David Grzywacz

Insect pathogens as biological control agents: Back to the future.

The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 1years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance. Insect pathogenic viruses are a fruitful source of microbial control agents (MCAs), particularly for the control of lepidopteran pests. Most research is focused on the baculoviruses, important pathogens of some globally important pests for which control has become difficult due to either pesticide resistance or pressure to reduce pesticide residues. Baculoviruses are accepted as safe, readily mass produced, highly pathogenic and easily formulated and applied control agents. New baculovirus products are appearing in many countries and gaining an increased market share. However, the absence of a practical in vitro mass production system, generally higher production costs, limited post application persistence, slow rate of kill and high host specificity currently contribute to restricted use in pest control. Overcoming these limitations are key research areas for which progress could open up use of insect viruses to much larger markets. A small number of entomopathogenic bacteria have been commercially developed for control of insect pests. These include several Bacillus thuringiensis sub-species, Lysinibacillus (Bacillus) sphaericus, Paenibacillus spp. and Serratia entomophila. B. thuringiensis sub-species kurstaki is the most widely used for control of pest insects of crops and forests, and B. thuringiensis sub-species israelensis and L. sphaericus are the primary pathogens used for control of medically important pests including dipteran vectors. These pathogens combine the advantages of chemical pesticides and MCAs: they are fast acting, easy to produce at a relatively low cost, easy to formulate, have a long shelf life and allow delivery using conventional application equipment and systemics (i.e. in transgenic plants). Unlike broad spectrum chemical pesticides, B. thuringiensis toxins are selective and negative environmental impact is very limited. Of the several commercially produced MCAs, B. thuringiensis (Bt) has more than 50% of market share. Extensive research, particularly on the molecular mode of action of Bt toxins, has been conducted over the past two decades. The Bt genes used in insect-resistant transgenic crops belong to the Cry and vegetative insecticidal protein families of toxins. Bt has been highly efficacious in pest management of corn and cotton, drastically reducing the amount of broad spectrum chemical insecticides used while being safe for consumers and non-target organisms. Despite successes, the adoption of Bt crops has not been without controversy. Although there is a lack of scientific evidence regarding their detrimental effects, this controversy has created the widespread perception in some quarters that Bt crops are dangerous for the environment. In addition to discovery of more efficacious isolates and toxins, an increase in the use of Bt products and transgenes will rely on innovations in formulation, better delivery systems and ultimately, wider public acceptance of transgenic plants expressing insect-specific Bt toxins. Fungi are ubiquitous natural entomopathogens that often cause epizootics in host insects and possess many desirable traits that favor their development as MCAs. Presently, commercialized microbial pesticides based on entomopathogenic fungi largely occupy niche markets. A variety of molecular tools and technologies have recently allowed reclassification of numerous species based on phylogeny, as well as matching anamorphs (asexual forms) and teleomorphs (sexual forms) of several entomopathogenic taxa in the Phylum Ascomycota. Although these fungi have been traditionally regarded exclusively as pathogens of arthropods, recent studies have demonstrated that they occupy a great diversity of ecological niches. Entomopathogenic fungi are now known to be plant endophytes, plant disease antagonists, rhizosphere colonizers, and plant growth promoters. These newly understood attributes provide possibilities to use fungi in multiple roles. In addition to arthropod pest control, some fungal species could simultaneously suppress plant pathogens and plant parasitic nematodes as well as promote plant growth. A greater understanding of fungal ecology is needed to define their roles in nature and evaluate their limitations in biological control. More efficient mass production, formulation and delivery systems must be devised to supply an ever increasing market. More testing under field conditions is required to identify effects of biotic and abiotic factors on efficacy and persistence. Lastly, greater attention must be paid to their use within integrated pest management programs; in particular, strategies that incorporate fungi in combination with arthropod predators and parasitoids need to be defined to ensure compatibility and maximize efficacy. Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are potent MCAs. Substantial progress in research and application of EPNs has been made in the past decade. The number of target pests shown to be susceptible to EPNs has continued to increase. Advancements in this regard primarily have been made in soil habitats where EPNs are shielded from environmental extremes, but progress has also been made in use of nematodes in above-ground habitats owing to the development of improved protective formulations. Progress has also resulted from advancements in nematode production technology using both in vivo and in vitro systems; novel application methods such as distribution of infected host cadavers; and nematode strain improvement via enhancement and stabilization of beneficial traits. Innovative research has also yielded insights into the fundamentals of EPN biology including major advances in genomics, nematode-bacterial symbiont interactions, ecological relationships, and foraging behavior. Additional research is needed to leverage these basic findings toward direct improvements in microbial control.

Quality Control of Fungal and Viral Biocontrol Agents - Assurance of Product Performance

An essential feature of the production of all microbial control agents is an effective quality control system. Well-defined product specifications with accompanying quality control procedures help to maximize product performance, ensure product safety, standardize manufacturing costs and reduce the risks of supply failure, thus building user confidence. A production system that does not have a quality control system is one whose output is uncontrolled and a lack of thorough quality feedback can result in batches of product with variable concentrations of active agent. This results in products with variable performance leading to control failures by users and serious loss of user confidence. Strict quality control procedures are not only essential for product consistency, but also for safety. Where quality control is inadequate, microbial contamination of the final product is inevitable. In most of such cases this will merely lead to a loss of efficacy due to dilution of the active ingredient by competing microorganisms, but also the potential of producing human pathogens must be ruled out. Recognition of contaminants and quantification of the degree of contamination are therefore important in determining any possible risk to human health. Many low technology production systems in use around the world have minimal or no quality control procedures. This is unacceptable and can damage the reputation of microbial control in addition to possibly posing health risks to those that produce or are exposed to the product. Two case studies from developing countries, are used to illustrate how the lack of quality control procedures can lead to the production of low viability, highly contaminated products with low or negligible concentrations of the active ingredient. However, it is also demonstrated that low technology production systems in developing countries can produce high quality products, provided appropriate quality control procedures are firmly implemented. It must be recognized that quality control procedures can be more complex and technologically demanding than the production procedures themselves, but it is largely on the effectiveness of these control procedures that the long-term acceptability of fungal and viral products depends. This paper details the quality control procedures considered necessary in the mass production of fungi and viruses for use as biocontrol agents, and attempts to suggest reasonable standards that can be achieved by all producers.

Wolbachia in a major African crop pest increases susceptibility to viral disease rather than protects

Wolbachia are common vertically transmitted endosymbiotic bacteria found in < 70% of insect species. They have generated considerable recent interest due to the capacity of some strains to protect their insect hosts against viruses and the potential for this to reduce vector competence of a range of human diseases, including dengue. In contrast, here we provide data from field populations of a major crop pest, African armyworm (Spodoptera exempta), which show that the prevalence and intensity of infection with a nucleopolydrovirus (SpexNPV) is positively associated with infection with three strains of Wolbachia. We also use laboratory bioassays to demonstrate that infection with one of these strains, a male-killer, increases host mortality due to SpexNPV by 6-14 times. These findings suggest that rather than protecting their lepidopteran host from viral infection, Wolbachia instead make them more susceptible. This finding potentially has implications for the biological control of other insect crop pests.

The effect of natural sunlight on Spodoptera littoralis nuclear polyhedrosis virus

The effect of natural sunlight on Spodoptera littoralis (Boisduval) nuclear polyhedrosis virus (NPV) in Egypt was investigated. Wavelengths between 300 and 320 nm were shown to be responsible for almost all of the inactivation attributed to sunlight, although there was some deleterious effect of wavelengths between 320 and 400 nm and above 665 nm. When NPV was exposed to wavelengths between 400 and 665 nm in addition to wavelengths above 665 nm, no inactivation occurred. A simple linear regression equation relating solar UV dose below 320 nm to inactivation of NPV was obtained based on several experiments carried out over a 4‐year period. The survival curve follows the pattern of a single—hit, single—target model. The relationship also could be described as a bisegmented curve and it was concluded that this might be due to a proportion of the virus being inherently more stable to inactivation by sunlight or that two reactions are involved in the inactivation process.

The use of indigenous ecological resources for pest control in Africa

Reducing the losses from crop pests will help to increase food availability and boost economic growth in sub-Saharan Africa (SSA). However, the existing crop protection paradigm that relies on synthetic agrochemical pesticides has had only a marginal impact on the productivity of many poor smallholder farmers who constitute a major segment of agriculture in SSA. This is primarily because many of them are not able to afford or access these imported chemicals. A solution to this crop protection problem may be to harness biological resources that are locally available, such as endemic insect natural enemies and indigenous pesticidal plant materials. Two specific examples of this already under development in Africa are the use of the pesticidal plant, Tephrosia vogelii, and the harvesting of the endemic insect baculovirus, Spodoptera exempta nucleopolyhedrovirus (SpexNPV). Both of these can be produced locally and have shown promise in trials as inexpensive and effective tools for pest control in Africa and their use is currently being scaled up and evaluated by African networks of researchers. A focus on these systems illustrates the potential for using locally-available natural resources for improved crop protection in Africa. The consideration of these pesticidal plants and insect natural enemies in the wider context of natural capital that provide valuable ecosystem services (including pest control), will facilitate greater recognition of their true economic and societal worth. While both of these model systems show promise, there are also very significant challenges to be overcome in developing production, supply and marketing systems that are economically viable and sustainable. The regulatory environment must also evolve to accommodate and facilitate the registration of new products and the establishment of appropriate supply chains that share the benefits of these resources equitably with the local communities from which they are harvested.

The strain variation and virulence of granulovirus of diamondback moth (Plutella xylostella Linnaeus, Lep., Yponomeutidae) isolated in Kenya

A project to develop non-chemical methods of DBM control on brassica crops in Kenya has been exploring the use of endemic pathogens as potential control agents. Initial surveys for endemic pathogens identified P.xylostella granulovirus (PlxyGV) on farms in Kenya. Subsequently 14 genetically distinguishable isolates were identified from field collected material. These were purified and ranging bioassays showed these isolates were pathogenic to Kenyan strains of DBM with LC50’s varying from 2.36x10 6 to 3.95x10 occlusion bodies (OB) per ml for second instar DBM. One isolate (Nya-01) was selected and subsequently used for field trials in Kenya. The trials showed that unformulated PlxyGV applied at weekly intervals at a rate of 3.0 x10 OB/ha could control DBM on Kale more effectively than available chemical insecticides. After application, infection rates in DBM can reach 90%. Further field trials are currently underway to determine the lowest effective dose rate for this virus when applied as a formulation. Initial virus production studies using in vivo

High levels of genetic diversity in Spodoptera exempta NPV from Tanzania.

The African armyworm, Spodoptera exempta, is a major pest in sub-Saharan Africa. A nucleopolyhedrovirus (NPV) is often recorded in later population outbreaks and can cause very high levels of mortality. Research has been addressing whether this NPV can be developed into a strategic biological control agent. As part of this study, the variation in natural populations of NPV is being studied. An isolate of S. exempta NPV was cloned in vivo and found to contain at least 17 genetically-distinct genotypes. These genotypes varied in size from approximately 115 to 153 kb.

Reviving Chickpea Production in Nepal Through Integrated Crop Management, with Emphasis on Botrytis Gray Mold

This paper describes the current status of chickpea production and assesses the damage caused by the grey mould disease (Botrytis cinerea) in Nepal. It also discusses the effects of different integrated crop management components (host plant resistance, fungicide management, forecasting model-based fungicide application, row spacing variation, and seed treatment with Rhizobium) in the control of Botrytis grey mould and improvement of chickpea production in the country.

Mass production of entomopathogens in less industrialized countries

Production of a number of entomopathogenic agents as pesticides, mainly fungal and some viral agents, has been established in a number of countries in Asia, South America, and Africa using low-technology approaches, often by small-scale producers. Although these reduced technology systems can generate effective products, these systems also require the application of appropriate production methodology and rigorous monitoring if the appropriate quality and safety standards are to be reached. The role of governments in creating and maintaining a policy and regulatory environment that promotes local entomopathogen production remains important for success. The wider adoption of entomopathogen products in less industrialized countries may also require a shift in users from the ad-hoc reactive application of pesticides to more systematic integrated pest management approach—a shift already underway in the export horticulture sectors in some of these countries.

Pest Control: Biopesticides' Potential

The special section on Smarter Pest Control (16 August, p. 728) highlighted the threats that chemical pesticides pose to human health and the environment, and some of the smart alternatives, including genetically modifi ed (GM) crops. However, an important and emerging technology against insect pests was overlooked: biological pesticides.