 |  | Controlling Insect Pests of Stored Products Using Insect Growth Regulators and Insecticides of Microbial Origin (NRI, 1994, 58 p.) | ||
 |  | Section 4: Microbial control | ||
|  | (introduction...) | ||
| | Insect viruses | ||
| | Bacteria | ||
| | Protozoa | ||
| | Fungi | ||
|
| |||||||||||||||||||||||||||||||||||
The concept of microbial control of insects has existed for a long time. Viruses, bacteria, fungi and protozoa have been developed for a broad range of applications by numerous companies, but their use as grain protectants requires further investigation.
The use of naturally occurring viruses for the control of insect pests has long been considered to have great potential, as these viruses should be pest-specific and relatively safe to vertebrates. Recently, their introduction has attracted greater commercial interest due to developments in technology and closer collaboration between the disciplines of molecular biology, genetics, microbiology, and protein and nucleic acid chemistry.
The group of viruses which has attracted the most attention is the Baculoviridae. This group represent about 60% of the 1 100 insect viruses which had been isolated by 1990. Baculoviruses usually infect only a few closely-related insect species and frequently, only one susceptible host species is known (Huber, 1990).
Many obstacles to the use of viruses on stored food have been encountered. One of these has been the ethical question of treating material destined for human or animal consumption with insect pathogenic viruses. The general public often fear microbial agents because of their association with ailments such as the common cold, stomach flu, AIDS, etc. (Falcon, 1990). A prerequisite for the use of microbial control would be the implementation of a public awareness campaign to inform the public of the benefits of this type of control system. A possible by-product of such a campaign could be consumer pressure. This could act as an additional motivator to encourage the agrochemical industry to take more serious action on the development of alternative methods of pest control.
The Indian meal moth, Plodia interpunctella, is attacked by a granulosis virus. The experience gained in adapting this particular virus for commercial application highlights some of the difficulties which have been encountered routinely in the development of viral control methodologies. In 1979, Tinsley reviewed the suitability of viruses for insect pest control. He referred to preliminary trials in which a virus had been used at a rate of 8 billion granules/ml of water/100 g of nuts to produce a mean mortality of 98% on infected almonds and walnuts. This granulosis virus was thought to be worthy of further testing, especially on groundnuts stored in open air bag stacks in tropical countries.
Interest was shown in developing the granulosis virus from P. interpunctella (IMMGV) for use as a protectant in stored dried fruits and nuts. However, semipurified homogenates of larvae infected with the virus lost their infectivity through time, particularly at temperatures above 32°C. A freeze-dried, powdered formulation of IMMGV with a relatively long shelf life was then developed which could be applied as a spray or dust (Cowan et al., 1986).
Vail and Tebbets (1991) developed a modified rearing diet for P. interpunctella omitting glycerol and honey; 10-day old larvae inoculated with the granulosis virus and incubated at 26.7°C for 10 days were able to survive for at least one generation on this diet. The diseased larvae and diet were then harvested, homogenized in sterile distilled water, freeze-dried, and milled to a particle size which could be applied through a spray nozzle or as a dust. The LC99 for this formulation was 1 4 µg of granulosis virus formulation/g of diet.
Vail and Tebbets (1991) considered that the application rate for this formulation in the field should be the LC99, using the upper 95% confidence limits (LC99u95%cl). The estimated cost for an application rate at LC99 [14 µg/g] was US $ 2.45 compared with US $ 4.08 for the LC99u95%cl [25 µg/g]. The estimated production costs for the granulosis formulation were US $ 36 for 200 g, of which US $ 30 was for labour. It was estimated that at an application rate of 14 µg/g, a 200 g production lot would be sufficient to treat 14 000 kg of commodity. The cost of US $ 2.45-4.08 compared favourably with the cost of phosphine fumigation at US $ 2.58-4.98, or modified atmospheres at US $ 4.40-6.80. It was suggested that the formulation could be further improved by adjusting the time of inoculation and the larval densities used for production.
Commercial applications
Commercial trials were carried out on a dried fruit packing line in which the same formulation was applied to raisins. It was found that at 27°C, insect control could be achieved for up to 6 months; however at higher temperatures (32-38°C), efficacy was reduced after 1-3 months of storage (Vail and Tebbets, 1991).
The US Department of Agriculture is currently seeking a patent for a formulation using the granulosis virus of Plodia interpunctella (Falcon, 1990).
Discussion
The advantages and disadvantages of using viruses for insect control need to be examined as they may determine whether viral control of storage pests is a feasible option for the future. Several of these points also apply to other microbial control agents.
(i) Viruses are highly selective and generally species-specific. Unfortunately, this limits the market potential for manufacturers. Also, since most stored product infestations comprise several insect species, there would be a need for several different control techniques.
(ii) Viruses can be applied at very low doses as they will persist and multiply in the pest population to produce long-term control. However, like IGRs, viruses mostly affect the larval stage and are relatively slow acting. This can have serious economic implications if the larval stages of the pest cause the most damage.
(iii) The production techniques are labour intensive, which is a disincentive for commercial pesticide manufacturers. However, the technology involved is thought to be simple and therefore suitable for cottage industry production in Third World countries using local resources. Brazil, Guatemala, Thailand, Columbia and Zimbabwe have already exploited certain viruses for pest control in non-storage situations (Huber, 1 990).
(iv) Preparations have to be registered and subjected to the same regulations as conventional chemical pesticides. This is a very expensive procedure.
Many of the standard tests designed for chemical pesticides may also be inappropriate for viruses (Falcon, 1990).
(v) Naturally-occurring biological control agents cannot be patented which thus preclude the payment of royalties. There is a suggestion, however, that federal governments could establish central microbial pesticide development centres which could mass produce and formulate viruses into standardized experimental products available for distribution to cooperating insect specialists. Registration could then be completed at the centre so that the product could become available for production and marketing by private industries (Huber, 1990).
(vi) An alternative approach would be a 'non-patentable product licensing programme' by which private sector interests could pay fees and thus acquire exclusive rights to the development and marketing of a microbial pesticide for a specified time and for as long as significant development activity was sustained (Falcon, 1990).
A major criticism of virus research to date is that individual research bodies have isolated and selected particular strains from pest species for their own purposes only, so there are no standardized materials to work on.
Conclusion
Commercial use of insect viruses to control stored product pests is in its infancy. However, the success already achieved with the P. interpunctella granulosis virus indicates that if other pathogenic viruses can be isolated from the major insect pests of durable foodstuffs, the method could have considerable potential.
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).
Coccidia
Eugregarines
Neogregarines
Microsporidia
Conclusion
Several groups of Protozoa are of interest as agents for the natural control of insect pests.
Coccidia
Adelina tribolii infects several stored product pests and causes epizootics in laboratory and natural populations of Tribolium confusum (Brooks, 1988).
Eugregarines
Eugregarines are frequently encountered as commensals in the digestive tract of insects. Of those thought to be potentially pathogenic to their hosts, Ascogregarina spp. have received the most attention (Brooks, 1988). A. bostrichidorum has been isolated from Prostephanus truncatus collected in Tanzania. Examination of the gut of heavily infested larvae revealed that they contained masses of cysts and spores. However, the prevalence of infected larvae in the sample of 2 500 insects was only 2%. (Purrini and Keil, 1989).
Neogregarines
Neogregarines occur naturalIy in Lepidoptera, Coleoptera and Orthoptera. Some are highly pathogenic and have been considered as potential control agents against species belonging to these three orders.
Farinocystis tribolii is a parasite of Tribolium destructor, T. molitor, T. castaneum and T. confusum. Farinocystis spp. have also been isolated from Prostephanus truncatus (Schulz and Laborius, 1987). An infection can be spread by the dispersal of spores from dead larvae during the handling or processing of an infested commodity, or by adults feeding on the bodies of the dead larvae. F. tribolii infection results in a slow decline of Tribolium spp. in laboratory cultures. It has also been shown to increase significantly the susceptibility of T. castaneum larvae to the insecticides malathion, chlorpyrifos-methyl, fenvalerate and cypermethrin (Rabindra et al., 1988).
Mattesia trogodermae has been isolated from Trogoderma granarium. This protozoan is cosmopolitan, occurring as a common pathogen of laboratory and natural colonies of Trogoderma spp. (Brooks, 1988). Baits have been used to introduce M. trogodermae into populations of T. glabrum. It has been shown that males surface-contaminated with spores will inoculate females while mating. The efficiency of spore transfer can be increased by releasing a natural female sex pheromone at the site where the males become contaminated.
M. trogodermae is regarded as a potentially useful control agent specific to Trogoderma spp. It is non-pathogenic to vertebrates, and it is relatively easy to produce and isolate in usable quantities (Henry, 1981). Extensive acute oral or acute inhalation tests on M. trogodermae showed no evidence of infection or pathological effects in rats. Tests on non-target species were also negative.
Mattesia spp. have also been detected in Prostephanus truncatus collected from farm-stored maize in Togo. Studies on the distribution and infection rate, which was 1-6% in Togo, indicated that artificial enhancement of the infection source would be necessary for effective control by Mattesia (Leliveldt et al., 1988).
Microsporidia
Nosema species
Nosema spp. have been isolated in Prostephanus truncatus (Schulz and Laborius, 1987). Most published articles however, relate to the use of N. whitei in Tribolium spp.
Laboratory efficacy experiments
Al-Hafidh (1985) investigated the toxicity of N. whitei in first instar larvae of T. castaneum and found the LD50 to be 2.41 million spores/g. It also reduced fecundity and fertility and increased adult mortality. Further investigations into the effects of N. whitei on the physiology and behaviour of T. castaneum confirmed the observed reduction in fecundity and fertility in infected insects (Armstrong and Newton, 1985; Armstrong and Bass, 1986; Khan and Selman, 1988).
Onstad and Maddox (1990) created a simulation model of a T. confusum population infected with N. whitei. The model indicated that the infection could suppress the population to less than 10% of the original number in 300 days. Validation trials (over 60 days) showed that the predicted adult population was correct, but other developmental stages were only predicted accurately for the first 30 days of the 60-day trial.
Conclusion
Several pathogenic protozoan species have been isolated and identified from insect pests of durable foodstuffs. However, their use as control agents is in the early stages of development and requires extensive research before they can be recommended for use as grain protectants.
Beauveria bassiana
Utilization of other fungal
species
Metarhizium anisopliae
Avermectins
Conclusion
Over 400 species of naturally occurring entomopathogenic fungi have been identified. Most do not have to be ingested by the insect to cause death. Fungal spores stick to the surface of the insect, germinate, and send out hyphae which penetrate the cuticle and invade the haemocoel. Death either occurs rapidly, due possibly to the production of complex toxic metabolites, or more slowly, due to hyphal proliferation and disruption of organs. The invading fungus then sporulates and re-enters the ambient environment to establish subsequent infections.
Entomopathogenic fungi were the first micro-organisms to be used as microbial insecticides. Only a few species have been mass produced, generally by government agencies rather than by private industries. Fungi are currently used regularly as microbial insecticides only in a limited number of countries such as Brazil, the former Soviet Union, the former Czechoslovakia, and China. Verticillium lecanii has been registered for use in the UK, and Hirsute//a thompsonii has been registered in the US (McCoy et al., 1988).
Beauveria bassiana
The application of Beauveria bassiana to glasshouse and field crops has been studied extensively. However, few researchers have considered its application for the control of storage pests.
Laboratory efficacy experiments
Laboratory trials were undertaken by Searle and Doberski (1984) to investigate the use of B. bassiana isolated from a soil sample against Oryzacphilus surinamensis. Humidity was found to be more critical than temperature, or inoculation rate. At 100% r.h., infection occurred rapidly within 20 days, whereas at lower humidities very little infection was observed. Tests were also carried out to determine the effect of temperatures between 7° and 25°C. The highest adult mortality occurred at 25°C and 100% r.h.; under these conditions 100% mortality had occurred within 13 days.
It was concluded that in grain stored at, or below, the recommended moisture content of 14% (70% r.h.), the fungus would be unlikely to control O. surinamensis populations.
Commercial applications
A method of surface fermentation has
been developed in the former Czechoslovakia for the mass production of two
preparations of B. bassiana known as Boverol and Boverosil. In trials using
Sitophilus zeamais, Oryzeaphilus surinamensis and Tribolium castaneum, Boverosil
was more effective than Boverol. 7: castaneum was the least susceptible of the
three stored-product pests studied (Frydocva et al., 1989).
The preparation, Boverosil, combined with the insecticide pirimiphosmethyl, has been registered in the former Czechoslovakia for the treatment of empty stores and silos against residual infestations of stored product pests.
Hluchy and Samsinakova (1989) examined the effects of a batch of Boverosil containing 50% dry fungal material and 50% amorphous silica gel (50-1 µg) against adult Sitophilus granarius and larvae of Galleria mellonella. S. granarius was the least susceptible; a dose which produced 50% mortality in G. mellonella larvae produced only 3% mortality in S. granarius adults. The LC50 appeared to be in the range of 2 x 10 8 and 5 x 108 conidia/ml.
In Iraq investigations were carried out to determine inoculation spray rates for Ephestia cautella larvae in stored dates. The results indicated that 300 000-400 000 spores/cm3 were needed to produce 96-98% mortality (Jassim eta/., 1988).
Utilization of other fungal species
Studies by Schulz and Laborius (1987) on natural fungal parasites of Prostephanus truncatus strains from Costa Rica showed that several microfungi can be isolated from dead adults. Taxonomically, all the fungal isolates belonged to the Deuteromycotina; most were species of Aspergillus and Penicillium and included the mycotoxigenic A. flavus.
Pathogenicity and virulence of spore suspensions were tested in adult P. truncatus by topical application. Virulence differed considerably between the isolates; after 4 days of incubation, mortalities varied from 13.3% to 100%. These preliminary experiments highlighted the problems involved in developing techniques using micro-organisms. The potential of fungi for the regulation of natural populations of P. truncatus remains unclear.
Metarhizium anisopliae
Description
(Bayer AG)
Toxicology Acute oral LD50 for rats, >2000 mg/kg
Metarhizium anisopliae is an entomopathogenic fungus with a worldwide distribution. It can be cultivated on both solid and liquid sterile media. An insecticide, code name BlO 1020, has been developed from a wild-type strain. It is produced by a special patented fermentation procedure in the form of pellets which are dried to granules; they have a shelf life of at least six months, particularly if stored at low temperatures. The optimum growth temperature for the fungus is 25°C. The product is very effective against Coleoptera and Lepidoptera, and has been tested against pests of ornamental crops (Reinecke et al., 1990).
Laboratory efficacy experiments
Rodrigues and Pratissoli (1990) carried out small-scale laboratory trials to evaluate the pathogenicity of Beauveria brongniartii and M. anisopliae for Sitophilus zeamais and Acanthoscelides obtectus. Adult insects were dipped in conidial suspensions (10 8 conidia/ml), returned to maize or beans, respectively, and retained at 25°-28°C and 60% r.h. B. brongniarti caused 89% mortality in adult A. obtectus within 15 days and 47% mortality in adult S. zeamais; M. anisopliae caused less than 50% mortality in either species.
Discussion
The major drawbacks to the use of fungi for insect control are thought to be their poor stability in storage situations, and their high dependence, for efficacy, on climatic conditions in agricultural situations (Kirschbaum, 1985). Commercial formulations cannot be stored at room temperature so must either be shipped fresh after manufacture or stored under refrigeration, both of which may prove difficult.
A successful rate of infection depends on spore germination. This requires optimum temperatures and relative humidities in excess of 80%. Consequently, fungi are only regarded as suitable for application in humid tropical climates and greenhouse situations. If the problems of temperature and moisture requirement necessary for conidial discharge and spore germination could be overcome, use of fungi might be a practical method for controlling insects in stored products.
Avermectins
Description
(Merck Sharp & Dohme Research Laboratories)
Toxicology Acute oral LD50 for rats, >5.0 g/kg Acute dermal LD50 for rats, >2.0 g/kg
The avermectins are a mixture of natural products produced by a soil actinomycete, Streptomyces avermitilis. They are a family of macrocyclic lactones which consist primarily of four major components (A1 a, A2a, B1 a, B2a) and four homologous minor components (A1 b, A2b, B1 b, B2b). Avermectins designated as A1, A2, B1 and B2 refer to mixtures of the homologous pairs containing at least 80% of the major component.
The avermectins have nematicidal, acaricidal and insecticidal activity. Their insecticidal properties were first demonstrated in laboratory assays against Tribolium confusum. On the basis of its high intrinsic toxicity to arthropods compared to the other natural avermectins and synthetic variants, avermectin B1 was selected for crop protection purposes (Lasota and Dybas, 1991).
The selective toxicity of avermectins to specific invertebrates is believed to be due at least partially to the differential distribution of gamma-aminobutyric acid (GABAergic) neurons which, in mammals, are restricted to the central nervous system (Lasota and Dybas, 1991 ).
Laboratory efficacy experiments
Beeman and Speirs (1984) carried out tests with avermectin B1 against a range of storage pests. It caused 100% mortality in parent adult Sitophilus oryzae, Rhyzopertha dominica and Oryzacphilus surinamensis exposed to a dose of 320 ppb on wheat. Tribolium castaneum was more tolerant; at a dose of 2.6 ppm, only 36% mortality occurred although at 160 ppb the insects appeared sluggish.
Suppression of F1 progeny was achieved at doses of 10 ppb in Sitotroga cerealella, 20 ppb in R. dominica, 160 ppb in S. oryzae and O. surinamensis, and 640 ppb in Plodia interpunctella. The half-life decay for avermectin B1 on wheat at 26.7°C and 60% r.h. was 3-6 months.
Commercial application of avermectins
Annual sales of the isolated avermectin toxin and its analogues are worth about US $ 200 million. However, sales are primarily for the control of veterinary pests and only limited use is made of the products for insect control in pre-harvest crops. Avermectin research has largely been confined to investigations using the entire organism of Streptomyces avermitilis for the production of avermectins, or using the related S. bikokenkinki, for the production of milbemycins. Attempts to synthesize new avermectin-like compounds have proved unsuccessful. However, chemical modification of the natural toxins has led to commercial success with 22,23 dihydroavermectin B1 which is used in the animal health sector (Jutsum et al., 1989).
In 1983, the high cost of avermectins and their high level of mammalian toxicity, precluded them from registration as stored grain protectants. Since then, however, their use in veterinary and public health areas has been pursued, and avermectin has been registered in the US as an outdoor control agent for the imported fire ant. In 1986, when the product Affirm was registered, technical avermectin was considered to be highly toxic to birds, fish, aquatic invertebrates and mammals. However, its use was approved for fire ant control because of the low toxicity of the bait formulation in mammalian acute toxicity trials and the rapid rate of hydrolysis which occurred. Tolerance data were not required then as the registered use did not include crop or food use.
Merck Sharp & Dohme Research Laboratories have indicated that although they are seeking to increase the range of food crops on which the use of avermectin is approved, they have no current plans to develop avermectins as grain protectants. Insufficient information is available to recommend their use as candidate protectants of durable foodstuffs at the present time.
Conclusion
Fungi are unlikely to be generally useful for the control of storage pests because the climatic conditions which usually prevail in storage situations are unsuitable. Temperatures are frequently far higher than the 25°C mentioned by Searle and Doberski (1984), and humidities in the commodity are generally at, or below 70%; the r.h. may be as low as 20% in some tropical climates. Overall, fungal control of storage pests may warrant further investigation but it shows no clear promise at the moment.
 |
 |