc. In addition to these agricultural pests, B.thuringiensis has also been used against pests of forests, shade trees and ornamental plants.
Effective control of the Gypsy moth is reported in the field tests with Thuricide 90 TS. Use of the bacterium for the control of stored grain pests is only in an experimental stage, because it is too expensive and perhaps it would not kill the whole range of insects found in the godowns or bins.
Recent reports indicate that insects have the capacity to develop resistance to B. thuringensist. Within the last few years, at least 16 insect species have been selected for resistance to Bt delta- endotoxins. These include Aedes aegypti (Walker), Choristoneura fumiferana (Clemens), Chrysomela scripta (Fabricius), Culex quinquefasciatus Say, Ephestia cautella (Walker), Ephestia kuehniella Zeller, Heliothis virescens (Fabricius), Homoeosoma electellum Hulst, Leptinotarsa decemlineata (Say), Ostrinia nubilalis (Hubner), Pectinophora gossypiella (Saunders), Plodia interpunctella (Hubner), Plutella xylostella (Linnaeus), Spodoptera exigua (Hubner), Spodoptera littoralis (Boisduval) and Trichoplusia ni (Hubner).
Among these reported cases of resistance, only three involve resistance among wild populations. The Indian meal moth, P. interpunctella, evolved low levels of resistance in grain bins because of treating the grain with Bt. The diamondback moth, P. xylostella is possibly the most notable because it evolved high levels of resistance in the field as a result of repeated use of Bt in intense control programmes. Recently, the cabbage looper, T. ni has been reported to develop resistance to Bt in vegetable greenhouses in British Columbia, Canada.
The reports of development of resistance in the field populations of P. xylostella are essentially from the countries where B. thuringiensis is extensively used, viz., China 1000 tons, Philippines 300 tons, Malaysia 250 tons, and North America about 1000 tons per annum. In Japan also, high level of resistance to B. thuringienis was observed in P. xylostella collected from watercress plant. This occurred because watercress was grown throughout the year with a frequent use of B. thuringiensis formulations (15-20 times a year).
In India, the populations collected from Tamil Nadu showed significantly high resistance to B. thuringiensis, whereas populations obtained from Delhi, Uttar Pradesh, Punjab and Maharashtra showed a high level of susceptibility despite their high level of resistance to many synthetic insecticides. Increased susceptibility to B. thuringiensis has been reported to be associated with synthetic insecticide resistance possibly involving monooxygenases in increasing susceptibility of resistant insects to B. thuringiensis.
The first report of development of field resistance to Bt in T. ni populations came from the commercial vegetable greenhouses in British Colombia, Canada. In a 3-year survey initiated in 2000, several greenhouse populations were surveyed multiple times within a growing season to monitor the rate at which resistance developed within a year in response to the grower Bt sprays.
Bt resistance levels were directly correlated to the amount of Bt applied, and Bt resistance was observed to evolve repeatedly within one year as a consequence of grower spray programmes. The application of high doses of Bt greatly intensified the rates of resistance evolution. It is likely that the greenhouse environment plays a significant role in contributing to the development of resistance to Bt.
The favourable environmental conditions and longer growing season in greenhouses increase the exposure period of T. ni to Bt, thus increasing the selection intensity for Bt resistance. Greenhouses probably also enhance Bt persistence by protecting Bt from sunlight degradation and rain. Furthermore, once resistance is present, the problem may be exacerbated by the application of high doses.
Resistance management strategies have been proposed as a means to decrease the rate at which resistance evolves and hence, to keep the frequency of resistance genes sufficiently low for insect control.
Some of the strategies are as follows:
i. An effective monitoring programme to detect as early as possible, shifts in pest susceptibility that could be abated in the initial stages.
ii. Use of mixtures of toxins with different mechanisms.
iii. Use of synergists to increase toxicity.
iv. Mosaic application to resort to time alterations rather than space alterations.
v. Rotation of toxins to reduce the frequency of resistant individuals.
vi. Ultra-high doses of toxins that kill resistant heterozygotes and homozygotes.
vii. Refuges to facilitate survival of susceptible individuals.
viii. Generally, resistance to Bt is the consequence of a mutation(s) that alters an insect midgut receptor protein(s), so that it no longer binds to the Cry protein. However, if a toxin gene was engineered, so that toxin is bound to other midgut cell surface proteins, then resistance might be less likely to arise.
Fungi:
There are over 72,000 described species of fungi and the total number of species in the world may be as high as 1.5 million. Fungi belonging to four groups, viz., Phycomycetes, Ascomyetes, Basidiomycetes and Deuteromycetes attack different species of insects. In contrast to other pathogens like bacteria and viruses that pass through the gut wall from contaminated food, fungi mainly infect their hosts through the integument. If ingested by the insect, the fungal spores do not germinate in the gut and are voided in the faeces.
Infection, therefore, results from contact between a virulent infectious inoculum and a susceptible insect cuticle, its germination, the penetration of the germ tube through the integument and finally spread of the fungus through the host tissues. Usually high humidity is required for the successful germination of the spores and sunlight has an adverse effect on them. Therefore, the atmospheric humidity or more precisely, the microclimate surrounding the fungal spores is a very important factor for the successful use of fungal pathogens.
Some of these fungi are known to produce toxic substances like ‘aflatoxin’ from Aspergillus flavus Link, ‘beauvericin’ from Beauveria bassiana (Balasamo) Vuillemin, and ‘destruxin A and B’ from Metarhizium anisopliae (Metchnikoff) Sorokin, which are also responsible for causing death of the host insects, but they are equally harmful to the mammals. There are, however, no fungal toxins under development at present for pest control.
With most entomopathogenic fungi, disease development involves nine steps:
i. Attachment of infective units like conidia or zoospores to the insect epicuticle.
ii. Germination of the infection unit on the cuticle.
iii. Penetration of the cuticle, either directly by germ tubes or by infection pegs from appresoria.
iv. Multiplications of the yeast phase-hyphael bodies in the haemocoel.
v. Death of the host.
vi. Growth in the mycelial phase with invasion of virtually all host organs.
vii. Penetrations of hyphae from the interior through the cuticle to the exterior of the insect.
viii. Production of infective units on the exterior of the insect.
More than 500 species of insects have been observed to be infected with fungi. Fungal epizootics have often been observed in the field among pest populations. In fact, the first record of insect disease is that of Metarhizium anisopliae (Metschnikoff) Sorokin in the wheat cockchafer, Anisoplia austriaca Herbst, in USSR in 1879.
The common fungal diseases are caused by the green (Metarhizium) and the white (Beauveria) muscardine fungi and they have a wide host range. Species of Entomophthora and Coelomomyces have been tried against aphids and mosquitoes in different parts of the world.
During the past few years, concerted efforts have been made to develop fungi as microbial insecticides, viz., Verticillium lecanii (Zimmerman) Viegas and Hirsutella thompsonii Fisher for the control of aphids and scales, and also the citrus rust mite, Phyllocoptruta oleivora (Ashmead). The latter is a specific fungal pathogen of Acarina, particularly of the eriophyid and tetranychid mites infesting citrus plants.
Abbott Laboratories, USA have developed a safe commercial formulation of the conidia of H. thompsonii. Another fungus, Nomuraea rileyi (Farlow) Samson, is also being developed as microbial insecticide for many species of caterpillar pests.
Main target pests of commercially available mycoinsecticides:
1. Benuveria Bassiana:
Product:
Botani Gard, Boverol. Naturalis-L, Proecol, Mycotrol, Beauverin, Bio-Power
Target Pests:
Lepidoptera (diamondback moth, beet armyworm, cabbage looper, cutworm, etc.), Coleoptera (scarab beetle grubs, weevils, coffee berry borer, cutworms, etc.), Heteroptera (psyllids, stinkbug, plant bugs, leafhoppers, mealybugs, aphids, whitefly, etc.), Thysanoptera (western flower thrips)
2. Beauveria Brongniartii:
Product:
Betel, Schweizer, Beauveria
Target Pests:
Lepidoptera (diamondback moth, beet army-worm, cabbage looper, cutworm, etc.)
3. Lecanicillium Lecanii:
Product:
Mycotal, Bio-Catch, Vertalec
Target Pests:
Heteroptera (stinkbug, aphids, whitefly, plant and leafhoppers, mealybugs), Thysanoptera (western flower thrips, onion thrips)
4. Mctarhizium Anisopliae:
Product:
Bio-Magic, Bio-Catch-M
Target Pests:
Coleoptera (scarab beetle grubs, weevils), Blattodea (termites), Heteroptera (leafhoppers), Orthoptera (grasshoppers), Lepidoptera (cutworms)
5. Mctarhizium Flavoviridc Var. Flavoi’iride:
Product:
BioGreen, BioCane
Target Pests:
Coleoptera (scarab beetles, weevils), Orthoptera (grasshoppers and locusts), Blattodea (termites)
6. Isaria Futnosorosea:
Product:
Preferal, Priority, FuturEco, Nofly
Target Pests:
Heteroptera (whiteflies, aphids, etc.), Lepidoptera (tomato moth), Acari (rust mites, spider mite, etc.)
7. Paecilomyces Lilacinus:
Product:
Bio-Nematon
Target Pests:
Nematodes (Root knot, cyst, lesion, burrowing).
The fungal spores can be applied as dusts, sprays or granules. The methods of culturing fungi have been studied in detail. The spores of B. bassiana (Balsamo) Vuillemin and M. anisopliae can easily be mass-produced on simple media containing wheat corn and/or potato products. There have been some difficulties in the mass production of the fungi.
Entomophthora require some special media like coagulated egg yolk. Colonization is usually attempted with relatively small inocula in the form of diseased insects or cultured material. For the use of microbial insecticides large amounts of material are needed, which can be applied satisfactorily with equipment designed for application of chemical insecticides.
Timing of application must, of course, coincide with the presence of susceptible stages of the hosts, but timing should also be related to the environment. Application immediately after rain or irrigation would be better than in the dry weather. Besides, treatment in the evening will save the fungi from sunlight.
There seems to be a wide scope for the utilization of insect pathogen fungi in India. Locally available materials can be used for mass production of fungal pathogens and then for extensive field trials, particularly on pests of rice, cole crops and other irrigated crops. It has been determined that the conidia of M. anisopliae can initiate mycosis resulting in the death of host insects among the field population of Pyrilla perpusilla (Walker) when sprayed immediately after irrigating the sugarcane crop.
The use of Entomophthora could cause 2.5-28.0 per cent infection among the mustard aphid, Lipaphis erysimi (Kaltenbach) under field conditions. Cephalosporium lecanii Zimmermann was found highly infective for controlling the coffee green bug, Coccus viridis (Green) in the field.
Before any wide-scale operation involving the use of fungal pathogens in any country is planned, these organisms must be thoroughly evaluated for their safety to vertebrates. Many of these fungi can be a potential source of allergenic and toxic reactions.
Protozoa:
These are unicellular microscopic organisms which form cysts or spores and these adaptations play an important role in the epizootiology of infections among insect populations. Several species of protozoa are associated with insects with different degrees of infection. Nosema apis Zander causes nosema disease in honey bee and Nosema bombycis Naegeli causes the pebrine disease in silkworm.
Protozoan diseases are generally chronic in nature and take quite some time to kill their hosts, hence they are often debilitative on insect populations and also the spread of the disease is slow.
Since the efficiency of host’s reproduction and other physiological functions are generally reduced, protozoan disease ultimately leads to reduction in pest populations. Glugea pyraustae (Paillot), pathogenic to Ostrinia nubilalis (Hubner), is the most important factor maintaining corn borer populations at a level which facilitates economic control by other means.
In USA, storage pests are excellent hosts for protozoan infections and this fact mostly goes unnoticed. Moreover, such pathogens cannot be utilized in a country like India where deliberate mixing of any insecticidal agent with food grains is not permissible and yet people do mix them.
Protozoa are generally non-specific in nature. Further, most protozoa are difficult to culture on artificial media. In spite of such limitations, considerable efforts have been made in the past few years to develop a microsporidian, Nosema locustae Canning, for the long time control of grasshoppers.
This can be produced and applied effectively and efficiently, and can be regarded as safe microbial control agent for use. Similarly, another microsporidian, Vairimorpha necatrix (Kramer), has the high virulence, wide host range (covering as many as 36 lepidopteran pests), can also be mass produced at a reasonable cost and can be stored for short periods.
Thus, V.necatrix is primarily, if not exclusively, a pathogen of phytophagous Lepidoptera. Though there are quite a few protozoan pathogens of insect pests reported from India yet none of them has been tried under field conditions for pest control.
Rickettsiae:
Rickettsiae are the microorganisms which like viruses, are known to be obligate pathogens, though they also have many features characteristic of bacteria. They possess an active metabolism, which is so heterotrophic that their cultivation on simple artificial media is not usually possible.
Rickettsiella melolonthae Wille & Martignoni, and Rickettsiella popilliae Dutky & Gooden are known to cause disease in the populations of Melolontha melolontha (Linnaeus) and Popillia japonica Newman, respectively. Because of their low host specificity and pathogencity for vertebrates, the use of these organisms in microbial control is not considered possible at present.
Viruses:
A virus is an entity whose genome is an element of nucleic acid, either DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) which is reproduced inside the living cells. It uses its host’s synthetic machinery to direct the synthesis of the specialized particle called the virion (nucleic acid & coat) which contains viral genome.
There are six main groups of viruses recognized as causing diseases in insects and mites. These are the baculoviruses (Baculoviridae), cytoplasmic polyhedrosis viruses (Reoviridae), entomopoxviruses (Poxviridae), irridoviruses (Iridoviridae), densoviruses (Parvoviridae) and small RNA viruses (unclassified).
These viruses occur naturally and produce diseases in Lepidoptera, Hymenoptera, Coleoptera, Diptera and several other smaller groups. Of these known viruses, many are closely related to those which are pathogenic to man, domestic animals, a wide range of invertebrates and plants. Only viruses in the Baculovirus group have no such dangerous relationships.
Approximately 60 per cent of the 1200 known insect viruses belong to the family Baculoviridae and it is estimated that such viruses could be used against nearly 30 per cent of all the major pests of food and fibre crops. Baculovirus infections have been described in over 700 species of invertebrates including Lepidoptera (455), Hymenoptera (31), Diptera (27), Coleoptera (5), Neuroptera (2), Trichoptera (1), Thysanura (1), Siphonaptera (1), besides Crustacea.
Most of the insect viruses, unlike the plant viruses, have an inclusion body around the virions. Insect viruses, being obligate pathogens, need to be cultivated on live insect hosts or in live cells through tissue culture.
Most baculoviruses infect only the larval stages of susceptible insects, exceptions are NPVs that infect Hymenoptera and the nonoccluded virus of Oryctes. These viruses infect the adult as well as the larval stage. Baculoviruses must be ingested to infect the larvae, so they are used mainly to control open-feeding species. The occlusion bodies dissolve in the highly alkaline host mid-gut and the virus infects the host epithelial cells.
The virus replicates within the nuclei of susceptible tissue cells. Tissue susceptibility varies greatly between viruses with some NPVs being capable of infecting almost all tissue types and most GVs being tissue-specific replications (e.g., fat body cell only). The budded virus initiates infection to other tissues in the hemolymph, i.e., fat bodies, nerve cells, haemocytes, etc.
The cells infected in the second round of virus replication in the insect larva also produce budded virus, but in addition occlude virus particles within polyhedra in the nucleus. The accumulation of polyhedra within the insect proceeds until the host consists almost entirely of a bag of virus.
In the terminal stages of infection, the insect liquefies and thus releases polyhedra, which can infect other insects upon ingestion. A single caterpillar at its death may contain over 109 occlusion bodies from an initial dose of 1000.
The infected larvae exhibit negative geotropism before succumbing to the virus infection, thereby facilitating widespread dissemination. The speed with which death occurs is determined in part by the environmental conditions. Under optimal conditions, target pests may be killed in 3-7 days, but death may be caused in 3-4 weeks, when conditions are not ideal.
The viruses, in general, lack quick knockdown effect and they take about one week for causing mortality. They are easily inactivated by sunlight and ultraviolet rays but they are known to accumulate and survive in top layer of soil. They are highly host specific and each species of insect will require specific virus of its own.
In general, the early instars of insects are more susceptible and require the correct timing of application. From the point of view of microbial control, the baculoviruses (nuclear polyhedrosis viruses and granulosis viruses) seem to have more value.
To some extent cytoplasmic polyhedrosis viruses too have been investigated but these are treated with caution. During the last decade, considerable work has been carried out on the practical use of these viruses, including application technology, safety testing, field testing and their use in pest management.
The use of nuclear polyhedrosis virus (NPV) for the control of soybean caterpiller, Anticarsia gemmatalis (Hubner), in Brazil, represents the world’s largest programme for the use of an entomopathogen to control a pest on a single crop. The use of the viral biopesticide has increased from 500,000 ha in 1986-87 to 1 million ha in 1989-90, 1.5 million ha in 1995 and 2.0 million ha at present.
The current annual savings at the soybean grower level are over US$ 14 million. Most importantly, this programme has avoided spraying of 23 litres of chemical pesticides resulting in considerable ecological benefits to the society. With recent achievements in commercial production of virus under controlled conditions, it is-expected that the use of the biopesticide may very soon reach 4 million ha per year.
Spectacular results have been achieved through the use of baculoviruses in sawfly populations in USA and Canada. The other important viruses which have undergone extensive field testing include nuclear polyhedrosis viruses of cotton bollworm, Helicoverpa sp.; cabbage loopper, Trichoplusia ni (Hubner); cotton leaf worm, Spodoptera littoralis (Boisduval); alfalfa caterpillar, Colias eurytheme Boisduval; the spruce budworm, Choristoneura fumiferana (Clemens); gypsy moth, Lymantria dispar (Linnaeus); codling moth, Cydia pomonella (Linnaeus); tent caterpillars, Malacosoma spp.; the European spruce sawfly, Gilpinia hercyniae (Hartig); the European pine sawfly, Neodiprion sertifer (Geoffroy), and a few other species of Neodiprion; granulosis viruses of the cabbage butterfly, Pieris rapae (Linnaeus); codling moth, C.pomonella, and the spruce budworm, C. fumiferana.
Several baculoviruses have been incorporated into integrated control programs for certain crops throughout the People’s Republic of China. These are the nuclear polyhedrosis viruses of Spodoptera litura (Fabricius) and Helicoverpa armigera (Hubner) (cotton), Sphrageidus similis (Fuessly) (mulberry) and Euproctis pseudoconspersa Strand (tea).
The first baculovirus comprehensively tested for safety was that of the nuclear polyhedrosis virus of Helicoverpa spp., during 1965-71, which had paved the way for granting exemption from a residue tolerance in 1973 by the Environment Protection Agency of USA. Subsequently, the product, ‘Elcar’ containing this virus was registered in 1975. At present more than 10 viral insecticides have been registered in various parts of the world.
Due to abundant and inexpensive labour, baculoviruses have been introduced into developing countries and are thus ideally suited for production.
There are four strategies for using viral insecticides:
(i) The virus spreads from limited applications and permanently regulates the insect population through a classical biological control.
(ii) An epizootic is established through vertical and horizontal transmission, but reapplication may be necessary because control is not permanent.
(iii) A vertical inoculum in the environment is conserved and reactivated through environmental manipulation.
(iv) Repeated applications are used to control an insect population because there is no horizontal transmission of the virus-a strategy, which is widely used because of its effectiveness.
The prospects of microbial control of agricultural pests through the use of baculoviruses seem to be very good in India. This is evidenced by the fact that more than 30 species of insect pests have been reported to suffer from either the nuclear polyhedrosis virus or the granulosis virus infections. Extensive fieldscale testings have been carried out with nuclear polyhedrosis viruses of S.litura, Mythimna separata (Walker), Spilarctia obliqua (Walker) and H.armigera.
To develop these viruses for their further use as viral insecticides the three requisites that have to be fulfilled are:
(a) Characterization of the virus,
(b) Field efficacy, and
(c) Safety evaluation.
Once these requirements are fulfilled then the candidate virus can be commercially exploited. Research work has already been done on the development of nuclear polyhedrosis viruses for the control of S. litura and S. obliqua which are serious polyphagous pests in India.
Various aspects like symptomatology, extent of natural incidence, production of the virus inclusion bodies, transovarial transmission, gross infectivity, effect of storage, surface disinfections, resistance to alkalies and temperature, and virulence of these viruses, besides their characterization, have already been investigated in detail.
The nuclear polyhedrosis virus of S. obliqua is the only baculovirus which has so far been exhaustively safety-tested keeping in view the recommendations on safety evaluation of viral agents laid down by the World Health Organization.
Results of preliminary studies on the safety testing of nuclear polyhedrosis viruses of M. separata, Amsacta albistriga (Walker) and H. armigera also indicated the safe nature of these viruses.
It is thus likely that the viruses might be developed in future as viral insecticides in India. The three summum bonum of the application and practice of viral control are- (a) it is safe, (b) it can be easily and economically produced with locally available material and skill, and (c) it gives desired level of control.
Nematodes:
The entomopathogenic nematodes have received increased attention as biological control agents in recent years. Beneficial nematodes are microscopic (0.6 mm) roundworms found associated with most of the insect orders. The two major groups of entomopathogenic nematodes that attack insects are Steinernema and Heterorhabditis. There are about 55 species of Steinernema the world over and a dozen of them occur in India.
Among Heterorhabditis, about 12 are on record and the most common in India is H. indica. The nematode-bacterium complex has attained the status of a potential biopesticide because of their impressive attributes. They are unique due to their symbiotic relationship with bacteria in the genera Xenorhabdus or Photorhabdus. All species of Steinernema are associated with bacteria of the genus Xenorhabdus and all Heterorhabditis with Photorhabdus species.
Steinernema and Heterohabditis are obligate pathogens in nature. The major difference between Steinernema and Heterorhabditis is that Heterorhabditis adults are hermaphrodites in the first generation but amphimictic in the following generations, whereas Steinernema adults are always amphimictic (Fig. 5.5).
The only stage that survives outside of host is the non-feeding third stage infective juvenile (IJ) or dauer juvenile. The IJs carry cells of their bacterial symbiont in their intestinal tract. After locating a suitable host, the IJs invade it through natural openings (mouth, spiracles, and anus) or thin areas of the host cuticle (common only in Heterorhabditis) and penetrate into the host hemocoel.
The IJs recover from their developmental arrestment, release the symbionts, and bacteria and nematodes cooperate to overcome the host’s immune response. The bacteria propagate and produce substances that rapidly kill the host and protect the cadaver from colonization by other microorganisms.
The nematodes start developing, feed on the bacteria and host tissues metabolized by the bacteria, and go through 1-3 generations. Depleting food resources in the host cadaver leads to the development of a new generation of IJs that emerges from the cadaver in search of a new host.
Each nematode species is specifically associated with one symbiont species, although a symbiont species may be associated with more than one nematode species. This specificity has been demonstrated to operate at two levels, (i) Although the nematode can develop on other bacteria, but reproduction occurs on their natural symbiont. (ii) Natural symbiont cells are retained better than cells of other bacteria.
In this association, the nematode is dependant upon the bacterium for- (i) quickly killing its insect host, (ii) creating a suitable environment for its development by producing antibiotics that suppress competing secondary microorganisms, and (iii) transforming the host tissues into a food source. The bacterium requires the nematode for (a) protection from the external environment, (b) penetration into the host’s hemocoel, and (c) inhibition of the host’s antibacterial proteins.
More than 300 nematode-insect relationships have been described to-date. However, mainly two families of the nematodes, Heterorhabditidae and Steinernematidae, have been extensively used to develop commercial formulations. The first commercially available formulation was Romanomermis culicivorax Ross & Smith, which was registered against mosquitoes in USA in 1976. The registration was subsequently cancelled due to the success of alternative products.
Currently, there are eight species of nematodes (7 for insect pests and 1 for slugs) that are available for pest control. These are from three genera and are being sold/and or manufactured by over a dozen companies offering 36 different products. The attributes of these nematode based formulations that make them amenable to production and use are ease of mass production, efficacy comparable to synthetic pesticides and safety to non-target organisms.
In general, steinernematids and heterorhabditids have shown excellent results in habitats where high humidity could be maintained, e.g., soil inhabiting insects and borers. Steinernema glaseri is still marketed in USA for grub control by Praxis. S. riobrave has recently been registered by Thermo Trilogy for the control of mole crickets, Scapteriscus spp.; sugarcane rootstalk borer, Diaprepes abbreviatus (Linnaeus); citrus root weevil, Pachnaeus litus (Germar), and the blue-green weevil, P. opalus (Oliver).
S. scapterisci, which was isolated from mole cricket in South America, was originally produced by BioSys (now Thermo Trilogy), who licensed it to Ecogen. There are two species of heterorhabditids that are currently commercially available. H. megidis is sold mostly in Europe for weevil and soil insect control. H. bacteriophora is effective against a wide range of soil insects but is sold primarily for the control of Japanese beetle.
In India, both steinenematidis and heterorhabditids are marketed by Bio-Sense Crop Protection (Eco-Max Agrosystems Ltd.), Mumbai. Steinernema sp. is commercially available for the control of American bollworm, pink bollworm, tobacco caterpillar, white grub, shoot borer, stem borer, tuber moth, spotted bollworm and leaf eating caterpillar on crops like cotton, groundnut, sugarcane, rice, potato, tomato, chilies, okra, sunflower and legumes. Heterorhabditis sp. is available for the control of white grub, flea beetle, grey weevil, and red palm weevil on groundnuts and coconut.