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Review, Program, Program Abstracts, Poster Abstracts, Photographs

Program and Abstracts for the

THIRD INTERNATIONAL SYMPOSIUM ON ENTOMOPATHOGENIC NEMATODES AND
SYMBIOTIC BACTERIA

SEPTEMBER 4-7, 2003
Arden Shisler Conference Center
Ohio State University, Wooster, Ohio 44691, USA

ORGANIZERS
Parwinder S. Grewal, Ohio State University, Wooster, Ohio, USA
Harry K. Kaya, University of California, Davis, California, USA
Heidi Goodrich-Blair, University of Wisconsin, Madison, Wisconsin, USA
Steve Forst, University of Wisconsin, Milwaukee, Wisconsin, USA
Susan Bornstein-Forst, Marian College, Marian, Wisconsin, USA

SPONSORS

USDA-NRI; OARDC/OSU; New England BioLabs; e-nema; SDS Biotech; IBCS
Certis; USA; BioLogic

PROGRAM

Day 1 (September 4, 2003, Thursday)
8:00 – 9:15 Registration and Continental Breakfast
9:15 – 9:30 Opening remarks

9:30 - 11:40             Session I Biodiversity
Moderator:                Ralf Ehlers
9:30 – 9:55                Erko Stackebrant – Bacterial biodiversity
9:55 – 10:20              Noel Boemare – EPB phylogeny, systematics, and biodiversity
10:20 – 10:45            Andras Fodor – Molecular and gnotobiological approaches to study cospeciation
10:45 – 11:10            Patricia Stock – EPN phylogeny, systematics, and biodiversity
11:10 – 11:40            Discussion: Byron Adams
11:40 – 12:40            Lunch

12:40 – 3:15            Session II Symbiosis
Moderator:                Susan Bornstein-Forst
12:40 – 1:05              Steve Forst –Attachment and motility in Xenorhabdus
1:05 – 1:30                Heidi Goodrich-Blair – Molecular approaches to study symbiosis
1:30 – 1:55                Helen Bennett – Symbiosis in Photorhabdus
1:55 - 2:20                 Ralf Ehlers – Nematode biology and reproduction
2:20 – 2:45                Creg Darby – Applying C. elegans techniques
2:45 - 3:15                 Discussion: Todd Ciche
3:15 – 3:45                Break

3:45 – 6:45             Session III Pathogenicity and genomics
Moderator:               Heidi Goodrich-Blair
3:45 – 4:10               Frank Kunst – Photorhabdus genome project
4:10 - 4:35                Richard ffrench-Constant – Photorhabdus virulence
4:35 – 5:00               Mark Blight – Genomics applied to virulence
5:00 - 5:25                Eric Pearlman – Wolbachia/Onchocerca and river blindness
5:25 - 5:50                Li Tan – Virulence of Moraxella osloensis to slugs
5:50 - 6:15                Brad Goodner – Genomics and undergraduate education
6:15 - 6:45                Discussion - Steve Forst

6:45 – 7:30: Round Table Discussion:
Transition from symbiosis to pathogenesis

7:30 Mixer (an international night of fun and fellowship, Fisher South Lobby)

Day 2 (September 5, 2003, Friday)

8:00-10:35             Session IV Nematode physiology, genetics, and molecular                                     biology
Moderator:              Dawn Gouge
8:00 - 8:25               Ann Burnell – Genetics and molecular biology
8:25 - 8:50               Denis Wright – Storage reserves and infectivity
8:50 - 9:15             Itamar Glazer – Anhydrobiosis
9:15 – 9:40              Susan Bornstein-Forst – In-host desiccation of nematodes
9:40 – 10:15            Ganpat Jagdale – Thermal biology
10:15 – 10:35          Discussion – Parwinder Grewal
10:35 – 11:00          Break

11:00 – 1:10          Session V Behavioral ecology
Moderator:              Patricia Stock
11:00 - 11:25            Jim Campbell – Foraging behavior
11:25 - 11:50            Christine Griffin – Infection behavior
11:50 - 12:15            Arne Peters – Host recognition and penetration
12:15 – 12:40            Harry Kaya – Ant deterrent
12:40 - 1:10              Discussion – Ed Lewis
1:10 - 2:10                Lunch

2:10 - 4:20             Session VI Population dynamics and modeling
Moderator:               Lynn LeBeck
2:10-2:35                  Mary Barbercheck – Competition and displacement
2:35-3:00                  Parwinder Grewal – Metapopulation biology
3:00-3:25                  Robin Taylor - Estimating entomopathogenic nematode abundance
3:25-3:50                  Casey Hoy - Stochastic and spatially explicit models
3:50-4:20                  Discussion – Robin Stuart

Day 3 (September 6, 2003) Saturday

8:00-1:00             Session VII Implementation around the world
Moderator:             Mary Barbercheck
8:00 - 8:25              Mike Wilson – Western Europe
8:25 – 8:50            Tamas Lakatos – Central Europe
8:50 - 9:15              Satoshi Yamanaka – Japan
9:15 – 9:40            Huaiwen Yang – China
9:40 – 10:05            Sudharshan Ganguly – Indian subcontinent
10:05 – 10:30          Ho Yul Choo – Korean Peninsula
10:30 – 10:50          Break
Moderator:            Elizabeth DeNardo
10:50 – 11:15          Albert Pye – North America
11:15 - 11:40           Mayra de la Torre M. – Central America
11:40 - 12:05           Marineide Aguillera – South America
12:05 - 12:30           Alfred Alumai – Africa
12:30 – 1:00            Discussion – Harry Kaya and Ralf Ehlers
1:00 – 2:00              Lunch

2:00-3:45             Session VIII Application technology
Moderator:            Michael Klein
2:00-2:25                Jane Patterson-Fife – Application equipment
2:25-2:50                Simon Piggott – Foliar application
2:50-3:15                David Shapiro-Ilan – Soil application
3:15-3:45                Discussion – Dawn Gouge

4:00 - 6:00 Poster Session (refreshments) – Susan Bornstein-Forst, Organizer

8:30-12:00             Session IX Successes and failures
Moderator:             Denis Wright
8:30-9:55                 Larry Duncan – Soil insects
8:55-9:20                 Lerry Lacey – Insects in cryptic habitats
9:20-9:45                 Albrecht Koppenhofer – Turfgrass and pastures
9:45-10:10             Marek Tomalak – Glasshouse and mushrooms
10:10 – 10:30          Break
10:30-10:55             Rob Van Tol - Nursery and tree applications
10:55-11:20             Michael Samish – Veterinary and livestock pests
11:20-11:45             Guy Belair – Vegetable crops
11:45-12:10             Peter Torr - Forestry
12:10-12:40             Discussion: Ramon Georgis

Erko STACKEBRAANDT, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany

As the majority of environmental microbiologists may not be interested in prokaryotic systematics or even interested in taxonomic problems, a few basic facts need to be stated at the beginning. Firstly, is it is not possible to just a describe a prokaryotic species without consultation of the International Code of Nomenclature Bacteria which governs rules recommended by the International Committee of Systematics of Bacteria/Prokaryotes (ICSP). It was an ICSB/P initiative that led to the implementation of the Approved Lists of Bacterial Names in 1980 which overnight reduced the number of prokaryotic species from tens of thousands of species to about 2500 in 272 genera and 65 families. Today the number of valid species is more than 6.100 in more than 1150 genera and about 160 families (see www.bacterio.cict.fr). Certainly, species descriptions can as well be published in journals other than the IJSEM (effective publication) but in order to be established as a valid species the species name, together with the indication of the type strain number, must appear in the IJSEM validation lists. In order to guarantee access to new type strains these must be deposited in two different public collections in two different countries. This mechanism prevents restrictions in handling materials due to commercial interests of depositors.

Considering the low number of described species and the effort needed for their maintenance the as yet uncultured organisms pose would pose an enormous challenge to the diverse range of culture collections and resource centres. Collection managers are presently not prepared to even double our inventory, neither in terms of taxonomic expertise, nor by space, nor by maintenance. In order to highlight the order of the problem few numbers should be brought to memory: the total number of prokaryotic cell on Earth has been estimated to be 2.5 10³⁰. This astronomically high number is believed to represent numerous species, and educated guesses range from 40.000 to10⁹ species as derived from estimates about in number of different genomes in about 2.000 different types of microbial communities worldwide.

The problem, taxonomists are facing at present is actually not the desire to cultivate the "uncultured" organisms but to cope with the recognized novelty among the cultured ones. In other words, today taxonomists are not even in a position to rapidly respond to the demand of describing new species of the culturable portion of prokaryotic diversity. Phylogenetic analysis has provided microbiologists with rapid assessment of novelty but not more than about 300 new species are described each year: this is due to a number of facts, three of which are most obvious:
1. The number of taxonomists worldwide is low.
2. The description is complex, requiring analysis at the genetic and epigenetic level.
3. The description is expensive. Costs involved in the description of a single type strain of a new species of the genus Bacillus (including salaries, overhead and the like) can be as high as approximately 10.000 €.
4. The description of about 6.000 type strains available today would then have accumulated to several tens of million Euros. The future perspective is somewhat frightening, as, unless as yet unforeseeable changes in the mode of species description are introduced; a sum of about 6.0 billion Euros needs to be generated for describing the anticipated number (conservative guess) of 600.000 type strains.
5. The long-term maintenance of cultures is expensive. The number of type strains housed in the DSMZ is around 4.400. The financial support to maintain these strains and an average of two additional strains of the species is around 2.5 million €uros annual total costs.

In the long run, however, the collection and maintenance strategy need to be changed and the tempo at which this may happens will depend upon (1) the basis of the progress at which the description rate of species is increasing, and (2) the harmonization process among collections, including selection of resources based on research strength, i.e., on taxon/material-based decisions. Even the large service collections will have to reconsider their present mission to cover a great part of the type strains as the return of some of the expenses by fees will depend on the use of these novel strains in academia and biotechnology. This may require an even higher increase in the research budget for academic facilities, a prognosis that is as unforeseeable as the dramatic increase of budget of collections of microorganisms.

Entomopathogenic bacterial symbionts of Steinernema and Heterorhabditi: systematics, phylogeny, and biodiversity

Noël BOEMARE, Ray AKHURST, Sylvie PAGES, Mathieu SICARD, Laboratoire de Pathologie comparée, C.P. 101, Université Montpellier II, INRA-CNRS n° 2209, F-34095 MONTPELLIER CEDEX 5, France

Entomopathogenic nematodes are able to infect a broad host range of insects, but in terms of symbiosis the relationship between the host nematode and its symbiont is very close. This is demonstrated by taxonomic studies using morphological, biochemical, and molecular analyses of conserved genes of both partners, and may be verified by gnotobiological experiments that test the association with the previously identified micro-organisms and axenic nematodes. Within Steinernematidae, Xenorhabdus are located in a special gut vesicle, and within Heterorhabditidae, Photorhabdus are housed in the anterior part of the gut of the young infective juveniles. All the experiments reported to date of symbiont isolation from nematodes indicate the presence of Xenorhabdus in Steinernema, and Photorhabdus in Heterorhabditis. Although these Enterobacteriaceae are carried in the nematode gut, they multiply in the body cavity of the parasitized insect. In nutritional terms they are rather entomophilic than nematophilic, but they are perennially maintained through the generations in these specific helminthic niches.

Systematics and Phylogeny of the symbionts. Xenorhabdus and Photorhabdus are chemoheterotrophic bacteria with respiratory and fermentative metabolism, and they belong to the family of Enterobacteriaceae. We consider them as being atypical Enterobacteriaceae because most of Xenorhabdus and Photorhabdus are nitrate-reductase negative (similar to only some strains of Erwinia and Yersinia), and Xenorhabdus are catalase negative (some strains of Shigella dysenteriae O group 1 are the few other examples in this family). In terms of phenotypic characters, Photorhabdus strains are always positive for bioluminescence and catalase activity. Very few strains of Photorhabdus do not produce light of the numerous isolates recognized today in the world. Analyses of the 16S rDNA established that Photorhabdus has a specific CAAG sequence and all Xenorhabdus strains an UC pair at the position 450 (E. coli numbering). Examination of the revised RDP (Ribosomal Database Project) Tree, shows that both genera branch deeply inside the family Enterobacteriaceae, and are sister genera which do not share any common ancestor. Proteus vulgaris is the nearest phylogenetic neighbor with similarity values between 93.5 and 95.1 % with the former genera. Today, taxonomic studies have defined clear groups within Xenorhabdus, with 5 described species and potentially 4 other in course of definition. Within the Photorhabdus genus 3 species have been described and 4 sub-species; potentially two other species and 4 sub-species could be yet defined.

Gnotobiology. Taxonomic studies of symbionts and their host nematodes have defined that every species of these entomopathogenic nematodes possess a specific symbiont species. This specificity was analyzed by using gnotobiological experiments. Today Steinernema axenic rearing is possible, but a substitute diet has not yet been discovered for Heterorhabditis. For Heterorhabditis we are able only to disinfect eggs and then combine them immediately with symbionts. Several examples have been reported with this kind of experiment and they mainly have established the specificity of each symbiont for its host and the difficulty of establishing heteroxenic associations. When these heteroxenic associations are viable over several generations, most of the examples are with a closely related bacterial strain. At this stage however, we have to compare the number of nematodes produced, and the quality in physiological and pathological terms, from those of the holoxenic animals. The most reliable test is the retention test of the symbiont, meaning that we have to probe over several generations the keeping of symbionts in the resting stage.

Taxonomic correspondence between symbionts and hosts. On the basis of all of the above experiments, it is now clearly established that X. nematophila is the symbiont species of S. carpocapsae, X. poinarii of S. glaseri and S. cubanum, X. beddingii of another unnamed Steinernema sp. as it was previously published. X. bovienii is a species in which bacteriological studies have not led to distinguishing parallel differences among the four host species: S. affine, S. intermedium, S. kraussei, S. feltiae. Photorhabdus luminescens is harbored by H. bacteriophora and H. indica, while P. temperata by H. megidis, H. downesi, H. zealandica. Two subspecies of Photorhabdus asymbiotica, that are clinical opportunistic bacteria isolated in US and in Australia, and that are not harbored by nematodes, are in course of definition.

We believe that the similarities between the two sister genera, Xenorhabdus and Photorhabdus, are the result of a convergent evolution between two different bacterial genera associated with two phylogenetically different nematode genera, Steinernema and Heterorhabditis, respectively. They share several common properties apparently linked with nematode symbiosis, but all the present recorded bacteriological and gnotobiological data indicate that they are different.

András FODOR, Department of Genetics, Eötvös University, H-1117 Budapest, Pázmány Peter sétány 1/C, Hungary

Heterorhabditis and Steinernema spp. are symbiotically associated with bacteria of the genera Photorhabdus and Xenorhabdus. In choice experiments on agar media the attraction of the nematodes H. bacteriophora, H. indica, H. megidis, S. feltiae, S. glaseri and S. carpocapse to different bacterial colonies was investigated. The Heterorhabditis spp. migrated to the bacterial colonies of Photorhabdus spp., whereas X. bovienii, Enterobacter cloacae or Bacillus cereus were not attractive. Heterorhabditis spp. could not distinguish between their own symbiont and Photorhabdus spp. isolated from other nematode species. The behaviour of H. megidis was inconsistent in choice experiments with P. temperata and P. luminescens subspecies akhurstii and laumondii. S. feltiae and S. glaseri were more attracted by X. bovienii than by P. luminescens, E. cloacae or B. cereus, whereas S. carpocapsae nematodes also migrated to colonies of P. luminescens and few to E. cloacae. When exposing S. feltiae to X. bovienii, X. poinarii and X. nematophila the majority of the juveniles migrated to the colonies of the specific symbiont. S. carpocapsae did not distinguish between the different symbiont colonies and S. glaseri was more attracted to X. bovienii than to X. poinarii and X. nematophila. Mixed cultures of H. bacteriophora and Rhabditis veechi, a free living soil nematode, could be separated by their preferences to different bacteria. H. bacteriophora mirgated to P. luminescens colonies whereas Rhabditis veechi preferred E. cloacae over B. cereus colonies. Choice trials can thus be an useful tool for the separation of EPNs from other soil nematodes, which are often isolated together with EPN.

Systematics, diversity and biogeography of entomopathogenic nematodes: A decade of exciting research accomplishments

S. Patricia STOCK, Division of Plant Pathology and Microbiology, Dept. Plant Sciences. The University of Arizona, Tucson, Arizona, USA

The field of entomopathogenic nematology has witness an exponential growth over the past decade,. The impetus for research in entomopathogenic nematodes (EPN) and their symbionts has mainly been motivated by their biological control potential. Thus, much of the focus in EPN research has been on applied aspects relating to pest control. In this respect, a worldwide search for species and isolates better adapted to different climatic conditions and insect hosts has significantly increased over the past decade. As a result, thousands of new isolates and many new species have been recovered and wait to be characterized. This explosive growth of taxa has promoted and demanded the search for new and improved taxonomic tools for species identification and diagnostics. At the same time, the availability of such diverse pool of taxa has stimulated research on more basic topics such as elucidation of their evolutionary relationships, understanding of their biological diversity, and interpretation of geographical patterns of distribution. This presentation will review the current state of EPN taxonomy, phylogenetic relationships, and summarize our current knowledge on biological diversity.

Steve FORST, H. He. and D. KIM, Department of Biological Sciences, University of Wisconsin, Milwaukee, WI 53201, USA

To better understand the interaction between X. nematophila and S. carpocapsae the mrx fimbrial operon was studied. The mrx-minus strain of Xenorhabdus was able to effectively colonize the nematode gut vesicle. However, competitive colonization experiments revealed that the mrx strain was not recovered suggesting that fimbriae are required for efficient release of the bacteria from the nematode. Swarming motility was also studied. The regulatory protein, OmpR, controls motility by negatively regulating the flagellar master regulatory operon, flhDC. An ompR mutant strain demonstrated precocious flagellation, swarming behavior, cell elongation and exoenzyme secretion and was fully virulent towards fourth instar Manduca sexta. The role that OmpR plays in symbiosis is presently being investigated.

Heidi GOODRICH-BLAIR, Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA

We have identified ten genes affecting Xenorhabdus nematophila colonization of Steinernema carpocapsae nematodes. Six encode proteins with predicted functions in regulation or metabolism. Two of these are aroA and serC which encode enzymes required for aromatic amino acid and serine biosynthesis respectively. These enzymes are also both required for synthesis of an iron-binding siderophore. We have determined that it is siderophore, rather than amino acid, biosynthesis that is essential for X. nematophila colonization of nematodes. Other genes we have identified as being required for colonization include one that encodes a putative regulatory RNA, NilD RNA (nematode intestine localization). Current experiments are aimed at understanding the role of NilD RNA in colonization and the stimuli affecting its expression. Finally, we have identified an additional three genes required for colonization, nilA, nilB, and nilC, that encode a ~10-kDa protein of unknown function, a b-barrel outer membrane protein, and an outer membrane lipoprotein. Membrane localization suggests that NilA, NilB and NilC function to link an aspect of the external environment to the inner cell. Such a function could be nutrient acquisition, adhesion, signal sensing, or some combination of these. Experiments are underway to examine sub-cellular localization of each protein and their possible association with each other or with nematode factors. Furthermore, we are assessing whether NilA, NilB and/or NilC affect the expression of other genes, and how they themselves are regulated.

Lipopolysaccharides are cell membrane structures that have been shown to be important in bacterial host interactions. In this talk I will describe the role of LPS in both the symbiotic and pathogenic host interactions of Photorhabdus luminescens TT01.

Ralf-Udo EHLERS, Institute for Phytopathology, Department for Biotechnology and Biological Control, Christian-Albrechts-University Kiel, Klausdorfer Str. 28-36, 24223 Raisdorf, Germany

Development and reproduction of entomopathogenic nematodes is impossible without the presence of their symbiotic bacteria. Exit from the dauer stage (DJ) of H. bacteriophora (recovery) is induced by a chemical signal excreted by the symbiotic bacterium P. luminescens. This signal is composed of at least 2 compounds, one of less than 20 kDa and are negatively charged and another of 5 kDa. Bacteria also secrete an antagonistic signal which inhibits nematode recovery. Since DJ recovery depends on the presence of the bacterial food signals, the percentage of recovering DJ is influenced by the bacterial density and the bacterial growth phase. The response to the food signal differs from batch to batch. Culture conditions during inoculum production have an impact. At low population density and enough bacteria supply the hermaphrodites lay many eggs into the medium. DJ developing from eggs laid into the medium better respond to the food signal than DJ developing inside the uterus by endotokia matricida. The pH (between 4-12) and the CO₂-concentration are positively correlated with DJ recovery. High temperature can inhibit DJ recovery. During the pre-dauer stage the nematodes harbour their symbiont cells in the intestine. Secondary form cells are not retained by the dauer juveniles of H. bacteriophora. Alternative developmental pathways to amphimictic or automictic individuals is also influenced by the bacterial density, as well as the start and progress of the endotokia matricida. Symbiosis is a matter of communication between the two organisms. We only start to unravel the interactions necessary to make the symbiosis a success for nematode and bacterium.

Symbiosis from the nematode side: prospects for genetic analysis of Steinernema and Heterorhabditis

Creg DARBY, Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

The Steinernema and Heterorhabditis genes that play roles in symbiosis are entirely unknown, and genetic systems for analyzing these nematodes do not exist. To find nematode symbiosis genes despite the lack of genetic and genomic data, we are adapting two techniques developed for C. elegans. RNA interference mimics mutations by transiently lowering expression of a specific gene; transposon mutagenesis creates bona fide mutations by inserting heterologous DNA into the genome. In both techniques, once a phenotype is obtained the identity of the relevant gene can be ascertained immediately.

Genome analysis of Photorhabdus luminescens, an endosymbiont of entomopathogenic nematodes

Eric DUCHAUD¹, Alain GIVAUDAN², Noël BOEMARE² and Frank KUNST¹. ¹Institut Pasteur, Laboratoire de Génomique des Microorganismes Pathogènes, Paris, France; ²Laboratoire de Pathologie Comparée, INRA Montpellier, France

The genus Photorhabdus belongs to the family Enterobacteriaceae that comprises intestinal bacteria living in symbiosis with entomopathogenic nematodes (EPNs) of the genus Heterorhabditis. Most of these bacterial species are orally toxic or pathogenic for insect larvae when injected into the hemocoel (Forst et al., 1997; Marokhazi et al., 2003). While most of the insect symbionts are endocytobionts and not culturable, Photorhabdus (Fischer-Le Saux et al., 1999), has the advantage to grow on standard media. Symbionts of EPNs encounter two different situations in their life cycle: they survive in the gut of their nematode host, and once inoculated they multiply in the body cavity of insects, killing the insect host due to septicaemia. These bacteria are now recognized as potentially important since Photorhabdus genes encoding entomotoxins may be useful to create transgenic plants for crop protection (Bowen et al., 1999).

We have recently completed the genome sequence of P. luminescens (5.68 Mb). The analysis of the genome sequence revealed the presence of i) many repeated elements, including > 300 copies of ERIC-like sequences (Hulton et al., 1991), Rhs-like elements (Wang et al., 1998), and mobile elements including phage- and transposon-like sequences; ii) putative virulence genes including a type III secretion system, tc (toxin complex) genes encoding entomotoxins, antibiotics, RTX-like toxins, hemolysins, and a gene possibly encoding a homologue of juvenile hormone esterase which has been shown to possess mosquitocidal activity. The genome analysis will allow to highlight the particularly interesting properties of this bacterium for fundamental and applied research, such as the studies of host-bacterial interactions (symbiosis and pathogenesis), the mechanisms of enzyme secretion and production of specific metabolites.

References

Bowen D. et al. 1999. Science 280 : 2129-2132.
Fischer-Le Saux, M. et al. 1999. Int. J. Syst. Bacteriol. 49 : 1645-1656.
Forst S. et al. 1997. Annu. Rev. Microbiol. 51 : 47-52.
Marokhazi J. et al. 2003. J. Bacteriol. 185/4648-4656.
Hulton, C. S. et al. 1991. Mol. Microbiol. 5 : 825-834.
Wang, Y. D. et al. 1998. J. Bacteriol. 180 : 4102-4110.

Richard fFRENCH-CONSTANT, Department of Biochemistry, University of Bath, Bath, UK

An update of the microarray work looking at the composition of tc genes required for oral toxicity. Identification of genes specific to Photorhabdus asymbiotica human isolates and absent from insect infecting Photorhabdus; using a genomic subtraction technique.

Slim TOUNSI, Andréa de Lima PIMENTA and Mark BLIGHT, Laboratoire de Pathogenèse Comparée, Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris Sud, 91405 Orsay, France

Many Photorhabdus strains have now been isolated from human lesions in both North America and Australia and are designated Photorhabdus asymbiotica, since no known nematode symbiont has been associated with them. We are interested in understanding what adaptive mechanisms may have been acquired by these strains in order that they are now capable of infecting humans. Could they have simply adapted existing virulence determinants or obtained novel ones via horizontal transfer from established human pathogens?

Our approach was to perform a genomic DNA subtractive hybridisation between two Australian Photorhabdus asymbiotica isolates (9800946 and SN98-1) from human infections and two geographically related Photorhabdus luminescens strains (HV16/2 and Q617/2) specific to insect infections (strains kindly supplied by Dr. Ray Akhurst, CSIRO, Canberra, Australia). Following subtractive hybridisation, 218 cloned fragments were analysed by ³²P-dCTP labelled genomic DNA probed dot-blots. 25% of these clones gave specific hybridisation signals to only the P. asymbiotica probe, indicating sequences potentially specific to P. asymbiotica. The remaining 75% showed weak hybridisation signals, indicating possibly divergent sequences between the human and insect strain isolates. In silico analysis of these clones with the P. luminescens TT01 and P. asymbiotica ATCC43494 genomes (Dr. Nick Waterfield, University of Bath, UK) indicated that 89% of the clones are common to all tested P. asymbiotica (9800946, SN98-1 and ATCC43494) and insect strains (HV16/2, Q617/2 and TT01) 11% are specific to the 9800946 and SN98-1 genomes and 7% totally specific to only the three P. asymbiotica strains. Of these 7% (16 clones), 7 showed homology in database searches with known virulence factors from human pathogens.

We initially selected one clone (N° 214) for further analysis due to homology with sopB from Salmonella spp. SopB is a 65kDa protein encoded within the SPI5 and is secreted to the medium by a Type III transport pathway (inv) located in SPI1 and has been shown to be a host-cell invasion factor. An analysis of the secreted protein profiles between the P. asymbiotica isolates demonstrated multiple differences, including the presence of a 65kDa protein. This protein was purified from the supernatant of P. asymbiotica SN98-1 and micro-sequencing of an internal protease digestion product revealed it to indeed be SopB. We now intend to make knock-out mutants of sopB in P. asymbiotica and to investigate their effect upon virulence to human macrophage cell-lines and a mouse infection model. Could sopB be a factor acquired by P. asymbiotica through horizontal transfer, enabling the bacteria to broaden their host range to include humans?

Eric PEARLMAN, Center for Global Health and Diseases and Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio 44106, USA

Virulence mechanisms of the nematode Phasmarhabditis hermaphrodita and its associated bacterium Moraxella osloensis to the gray garden slug Deroceras reticulatum

Li TAN* and Parwinder S. GREWAL, Department of Entomology, Ohio State University, OARDC, Wooster, OH 44691, USA, *Present address: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

Moraxella osloensis, a gram-negative bacterium, is associated with Phasmarhabditis hermaphrodita, a lethal slug-parasitic nematode that has potential for the biocontrol of mollusk pests, especially the gray garden slug Deroceras reticulatum. We discovered that the shell cavity in the posterior mantle region of D. reticulatum served as the main portal of entry for P. hermaphrodita. Only dauer stage of the nematode can serve as an infective stage in the natural environment. Aged M. osloensis cultures were pathogenic to D. reticulatum after injection into the shell cavity or hemocoel of the slug. P. hermaphrodita vectors M. osloensis into the shell cavity and the bacterium is the main killing agent in the nematode/bacterium complex. We also discovered that M. osloensis lipopolysaccharide (LPS) was an endotoxin that was active against the slug. Purified M. osloensis LPS had a lethal injection toxicity but no contact or oral toxicity against the slug. Toxicity of M. osloensis LPS resides in the lipid A moiety but not in the polysaccharide moiety. The LPS was a rough-type LPS with an estimated molecular weight of 5,300. Coinjection of galactosamine with the LPS increased its toxicity to D. reticulatum by 2-4 fold. The galactosamine-induced sensitization was reversed completely by uridine. We further discovered that 1 or 2-day M. osloensis cultures were non or less pathogenic whereas 3 to 5-day M. osloensis cultures were more pathogenic to the slug. The average yield of M. osloensis LPS per bacterium did not differ among the 1 to 5-day cultures. However, M osloensis cells from the 3-day cultures produced more outer membrane proteins than those from the younger or older cultures. The intensity and pattern of M. osloensis aggregation changed with time of culture. Pili-like projections were rarely present on the bacterial surfaces of M. osloensis from 1-day cultures, but reached maximal density in 3-day cultures. The temporal expression of the pili-like projections correlates with the temporal pattern of M. osloensis virulence to D. reticulatum. The changes of M. osloensis pathogenicity against D. reticulatum during culture strongly correlate with structural changes in the bacterial cell wall.

Genomics has certainly transformed biology, but it also has the potential to revolutionize education. Over the past six years, undergrads at University of Richmond and Hiram College have been involved in several collaborative genome projects, including the published Agrobacterium tumefaciens C58 genome. I will present the strategies we are using within the framework of courses and through independent research teams. I will also provide an update on the recently funded project to sequence two Xenorhabdus strains.

A.M. BURNELL, I. DIX, K.M. DOLAN and D.M. O'HALLORAN,Institute of Bioengineering and Agroecology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland

Techniques of classical genetics - mutagenesis, hybridization and artificial selection have been successfully used in entomopathogenic nematode (EPN) strain improvement programmes. By contrast, the techniques of molecular genetics have not been widely applied to EPN, except in the area of molecular diagnostics and in studies of molecular phylogeny. We are now entering a new phase in EPN research in which the tools of molecular genetics will be increasingly used to address a range of biological questions, both fundamental and applied. Techniques for the cloning of differentially expressed genes (e.g. suppression subtractive hybridization) can now be readily used to identify EPN genes whose expression is restricted to a particular developmental stage, or whose expression is induced following an environmental stimulus or signal. Such studies provide a rapid means of investigating the physiological/biochemical strategies used by EPN at different developmental stages and may also identify novel genes or processes in these nematodes. Specific genes can also be targeted using degenerate PCR. We have investigated differential gene expression in H. bacteriphora IJs during the early infection phase in G. mellonella and we have also used degenerate PCR to isolate H. bacteriophora G-protein a-subunit genes. An overview of the results obtained in these studies will be presented.

EPN belong to the same family as Caenorhabditis elegans whose genome has been fully sequenced and annotated. In principle, the molecular tools which have been developed for C. elegans could be developed and applied to studies on EPN but, in practise, such technology transfer has been rare. The main problem is one of manpower, resources and research focus. The C. elegans community work on a single strain (Bristol N2) of C. elegans, whereas EPN researchers work on a large number of species and strains in two nematode genera, and have a more applied focus. Certain C. elegans protocols, e.g. transposon mutagenesis, RNAi (for gene silencing) and genetic transformation may however require substantial research and development to obtain a working system for EPN. It has been reported that H. bacteriophora can be successfully transformed by microinjection using a reporter construct under the control of a promoter from the C. elegans hsp-16 heat shock gene. It has also been reported that Steinernema feltiae was successfully transformed with a trehalose phosphate synthase (TPS) gene under the control of the C. elegans hsp-16 heat-shock promoter. However, genetic transformation techniques are not yet routinely in use for either Heterorhabditis or Steinernema and there are no reports of the successful application of RNAi in these nematodes. Our attempts to develop genetic transformation and RNAi protocols for S. carpocapsae will also be reported.

Storage reserves and infectivity

Denis J. WRIGHT, Department of Biological Sciences, Imperial College London, Silwood Park campus, Ascot, Berkshire SL5 7PY, UK

The infective dauer juveniles (IJ) of Steinernema and Heterorhabditis species are typical facultative aerobes with relatively high lipid to glycogen ratios. Neutral lipids are the primary energy stores with triacylglycerols by far the most abundant form of lipid. Freshly emerged (in vivo) IJ of different Steinernema spp. vary considerably in the total amount of neutral lipid they contain and this appears to be related primarily to differences in body size. The glycogen content per unit weight of freshly emerged IJ appears to be more variable between species. Neutral lipid consumption by IJ stored in water can vary considerably between species and the rate of decline in lipid stores can usually be correlated closely with the decline in their infectivity and with their longevity. The rate of glycogen consumption shows a similar pattern to neutral lipids between species. For example, glycogen and lipid consumption by S. feltiae and S. glaseri at 25ºC is much slower compared with S. carpocapsae and S. riobrave. In S. carpocapsae, glycogen can act as a limited late energy store prolonging infectivity after neutral lipids have been depleted. The factors influencing the amount and composition of energy stores in IJ in vivo and in vitro and their rate of consumption will be considered, particularly in relation to storage time (shelf-life). Knowledge of intermediary metabolism in dauer juveniles of Caenorhabditis elegans and entomopathogenic nematodes will be briefly reviewed.

Anhydrobiosis- revealing stress tolerance mechanism in entomopathogenic nematodes: a genomic approach

Itamar Glazer¹, Tali Zitman-Gal¹, Hinanit Koltai², ¹Dept. Nematology, ²Dept. Genomics and Bioinformatics, Volcani Center, Bet Dagan 50250, Isreal

All nematodes are aquatic organisms and need a film of water surrounding their body in order to move. Dry conditions adversely affect nematode motility and survival. The natural habitat for entomopathogenic nematodes (EPN), the soil is a difficult environment for persistence of any organism considering its complexity of physical, chemical and biological components. Dehydration has been identified as one of the key components affecting EPN persistence and efficacy. Despite the vast progress in the studies on EPN little is known about the mechanisms of survival. Nevertheless, EPN have been isolated from soils throughout the world in ecosystems ranging from sub-arctic to arid and temperate to tropical climates. In the presentation the current knowledge on behavioral and physiological adaptation of EPNs to dehydration. Special emphasis will be given to the recent advances in molecular basis for the tolerance mechanisms to dedication and induction of anhydrobiotic state. We used the EPN Steinernema .feltiae IS6 as target nematode to study the these mechanisms. We utilized advance genomic and bioinformatics approaches. Using cDNA subtractive hybridization we identified IS6 genes that are differentially expressed during exposure to desiccation stress. One hundred and ten genes were identified, among them Late-Embryogenic-Abundant gene (Sf-LEA) and aldehyde dehydrogenase (Sf-ALDH) , both are known to be involved in response to water stress in other organisms. Furthermore, using real-time PCR we detected a significant increment in the steady state level of the genes transcription products upon 8 hours of nematodes exposure to desiccation, and further increase upon 24 hours of desiccation. Future studies of desiccation tolerance, including identification of additional desiccation-related genes and study of their biological roles and regulation, will shed light on the genetic and biochemical alterations evolved in environmental-stress tolerant organisms.

The in host desiccation response of Steinernema carpocapsae A10 in Galleria mellonella

This study attempts to examine desiccation stress using a laboratory method that would mimic an actual field response. Galleria mellonella hosts were infected with the entomopathogenic nematode Steinernema carpocapsae A10 and were allowed to air-dehydrate in an environmental chamber for up to 56 days at 23⁰C. Host carcasses were rehydrated at 10, 17, 24, 31, 37, and 44 days post infection on water-saturated filter paper and placed into White traps to collect and count emergent EPN. Weight loss for each Galleria carcass was recorded with a total loss of 86% by day 44 post-infection. There was no significant loss of weight in controls, which were kept moist throughout the experiment. Emergent infectious juveniles (IJ) per host were counted for each group with an apparent peak coinciding with dehydrated hosts from the 24-day post infection time interval and a significant drop in numbers at 37 days post infection. At the end of 44 days a measurable number of IJs were obtained from fully desiccated hosts. IJ populations from each time interval were tested for infectivity, and resistance to temperature and pH stresses. Survival under secondary stress conditions is not increased through prior exposure to desiccation stress. Total aqueous soluble proteins were extracted from IJs collected from control and desiccated hosts and were analyzed using 10% SDS Laemmli gels. A novel protein of 37kDa is over-expressed under conditions of host desiccation.

Ganpati B. JAGDALE and Parwinder S. GREWAL, Department of Entomology, Ohio State University, OARDC, Wooster, OH 44691, USA

Entomopathogenic nematodes have been isolated from a wide range of habitats, where they face a challenge of daily and seasonal temperature fluctuations. We explored biochemical changes and consequent environmental tolerance of cold-adapted Steinernema feltiae, an intermediate S. carpocapsae, and warm-adapted S. riobrave during recycling or acclimation to different temperatures. Fatty acid composition of total lipids and phospholipids changed adaptively with recycling temperatures. The unsaturation indices of lipids increased as temperature decreased. Recycling temperatures also influenced the activities of glucose-6-phosphate dehydrogenase and hexokinase in an adaptive fashion. Isozyme patterns of malate dehydrogenase (MDH), mannose-6-phosphate isomerase (MPI) and phosphoglumutase (PGM) were also affected. S. feltiae synthesized additional isozymes of MPI, MDH and PGM in response to cold temperatures while S. carpocapsae synthesized isozymes of MDH in response to warm temperatures. All three species accumulated trehalose when acclimated at either 5 or 35^oC, but the amount of trehalose accumulation differed by species and temperature. S. riobrave and S. carpocapsae accumulated high levels of trehalose when acclimated at 35^oC, and S. feltiae at 5^oC. Heat tolerance increased in acclimated S. carpocapsae and S. feltiae, but not in S. riobrave. Freezing tolerance increased in acclimated S. carpocapsae and S. riobrave, but not in S. feltiae. Desiccation tolerance of S. feltiae in 25% glycerol at both 5and 35^oC was enhanced by both cold and warm acclimation and the enhanced desiccation tolerance was positively correlated with the acclimation induced trehalose accumulation. At 5^oC, desiccation tolerance of S. carpocapsae was enhanced by either cold or warm acclimation, but at 35^oC it was increased by only cold acclimation. Similarly, at 5^oC, desiccation of S. riobrave was enhanced either by cold or warm acclimation, but at 35^oC, it was increased only by warm acclimation.

J. F. CAMPBELL, USDA-ARS, GMPRC Biological Research Unit, Manhattan, KS 66502, USA

All parasites must bridge the gap between hosts and many adopt active behavioral mechanisms with which to facilitate the process of searching for a new host. Among entomopathogenic nematode species there is a great deal of variation in the expression of behavioral traits by infective stages. The proximate and ultimate causation of some of these behavioral traits will be discussed within the framework of adoption of different foraging strategies. The implications of understanding foraging behavior in terms of improving biological control will also be discussed.

There is evidence that infective juveniles of entomopathogenic nematodes have complex infections strategies. Elements of these strategies include the decision whether to invade a host based on its current infection status, and strategies that are less dependent on immediate environmental conditions. The latter includes the “phased infectivity” hypothesis which states that not all IJs are equally infective, and that infectivity of a population can change adaptively over time. In the original statement of the hypothesis by Hominick and others, it is suggested that a proportion of IJs is dormant or temporarily non-infectious, and that this proportion may change over time. According to this hypothesis, an observed increase in the proportion of IJs infecting under standard conditions may be explained by a switch in some of the IJ population from a non-infectious (or dormant) state to an infectious one. An alternative explanation for an observed increase in the proportion of a population invading is that IJs have different levels of infectivity, and this level may increase (as well as decrease) with time. The evidence for phased infectivity, and for the existence of a non-infectious proportion, will be reviewed, and the ecological and applied significance summarised

Host recognition and penetration

It is widely accepted in parasitology, that there are specific token stimuli from the host that trigger a cascade of events resulting in the penetration of the infective units into the target tissue of the host. Evidence supporting this hypothesis for entomopathogenic nematodes is the differential response of S. carpocapsae infective juveniles to cuticle contact with host and non-host arthropods. Other behavioural changes include head thrusting and change from cruising to localised movement. Physiological changes are the secretion of proteins which are likely to be involved in the penetration process. Nematodes penetrate into the insects via natural openings and via the cuticle, preferably at poorly sclerotized sites. Which way is taken largely depends on the insect host but also on the habitat and the nematode species. Penetration via the spriacles and the anus can be enhanced markedly by covering the target insect with a liquid film containing nematodes, whereas penetration via the skin is diminished in substrate lacking mechanic support for the nematodes penetrating. Ultimately, the IJs must penetrate either the integument, the trachea-wall or the peritrophic membrane and the gut wall to enter the insect’s haemocoel. There is a mechanical element in the penetration process. Heterorhabditis spp. use a distal tooth to rip the insect’s integument and Steinernema spp. simply press their head against the barrier enclosing the insect’s haemocoel. Proteolytic enzymes are probably involved since blocking protease activity decreased the penetration potential in S. glaseri. Also, enzymes were produced in IJs of S. carpocapsae shortly before penetration took place. Insects protect themselves from penetration by the structure of the cuticle, the spiracles and the peritrophic membrane. Frequent defecation or rigurgitation are mechanisms to expel nematodes from the intestine. There are no mechanisms to expel nematodes that have successfully entered the tracheal system. Implications of host recognition and penetration behaviour for improving biocontrol strategies are discussed.

Harry K. KAYA, Department of Nematology, University of California, Davis, CA 95616, USA

The ability of Xenorhabdus nematophila and Photorhabdus luminescens, the symbiotic bacteria of the nematodes, Steinernema carpocapsae and Heterorhabditis bacteriophora, respectively, to produce an ant deterrent factor(s) (ADF) was tested in vivo and in vitro. ADF activity is present in the supernatants of bacterial cultures and that the amount of ADF repellency detected depends the bacterial strain, form, and age. Several biochemical characteristics of ADF were determined. The factor(s) is filterable, heat stable, and acid sensitive and is eluted through a 10-kDa cut-off membrane. ADF appears to be comprised of a small, extra-cellular, and possibly non-proteinaceous compound(s). These findings demonstrate that the symbiotic bacteria of some species of entomopathogenic nematodes produce a compound(s) that deters scavengers such as ants and could protect nematodes from being eaten during reproduction within insect cadavers. Isolation of the gene responsible for ADF activity is being pursued.

Session VI. Population dynamics and modeling

M. E. BARBERCHECK , Department of Entomology, Pennsylvania State University, University Park, PA 16802, USA

Competition is a mutually negative interaction between two or more species (interspecific) or individuals (intraspecific) that does not involve mutual predation. Classical competition theory (1960's and early 70's) predicts that coexisting species that share limiting resources should compete. For coexistence of competing species to continue, the species should diverge in resource use, thereby reducing niche overlap. The resulting pattern, termed "competitive displacement" consists of a regular segregation of species in resource space. A reoccurring controversy in ecology addresses the relative importance of competition and predation in determining the characteristics of organisms, populations, and communities. This presentation will examine some of the research on entomopathogenic nematodes that has examined intra- and interspecific competition.

Metapopulation biology

Parwinder S. GREWAL, Department of Entomology, Ohio State University, OARDC, Wooster, OH 44691, USA

According to common wisdom in ecology, the distribution of species’ abundances in space reflects the match between the environment and the species’ ecological requirements. Spatial ecology challenges a strict interpretation of this habitat-organism relation, as species may exhibit complex spatial patterns in uniform environments. Metapopulation biology is concerned with the dynamic consequences of migration among local populations and the conditions of regional persistence of species with unstable local populations. Thus, a metapopulation is defined as a population of unstable local populations, inhabiting discrete habitat patches. If dispersal is low, then subpopulations remain genetically distinct and weekly selected deleterious alleles can reach high frequencies in local populations. This can lead to inbreeding depression and extinction of local populations. The semi-isolation of subpopulations means that they are likely to differ with respect to the deleterious alleles they harbor. Therefore, benefits accrue among the hybrid offspring of residents and immigrants, as the bad effects of any recessive alleles they receive from one parent are likely to be masked by the alleles from the other parent. One of the hallmarks of metapopulations is the appearance and disappearance of subpopulations from habitat patches as a result of frequent extinction and recolonization. The apparent disappearance of entomopathogenic nematodes soon after their application to the soil has been well documented. However, the nematodes do perpetuate at certain locations naturally. Therefore, elucidating the factors/processes that prevent the extinction of nematode populations is important to develop novel conservation approaches for the use of entomopathogenic nematodes. We explored the possibility of the existence of a metapopulation dynamics in natural populations of the entomopathogenic nematode, Heterorhabditis bacteriophora on a low maintenance turfgrass site, a golf course rough area of approximately 200 m². We discovered that the nematode populations isolated from this area different in several important phenotypic traits. These populations showed differences in infective juvenile longevity and tolerance to major environmental stresses including heat (survival at 40^oC for 2 h), ultraviolet (UV) radiation (original virulence remaining after exposure to 302 nm UV for 5 min), hypoxia (survival at approximately 0% dissolved O₂ at 25^oC for 96 h), and desiccation (survival in 25% glycerol at 25^oC for 72 h). Intrinsic infective juvenile longevity, defined as the number of weeks to 90% mortality (LT₉₀) estimated using probit analysis of nematode survival at 25^oC varied between 11 to 16 weeks among the populations and survival after exposure to different stresses varied between 25-100%. The nematode populations also showed differences in the isozyme patterns for several metabolic enzymes when analyzed through a cellulose acetate gel electrophoresis. These phenotypic differences in nematode populations from such a small area strongly suggest that the population structure of heterorhabditid nematodes be highly fragmented. However, the presence of several common bands in the isozyme patterns of several of these populations, together with observations on gene flow patterns in field populations of H. marelata underscore the existence of a metapopulation dynamics in the natural populations of heterorhabditids.

R.A.J. TAYLOR, Corrie YODER, and Parwinder GREWAL, Department of Entomology, Ohio State University, OARDC, Wooster, OH 44691, USA

Koppenhofer's data and method for estimating entomopathogenic nematode abundance in the field is re-examined. The data will be shown to contain other ecologically interesting information which suggests a refinement to Koppenhofer's method. This refinement is tested with new experiments.

Stochastic and spatially explicit simulation models: template and package for research on entomopathogenic nematode population dynamics

Casey W. HOY, Department of Entomology, Ohio State University, OARDC, Wooster, OH 44691, USA

Although the quantitative ecology of insects and microbes has been well-studied, mathematical modeling of entomopathogenic nematode population dynamics has received attention only recently. This paper will review recent work on modeling nematode population dynamics and discuss a generalized framework for ongoing research in this area. Recent modeling in our laboratory includes a spatially explicit and stochastic simulation model for agricultural landscapes. Such a model can be used as a template for research on the relative importance and detailed biology of mortality rates and probabilities of host infection in nematode population dynamics. The analysis of the model will be summarized along with progress on empirical work spurred by model predictions and additional hypotheses for continued studies.

Michael WILSON, School of Biological Sciences, University of Aberdeen, Aberdeen AB24 3UU, Scotland, UK

Nematode-based biological control agents are now sold throughout western Europe in a broad range of market segments. Several companies are producing Steinernematid or Heterorhabditid nematodes for both domestic use and for export. Traditional key target pests have been the black vine weevil and sciarid fly larvae in protected ornamental crops and mushrooms. More recently a wider range of pests have been targeted including Scarab beetle larvae in turf and leaf miners in glass-house tomatoes. In addition to entomopathogenic nematodes, the slug parasitic nematode Phasmarhabditis hermaphrodita is also sold. This nematode can control a range of slugs but is particularly effective at controlling the gray garden slug, Deroceras reticulatum. Until recently this has been primarily sold for use in home gardens, but now it is sold to control slugs in Brussels sprouts, iceburg lettuce and orchids, particularly in the Netherlands.

Perspectives and problems of the use of EPN/EPB as biological control agents in the Hungarian horticulture

Tamas LAKATOS¹ – Andras FODOR², ¹Research and Extension Centre for Fruit Growing, Ujfeherto, Hungary, ² Department of Genetics, Eötvös University, Budapest, Hungary

The most harmful insect pest in the Hungarian fruit production is the Melolontha melolontha. The grubs of Melolontha melolontha damage the root system of the trees and may cause destruction of the newly planted young trees. This problem concern mainly the orchards in light sandy soils, and about 60 % of the Hungarian orchards were established in this type of soils. In Integrated Fruit Production (IFP) there is not possibility to use pesticides in the soil, consequently there is not chemical control of grubs. The only solution is the biological control and EPNs are the most important candidates as effective control agents. There is an ongoing Hungarian project to elaborate an effective product against grubs of Melolontha melolontha based on the nematode collection of Eötvös University. The details of the project will be presented. Generally, the main problems with elaboration and introduction a new nematode product in Hungary are: the lack of systematic and reliable information about the Hungarian nematodes fauna, the unpredictable regulation, the high estimated cost of a nematodes product and the ‘complicated’ application techniques. There is an increasing interest in antimicrobial metabolites of EPBs in Hungary. Fireblight caused by Erwinia amylovora has become the most important bacterial disease of apple since 1996, the first time isolation of the E. amylovora in Hungary. The standard chemical control of the disease is the streptomycine. Recently, there are some promising experiment with a metabolites of EPBs, offering effective therapy instead of or in addition to streptomycine.

Satoshi YAMANAKA, SDS Biotech K.K. Tsukuba Research & Technology Center, Midorigahara 2-1, Tsukuba City, Ibaraki, Japan 300-2646

In 1984, SDS initiated the development of EPN products in Japan. The first registration of Steinernema carpocapsae – based product Biosafe was approved in 1993. In 2000, SDS received the registration of S. galseri -based product Biotopia. Biosafe was introduced in turf market for the control of Hunting Billbug and Lepidoptera larvae such as lawn grass cutworm and blue grass webworm. The sales volumes of Biosafe grow steadily from 1993 to 2000. S. carpocapsae produced effective results against the billbug, the most common pest golf courses in Japan. At that time, there were no effective chemical insecticides against the billbug. S. glaseri was developed as an ideal turf insecticide against white grubs. In recent years, further research led to the introduction of Biosafe in the agriculture market. However, in recent years, the sales of biological products in turf market declined due to slow economy and the introduction of new chemistries. As a result, SDS decided to expand the usage of Biosafe into agriculture crops . In 2002, Biosafe received approval for use in strawberry on the common cutworm, in fig on yellow spotted longicorn beetle larvae , in flowers on black vine weevil and in sweet potato on sweet potato weevil & west Indian weevil. A new product specification based on the number of nematodes per certain weight was developed. Various product development and marketing strategies are in progress to increase the sales volume of Biosafe and Biotopia . Research is in progress on the use of Biosafe against peach fruit moth and oriental fruit moth in orchard and as well as against the red palm weevil. The efficacy of Biotopia against cutworms in turf is being investigated.

Huaiwen YANG, Institute of Biological Control, Chinese Academy of Agricultural Sciences, Beijing, China

Sudershan GANGULY and Vishal S. SOMVANSHI, Division of Nematology, Indian Agricultural Research Institute, New Delhi-110012, India

Entomopathogenic nematodes (EPN) belonging to the families Steinernematidae and Heterorhabditidae, are soil dwelling insect killers, having high biocontrol potential for managing several insect pests of agricultural crops as well as household pests. In some of the developed countries, the formulations of EPN are commercially available for applying against the insect pests of pastureland, horticultural and important field crops. Till 1990, there were 13 species of EPN which has now grown up to 41, thus indicating the tremendous increase in awareness and thrust on these nematodes during the last one decade. Presently, there are 32 known species of Steinernema, 8 of Heterorhabditis and one of Neosteinernema, of which 10 species have been described from USA, 4 each from China and Vietnam, 3 from Argentina, 2 each from Pakistan, Russia and India, and one each from other countries.

EPN research in India initiated in 1966 and till 1987 there was a lot of work on the efficacy of exotic strains against the local insect pests of rice, sugarcane and other field crops under laboratory conditions as well as in microplots. Due to the poor adaptability of those strains under Indian conditions, the results on field efficacy were not found consistent and therefore a need to search for indigenous strains of EPN was felt. Resultantly, several strains were isolated, thus leading to the descriptions of Heterorhabditis indica Poinar et al, 1992 from Tamil Nadu; Steinernema thermophilum Ganguly & Singh, 2000 from New Delhi; and identification of Steinernema abbasi, S. bicornutum, S. carpocapsae, S. feltiae, S. glaseri, S. riobrave, S. tami and Heterorhabditis bacteriophora. Several strains are yet to be identified.

S. thermophilum has been found to infect several insect species belonging to 6 orders. It can infect the host at wide range of soil moisture (3-16 % w/w, with 9% being the optimum), and adapts intermediate foraging strategies. Though heat tolerant, foliar spray of S. thermophilum has been found to be very effective, causing 37 to 45 per cent mortality against the diamond back moth ( Plutella xylostella ) on cabbage under field conditions even during the extreme winter when the minimum temperature recorded was 5⁰C. The mass production and formulation technologies have to be immediately strengthened in order to incorporate the EPN component in the IPM schedules. The symbiotic bacterium associated with S. thermophilum, is being characterized and has been found to be different from other species of Xenorhabdus. Efforts are also being made to exploit the insect toxicity of Photorhabdus luminescence isloted from H. indica. Several centers in the country have started working on EPN, but organized research is being pursued only at IARI (New Delhi), PDBC, (Bangalore), and GAU (Anand). Indian Council of Agricultural Research has stressed the need for a Network Project on EPN for maintaining the coordination among the researchers and making it more effective.

India is blessed with rich biodiversity resources due to its varied geographic, climatic and weather conditions based upon which the country has been divided into 15 agro-climatic and 21 agro-ecological zones. It is therefore speculated that the EPN biodiversity existing in India, would cater to the EPN demands for most of the tropical and subtropical parts of the world, in future, and perhaps without any need for genetic improvement.

Ho Yul CHOO and Dong WOON LEE, Department of Applied Biology and Environmental Sciences, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Gyeongnam, 660-701, Republic of Korea

Korean consumers have changed their consuming patterns for agricultural products. The safe agricultural products are preferred regardless of price. Environmentally friendly control of insect pests, thus, has recently received attention in Korea. Because entomopathogenic nematodes (EPN) have proved as promising control agents of many Korean important insect pests, researches have made in taxonomy, ecology, pathogenicity, utilization, and production. In Korea, EPNs have isolated and screened highly virulent Korean EPNs with investigation of ecological characters of them to use efficiently. The utilization and commercialization have also being made. Many species and strains have isolated from forest, agricultural fields, golf courses, seashores, and riparian. These isolates have continuously tested to the great wax moth larvae and white grubs to screen highly virulent EPN and virulent EPNs have used against Korean economic insect pests in greenhouses, sustainable agriculture fields, vegetable fields, and golf courses. Many positive results have obtained in greenhouses, vegetable fields, and golf courses. In addition, application strategies have also studied in golf courses and stored products. In recent, EPNs have started to be widely used in the fields and some EPNs have produced in private company thereby and commercialized for greenhouse pests.