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In this article we will discuss about the protection of crops by the use of bio-pesticides.
1. Microbial Herbicides:
The use of endemic or exotic plant pathogens to kill weeds is the efforts of phytopathologists. The microbial origin of herbicides is definitely a major contribution and an alternative to chemical weedicides.
Some of the successes with exotic pathogens have been the use of Puccinia chondrillina from Southern Europe to control skeleton weed (Chondrilla juncea) in Australia, while the use of Cercosporella riparia to control Ageratina riparia introduced in Hawaii from Jamaica, and the use of introduced rust Phragmidium violaceum to control wild black berry (Rubus spp.).
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Similarly, the use of Cercospora rodmanii to control water hyacinth and Colletotrichum gloeosporioides to control Aeschynomene virginica, the host specific pathotype Phytophthora citrophthora to control milkweed vine (Morreria odorate) have been established. Such type of weedicides needs a close cooperation, whereas the use of endemic microbial herbicides needs cooperation from various agencies.
2. Bacterial Insecticides:
Bacteria are often associated with plant and human diseases. However, there are certain bacteria such as Clostridium acetobutylicum (acetone-butanol production), Bacillus licheniformis (antibiotic- bacitracin production), B. megaterium (vitamin B12 production) and Pseudomonas fluorescens (2-ketogluconate) are some of them which are used for industrial product formation.
In addition, there are certain bacteria which are of immense importance to mankind. They are pathogenic to insects, pests and other pathogens and kill these wide range of parasitic organisms.
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The majority of bacteria isolated from insects can be regarded as facultative pathogens. Interests in the use of bacteria as biological control agent was stimulated by an increasing number of insects and pests. Some of the bacteria which are being used as bacterial insecticides are listed in Table 34.3.
(i) Pseudomonads as Bacterial Insecticides:
Pseudomonas aeruginosa is among the most frequently described pathogen causing disease in insects. It is still unknown that certain strains pathogenic to insects by feeding differ from those that are potentially pathogenic to man. P. aeruginosa that produces toxic enzymes has also been used as a model organism to study the mechanism of insect pathogenicity and immunity.
Non-fluorescent pseudomonads isolated from insects include Pseudomonas alcaligens, P. cepacia, P. maltophila and P. acidovorans. Aeromonads occur in laboratories devoted to the culture of insects in aquatic habitat. Some aeromonads such as Aeromonas hydrophila and A. formicans are pathogenic to insects.
Pseudomonas cepacia is known to be a versatile bacterium of soil, as a plant pathogen and a human pathogen as well as a broad spectrum antagonist to plant pathogens through the production of various types of antibiotics such as pyrrolnitrin. P. cepacia is a Gram-negative bacterium that has been reported to produce siderophores. This bacterium also acts as plant growth promoting rhizobacteria (PGPR).
Suppression of plant diseases may involve secretion of siderophores or antibiotics and/or aggressive root colonization by organisms that displace or exclude deleterious rhizosphere microorganisms.
Pseudomonas fluorescens is one of the most important biological control agents of many plant disease causing organisms. These are also common PGPR that secrete siderophores. Seed inoculation with these organisms helps in inducing growth and suppression of diseases.
It produces fluorescent siderophores called pyoverdine or pseudobactrin which is characteristic of the fluorescent pseudomonads. The siderophores are low molecular-mass, water soluble, high affinity Fe (III) chelators.
Siderophores are secreted under iron-limiting conditions as a means to secure available iron present at low concentration in soil. The ability of certain pseudomonads to utilize a wide range of ferric siderophores as a source of metabolic iron may contribute to their competitiveness and survival in the soil.
(ii) Bacillus Species as Bacterial Insecticides:
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(a) Bacillus Thuringiensis:
This bacterium is now widely known as Bt. It is the most important bacillii reported to kill a wide range of insects like moths, beetle, mosquitoes, flies, aphids, insects, ants, termites, midges, butterflies even some pathogenic fungi such as Pythium ultimum and Fusarium oxysporum f.sp. lycopersici, depending upon the host strains of the bacterium. Some of the Bt strains are pathogenic to cockroaches, snails and protozoans.
Due to a wide range of host killing, Bt occupies a tremendous significance in agriculture due to exo- and endo-toxin production and its related ease of mass production in submerged fermentation on relatively cheap media besides many chemical compounds designed for use in controlling economically and biomedically important insects.
The Bt bacterium was first discovered by a Japanese scientist Ishiwata in 1902 who isolated a bacterium from diseased or unhealthy silk larvae, and was named as Bacillus satto. In the year 1912, a German microbiologist also isolated this bacterium from infected insects from a flour mill.
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He named the bacterium which caused the insect disease, as Bacillus thuringiensis. After a gap of 50 years, the importance of this organism was realized and development of commercial formulation of Bt as biopesticide in USA was carried out in 1960. Now hundreds of Bt strains are commercially available round the world and all the major insect pests are susceptible to these strains.
The mechanism of action of Bt endotoxins on insect is quite interesting. The endotoxin is the protein. The crystalline proteins upon ingestion by the insect larvae are solubilized under highly alkaline conditions prevalent in the midgut.
The toxins are digested by the enzymes, called proteases into active fragments. These active fragments bind to receptor proteins present in the gut epithelial membrane. Upon binding, the toxin molecules form pores in the gut membrane. As a result the osmotic equilibrium of the cell is disturbed, the cells swell and burst. This results into the death of insect larvae.
Now, more than 50 Bt genes have been isolated, cloned and characterized. Making use of microorganisms and genetic engineering, transgenic microbes and transgenic plants are developed.
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Many crop plants are colonized by harmless bacteria. Such bacteria are identified and genetically transformed by vectors carrying Bt genes. These bacterial formulations are then sprayed on the crop which provide a protective cover to the crop.
Certain crop plants are also being protected against insects by genetic mediation i.e. transgenic plants (resistant to insects) with Bt genes for example bollworm resistant cotton, stem borer resistant rice, com borer resistant maize, potato beetle and tuber moth resistant potato, tomato resistant to pinworm, etc. At IARI, New Delhi, Bt transgenic cabbage and cauliflower plants have been developed.
Apart from crop plants, many forest tree species are also being transformed using Bt genes. Now Bt technology is being used in expressing two different kinds of Bt genes in transgenic plants or microorganisms. This technique helps in preventing insects from developing resistance to Bt toxin proteins.
Bt formulations are being used to control Aedes and Anopheles mosquitoes, black fly which spread yellow fever, malaria and blindness respectively.
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Now-a-days Bt genes are being expressed in aquatic bacteria and cyanobacteria so that during their propagation in lakes, the breeding of mosquito could be checked. Scientists in Anna University, Chennai are engaged in research on Bt. Bt strains are also being developed towards killing of liver flukes, tapeworms, etc. which cause disease in cattle and human.
Bt is produced commercially in the form of powder the composition of which is given in Table 34.4.
All the ingredients are mixed to form powder thoroughly; the powder was administered to the insects by mixing it into their diet, with larvae being allowed to feed on the powder diet mixtures. The effect of this exposure was judged by a single criterion, death.
The calculations of potentials of dry powders of δ-endotoxins of B. thuringiensis are given below:
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LC50 Standard/ LC50 test sample × Potency of standard (IU/mg)
(b) Toxins Produced by B. thuringiensis:
Bt produces several toxins, four of which will be considered here: α-exotoxin (heat- labile toxin); β-exotoxin (fly-factor or heat-stable exotoxins); δ-endotoxin (crystalline toxin or just crystal or parasporal body) and louse factor.
The α-exotoxin identifies as lecitherase c water soluble, heat labile and toxic to insect while, β- exotoxin defined as adenine nucleotide and ATP analogue and given the name “thuringiensin” which is water soluble heat-stable toxin. The 6-endotoxin in the crystal of Bt has a limited activity spectrum. It is produced during sporulation.
In 1974, Ginrich et al reported that four species of mammal – biting lice were susceptible to powders containing the spore endotoxin complex of Bt-var Kurstaki (HD-1), an isolate of Bt that does not produce β-exotoxin, a toxin to which these lice are susceptible. It seems improbable that the endotoxin could be responsible for the action. Hence, the toxicity to lice was due to a new chemical which was called the “louse-factor”.
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(c) The Other Bacteria which Control Insects:
Among the species of Bacillus, there are other obligate spore-forming bacteria such as Bacillus papilliae and B. lentimorbus, facultative spore former such as B. cereus which are pathogenic to insect.
The control of Japanese beetle by inducing milky disease with B. lentimorbus and B. papilliae has been reported. E. coli is reported from flies as surface contaminant. The strains of E. blattae have been isolated from cockroach. Strains of Enterobacter, Serratia and Klebsiella are frequently reported as facultative insect pathogens.
Serratia liquefaciens and S. marcescens control lab insects in culture due to unsatisfactory nutrition and physical conditions in the maintenance. Alcaligenes odorans has been isolated from insects where this bacteria acts as facultative pathogen.
(d) Bacteria associated with nematodes:
Relationship between the insect (host) and the nematode gut has been studied by Poiner (1979). Nematode larvae entering an insect body were reported to cause septicaemia by dissolving the insect gut wall and thus introducing bacteria to the haemolymph.
An entomocidal (insect-killing) bacterium named Achromobacter nematophilus was described initially from the nematode Neoplectana carpocapsae and regarded as a commensal symbiont of the nematode. The nematode genera, Neoplectana and Heterorhabditis carry in its gut bacterial species named Xenorhabdus nematophilus and X. fluorescens belong to family Entero- bacteriaceae. Even one bacterium from each species is enough to kill insect.
The advantages of using bacterial insecticides are:
(i) Permanency,
(ii) Economic feasibility due to low cost,
(iii) Environmental safety, and
(iv) Absence of development of resistance.
3. Virus Insecticides:
There are viruses or their products commercially exploited in place of chemical insecticides. Viruses of the family Baculoviridae are pathogenic to arthropods. Viruses contain lipid envelope with circular, double stranded DNA genome of 80-150 Kilo base pairs depending upon the viruses.
Naked viral DNA is infectious per se. Baculoviruses are restricted in their host ranges. They do not infect vertebrates, non-arthropod invertebrates, microorganisms or plants. Indeed baculoviruses infect only a few arthropod species. Their use dates back to the 19th century. But the commercial use of virus insecticides has been limited by their high specificity (limited host range) and slow action.
Viruses most frequently considered for control of insects (usually saw flies and lepidoptera) are the occluded viruses, namely NPV, cytoplasmic polyhedrosis (CPV), granulosis (GV) and entomopox viruses (EPN).
4. Entomopathogenic Fungi:
Many entomopathogenic fungi overcome their hosts only after limited growth in the haemocoel so toxins are presumed to cause host death. The importance of toxins to the virulence of an entomopathogenic fungus is difficult to evaluate.
Because toxin production in the host must be preceded by following activities:
(i) Attachment of the infective unit on the cuticle,
(ii) Germination of infective unit,
(iii) Penetration followed by formation of germ tube and appearance of infection pegs from appressoria,
(iv) Multiplication of hyphal bodies in the haemocoel,
(v) Production of toxic metabolites,
(vi) Death of the host,
(vii) Growth in the mycelial phase with invasion of all host organs,
(viii) Penetration of hyphae from the interior through the cuticle to the exterior of the insect,
(ix) Production of infective units on the exterior of the insects.
(a) Metarhizium Anisopliae:
This fungus produces destruxin B and desmethyl destruxin B in silk worm larvae. These toxins are referred to as vivotoxins. In addition to low molecular weight compounds, proteases and other enzymes are produced by entomopathogenic fungi. Injection of Entomophthora species cultures into Galleria mellonella larvae caused blackening similar to that noted in infected larvae.
Metarhizium culture filtrate is toxic to coleoptera haemocytes in vitro producing changes in organelles. The extract of mycelium is toxic if it comes in the contact of adult house fly.
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The solvent extracts of Metarhizium kill silk worms if it is injected into intrahaemocoelic injection. Six cyclodepsipeptides with five member amino acids viz., β-alanine, alanine, valine, isoleucine and proline have been isolated from filtrates of M anisopliae cultures.
Cytichalasins are the other fungal metabolites. Their origin is from phenylalanine or tryptophan linked to C14 or C18 polyketide chain. Their activity includes inhibition of cytoplasmic cleavage in cultured mammalian cells.
(b) Beauveria Bassiana and B. Brongriartii:
B. bassiana and B. brongriartii are the other fungal species that attack insects. Beauvericin is a depsipeptide. It comprises of a cyclic repeating sequence of 3 molecules of N-methyl phenylalanine alternating with three molecules of 2-hydroxy isovaleric acid. It has also been isolated from mycelium of Paecilomyces fumosoroseus.
Beauverolides and bassianolide are the other cyclodepsipeptides. The beauverolides H and I were isolated from B. bassiana, while bassianolide which is composed of 4 molecules each of L-N methyl leucine and D-α-hydroxy isovaleric acid has been isolated from B. brongriartii.
The isorolides A, B, and C are also cyclodepsipeptides which were found in B. brongriartii. Two very similar pigments, tanellin and bassianin, produced in vitro by some strains of both B.bassiana and B. brongriartii are concentrated in the mycelium rather than released into the medium.
The dibenzoquinone pigment oosporein, produced by many isolates of Beauveria, probably accounts for the reddish colour of infected caterpillars. B. brongriartii converts 20% of the original solids in a peptone medium into oxalic acid. This acid is a general poison. Oxalate crystals have been noted on the surface of insects killed by B. bassana.
(c) Verticillium Lecanii:
V. lecanii is non-fastidious and can grow on all conventional media meant for culturing of fungi. The most frequently recorded hosts are scale insects and aphids. Much less reports of hosts have been recorded in other orders. V. lecanii sometimes hyperparasitizes phytopathogenic fungi, mostly rusts and powdery mildews.
The control of aphids and seales is possible by using V. lecanii conidia or blastospores suspended in phosphate buffer containing 0.02% Triton X-100 as wetting agent. After evening sprays, plants were covered with polythene blackout sheets to restrict day length for flower initiation.
The appraisal of V. lecanii as a microbial insecticide is based on the choice of infectious material i.e. between conidia and blastospores. Production of conidia on agar is too expensive and also difficult to ensure culture purity. Alternatively, the conidia can be produced on a cheap granular solid substrate. V. lecanii is reported to be a promising biological control agent against aphids of chrysenthemum in green houses.
(d) Hirsutella Thompsonii:
H. thompsonii a deuteromycetous fungus is a potential killer of citrus rust mite (Phyllocoptrula oleivora). Most of the species are pathogenic to invertebrates. The fungal strains have also been reported from the citrus bud mite, Eriophyes sheldone and coconut flower mite, E. guerreronis.
In the USA, a commercial formulation of conidia has been introduced by Abbott Laboratories, North Chicago, Illinois. H. thompsonii can be cultured on agar media.
The spore suspension is mixed in semisolid medium containing wheat bran (60 g) and distilled water (60 ml) to which 250 ppm per litre of chloramphenicol is added. The flasks are incubated at 25°C for 2 weeks to get bran- fungal mat. It is air dried and blended, finally stored at 18-20°C.
(e) Nomuraea Rileyii:
It is the Ascomycetous fungus for which first effort of mass-production was made for applying as entomopathogenic agent. This fungus usually is found to induce extensive epizootics in caterpillar pests on cabbage, clover, soybean and velvet beans and thus is a potential agent for use as a microbial insecticide.
The natural epizootics were observed on Bombyx mori, Peridroma saucia, Leptinotarsa decemlineata. The unidentified compounds were extracted from mycelium produced in submerged culture.
(f) The Other Fungi:
Aspergillus ochraceus, Paecilomyces fumosoroseus, Fusarium solani Trichoderma harzianum, T. reesei, Gliocladium virens, etc. have also been reported for other microbial pesticides.
