The Entomopathogenic Nematode-Bacterium Complex: Biology, Life Cycle and Vertebrate Safety

This paper reports on several aspects of the taxonomy and biology of the symbiotic bacteria, Xenorhabdus spp. and Photorhabdus spp., associated with entomopathogenic nematodes (EPNs), which may be used to define the boundaries with pathogenic bacteria of medical, veterinary or agronomic importance. All the result of tests undertaken to assess the effects of these bacteria on warm-blooded vertebrates were negative, indicating that the bacteri would pose no hazard to vertebrates in practice. Non-symbiotic microorganisms are also associated occasionally with EPNs, and some of them, e.g Providencia rettgeri, belong to taxa which include opportunistic pathogens of man. This review emphasizes that the relationship between these non-symbiotic bacteria and nematodes cannot be considered to be a risk for humans because they do not support the growth of nematodes during long-term mass rearing or they do not persist during storage of nematodes, so their purposeful use is necessarily excluded for industrial production. Good practice by biotechnology laboratories can avoid such contaminants. However, commercial producers should be aware of the possible occurrence of human opportunistic pathogens and prevent such contaminations by establishing monoxenic symbiotic cultures. The pathogenic properties of both partners of the normal bacterium-EPN complex are examined with respect to the risks they pose to human and vertebrate health and to the environment. It is concluded that no risk to warm-blooded animals or plants is related to the use of EPNs in biological control.     

Insect-parasitic nematodes: from lab curiosities to model organisms

Interest in studying insect-parasitic nematodes was originally focused on their potential as biological control agents of insects and other arthropod pests. Now, after 30 years of intense basic and applied research, realization of the practical use of insect-parasitic nematodes, particularly of entomopathogenic nematodes and their symbiotic bacteria, has spurred developments across a far broader scientific front. We are now entering a new era of discovery in which tools of molecular genetics are being increasingly used to address a range of biological questions. The knowledge gained from these efforts will directly benefit the practical application of insect-parasitic nematodes as more effective biopesticides. Moreover, these studies will advance these nematodes as unique and intrinsically interesting biological model systems not only for basic research but also in applied fields such as plant health, human medicine, pharmaceutical bioprospecting, and genetic engineering. In this review, the past and current state of insect-parasitic nematode research is summarized. Future research priorities and goals are identified and discussed.     

Identification, Pathogenicity and Comparative Virulence of Fusarium spp. Associated with Diseased Euphorbia spp. in Europe

Fusarium spp. isolated from diseased Euphorbia spp. in Europe were assessed for pathogenicity to North American accessions of leafy spurge ( Euphorbia esula/virgata ). Of the nine strains of Fusarium spp. isolated from diseased E. stepposa or E. virgata in the Caucasus region of Russia and E. esula/virgata in southern France, all were pathogenic to leafy spurge. There were significant differences in virulence among strains. Four strains, including the two that were most virulent, were identified as F. oxysporum . Four of the five other strains were identified as F. solani and one was identified as F. proliferatum . Three of the four most virulent strains to leafy spurge were isolated from E. stepposa . The most virulent strain was associated with root damage caused by insect biological control agents, as found earlier with domestic strains of Fusarium spp. pathogenic to leafy spurge. Two strains identified as F. solani were vegetatively compatible. It was concluded that further screening of a larger set of strains of foreign Fusarium spp. under quarantine conditions in the US or in limited overseas facilities would be justified, and could yield promising biological control agents for leafy spurge.     

Vibrating membrane filtration for recovery and concentration of insect killing nematodes

New experimental data are reported that demonstrate the use of a novel vibrating membrane filter (VMF) for the combined recovery and concentration of two species of nematodes, S. feltiae and P. hermaphrodita, from mature liquid fermentation cultures. The disk membrane module had a working surface area of 0.2 m(2) and was operated at a constant flow rate of 0.2 m(3) h(-1). The recovery of the viable nematodes from the spent media and nonviable nematodes was assisted by an independently imposed oscillatory motion of the disk assembly, which produced an intense shear field at the membrane surface with calculated mean values on the order of 10(4) s(-1). Adult (nonviable) nematodes in the fermentation culture were preferentially dissolved in a detergent (sodium dodecylsulfate) and successfully separated from the juveniles using the VMF equipment. Permeate fluxes on the order of 15 to 30 L/m(2/)h were achieved for an operating transmembrane pressure of 800 mbar. Industrial-scale liquid fermentation for the manufacture of nematodes as biopesticides produces the viable nematode life stages in low-concentration suspension containing large quantities of spent media and other waste material. The VMF equipment provided a flexible operation for separation, cleaning, and concentration of viable nematodes from the fermentation broths.     

The development, regulation and use of biopesticides for integrated pest management

Over the past 50 years, crop protection has relied heavily on synthetic chemical pesticides, but their availability is now declining as a result of new legislation and the evolution of resistance in pest populations. Therefore, alternative pest management tactics are needed. Biopesticides are pest management agents based on living micro-organisms or natural products. They have proven potential for pest management and they are being used across the world. However, they are regulated by systems designed originally for chemical pesticides that have created market entry barriers by imposing burdensome costs on the biopesticide industry. There are also significant technical barriers to making biopesticides more effective. In the European Union, a greater emphasis on Integrated Pest Management (IPM) as part of agricultural policy may lead to innovations in the way that biopesticides are regulated. There are also new opportunities for developing biopesticides in IPM by combining ecological science with post-genomics technologies. The new biopesticide products that will result from this research will bring with them new regulatory and economic challenges that must be addressed through joint working between social and natural scientists, policy makers and industry.     

Biological control potential of plant growth‐promoting rhizobacteria suppression of Meloidogyne incognita on cotton and Heterodera glycines on soybean: A review

Over the past decade, we have seen an increasing market for biopesticides and an increase in number of microbial control studies directed towards plant‐parasitic nematodes. This literature survey provides an overview of research on biological control of two economically important plant‐parasitic nematodes, Meloidogyne incognita (Kofoid & White) Chitwood (southern root‐knot nematode) and Heterodera glycines Ichinohe (soybean cyst nematode) using spore‐forming plant growth‐promoting rhizobacteria (PGPR). In this review, the current biological control strategies for the management of those cotton and soybean nematodes, the mechanism of using BacillusPGPR for biological control of plant‐parasitic nematode including induced systemic resistance and antagonism and the future of biological control agents on management of plant‐parasitic nematodes are covered.     

Bt: mode of action and use

The insecticidal toxins from Bacillus thuringiensis (Bt) represent a class of biopesticides that are attractive alternatives to broad-spectrum "hard" chemistries. The U.S. Food Quality Protection Act and the European Economic Council directives aimed at reducing the use of carbamate and organophosphate insecticides were expected to increase the use of narrowly targeted, "soft" compounds like Bt. Here we summarize the unique mode of action of Bt, which contributes to pest selectivity. We also review the patterns of Bt use in general agriculture and in specific niche markets. Despite continued predictions of dramatic growth for biopesticides due to US Food Quality Protection Act-induced cancellations of older insecticides, Bt use has remained relatively constant, even in niche markets where Bt has traditionally been relatively high.     

Updated assessment of potential biopesticide options for managing fall armyworm (Spodoptera frugiperda) in Africa

The fall armyworm (FAW, Spodoptera frugiperda) has recently spread to many countries in Africa, the Near East, Asia and the Pacific. In sub-Saharan Africa (SSA), more than 300 million people depend on FAW’s preferred host plant, maize, as a staple crop. Hence, the spread of FAW in SSA has the potential to negatively affect livelihoods and food security. Many farmers have responded to FAW by increasing their use of synthetic pesticides, but these are not always used safely or effectively. More information on sustainable alternatives to high-risk synthetic pesticides is needed to inform decisions by farmers and policy makers. In a previous paper, the authors responded to this information need by identifying fifty biopesticides which had been registered for FAW management in one or more of 30 countries in FAWs native region and Africa. For each biopesticide identified, detailed profiles with information on their efficacy against FAW; associated human health and environmental hazards; their agronomic sustainability; and whether or not they are practical for use by smallholder farmers were developed Research for development (R4D) efforts is ongoing in Africa and Asia for development and use of biopesticides for FAW management. Hence, in this study the authors assessed the current state of knowledge and documented how information gaps have been filled (or not) since the previous paper was published. The authors found that for many biopesticides there is a growing body of information on their efficacy in the field in Africa and increased availability of commercialized products. They also note remaining information gaps, particularly the compatibility of the biopesticides with other recommended management practices, and cost-benefit analyses, important for developing and implementing sustainable IPM. An updated list of priority biopesticides for research, development and promotion is provided.     

Production technology for entomopathogenic nematodes and their bacterial symbionts

Entomopathogenic nematodes (genera Steinernema and Heterorhabditis) kill insects with the aid of mutualistic bacteria. The nematode–bacteria complex is mass produced for use as biopesticides using in vivo or in vitro methods, i.e., solid or liquid fermentation. In vivo production (culture in live insect hosts) is low technology, has low startup costs, and resulting nematode quality is high, yet cost efficiency is low. In vitro solid culture, i.e., growing the nematodes and bacteria on crumbled polyurethane foam, offers an intermediate level of technology and costs. In vivo production and solid culture may be improved through innovations in mechanization and streamlining. In vitro liquid culture is the most cost-efficient production method but requires the largest startup capital and nematode quality may be reduced. Liquid culture may be improved through progress in media development, nematode recovery, and bioreactor design. A variety of formulations is available to facilitate nematode storage and application. Journal of Industrial Microbiology & Biotechnology (2002) 28, 137–146 DOI: 10.1038/sj/jim/7000230 Received 16 August 2001/ Accepted in revised form 10 November 2001     

Microbial antibiotics take the lead in the fight against plant pathogens

The plant microbiome is essential for plant fitness and health. Antibiotics produced by plant-associated bacteria have been shown to play an important role in protecting plant hosts against phytopathogens. Here, we highlight the strong biotechnological potential of (i) antibiotic producing plant-associated bacteria as biocontrol agents and (ii) the heterologous expression of antibiotic biosynthetic gene clusters in non-pathogenic plant-associated bacteria. We also provide the complete list of the active substances based on bacteria, fungi, and viruses currently approved or pending approval in the European Union, as an indication of the significant emergence and biotechnological applicability of biopesticides. Further progress in this field of research will enable the development of novel biopesticides for the biocontrol of agricultural pests.     

Mosquitocidal activity of anthraquinones isolated from symbiotic bacteria Photorhabdus of entomopathogenic nematode

Two anthraquinones were isolated from the symbiotic bacteria Photorhabdus temperata of entomopathogenic nematodes Heterorhabditis spp. by repeated column chromatography. They were abundantly present in the culture medium and identified as 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone and 3-methoxychrysazine by spectral analysis. The isolated anthraquinones were highly lethal to larvae of Culex pipiens pallens. Our results suggest that anthraquinones might be useful as biopesticides for the biological control of mosquitoes.     

Plant parasitic nematode proteins and the host parasite interaction.

This review focuses on the proteins and secretions of sedentary plant parasitic nematodes potentially important for plant-nematode interactions. These nematodes are well equipped for parasitism of plants. Having acquired the ability to manipulate fundamental aspects of plant biology, they are able to hijack host-cell development to make their feeding site. They feed exclusively from feeding sites as they complete their life cycle, satisfying their nutritional demands for development and reproduction. Biochemical and genomic approaches have been used successfully to identify a number of nematode parasitism genes. So far, 65 204 expressed sequence tags (ESTs) have been generated for six Meloidogyne species and sequencing projects, currently in progress, will underpin genomic comparisons of Meloidogyne spp. with sequences of other pathogens and generate genechip microarrays to undertake profiling studies of up- and down-regulated genes during the infection process. RNA interference provides a molecular genetic tool to study gene function in parasitism. These methods should provide new data to help our understanding of how parasitic nematodes infect their hosts, leading to the identification of novel pathogenicity genes.     

Microbial and viral chitinases: Attractive biopesticides for integrated pest management

The negative impact of the massive use of synthetic pesticides on the environment and on human health has stimulated the search for environment-friendly practices for controlling plant diseases and pests. Among them, biocontrol, which relies on using beneficial organisms or their products (bioactive molecules and/or hydrolytic enzymes), holds the greatest promise and is considered a pillar of integrated pest management. Chitinases are particularly attractive to this purpose since they have fungicidal, insecticidal, and nematicidal activities. Here, current knowledge on the biopesticidal action of microbial and viral chitinases is reviewed, together with a critical analysis of their future development as biopesticides.     

Potential of Lecanicillium spp. for management of insects, nematodes and plant diseases

Fungi in the genus Lecanicillium (formerly classified as the single species Verticillium lecanii) are important pathogens of insects and some have been developed as commercial biopesticides. Some isolates are also active against phytoparasitic nematodes or fungi. Lecanicillium spp. use both mechanical forces and hydrolytic enzymes to directly penetrate the insect integument and the cell wall of the fungal plant pathogen. In addition to mycoparasitism of the plant pathogen, the mode of action is linked to colonization of host plant tissues, triggering an induced systemic resistance. Recently it was demonstrated that development of Lecanicillium hybrids through protoplast fusion may result in strains that inherit parental attributes, thereby allowing development of hybrid strains with broader host range and other increased benefits, such as increased viability. Such hybrids have demonstrated increased virulence against aphids, whiteflies and the soybean cyst nematode. Three naturally occurring species of Lecanicillium, L. attenuatum, L. longisporum, and an isolate that could not be linked to any presently described species based on rDNA sequences have been shown to have potential to control aphids as well as suppress the growth and spore production of Sphaerotheca fuliginea, the causal agent of cucumber powdery mildew. These results suggest that strains of Lecanicillium spp. may have potential for development as a single microbial control agent effective against several plant diseases, pest insects and plant parasitic nematodes due to its antagonistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecanicillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes.     

Combining Pest Repellent Plants and Biopesticides for Sustainable Pest Management in Chinese Kale

The objective of this investigation was to study the use of pest repellent plant (PRP) species and biopesticides for sustainable pest management in Chinesekale(Brassica oleracea L. var. alboglabra Bailey). Ten selected treatments composed of three PRP species namely citronella grass - (CG) [Cymbopogon nardus L. (Rendle)], sweet basil - (SwB) (Ocimum basilicum L.) and sacred basil - (SaB) (Ocimum sanctum L.)] were intercropped with Chinese kale, integrated with two commonly used biopesticides, viz. Bacillus thuringiensis (Bt) and neem extract (Azadirachta indica) and a control were tested in a randomized complete block design (RCBD) with 3 replicates. The insect pest species infested in Chinese kale and their populations, % pest damage, fresh weight and quality of yield were investigated. The results showed that diamondback moth - (DBM) [Plutella xylostella (Linnaeus)] density was lowest in CG alone, CG+SaB, SwB+biopesticides and SaB+biopesticides, relatively low in SwB alone, CG+SaB+SwB, and CG+biopesticides, and moderate in biopesticides and sacred basil alone, and highest in the control. The densities of common cutworm - (CCW) [Spodoptera litura (Fabricius)], cabbage webworm - (CWW) [Hellula undalis (Fabricius)] and flea beetle - (FBT) (Phyllotreta sinuata Steph.) were not significantly different among treatments. The overall pest damage was lowest in CG+biopesticides treated plots followed by SwB+ biopesticides, SaB+biopesticides and biopesticides alone, and CG+biopesticides gave the best quality of yield of Chinese kale, and hence such combinations could be integrated for managing crucial insect pests, i.e. DBM and to minimize/avoid the use of highly toxic synthetic pesticides in Chinese kale.     

Increasing ecological heterogeneity can constrain biopesticide resistance evolution

Microbial biopesticides containing living parasites are valuable emerging crop protection technologies against insect pests, but they are vulnerable to resistance evolution. Fortunately, the fitness of alleles that provide resistance, including to parasites used in biopesticides, frequently depends on parasite identity and environmental conditions. This context-specificity suggests a sustainable approach to biopesticide resistance management through landscape diversification. To mitigate resistance risks, we advocate increasing the range of biopesticides available to farmers, whilst simultaneously encouraging other aspects of landscape-wide crop heterogeneity that can generate variable selection on resistance alleles. This approach requires agricultural stakeholders to prioritize diversity as well as efficiency, both within agricultural landscapes and the biocontrol marketplace.     

Neutral density liquid formulations for nematode-based biopesticides

Entomopathogenic nematodes are widely used as biological insecticides for agricultural and horticultural pests. In the vast majority of cases, commercial products contain nematodes partially dehydrated on to inert solid carriers. Nematode survival in these products is generally poor, they are difficult to handle and are not suitable for use with all nematode species. We have developed a non-viscous, non-adhesive and non-toxic liquid formulation for nematode storage and transport based on neutral density colloidal silica suspensions. Survival and virulence of nematodes stored in this formulation without aeration was superior to nematodes stored in aerated quarter strength Ringer's solution.     

Biocontrol: The Potential of Entomophilic Nematodes in Insect Management

A review of the development of entomophilic nematology and a commentary on the potential of entomophilic nematodes in controlling insect pests. The paper considers some of the major contributions to our knowledge of entomophilic nematology; factors involved in insect pest management and how they are applicable to the use of nematodes; nematodes which are most promising as biological control agents; and problems to be solved to facilitate the use of entomophilic nematodes in insect management.     

From Augmentation to Conservation of Entomopathogenic Nematodes: Trophic Cascades, Habitat Manipulation and Enhanced Biological Control of Diaprepes abbreviatus Root Weevils in Florida Citrus Groves

The use of entomopathogenic nematodes (EPN) for management of the root weevil, Diaprepes abbreviatus, in Florida citrus groves is considered a biological control success story and typically involves augmentation in which EPN are applied inundatively as biopesticides to quickly kill the pest. However, recent evidence indicates that efficacy of EPN applications in Florida citrus depends on soil type. They are very effective in the well drained coarse sands of the Central Ridge but often less so in poorly drained fine-textured soils of the Flatwoods. Moreover, groves on the Central Ridge can harbor rich communities of endemic EPN that might often suppress weevil populations below economic thresholds, whereas Flatwoods groves tend to have few endemic EPN and frequent weevil problems. Current research is examining the ecological dynamics of EPN in Florida citrus groves, the potential impact of EPN augmentation on soil food webs, especially endemic EPN, and whether habitat manipulation and inoculation strategies might be effective for conserving and enhancing EPN communities to achieve long-term control in problem areas. Conservation biological control could extend the usefulness of EPN in Florida citrus and be especially appropriate for groves with persistent weevil problems.     

The role of parasite release in invasion of the USA by European slugs

Several species of European slugs are invasive in the USA, threatening native species and damaging agricultural and horticultural crops. One possible explanation for the success of these invaders is parasite release. To test this hypothesis we collected European slugs in part of their native range (United Kingdom) and in the USA and compared prevalence, distribution and species richness of their nematode parasites. All slugs were dissected and examined for the presence of nematodes. In the UK, nematodes were present at 93% of study sites and 16.4% of all slugs examined were associated with nematodes whereas in the USA the respective figures were 34% of sites and 5.4% of slugs. Nematode species richness was greater in the UK with 12 species being found, seven of which were thought to be truly parasitic as opposed to being phoretic or necromenic. Nine species of nematode were found in the USA, four of which were truly parasitic. Four of the ten European slug species examined in the USA, were entirely free of truly parasitic nematodes whereas all were infected by nematodes in some sites in the UK. There was a significant difference in the prevalence of truly parasitic nematodes in five of these species when comparing their home versus invasive range. A significant difference in parasite prevalence was observed when comparing native and introduced slug species in the USA, however, this was not significant in the UK. Our data support a role for parasite release during the invasion of the USA by European slugs.