BCAs have been used or are being used on a commercial scale to control plant pathogens. There are many more examples, which have shown promise in experimental trials, but have yet to make a significant impact in the market place.
1. Bacillus Thuringiensis:
About 100 species of bacteria infect insects. Nearly all the entomapathogenic bacteria are from the class Schizomycetes, order Eubacteriales. Although species of both sporulating and non-sporulating bacteria are considered potential candidates for development into bacterial insecticide, more success has been achieved with the spore formers, especially species of the genus Bacillus, family Bacillaceae.
In fact, four species of Bacillus (B. thuringiensis, B. popilliae, B. lentimorbus and B. moritai), account for nearly one-half of all the trade-named microbial products in existence. There are two insecticides produced commercially from Bacillus thuringiensis (Bt) and Bacillus popilliae to control caterpillar pests, mosquitoes and black flies, Japanese beetles, etc. Bacillus sphaericus is considered to be a highly promising pathogen for mosquito control but is not yet in commercial production.
(Bt) based insecticidal formulations are available in USA, France, Japan, and many other developed and developing countries. The toxicity of Bt strains has been attributed to a glycoprotein, protoxin, often referred to as delta endotoxin, which is produced during sporulation/fermentation. It is a crystalline product. When the larvae ingest Bt spores, they are activated within the gut to produce toxins. The histological studies conducted on the mid-gut epithelial cells of Aedes aegypti revealed that the toxins disrupted the cells, which became distorted and eventually burst.
Production of Bacillus Thuringiensis (Bt):
Bt was discovered in silkworms in Japan in the early part of the twentieth century. It first became a commercial product in France in 1938 and has been on sale ever since. The bacterium produces a proteinaceous crystal, which becomes insecticidal after digestion in the alkaline environment found in the mid-guts of susceptible species.
The two commercial strains are Kurstaki (used against caterpillars) and israelensis (H-14), (used against mosquitoes and black fly). Some strains of Bt produce a soluble toxin called β-exotoxin, which is highly toxic to houseflies and does not need to be ingested. Unfortunately, the β-exotoxin has slight mammalian toxicity. It is produced in liquid fermentations after which it is concentrated and formulated into a variety of liquid and powder forms to be used.
Problems in Bt Production: Although the fermentation of Bt presents few conceptual problems, its cost will determine the competitiveness of the company making it. Costs may be reduced at least 30% over by the use of inexpensive raw materials, by faster fermentations, and by simplification of the concentration steps used after the fermentation.
At Microbial Resources, Bt H-14 is made into five separate products:
(i) A wet-table powder for formulation at the point of use,
(ii) A flow-able liquid for general use,
(iii) An emulsion for rapid dispersion over large areas of water,
(iv) A sand-granule for aerial application through foliage,
(v) An ultra-low volume liquid for efficient applications by aircraft when there is no canopy.
Stability of Bt:
It is essential to know about the stability of the microbial biocide. With no additives Bt H-14 loses half of its activity in 0.05 months at 42° C.
2. Agrobacterium Radiobacter (Agrocin):
Crown gall is plant tumour caused by the soil-borne bacterium Agrobacterium tumefaciens; galls occur on the roots or often at the crown of the plant between roots and shoot. The pathogen has a wide host range but the disease is especially important in some fruits such as peaches, on grapevine, and in woody ornamentals such as roses.
A highly effective biocontrol method has been developed using a related non-pathogenic bacterium, Agrobacterium radiobacter, to treat roots during transplanting. The BCA is commercially available and gives cheaper, more effective control than antibacterial chemicals.
Not all strains of A. radiobacter are able to protect plants from the disease.
Effective strains of A. radiobacter possess two following important properties:
1. They are able to colonize host roots to a higher population density than ineffective strains.
2. Biologically active strains produce an antibiotic, agrocin, which is toxic to A. tumefaciens. This molecule is a ‘rogue’ nucleotide, which interferes with DNA synthesis. Agrocin production is encoded by a plasmid, which also carries genes for insensitivity to the toxin.
As plasmids are often able to transfer between bacterial strains and even species, one concern with the use of this BCA is the possibility that the genes for agrocin insensitivity might ‘jump’ into the pathogen, rendering it immune to the toxin.
3. Pseudomonas Fluorescens (Phenazine):
Damping-off disease caused by Pythium species and Rhizoctonia solani, and Gaeumannomyces graminis (in cereals) soil-borne fungal pathogens. These pathogens infect seeds and roots are a serious constraint to agricultural production. They affect crop establishment, leading to patchy growth and delayed development.
One feature common to the infection cycle of these diverse pathogens is the need to colonize the zone surrounding seeds or roots prior to penetration of host tissue. Interfering this step by introducing aggressive microbial competitors, or to manipulate resident microbial populations can reduce infection.
The decline to take-all disease in cereal monocultures coincides with changes in the microbiology of the rhizosphere. Bacteria isolated from this zone (rhizobacteria) have been intensively studied as potential biocontrol agents for take-all and several seedling diseases.
The most promising candidate strains are almost invariably isolates of Pseudomonas fluorescens, which are well adapted to growth in the rhizosphere. Several strains of P. fluorescens have been tested in plot and field trials for the control of soil-borne fungal pathogens with varying degrees of success. To date only one commercial product based on this bacterium has been launched.
4. Bacillus (Bacteria):
Bacillus is an alternative to P. fluorescens and has been used for seed treatment. Bacillus produce resistant endospores, which can be kept as a dry formulation for long periods without losing viability. This property might also aid use in situations exposed to drying, such as on leaves and other aerial plant surfaces.
5. Protozoa:
The phylum Protozoa consists of about 15,000 free-living and parasitic species; about 300 members of the sub-phyla Ciliophora (ciliates), Cnidispora (microsporidians) and Sporozoa (sporozoa) and the super classes Mastigophora (flagellates) and Sarcodina (amoebae) have been isolated from insects. These members produce entomopathogens.
Mode of Action of Protozoa:
Infection of insect hosts by species usually follows ingestion of the infective entity (e.g., spore, cyst, or infectious cell). The following describes the infection process in a typical Nosematide. The host is invaded via the reverted polar filament and of ingested spore. The polar filament penetrates the peritrophic membrane of the gut and injects the sporoplasm into susceptible cells of the epithelium.
The protozoa then develop in the cytoplasm of the primary tissue by asexual reproduction and finally produce spores. Presence of protozoa destroys the cells and in many cases the primary tissue or organ. Quick kill of the host is rare except when the host is exposed to a heavy dose or a secondary bacterial septicaemia is encountered.
6. Fungi:
Over 400 species of fungi are known to attack insects and mites. Fungi usually infect their hosts by direct invasion and are, therefore, able (unlike most bacteria and viruses) to attack pests without first being ingested. In addition by their ability to sporulate on the dead bodies of their houses, also control the insect.
The timely application of sufficient fungal propagules at an early stage can control pests to sub-economic levels for the duration of the crop. However, to be effective, fungi have fairly stringent requirements for humidity and temperature. If the humidity falls, or the temperature varies too far from the fungus, optimum, control of pests may be sluggish or non-existent and the desired epidemic (termed epizootic) may fail to develop.
Trichoderma:
Nectria galligena, which causes silver-leaf disease of fruit trees. It grains entry to the host via pruning wounds. These sites can be treated with a fast-growing antagonist, such as formulations of the saprophytic fungus Trichoderma, to prevent infection by the pathogen.
Peniophora (Phlebia) Gigantea (Basidiomycete):
Heterobasidion annosum is a soil-borne fungus and causes serious problem in forestry plantations and causes root and butt rot of conifers. The pathogen is not aggressive to intact trees but instead exploits cut tree stumps to establish a food base from which it can spread along the roots and infect adjacent hosts. As forestry practice involves frequent thinning and falling of trees, this provides the pathogen with ample opportunity to gain access.
The use of chemical fungicides declines the pathogen problem to an ineffective level. Biocontrol agent as microbial antagonists has been tried. The most effective agent proved to be another wood-rotting basidiomycete, Peniophora (Phlebia) gigantea. Spores of this fungus are painted or sprayed onto cut stumps.
Around 106 spores are used per square metre of stump surface, which ensures rapid colonization of the timber by the antagonist, thereby occupying the infection court and denying access to the pathogen. In UK this treatment has provided cost-effective and environmentally safe control of the disease in pine plantations for more than 30 years, while in Scandinavia strains of the BCA have been developed for use on Norway spruce.
Metarhizium Anisopliae:
Nevertheless, fungi have great promise as economical pest control agents. Metarhizium anisopliae is the best known of all entomopathogenic fungi and was the first fungus to be produced on a large scale. It infects a wide range of species and has been successfully commercialized by a number of small companies in Brazil for the control of spittlebug in pasture and in sugar cane; it has been combined effectively with a virus to obtain control of the rhinoceros beetle on palms in the Pacific.
Beaveria Bassiana:
Another extensively researched fungus is Beaveria bassiana; its product has been formulated in the USSR to control Colorado potato beetle. Experiments in the United States have shown good control of this beetle to be possible. Verticillium lecanii has been produced commercially in the U.K. for several years. Strains of this fungus can control aphids and whitefly, sometimes for months at a time.
Hirsutella thompsonii was produced briefly in the United States by Abbott Laboratories for the control of mites in citrus; environmental conditions did not favor the growth of the fungus and it has not yet found a place in citrus pest control programmes. Nomuraea rileyi was investigated for many years by researchers in the U.S. primarily in collaboration with Abbott. This fungus can be very effective against caterpillars particularly in soybeans.
Members of Order Entomophthorales:
In addition to these, fungi from the Order Entomophthorales show good potential. Research at Rothamsted over many years has shown one particular strain to be effective against aphids in outdoor crops. These fungi are particularly difficult to grow outside of their inside host; recent work however has shown that it is possible to produce infective propagules in vitro so a product based on this fungus may not now be far off.
Arthropod Toxins from Fungi:
Tetranactin:
A macrotetrolide antibiotic was isolated from the fermented broth of Streptomyces aureus S-3466. It exerts a potent miticidal action (LC50 = 4.8 ppm) against the carmine spider mite (Tetranychus cinnabarinus) and concentrations of < 10 ppm were found lethal to related four mites such as two-spotted spider, European red mites etc. It is also known as an ionophore antibiotic because of its facile formation of complexes with alkaline cations.
Avermectins and Milbemycins – Multipurpose Biocides from Fungi:
These are members of the class of sixteen- ring macrolides, having a significant role in crop protection, treatment of parasitic diseases of animals and of humans. Natural avermectins fall into two groups, designated as A and B, the former possessing a methoxy-group at C-5 and the latter possessing a hydroxy group instead.
These groups are further sub-divided into the 1-series with a 22 double bond and a 2-series with a 22 double bond and a 2-series, which has an axial C-23- OH. A final sub-division into a and b series designates the presence of a sec-butyl or an isopropyl group respectively at C-25.
Avermectins:
Avermectins were isolated from an actinomycete found in soils of Kawana, Ito city, Japan. This novel actinomycete was named Streptomyces avermitilis MA-4680. Extensive work on this species, such as causing mutation with various agents and modification of the growing medium gave more than a fifty-fold improvement in yield from the mutated strain, S. avermitilis M-4848, which could produce 53 µg ml-1 of avermectins.
Mibemycins:
Mibemycins have relatively simpler structures but with a greater diversity of fictionalization. Over 20 naturally occurring milbemycins known can be sub-divided into α and β series, on the basis of respective presence or absence of tetrahydrofuranyl ring as a part of the structural skeleton. A comparison of structures of avermectins and milbemycins would reveal the only major difference as the lack of L-oleandrosyl – L- oleaandrosyl group at C-13 in the latter.
The name milbemycin originated from its high potency against mites of various orders. These exhibited a broad spectrum of activity against agricultural pests such as aphids, mites, tent caterpillars, intestinal worms and other parasites that prey on crops and livestock. They are promising as agricultural pesticides because of their potent activity without toxicity to plants and animals at effective dosages.
For example, milbemycin D was highly effective against all stages in the life cycle of the two-spotted spider mite and the motile stage of the citrus red mite. When applied in solution directly onto adult and nymphal populations of foliage, D showed LD50 of 0.3 ppm for the former and 0.03 ppm for the latter.
Milbemycins were isolated in 1974 from Streptomyces hygroscopicus subsp. aureolacrimosus strain B 41-146. Mutation of the strain by exposure to UV light, gave two mutants with very different milbemycin- producing characteristics. Strain Au-3 gave markedly higher yields of milbemycins D to H but did not produce milbemycins αs to α8, while strain Rf-107 gave only milbemycins J and K. A positive correlation which exists between the absorbance of UV light and anti-helminthic activity was made use of in isolation of these antibiotics.
Biological Activities:
A battery of in vivo antihelminthic assays maintained by parasitologists at Merck made use of the purified natural avermectin derivatives and discovered activities at the very low levels of 0.05 to 0.1 mg/kg against a wide spectrum of economically important nematodes, but not cestodes or trematodes. A laboratory animal test also demonstrated very interesting insecticidal properties against an ectoparasitic insect species upon systemic application to the host Avermectin B1 as the most interesting component.
The compound was further tested and found effective upon oral administration in sheep; and oral and parenteral application in cattle against all-important gastrointestinal parasites at doses from 0.1 to 0.025 mg/kg. Avermectins B1 was more effective than B2 on oral treatment of sheep and cattle for adult Haemonchus contortus and H. placei while B2 was more effective than B1 for Cooperia oncophora or parenteral injection in cattle. In particular, efficacy against Cooperia species decreased markedly upon parenteral treatment with B1.
Stability and Chemical Reactivity:
The solubility’s, stabilities and chromatographic behaviour of the avermectins have been studied with the help of thin layer and column chromatography on silica gel. The avermectins dissolve in most organic solvents but have an extremely low water solubility of about 0.006 to 0.008 ppm.
Mode of Action:
Strong evidence has been accumulated suggesting that avermectins and presumably milbemycins exert their antiparasitic effects by stimulating chloride ion conductance of axons mediated by the putative inhibitory neurotransmitter aminobutyric acid (GABA).
Since GABA is a neurotransmitter in the peripheral nervous system of a large number of nematodes, acarids and insects but is mainly concentrated in the central nervous system of vertebrates, this mode of action can be responsible for the selective toxicity toward parasites, provided that the avermectins do not penetrate the blood brain barrier readily.
Adult Ascaris become immobilized within few minutes upon injection of 1.5 µg of avermectins B1 retaining normal muscular rigidity capable of a reversible muscular tetanus upon injection of 50 µg of acetylcholine. These actions are not readily understood so far and further research on the effects of avermectins is ongoing.
Agricultural Applications of Avermectin:
Avermectin Bi (abamectin) was selected for development of an agricultural pesticide since it is the most potent acaricide and insecticide of the naturally occurring avermectins. It is highly toxic to the two-spotted spider mite at 0.02 to 0.03 ppm and to other mites including the citrus rust mite, citrus red mite and the strawberry mite at 0.02 to 0.24 ppm in laboratory studies.
In a wet formulation, it is active at 125 mg/ha against the imported red fire ant. It also has insecticidal activity against a number of pests of economic importance with somewhat reduced potency against the Southern armyworm and the Corn earworm. In a number of small-scale field tests, this derivative showed excellent control of armyworm species on celery, corn, alfalfa and chrysanthumum and of Corn earworm on soybean at 0.02 lbs/acre.
7. Viruses:
Viruses are strong regulators of insect populations and have often been identified as key determinants of the periodicity of upsurges of pests of such relatively undisturbed habitats as forests and pastures. Habitat disruption by normal agricultural practices generally diminishes the epizootic capacity of insect viruses but nevertheless economically telling epizootics do occur as for instance in Trichoplusia ni on cabbage and Phthorimaea opercullela on potatoes.
Occurrence of Viruses in Insects:
Viruses from eleven separate families occur in insects. Nine of these families include viruses with vertebrate hosts and it is unlikely that their insect-pathogenic members will be considered for use as pesticides until their possible impact on vertebrates has been better explored. The remaining two families are the Nudaurelia β viruses and the Baculoviridae of which the latter appear to be most frequent and to have the better characteristics for employed as pesticides.
Baculoviruses (Bvs):
Baculovirus infections are common among Lepidoptera and Hymenoptera sp., when larvae eat food contaminated with virus containing inclusions, they get infected. Once infected, these viruses have the ability to multiply in their host, sometimes keeping the target pest at a low level for several years making further control measures unnecessary. The bioactivity of baculovirus is attributed to the matrix protein hedrin, which dissolve in the dissolves in the insect gut releasing virus particles, which infect and multiply in gut epithelial cells.
In lepidopteran insects the infection quickly spreads to other tissues. Since the larvae are killed after three or more days after infection, slow rate of their kill is the only limitation. Like Bt, baculoviruses are also non-hazardous to other mammals, fish and wildlife. Being host-specific, the non-target organisms such as beneficial insects and other anthropods remain unharmed.
The virus particles are rod-shaped (250 m diameter) and the genome consists of circular double-stranded supercoiled DNA.
There are three sub-groups:
1. The nuclear polyhedrosis viruses (NPV) in which groups of virus particles are occupied in proteinaceous polyhedral inclusion bodies (PIBs).
2. The granulosis viruses (GV) where the ovoid inclusion bodies each contain only one virion.
3. The Oryctes types where no inclusion body is formed. Inclusion bodies are virus coded.
NPV can be used against cotton bollworm and tobacco bud worms at the rate 140-250 h/ha. The wettable of NPV developed by Sandoz (India) Ltd. contains at least 4 million polyhedral inclusion bodies (PIB) of Heliothis NPV per g of product.
The major problems with Bt and viral preparation are that they are systemic in nature, selective, less persistent and do not act on contact. However, being specific biodegradable, cost effective and of increased safety to environment, such products hold much promise. Moreover, with appropriate biotechnological and genetic engineering techniques such as mutations, gene cloning and process optimization, it should be possible to develop useful new strains.
With the help of these modern techniques, it should also be possible to improve, stabilize and deliver baculoviruses and Bt toxins to control wide varieties of insect species. Metarhizium amisopilae, fungi has been used effectively at 1011-1012 spores/ha against Cosmopolites sordidus, Lissorptnus brevirostris, Mocis sp, Plutella xylostella and Galleria mellonella in Cuba.
Time and Location and Mechanism of Viral Infection:
The prime susceptible life stage is the larva, which is infected following ingestion of virus. Under the alkaline conditions in the gut inclusion body protein disintegrates and releases virions to infect midgut secretary cells and other tissues. Inclusion body production is commonly 1 x 109 (NPV) and 1 x 1011 (GV) per larva whilst LD50s for neonate larvae can be.
Some very broad generalizations can be made about dosages. For instance the numbers of NPV PIBs currently required per hectare per year are, cotton – 1.5 x 1013; other field crops (cabbages, clover, maize, etc.) – 1.0 x 1013; broadleaved trees – 5 x 1012; coniferous trees – 4.5 x 1011 (Lepidoptera) and 5 x 1010 (sawflies).
The ultimate validity of these averages depends on future developments in formulation and application with reference to both viruses and other insect pathogens. Methods of use range from classical biological control to spraying which is the most effective and by which research during the past 30 years has shown it is possible to control over 40 species of Lepidoptera and diprionid sawflies.
BVs are particulate entities not amenable to the considerable dilutions at which many soluble chemical insecticides can be used so that for even the most infective-viruses this imposes constraints on spray volume and droplet size. Spray techniques should aim to achieve optimal coverage of neonate larval feeding sites.
Economic consideration in the use of BVs is the cost of their production. It is possible to culture viruses in vivo in many Lepidoptera, which can be fed on semi-synthetic diets under controlled and sterile conditions. Such a methodology can be industrialized as has been done for the NPV of Heliothis spp., cotton bollworm, and is being done for C. pomonella GV. This can reduce high original cost of production.
Neodiprion Sertifer Nuclear Polyhedrosis Virus:
Dr. Entwistle and coworkers of NERC Institute of Virology (Oxford) characterized the action of Neodiprion Sertifer Nuclear Polyhedrosis Virus (NPVs). The scientists of the U.S. Forest Service also did similar work including toxicological studies of the virus. Dr. Entwistle developed a product named VIROX after the telex number of the Institute. There are many more viruses that are known to be active against insects that can be produced and commercialized as cost- effective pest control agents.
One of the key reasons for the lack of cost- effectiveness is that viruses must usually be produced in vivo, i.e., in the host. The biotechnical revolution would be making a major contribution to insect control; if a way could be found to reduce viral production costs sufficiently to allow the use of larger, more effective quantities.
At present multiply enveloped NPVs (in which several nucleo-capsids share a common virus membrane) grow best in cell culture but few singly enveloped ones and GVs can be so cultured. Media development is required especially to find a replacement for expensive foetal bovine serum. Despite these problems a recent evaluation viewed the possibility of achieving economic in vitro production very favourably.
Use of new sensitive and effective detection methods have developed potential microbial pesticides from viruses. Toxicological studies that would have been impossible several years ago because they involved attempts to recover the virus from animal tissues using a bioassay as a detection method, now is relatively simple using DNA and other probes.
Substitution of persistent chemical pesticides with biocides offer the most desirable solution to the problem of preservation of terrestrial and aquatic ecology in the face of bio-resistance, bioaccumulation and bio-magnification associated with the use of synthetic and organic pesticides.