Nematodes and the Biological Control of Insect Pests

Nematodes and the biological control of pests.

1. Insect pests - Biological control. 2. Nematoda as biological pest

control agents. 3. Nematoda. I. Kaya, Harry K. II. Bedding, Robin,

1940-. III. Akhurst, Ray, 1947-.

Bibliography.

Includes index.

ISBN 0 643 05479 0.

© CSIRO 1993

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Preface

Environmental and public health problems arising from the use of chemical insecticides have led to an increasing demand for alternatives for insect pest control. Together with this, widespread public concern resulting in governmental bans on many of the most effective insecticides and development of insecticide resistance has severely reduced the range of useful insecticides available. Alternative control measures such as the biological control of pest insects with parasitoids and predators and microbial biocides have been in field use for several decades. Although these alternatives are being continually improved, most insect pests are nonetheless still controlled with chemical insecticides.

Recently there has been a proliferation of specialist biocontrol companies. More significantly, most ot the major chemical pesticide companies are now taking more than just a token interest in the field. In this climate, new alternatives to chemical insecticides are eagerly sought and amongst the most promising of these are the entomogenous and entomopathogenic nematodes.

The use of nematodes for controlling insect pests is at a stage of rapid development. Recent woodwasp outbreaks in Australia and South America have led to renewed research activity on Deladenus siricidicola, the nematode first used successfully for controlling an insect pest. Development of the most commercially attractive group, the entomopathogenic nematodes, has accelerated significantly during the last five years generating considerable interest in and research on nematodes for insect control Consequently, advances in the discipline have been rapid.

In recognition of the importance of these nematodes the XIX International Congress of Entomology, held in Beijing in July 1992, devoted two symposia to insect nematology. Eminent workers from various parts of the world were invited to speak on new developments in symposia entitled Entomogenous Nematodes for Biological Control, and Entompathogenic Nematode/Bacterium Symbioses. This book, which is based on the two symposia, presents the latest work on the biological control of insects using nematodes; it covers a range topics that will help provide a better understanding of the potentials and problems involved in developing these nematodes as biological control agents.

We thank the 23 authors who have contributed to this book for their expertise, patience and cooperation. We are greatly indebted to Li Li Ying who invited our participation in the Congress and encouraged the writing of this book, to Richard Milner for his comments on several chapters and to Sandy Smith and Chris Hunt who produced many of the illustrations. We are also grateful to Kevin Jeans, our publisher, for his patience and efforts well beyond the call of duty.

Robin Bedding

Ray Akhurst

Harry Kaya

Contents

Preface

1      An Overview of Insect-Parasitic and Entomopathogenic Nematodes

Harry K. Kaya, Robin A. Bedding and Raymond J. Akhurst

2      Biological Control of Sirex noctilio using the Nematode Deladenus siricidicola

Robin A. Bedding

3      Parasitism of the Brown Planthopper by the Mermithid Nematode Agamermis in Rice

Ho Yul Choo and Harry K. Kaya

4      Noctuidonema guyanense (Nematoda: Aphelenchoididae): Host Range and Pathogenicity to the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

C.E. Rogers, O.G. Marti, Jr., and A.M. Simmons

5      Biological Control of Tree Borers (Lepidoptera: Cossidae) in China with the Nematode Steinernema carpocapsae

Yang Huaiwen, Zhang Gangying, Zhang Shangao and Jian Heng

6      Development of a System for the Control of the Banana Weevil Borer, Cosmopolites sordidus with Entomopathogenic Nematodes

Neil L. Treverrow and Robin A. Bedding

7      Biological Control of Scarabs with Entomopathogenic Nematodes

Michael G. Klein

8      Control of the Peach Fruit Moth, Carposina niponensis, using Entomopathogenic Nematodes

Wang Jinxian

9      Post-application Biology of Entomopathogenic Nematodes in Soil

John Curran

10      Factors Affecting Physiological Fitness and Modes of Survival Employed by Dauer Juveniles and their Relationship to Pathogenicity

Christopher Z. Womersley

11      Ecological Genetics of Entomopathogenic Nematodes

Randy Gaugler

12      Soil Microorganisms Affecting Entomopathogenic Nematodes

Harry K. Kaya and Graham S. Thurston

13      Integrated Control of Insect Pests by Steinernema carpocapsae

Nobuyoshi Ishibashi

14      Temperature Responses of Entomopathogenic Nematodes: Implications for the Success of Biological Control Programmes

Christine T. Griffin

15      Bacterial Symbionts of Entomopathogenic Nematodes — the Power behind the Throne

Ray Akhurst

16      The Phages and Bacteriocins of Xenorhabdus spp., Importance for the Specificity of their Bacterial Host to the Nematodes Steinernematidae and Heterorhabditidae

N.E. Boemare, Boyer-Giglio, M.-H. Thaler, J.-O. and R.J. Akhurst

17      Fimbrial Attachment of Xenorhabdus nematophilus to the Intestine of Steinernema carpocapsae

Keith C. Binnington and Liz Brooks

Conclusion

Future Prospects for Entomogenous and Entomopathogenic Nematodes

Robin A. Bedding, Raymond J. Akhurst and Harry K. Kaya

Index

1

An Overview of Insect-Parasitic and Entomopathogenic Nematodes

Harry K. Kaya, Robin A. Bedding and Raymond J. Akhurst

Insect nematology is emerging as a major discipline within insect pathology and biological control. Interest in insect nematology can be attributed to (1) the successful use of nematodes in biological control of insect pests, (2) the ready availability of some nematode species for classical biological control, integrated pest management (IPM) and inundative or inoculative release programmes against major insect pests, and (3) the need to find alternative control tactics for chemical insecticides. Although there are eleven nematode families which offer potential for the biological control of insects, the recent focus has been on five families. Specifically, they are Mermithidae in the class Adenophorea and Phaenopsitylenchidae (formerly Neotylenchidae), Aphelenchoididae, Steinernematidae, and Heterorhabditidae in the class Secernentea.

Nematodes infect hundreds of different species from most orders of insects and affect their insect hosts in a variety of ways. Some insect-parasitic nematodes have little or no detectable effect on their hosts, whereas others reduce fecundity or cause sterility, reduce longevity, reduce flight activity, or delay development. There may also be other behavioural, physiological or morphological aberrations. Some insect parasitic nematodes such as mermithids eventually kill their hosts. In contrast, the entomopathogenic nematodes, Steinernema and Heterorhabditis, rapidly kill their hosts because of their associations with mutualistic bacteria of the genus Xenorhabdus. Thus, the insect-parasitic nematodes have been primarily utilised as classical biological control agents and the entomopathogenic nematodes are largely used as biological insecticides.

Historical Perspective

During the formative years of insect nematology from the mid-19th century to 1950, the science was primarily a descriptive one and included major contributions by such eminent nematologists as P. Bovien, J.R. Christie, N.A. Cobb, I.N. Filipjev, G. Fuchs, O.F.B. von Linstow, C.T.E. von Siebold, G. Steiner, and G. Thorne (Nickle and Welch 1984). Since 1950, many researchers, too numerous to identify individually, have contributed immensely to the discipline.

Steinernematids and Heterorhabditids

One of the most ambitious projects in the annals of biological control of insects dates back to the 1930s and early 1940s. This brief but significant contribution by Glaser and his coworkers to biological control by nematodes was the mass production in vitro of Steinernema (syn. Neoaplectana) glaseri for inoculative release against the Japanese beetle, Popillia japonica, in New Jersey, USA (Glaser and Farrell 1935; McCoy and Glaser 1936; Glaser et al. 1940). Despite the major effort that went into this project, it was curtailed for a number of reasons including the onset of World War II, the development of the milky disease bacterium, Bacillus popilliae, for biological control of the Japanese beetle, and the poor efficacy against the Japanese beetle grubs in field trials toward the end of the project. This poor efficacy may well have been due to the loss of the symbiotic bacterium (Gaugler et al. 1992). Glaser and his co-workers were unaware of the nematode’s mutualistic relationship with the bacterium and, in fact, did their utmost to minimise bacterial contamination of their mass production media. Although the project failed to accomplish its goal of controlling the Japanese beetle with nematodes, its concepts served as an inspiration for later workers.

The nematode/bacterial relationship was first alluded to by Bovien (1937) and Dutky (1937) but was not widely appreciated until Dutky (1959) specified some characteristics of the association including the existence of antibiotics produced by the mutualistic bacteria. The nematode/bacterial association was later examined in detail by Poinar and Thomas (1965, 1966, 1967) and the bacterial symbiont, formerly in the genus Achromobacter, was placed in the new genus Xenorhabdus by Thomas and Poinar (1979). Thereafter, Akhurst (1980) demonstrated phase variation in Xenorhabdus species, and Akhurst (1982, 1983a,b, 1986), Akhurst and Boemare (1988) and Boemare and Akhurst (1988) contributed considerably to our understanding of the taxonomy and biologies of the various species of these bacteria.

The most intensively investigated entomopathogenic nematode is Steinernema (syn. Neoaplectana) carpocapsae. This species, isolated in Czechoslovakia, was described by Weiser (1955) and subsequently shown to be conspecific with the ‘DD136’ nematode from the eastern USA (Dutky and Hough 1955) by hybridisation experiments (Poinar, 1967). This nematode (Tang 1958; Tanada and Reiner 1960, 1962; Welch and Briand 1961a,b) and S. glaseri (Hoy 1955) were tested for biological control against several insect species in the 1950s and early 1960s. Since then the number of field tests has increased significantly, much of the information is summarised by Poinar (1979), Klein (1990), and Begley (1990).

In the mid-1970s, the description of the genus Heterorhabditis (syn. Chromonema) (Poinar 1975, Khan et al. 1976) in the new family Heterorhabditidae (Poinar 1975) presented another nematode group which has since shown considerable promise for biological control (Klein 1990; Begley 1990).

By the early 1980s, the ability to mass produce nematodes in vitro with their symbiotic bacteria (Bedding 1981, 1984a) coupled with the successful control of plant-boring insect pests (Lindegren et al. 1981; Bedding and Miller 1981a; Miller and Bedding 1982) and of a soil pest, the black vine weevil (Bedding and Miller 1981b; Simons 1981), created a positive atmosphere for commercialisation of these nematodes. Unquestionably, the wide host range of these nematodes, the exemption from registration in many countries, and the need to find alternative methods for chemical insecticides for insect control were added incentives for commercialisation (Kaya 1985). The nematodes (Gaugler and Boush 1979; Poinar et al. 1982) and the bacteria (Poinar et al. 1982; Obendorf et al. 1983) were found to be safe to birds and mammals, and these data assisted in obtaining the exemption from registration in many countries. In addition, the nematode did not have a negative impact upon honey bees (Kaya et al. 1982). Hence, several cottage industries devoted to nematode production were established during the early 1980s with at least two companies growing into industrial scale operations. The availability of these commercially produced nematodes for testing against a number of different insect pests further encouraged their use as biological insecticides.

A significant contribution that provided a wide range of entomopathogenic nematode species and strains for commercial companies and researchers was the development of a very simple technique for their isolation (Bedding and Akhurst 1975). This ‘Galleria trap’ technique and its variants have been used to isolate a large variety of entomopathogenic nematodes from broad geographical, climatic and environmental situations (Mrácek 1980; Roman and Beavers 1983; Akhurst and Brooks 1984; Akhurst and Bedding 1986; etc.). Some of these new isolates are being developed by commercial companies or being tested by scientists for use against various pest species.

The great number of isolates is welcomed for they represent new species and strains and genetic diversity. However, they also created challenges for taxonomists because the morphological description of these nematodes involved laborious methods and was problematic. Cross-breeding tests (Poinar 1967; Akhurst and Bedding 1978) were used to solve some problems in the taxonomy of Steinernema but because of Heterorhabditis’ complicated life cycle, they have not been useful until very recently (Dix et al. 1992) for this nematode. With the use of modern tools and techniques, the problem of speciation in Heterorhabditis was first solved by electrophoretic analysis (Akhurst 1987) and then by restriction fragment length difference analyses (Curran and Webster 1989).

The nematodes were used successfully against the black vine weevil and certain plant boring insects, but the results with other insects were variable. The inconsistency emphasised the need to obtain more information on the ecological, physiological and genetic factors influencing their efficacy. Accordingly, studies on the effects of abiotic and biotic factors on the nematodes were initiated. Schmiege (1963), Kamionek et al. (1974), Kaya (1977), and Molyneux (1985) reported on the effects of temperature, and Molyneux and Bedding (1984) reported on the importance of soil water potential (pF) and texture. The dispersal of nematodes in soil was investigated by Moyle and Kaya (1981) and Georgis and Poinar (1983a,b,c), and the detrimental effects of ultraviolet light were demonstrated by Gaugler and Boush (1978). The significance of carbon dioxide as a major attractant for entomopathogenic nematodes was first shown by Gaugler et al. (1980). The compatibility of the nematodes with certain chemical pesticides was reported by Dutky (1974), Prakasa Rao et al. (1975), Kovàcs (1982), and Hara and Kaya (1982, 1983). The importance of biotic factors on these nematodes was shown by Poinar and Jansson (1986a,b), Epsky et al. (1988), and Timper and Kaya (1988) and of nematode and insect species differences with regard to nematode infectivity and insect susceptibility was first brought to our attention by Bedding et al. (1983). The interactions with various beneficial insects were first shown by Kaya (1978a,b). To improve host-finding by the natural isolates of nematodes, Gaugler et al. (1989a,b) demonstrated the potential of genetic selection for improving this trait. These and other studies indicated the diversity and complexity of entomopathogenic nematodes and demonstrated the need for careful management and proper application of them for effective control against insect pests.

In 1989, Gaugler and Kaya organised a seminal meeting of researchers in entomopathogenic nematology. It culminated in a multi-authored book entitled ‘Entomopathogenic Nematodes and Biological Control’ (1990) that has served as a bench mark for the discipline and has delineated major gaps in our knowledge of these nematodes and their associated bacteria. To progress beyond using entomopathogenic nematodes against other than the ‘easy’ insect targets, continued research on genetic, behavioural, physiological and ecological factors is required. These factors are addressed in various chapters throughout our book including genetics and behaviour in Chapter 13, physiology in Chapter 14, and basic and applied ecology in Chapters 7 and 9-12.

Phaenopsitylenchids

While the development of steinernematid and heterorhabditid nematodes is a recent success in insect nematology, a major contribution to classical biological control of insects occurred in the late 1960s and early 1970s. The stage for this biological control programme began near the turn of the 20th century when the European wood wasp, Sirex noctilio, was accidentally introduced into New Zealand. This insect was not recognised as a major pest until it devastated Monterey pine, Pinus radiata, in plantations from 1946 to 1948 (Talbot 1977). Subsequently, in 1952, the wood wasp was accidentally introduced from New Zealand to Tasmania, Australia and by 1961, was established on the Australian mainland. Although the insect did not cause damage for many years in New Zealand, Sirex immediately became a major pest in Australia. Meanwhile in New Zealand, Zondag (1962) had discovered that a phaenopsitylenchid nematode, Deladenus (syn. Beddingia) siricidicola, which probably arrived in New Zealand with its host, parasitised Sirex. He later attributed the collapse of Sirex populations to this nematode (Bedding 1984b). This finding prompted Australian scientists to begin an intensive worldwide search for nematode parasites (and other natural enemies) of Sirex in the early 1960s. Between 1965 and 1974 substantial information on the biology, taxonomy, mass production, formulation, and release of D. siricidicola was obtained. In Chapter 2, Bedding provides a fascinating account of this program including the recent resurgence of this insect in the ‘Green Triangle’ of Australia which was followed by a massive release of the nematode.

Aphelenchoidids

The association between aphelenchoidids and insects is primarily commensal, but two species, namely Acugutturus parasiticus from cockroaches (Hunt 1980) and Noctuidonema guyanense from noctuid moths (Remillet and Silvain 1988), are ectoparasites of insects. Only N. guyanense is being examined in detail and Rogers (Chapter 4) provides the latest status of this nematode as a potential biological control agent of the fall armyworm, Spodoptera frugiperda, and other closely related species.

Mermithids

Another nematode which gained recognition in biological control was Romanomermis culicivorax (syn. Reesimermis neilseni), a parasite of mosquitoes. Considerable research was conducted by Petersen and his co-workers (Petersen 1984, 1985) and others during the 1970s resulting in significant contributions on its biology, ecology, host range, and biological control attempts. In biological control, the inundative approach with the preparasitic stage of this nematode produced variable results (Petersen 1984, 1985). Inoculative releases of the postparasitic stage in rice fields produced partial control of mosquito larvae (Kerwin and Washino 1985).

With very few alternatives for biological control of larval mosquitoes, R. culicivorax was nonetheless commercialised for a brief period in the late 1970s. This commercial venture failed because of poor nematode storage and transport capabilities, poor economics of mass production and because of the success of Bacillus thuringiensis subspecies israelensis (Bti) as an effective mosquito larvicide. Ultimately, Hominick and Tingley (1984) concluded that this mermithid’s life cycle precludes it from being a very effective biological control agent because the environmental sex determination in the nematode prevents it from maintaining high rates of parasitism. In addition, the mosquitoes’ reproductive potential exceeds that of the nematode. However, Goldman et al. (1986) demonstrated that mosquito larvae can become resistant to Bti. Accordingly, this nematode, although not highly effective by itself, may still have potential as a control agent of mosquitoes in an IPM program.

Many more mermithid species occur in nature, and a search to find those that offer potential for biological control or for use in IPM programs may prove fruitful. For example, a major pest in many Asian rice fields is the brown planthopper, Nilaparvata lugens, which is attacked by the mermithid, Agamermis unka. Described by Kaburaki and Imamura in 1932, this mermithid can parasitise a high proportion of planthoppers in Korean rice fields (Choo et al. 1987, 1989). In Chapter 3, Choo and Kaya assess the impact of this mermithid on the brown planthopper in Korea.

Biology

Steinernematids and Heterorhabditids

Steinernematid and heterorhabditid nematodes, obligate pathogens in nature, are characterised by their association with a specific bacterium in the genus Xenorhabdus (Table 1). Each entomopathogenic nematode species has a specific natural association with only one Xenorhabdus species (Akhurst and Boemare 1990), although a Xenorhabdus species may be associated with more than one nematode species (Table 1). Steinernema has 14 recognised species and Heterorhabditis has five (Table 1). Both genera contain several undescribed species that are designated by code names.

The non-feeding, infective third-stage (dauer) juveniles of steinernematids and heterorhabditids carry their specific mutualistic bacteria in their intestines. The infective juveniles enter their insect host through natural openings (mouth, anus or spiracles) and penetrate into the haemocoel (Poinar 1990); the infective juveniles of heterorhabditids can also penetrate directly through the integument into the haemocoel (Bedding and Molyneux 1982). Once in the host’s haemocoel, the juveniles release the bacterial cells that propagate and kill the insect within 48 hours. The nematodes feed on the Xenorhabdus bacteria and host tissues, produce two or three generations, and emerge from the cadavers as infective juveniles to search for new hosts. At room temperatures, the life cycle for most steinernematids, from infection to emergence of the infective juveniles, ranges from 7-10 days and for heterorhabditids from 12-15 days.

Table 1 Entomopathogenic nematode species and their respective mutualistic bacterial species

A The species name of some steinernematids, particularly ‘carpocapsae’ and ‘feltiae’, causes considerable confusion in the literature. The species ‘carposcapsae’ has been referred to as ‘feltiae’ in the literature primarily between 1983 and 1989. The name ‘feltiae’ is valid and takes precedence over the species name ‘bibionis’ (Poinar 1990). To alleviate some of the confusion, some authors use ‘feltiae (=bibionis)’ in their papers.

B Possibly S. longicaudum.

C Akhurst and Boemare (1990) are of the opinion that the Xenorhabdus luminescens group should be in a separate genus from the bacterial species associated with steinernematids.

A fundamental difference between the reproductive strategies of the two families exists. Steinernematids require infection of the host by both male and female infective juveniles for reproduction although not for killing the host (i.e. a juvenile of either sex may infect and kill the host without reproduction). On the other hand, every Heterorhabditis infective juvenile is capable of reproduction because it develops into a hermaphrodite adult; eggs released by the hermaphrodite develop into dioecious adults which undergo another reproductive cycle whereas eggs retained within the hermaphrodite develop to the infective juvenile stage. The relationship between the nematode and bacterium is one of classical mutualism. The bacterium benefits because the nematode acts as a vector for the bacterium and provides it with a competitive advantage in the host insect against other microorganisms. This advantage occurs by the infective juvenile carrying its symbiont within the otherwise sterile intestine and releasing both an inhibitor of inducible antibacterial enzymes (Götz et al. 1981) and its mutualistic bacterium. The host’s phagocytes destroy contaminating bacteria that may be accidentally introduced into the haemocoel on the outside of the nematode or from the insect’s gut. The nematode is now able to establish a virtually monoxenic culture with its symbiont within the insect. The nematode benefits because the bacterium propagates causing septicaemia that kills the host quickly, converting the host tissues into nutrients suitable for the nematode, to use as a food source. The bacterial symbiont also protects these nutrients in the cadaver against secondary microorganism invasion by producing antibiotics with a wide range of activities. The interactions between the nematodes and bacteria are described in greater detail by Akhurst in Chapter 16.

Phaenopsitylenchids and Aphelenchoidids

Phaenopsitylenchids are characterised by an alternation of a parasitic heterosexual and one or more free-living heterosexual generations (Remillet and Laumond 1991). In the free-living stage, D. siricidicola, a parasite of woodwasps, utilises the symbiotic fungus of its host to multiply within the infested tree. Details of D. siricidicola’s biology are presented in Chapter 2.

The biology of the aphelenchoidid, Noctuidonema, is presented in Chapter 4. Briefly, this ectoparasite occurs on the cuticle of moths, especially on the posterior part of the abdomen, and feeds on the moth’s haemolymph by penetrating its stylets directly through the cuticle. The nematodes are transferred from moth to moth during mating.

Mermithids

Mermithids, ranging in length from 1 to 30 cm, parasitise many species of invertebrates, the majority of which are insects (Kaiser 1991). A variety of mermithid life cycles exists, but most species conform to a similar plan. A newly-hatched, second stage juvenile (preparasite) emerges, seeks its host, penetrates through the host’s cuticle using its stylet, and enters the haemocoel where nutrients are absorbed directly through the nematode’s cuticle and stored in the trophosome. When the parasitic phase is complete, the nematode emerges from the host, invariably killing it in the process, and commences its free-living stage. The postparasitic stage enters the soil (terrestrial mermithids) or the sediment (aquatic mermithids), matures, mates, and oviposits to complete the life cycle.

Conclusion

The five families of insect-parasitic and entomopathogenic nematodes considered here represent the most useful for controlling insect pests at present. Although the most spectacular biological control of an insect pest by nematodes has been achieved by Deladenus siricidicola, most research is currently directed towards the entomopathogenic nematodes because of their commercial appeal as biological insecticides. This appeal arises from their wide host range, particularly among soil dwelling pests and those in other cryptic habitats that are not amenable to control by other nondisruptive measures. Their relative ease of production, storage, and application and safety to birds and mammals also contribute to the high level of interest in these entomopathogenic nematodes. This book is slanted towards the entomopathogenic nematodes because of this current level of interest but endeavours to cover the most important issues in the control of insects in general.

References

Akhurst, R.J. (1980). Morphological and functional dimorphism in Xenorhabdus spp., bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis. Journal of General Microbiology 121, 303–309.

Akhurst, R.J. (1982). A Xenorhabdus sp. (Eubacteriales: Enterobacteriaceae) symbiotically associated with Steinernema kraussei (Nematoda: Steinernematidae). Revue de Nematologie 5,