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close this bookControlling Insect Pests of Stored Products Using Insect Growth Regulators and Insecticides of Microbial Origin (NRI, 1994)
close this folderSection 4: Microbial control
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View the documentInsect viruses
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View the documentProtozoa
View the documentFungi

Bacteria

Bacillus thuringiensis
Pseudomonas syringae
Other bacterial species

Bacillus thuringiensis

Description

(Abbott Laboratories, Solvay Duphar B.V., Novo Ind., Sandoz and Mycogen)

Toxicology Acute oral LD50 for rats Javelin >5000 mg/kg Thuricide > 13 000 mg/kg

Bacillus thuringiensis (BT) is a gram-positive, peritrichously flagellated rod-shaped bacterium which produces a parasporal crystal during sporulation. When ingested, this proteinaceous crystal is responsible for the toxic effect in susceptible Lepidoptera, Diptera and Coleoptera larvae.

The several isolates of BT are placed under 14 serotypes based on their flagellar or 'H' antigenic properties. These serotypes are divided into 19 varieties (Subramanyam and Cutkomp, 1985). All the varieties produce crystals which differ in shape and insecticidal potency. At least eight varieties of BT have been recovered from stored-product moth larvae following natural infections. These are as follows:

Larval source

BT variety

Corcyra cephalonica

var. galleriae

Ephestia cautella

var. kenyae

E. elutella

var.kurstaki morrisoni

E. kuehniella

var. kurstaki morrisoni

E. kuehniella

var. thuringiensis

Plodia interpunctella

var. galleriae

P. interpunctella

var. subtoxicus

Nemopogan granella

var. tolworthi

The histological symptoms in the infected host are enlargement, distension and disintegration of the midgut epithelial cells. Pathogenic spores germinate in the gut and the multiplying vegetative bacterial rods invade the haemocoel producing toxaemia and septicaemia. The external signs of the disease are larval sluggishness, flaccidity and dark brown spots on the cuticle. Cadavers which have turned dark brown are filled with bacterial spores.

Toxicity data for BT obtained from a single strain of a stored-grain moth species cannot be extrapolated to other populations of the same species with any degree of confidence. It is particularly necessary to evaluate the toxicity to local populations before control recommendations can be made. The dosage required to achieve a kill increases with larval age; early instar larvae are the most susceptible.

Commercial preparations

Commercial preparations of BT var. kurstaki have been developed for the control of Lepidoptera. BT can be applied as a wettable powder (WP), liquid formulation or dust. It was initially granted approval in the US in 1979 as Dipel WP for controlling moth infestations, particularly Ephestia cautella and Plodia interpunctella in stored grains and soya beans. Approval was subsequently extended to other formulations and for use on other commodities.

In 1988 the global retail insecticide market was estimated to be US $ 6075 million, of which BT sales were believed to be less than 1%. BT was used mostly in forestry, vegetables, maize production and public health, and sales consisted mainly of the whole organism (such as the product Dibeta) rather than the isolated toxin (Jutsum et al., 1989). BT sales are expected to increase in the future, mainly because of the development of new products with increased potency and a broader host spectrum. In 1990 it was possible to register a new BT product in the US in less than one year and for less than US $ 300,000.

Fermentation processes have been widely used for the commercial production of bacteria. Initially, semi-solid fermentation was used but this has largely been abandoned and replaced by deep-tank liquid fermentation. Unfortunately, this method is expensive in terms of initial capital investment and operational costs. However, careful monitoring of physical parameters during the fermentation process enables product quality to be maintained. Media can also be adjusted to optimize the quality and quantity of the active ingredient produced. The final potency and crystal toxin yield in BT fermentation beers is influenced by various factors. Genetically related strains, grown in the same medium under identical conditions, can produce different by-products and widely different yields. To ensure product consistency the growth medium and fermentation conditions must be carefully defined. Overall, the cost effectiveness of the process is governed by the cost of the medium relative to productivity as measured by the amount of toxin protein produced. In the US, all BT products must be labelled for their delta-toxin (active ingredient) content as a percentage of the total ingredients (Daoust, 1990).

Formulations developed by different companies can vary in toxicity. Toxicity is also influenced by the commodity to which the formulation is applied. Dust and wettable powder formulations can be used for either crop seed or stored food grain. The liquid formulations are easily applied in water, but in the US, their use is frequently limited to seed for planting. The dust consists of 5 g of formulation/kg of wheat flour. Generally, the formulation is mixed with the grain in augers, or other handling equipment, as the last layer of grain is elevated into the storage bin; alternatively, it is raked into the surface of the grain bulk. In either case, the recommended depth for effective control of Ephestia cautella and Plodia interpunctella is 10 cm. Both methods are labour intensive and alternative application means are being sought.

Field trials

The application of BT dust using high-velocity grain drying fans to draw airborne dust downwards from the overspace onto the grain bulk, has been assessed in grain bins at farm level. The initial trials proved promising; at the normal rate of air flow, 25% of the dust penetrated 2.5-12.5 cm into the corn and prevented infestation by Plodia interpunctella (McGaughey, 1986). Further testing was considered to be necessary, and it was suggested that the method may be more effective in commodities with large kernels or pods (McGaughey, 1987).

BT is compatible with most other protectants, seed fungicides and fumigants, but not methyl bromide. BT deposits remain active on grain indefinitely except at very high temperatures. In stores, they are usually protected from solar radiation as the ultra-violet (UV) content would cause rapid loss in activity.

Toxicity

BT is rated as a safe microbial insecticide which is harmless to vertebrates including man, and also harmless to beneficial insects such as bees. In the US, commercial BT is placed under the lowest toxicity category of the EPA, and an LD50 for rats has not been established. It is exempt from residue tolerances on all raw agricultural commodities in the US.
There have, however, been two recorded instances of mammalian toxicity associated with BT application. Various abnormalities were observed in sheep which had been fed on maize treated with 250 and 500 mg of formulation/kg. Endocardial and myocardial haemorrhages, and lesions in the heart, liver and lungs were reported. Histological examination revealed the presence of bacterial rods, subsequently identified as BT in the infected organs. It was thought that an inert material in the formulation may have created a route of entry for the bacteria, and further studies using pure spores were recommended (Subramanyam and Cutcomp, 1985). The second instance concerned a labourer who, when applying Dipel for the control of Lepidoptera, accidently splashed the formulation into his eye. A corneal ulcer developed which required treatment with gentamicin to cure the infection. Eye protection was recommended as a safety procedure for operators (Samples and Buettner, 1983).

High residues of BT on grain are not thought to pose toxic or physical problems as grain processing eliminates most of the spores (Subramanyam and Cutcomp, 1985).

Development of insect resistance

Resistance to BT developed in the laboratory amongst strains of Plodia interpunctella and Ephestia cautella reared on a treated diet. Resistance in P. interpunctella strains varied from double to 29-fold within three generations, and from 15-fold to 100-fold in 40 generations, under relatively low selection pressure. By contrast, resistance in E. cautella increased only seven-fold in 21 generations. Resistance was stable if selection was discontinued when resistance levels reached a plateau, but it declined if selection was discontinued earlier. Resistance was considered to be a partially recessive characteristic.

The ability of these moths to develop resistance, and the speed with which it developed in laboratory trials, caused much concern. Many scientists had believed that resistance to the spore and endotoxin was unlikely, but this trial showed that it could occur in one storage season in the US (McGaughey and Beeman, 1988)

Chiang et al. (1986) examined the defence reaction of midgut cells of Corcyra cephalonica during an infection using scanning and sectioning techniques. They found that following an infection, the epithelial cells become loose, the columnar cells swell, and new cells develop in the basal portion of the epithelium. A protective mucous layer covers the surface of the epithelium cells and thus protects the new cells from toxic attack. These defence mechanisms of the midgut cells prolonged the life span of the infected larvae.

Subsequent work has shown that the host spectrum and potency of BT isolates differs extensively. As many isolates have yet to be fully examined, it is thought that their introduction might overcome the short-term problems encountered if resistance develops in stored grain treatments (McGaughey, 1987).

BT screening programmes
Many major agrochemical companies have undertaken massive screening programmes to search for natural isolates which show better intrinsic activity and a broader spectrum of activity. An initial screening carried out by ZENECA Agrochemicals of more than 500 strains isolated from soil, insects, and grain samples, led to the isolation of the B. thuringiensis var. kurstaki strain (A20) which showed enhanced activity against various Lepidoptera of forestry and agricultural importance (Jutsum et al., 1989).

The discovery of a natural plasmid transfer system by Gonzales and Carlton (1982) led to the production of new BT strains with improved intrinsic activity and spectrum. This system also allowed the transfer of lepidopteran-active crystal genes into coleopteran-active BT strains for the generation of new hybrid clones active against both orders.

A new strain of BT belonging to the pathotype C was isolated from Tenebrio molitor by Krieg et al. (1983) and identified as belonging to a new subspecies, B. thuringiensis var. tenebrionis. It was hoped that this subspecies would have potential for controlling coleopteran pests (McGaughey, 1987). However, no further references to the subspecies and its effects on stored product pests have been found.

Many organizations, such as CINVESTAV in Mexico, are attempting to isolate BT strains for the control of stored-product Coleoptera. In 1988-89, CSIRO in Australia isolated 200 samples of BT; these were screened against Tribolium castaneum to find a bacterium effective against stored-product coleopteran pests (Beckett,1989).

B. thuringiensis is frequently indistinguishable from B. cereus by DNA/DNA hybridization and immunological assays. The characteristic which distinguishes the two species is the toxic, proteinaceous crystal produced only in B. thuringiensis during sporulation. One of the difficulties of using the crystal as a taxonomic trait is that it is an unstable characteristic which is normally coded for on a plasmid. When the ability to synthesize the parasporal crystal is lost, B. thuringiensis is indistinguishable from B. cereus. These plasmids are also capable of being transmitted to B. cereus strains, thereby converting B. cereus to a crystal-producing phenotype. These features have led several authorities to suggest that B. thuringiensis should be regarded as a variety of B. cereus (Kawanishi and Held, 1990).

Conclusion

The use of BT against storage pests has considerable potential and application of BT is a registered method for the control of lepidopteran storage pests. However, it has been shown in the laboratory that these pests can develop resistance to B. thuringiensis.

Therefore, the priority for the future is the adoption of an integrated pest management (IPM) programme which includes the use of BT where and when appropriate, and which is closely supervised by the authorities for resistance monitoring.

Pseudomonas syringae

The ability of ice-nucleating bacteria to reduce the cold hardiness of stored product pests has recently been exploited. The use of low temperatures to control stored product pests has been extensively studied because of the potential benefits to countries with low winter temperatures.

Laboratory efficacy experiments

Fields (1991 ) carried out preliminary tests to investigate the potential of Pseudomonas syringae which is used commercially in snow-making equipment at ski resorts. P. syringae (strain 31 a) is a common foliar bacterium isolated from maize leaves. It can be grown under conditions which maximize its ice-nucleating activity and then concentrated, freeze dried and killed with electron beam irradiation.

Pellets of P. syringae at 10, 100 and 1000 ppm were added to 8 g of wheat. Groups of 100 cold-acclimated or non-cold acclimated Cryptolestes ferrugineus were then added to the wheat and held at a range of temperatures (-10°C to -30°C) for various times. P. syringae greatly reduced the cold-hardiness of non-cold acclimated C. ferrugineus adults. Increasing the concentration of P. syringae raised the supercooling points of the treated insects; this reduced their tolerance of sub-zero temperatures and, therefore, increased cold-induced mortality. In insects treated with 100 and 1000 ppm of P. syringae, mortality of cold-acclimatized adults held at -10°C for 21 days was 61% and 64%, respectively, compared with 45% in the controls.

Toxicity

P. syringae has been used commercially for snow-making and toxicity data have been established. The live end-product has been shown to be non-toxic, with an acute oral LD50 for rats greater than 5 g/kg. It is non-pathogenic to other mammals and plants.

Conclusion

These preliminary trials indicate that P. syringae has potential for reducing the cold-hardiness of insect pests of stored products. However, practical applications would be limited to those situations where stored grain can be cooled in winter to sub-zero temperatures.

Other bacterial species

Other bacterial species isolated from the gut of Tribolium castaneum have been identified and examined for their pathogenicity. The four asporogenous species, Enterobacter aerogenes, E. cloacae, Proteus vulgaris and P. mirabilis, and two sporeformers, Bacillus subtilis and B. cereus, were administered orally to T. castaneum larvae by the diet dilution technique (at 0.01 ml of 1.0 optical density units). The rate of infectivity in terms of mortality was as follows: E. aerogenes and E. cloacae (94.3%); B. cereus (91.2%); the rest were below 50%. Larvae which survived the infection developed into adults. The three named bacteria were considered to be potential control agents by Kumari and Neelgund (1985).