Review, Program, Program Abstracts, Poster Abstracts, Photographs |
Program and Abstracts for the |
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
3:45
– 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)
7:00 - 8:00
Continental Breakfast
8:00-10:35
Session IV Nematode physiology, genetics, and molecular
biology
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
5:30
- 11:00
Banquet
7:00:8:00
Breakfast
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
6:00 – 9:00 Barbecue
(Secrest Arboretum)
Day 4 (September 7, 2003) Sunday [fisher
north lobby]
7:30 - 8:30
Breakfast
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
12:40-1:30
General discussion and concluding
remarks
PROGRAM ABSTRACTS
Day
1 (September 4, 2003, Thursday)
Session I. Biodiversity
Bacterial biodiversity: a
view from a culture collection manager
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.
Molecular and
gnotobiological approach to EPN/EPB cospeciation
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.
Day
1 (September 4, 2003, Thursday)
Session II Symbiosis
Sticking and swarming
in Xenorhabdus nematophila.
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.
Molecular approaches reveal genetic elements
required for symbiosis
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.
Symbiosis in Photorhabdus
Helen
BENNETT , Department of Biochemistry,
University of Bath, Bath, UK
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.
Nematode
biology and reproduction
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.
Day
1 (September 4, 2003, Thursday)
Session
III. Pathogenicity and genomics
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 Pathogčnes, 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.
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.
Photorhabdus virulence
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 Pathogenčse 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?
Wolbachia/Onchocerca
and river blindness
Eric PEARLMAN, Center for Global Health and Diseases and
Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio
44106, USA
No abstract.
Virulence
mechanisms of the nematode Phasmarhabditis hermaphrodita and its
associated bacterium Moraxella osloensis to the gray garden slug
Deroceras reticulatum
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.
Massively parallel
undergraduates for bacterial genomics
Brad GOODNER, Department of
Biology, Hiram College, Hiram, Ohio, USA
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.
Day
2 (September 5, 2003, Friday)
Session
IV. Nematode physiology, genetics,
and molecular biology
EPN genetics and molecular
biology
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.
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 25şC 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
Susan BORNSTEIN-FORST, Marian College, Marian, Wisconsin, USA
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.
Thermal biology
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 35oC, but the amount of trehalose accumulation differed by species and temperature. S. riobrave and S. carpocapsae accumulated high levels of trehalose when acclimated at 35oC, and S. feltiae at 5oC. 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 5 and 35oC was enhanced by both cold and warm acclimation and the enhanced desiccation tolerance was positively correlated with the acclimation induced trehalose accumulation. At 5oC, desiccation tolerance of S. carpocapsae was enhanced by either cold or warm acclimation, but at 35oC it was increased by only cold acclimation. Similarly, at 5oC, desiccation of S. riobrave was enhanced either by cold or warm acclimation, but at 35oC, it was increased only by warm acclimation.
Day
2 (September 5, 2003, Friday)
Session
V. Behavioral ecology
Foraging
behavior
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.
Infection behavior
Christine
GRIFFIN, National University of Ireland, Maynooth, Ireland
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
Arne
PETERS , e-nema GmbH, Germany
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.
Day
2 (September 5, 2003, Friday)
Competition and displacement
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.
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 40oC 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 25oC for 96 h), and desiccation (survival in 25% glycerol at 25oC
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 25oC 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.
Estimating
entomopathogenic nematode abundance
Stochastic and spatially
explicit simulation models: template and package for research on
entomopathogenic nematode population dynamics
Day 3 (September 6, 2003) Saturday
Session
VII. Implementation around the
world
Implementation in Western Europe
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.
Implementation of nematode
products in Japan
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.
Progress towards implementation
of entomopathogenic nematodes in China
Huaiwen YANG,
Institute of Biological Control, Chinese Academy of Agricultural Sciences,
Beijing, China
No abstract
Implementation of
entomopathogenic nematodes in India
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.
Implementation
of nematodes in Korean peninsula
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.
Nematode commercialization
in North America
Albert PYE, BioLogic Company,
Springtown Road, Willow Hill, PA, USA
No abstract
Entomopathogenic nematodes
in Mexico and Central America
Mayra
de la Torre, Centro de Investigación en Alimentación y Desarrollo A.C., Km 0.6
Carretera a la Victoria, 83000,
Hermosillo, Sonora, Mexico and CINVESTAV-IPN.
Native
strains of entomopathogenic nematodes (EPN) have been recovered in Mexico,
Guatemala and other Central American countries and have been used successfully
in laboratory test or field trials for biological control of white grub, coffee
berry borer, black flies, domestic flies and mosquitoes. Some small companies
produce EPN in vivo at low efficiency costs; however, commercial application has
been hindered by the reduced availability of EPN in these countries and their
high selling prices. Therefore, a successful commercialization depends on the
ability to produce sufficient quantities of products at competitive prices for a
full pest control program.
Even
when liquid culture is deemed to have the greatest cost efficiency, its broad
implementation has been denied for the need of a high level of technical
expertise and a large capital outlay (Shapiro-Ilan, 2003). Capital investment
and production cost could be diminished using engineering approaches to improve
the process technology. The two most important engineering aspects to take into
account in bioreactors for mass
production of EPN are oxygen transfer rate and hydrodynamics to allow mating and
to avoid mechanical damages of J2 (de la Torre, 2003).
Two
frame particle tracking velocimetry (PTV) studies in a bubble column showed that
the instantaneous velocities of S. feltiae
adults are 3 to 6 fold slower than those of the bubbles, except when a nematode
is attached to the bubble in its wave and leaves the bulk of nematodes.
Therefore, this nematode hardly will mate. In fact, high velocities of both
nematodes and bubbles appear to inhibit reproduction. Also, a different
distribution pattern of females and males seems to favor mating and reproduction
of S.
carcocapsae in an airlift reactor (Neves et al., 2001). Heterogeneous
distribution of females and males depends on both bioreactor design and
operation conditions. In a bubble column females accumulated in the bottom when
a porous stone was used as air disperser.
Bubble
columns, once the appropriate design and operation conditions are established,
present a real alternative to increase cost efficiency of submerged culture of
EPN, in addition small commercial facilities could be implemented in Mexico and
Central American countries, since they have expertise in biochemical engineering
and bubble columns are economical and very simple.
REFERENCES
De
la Torre, M.(2003) Biotechnol Adv 21(5): 407
Neves JM, Teixeira JA, Simoes N and Mota M. (2001). Biotechnol Bioeng 72:369
Shapiro-Ilan DI. (2003)Abstracts XXXVI Annual Meeting of the SIP. pg 37
Progress
towards the implementation of entomopathogenic nematodes in South America
M.
M. AGUILLERA1; E.A.B. De NARDO2; L.G. LEITE3;
L. MACHADO3; A MOINO Jr.4
1Universidade Federal
de Săo Carlos, CP 153, 13600-970, Araras, SP, Brasil, 2Embrapa –
Meio Ambiente, CP 69, 13820-000, Jaguariúna, SP, Brasil, 3Centro
Experimental do Instituto Biológico, CP 70, 13001-970, Campinas, SP, Brasil, 4Universidade
Federal de Lavras, CP 37, 37200-000, Lavras, MG, Brasil.
South
America, which is located on an extensive area comprising a
wide range of climatic conditions is thought to harbor a varied pool of
entomopathogenic nematodes (EPN) species and strains which in turn are adapted
to specific ecological niches and to some extent are likely to exert natural
biological control to either native or exotic insect pests.
Agricultural Nematology itself is a recently developed biological
sciences issue in South America. In the last few years this branch of science has been
developed especially due to profitable interchanging among governmental
institutions and to an increasing number of graduate programs being established.
Entomopathogenic Nematology is even more in its beginning but, following
a worldwide trend, efforts have been done to, as intensively as possible,
improve this area. Knowledge about
South American species occurrence, however, is still scarce.
So far, the only EPN species known to occur in South America are Steinernema
carpocapsae, S. feltiae, S. glaseri, S. rarum, S.
ritteri, S. scapterisci, Heterorhabditis argentinensis, H.
bacteriophora and H. hambletoni (species inquirendae). These species have been reported from the following South
American countries: Argentina, Brazil, Chile, Colombia, Suriname, Uruguay and
Venezuela. The most urgent need for
this region is to emphasize on systematic surveys and concurrently expand the
still small group of taxonomists to face the challenge of identifying native
species based on an ever increasing list of new species described from around
the world, by relying not only on morphometrics but also on molecular methods so
as to establish possible differences among populations.
The next most important step is to develop technology for economic mass
rearing for which relatively lower labor costs are to be considered as an
important component which could tremendously contribute for turning
implementation of EPN viable in a near future.
Other important issues include biology of species such as thermal and
soil activity ranges as well as foraging behavior.
Potential for controlling important insect pests is to be stressed and
expanded through laboratory, greenhouse and field tests using standard methods. Target insect pests include mainly Coleoptera, Lepidoptera,
Hemiptera, Isoptera, and Homoptera which cause economic losses in cultures such
as citrus, sugarcane, coffee, guava, some annual crops, mushrooms and ornamental
plants. Environmental impact on
benefitial organisms should also be stressed before field applications can be
implemented. As a general
conclusion, it is expected that soon other species native to South America can
be known and exploited accordingly either through inoculative and inundative
releases or through soil management practices aiming at increasing local
populations.
Pregress on entomopathogenic
nematode discovery and research in Africa
Alfred ALUMAI,
Department of Entomology, Ohio State University, OARDC, Wooster, OH 44691
No abstract
Day 3 (September 6, 2003) Saturday
Session VIII Application technology
Application
Equipment
Understanding the effects of the different physical phenomena (pressure, hydrodynamic stress, temperature) within a spray system is important to begin identifying the equipment characteristics and operating conditions that are least detrimental to entomopathogenic nematodes (EPNs). The general recommendation for EPN application has been common nozzle type sprayers with openings larger than 50 mm and operating pressures less than 2000 kPa (290 psi). These recommendations are most likely based on information from Steinernema carpocapsae, the most widely studied and available EPN, and may not be representative for all EPN species. Results from recent work indicate that S. carpocapsae nematodes were able to withstand greater pressure differentials and more intensive hydrodynamic conditions than other EPN species. Consequently, EPN species is an important factor to consider when defining spray operating conditions. Operating pressures within a spray system should not exceed 2000 kPa (290 psi) for S. carpocapsae and H. bacteriophora, and 1380 kPa (200 psi) for H.megidis. Other EPN species may require lower pressure. Common hydraulic nozzles (flat fan and cone) are suitable for spray application when following the manufacturer's recommendations. However, based on the flow field characteristics, the rotational flow of a cone type nozzle produces hydrodynamic conditions that are potentially less damaging to EPNs compared to the extensional flow developed within the narrow, elliptic exit orifice of the flat fan nozzle. Extensive recycling of the tank mix can cause considerable increases in the liquid temperature. Diaphragm and roller pumps are better suited for use with EPNs (i.e., minimal temperature increase) compared to the centrifugal pump, which contributes significant heat to the pumping system.
Foliar application
Simon
PIGGOTT, Becker Underwood, Littlehampton, Sussex, UK
No
abstract
Soil application technology
David
I. SHAPIRO-ILAN, USDA-ARS, SAA, 21 Dunbar Rd.
Byron, GA 31008, USA
The soil environment is the natural habitat for entomopathogenic nematodes, and thus offers great potential for successful biocontrol applications using these organisms. Nonetheless, numerous attempts to control soil insect pests with entomopathogenic nematodes have failed. To achieve successful applications in the soil environment a variety of abiotic and biotic factors must be considered. Critical abiotic factors during application include minimizing exposure to ultraviolet radiation, maintaining adequate soil moisture levels, and temperature. Soil parameters such as pH and texture can affect the success of application. Although entomopathogenic nematodes are compatible with an array of agrochemicals, certain pesticides and fertilizer can be detrimental and should not be applied simultaneously. Method of application and formulation can also affect field efficacy. Matching the appropriate entomopathogenic nematode species to the target pest is essential. Some of factors that should be considered include nematode virulence, persistence, search behavior, and environmental tolerance. Interactions with other biotic agents in the soil may also have an impact. Future research and development in soil application should include further comparisons of application methods, effects of soil biota on efficacy, and novel application technology. One novel approach to soil application is dissemination of nematode infected cadavers. Laboratory and greenhouse trials indicate greater nematode dispersal, infectivity and insect mortality when nematodes are applied in infected cadavers compared with application in aqueous suspension.
Session IX. Successes and failures
The role of habitat
management of soilborne insects with entomopathogenic nematodes
L.
W. DUNCAN, University of Florida,
Citrus Research and Education Center, Lake Alfred, FL 33850, USA
The
foremost biotic threat to citrus in Florida is a pest-disease complex caused by
the weevil Diaprepes abbreviatus and
fungi in the genus Phytophthora.
Due to deregulation of effective insecticides, citrus growers have relied
for more than a decade on commercial entomopathogenic nematode (epn) products to
help manage soilborne stages of the insect.
A large body of applied research make the pest-disease complex an good
model system regarding control of soilborne insects with epn.
The epn species used in Florida have changed over time in response to
research that revealed those species most virulent to the insect. Augmentation of epn to reduce population densities of both
the weevil and Phytophthora spp. can
be profitable in groves located on Florida’s central ridge.
However, efficacy has been inconsistent and often poor elsewhere.
Consequently, ongoing research is focused on the role of habitat in epn
efficacy. Endemic epn species are a
key factor regulating population density of D.
abbreviatus on the central ridge, in contrast to some orchards in other
regions. Regional differences in
soil texture may be causally related to the variable response to augmentation
with epn and the prevalence and diversity of endemic epn species.
Evidence of greater persistence of some endemic compared to exotic epn
raises the possibility that competition from exotic epn, which can reduce the
prevalence of endemic epn, may mitigate the value of nematode augmentation.
Therefore, augmentation of locally adapted endemic epn may be a means to
increase the current level of biocontrol. Density
dependent factors may govern the observed seasonality of natural control by epn.
Such factors also vary with habitat and include competition with free
living bactivorous nematodes in the weevil cadaver, antagonism by nematophagous
fungi, and parasitism by epn-phoretic Paenibacillus
species that reproduce in D. abbreviatus
and impair epn motility in soil.
Success
and limitations of entomopathogenic nematodes in cryptic habitats
Lawrence A. LACEY, USDA-ARS, Yakima Agricultural Research Laboratory, Wapato, WA 98951 USA
A
multitude of insect pests utilize cryptic habitats for a portion of their cycle.
These habitats include leaf litter, under bark, galleries, nut mummies,
buds and flowers, fruit bins, cracks and crevices of support structures and
several others. In the protection
of such locations entomopathogenic nematodes are less vulnerable to desiccation
and more likely to find a host than in exposed habitats. One of the best
examples of classical biological control of an insect pest in a cryptic habitat
is the use of the entomogenous nematode, Deladenus siricidicola, to
control a severe pest of pine, Sirex noctilio. In my presentation I will
focus on the use of entomopathogenic nematodes in cryptic habitats for control
of three lepidopterous pests of tree fruit, the codling moth, Cydia pomonella,
the navel orangeworm, Amyelois transitella, and the cherry bark tortrix, Enarmonia
formosana. Codling moth, is one of the most serious worldwide pests of
apple, pear and walnut. It has
traditionally been controlled using organophosphate and other broad spectrum
insecticides. The need for
alternative interventions has included development of biological control agents.
Codling moth utilizes cryptic habitats for most of its developmental stages.
Mature 5th instars exit the fruit and seek sites in which to spin their
cocoons such as under and within the bark of trees, cracks in wooden supports,
leaf litter and other cryptic habitats. Overwintering
by mature larvae that leave the fruit in late summer or early fall takes place
in these habitats and pupation ensues the following spring. Steinernema
carpocapsae was originally isolated from codling moth in the Czech Republic
and the eastern United States. Field trials of this nematode and other EPN
species against diapausing codling moth larvae in natural and artificial
substrates demonstrated the utility of EPNs for codling moth control.
In orchards the principal limiting factors of EPNs are low temperature
(below 15oC)
and desiccation. EPNs have also
been efficacious in controlling cocooned
codling moth larvae in fruit bins when bins are treated by submersion, kept damp
and at 15-25şC for 24 hours. Formulation
to retard desiccation and selection of cold tolerant nematode strains has
improved efficacy. The navel orangeworm is a serious pest of almond, pistachio
and fig in California. It utilizes
nut mummies for larval development and pupation sites in the fall, throughout
the winter and in the spring. Removal
of the nut mummies from trees and subsequent flail mowing or plowing destroys
many of the larvae, but significant numbers survive to infest nuts in the
following season. Recent research
conducted at the USDA-ARS lab in Parlier, California by J. Siegel et al. has
demonstrated the efficacy of EPNs, especially S. carpocapsae for control
of the navel orangeworm that exceeds that of sanitation and plowing.
The cherry bark tortrix was introduced into North America from Europe in
the late 1980's and has since spread from British Columbia to Northern Oregon.
It is a devastating pests of cherry and several other tree fruit
varieties and can kill heavily infested trees in a short period of time. Larva
feed on the cambium just beneath the bark and maintain a silken tube at the
opening of the gallery for removal of frass. Research has recently been started
in Washington State on the efficacy of EPNs for control of this pest. Initial
results are promising, especially when frass tubes are removed and a wetting
agent is added to EPN suspensions to facilitate entry into the opening of the
gallery.
Turfgrass and Pastures
Albrecht
M. Koppenhöfer, Rutgers
University, NewBrunswick, NJ, USA
Many
different species of white grubs (Coleoptera: Scarabaeidae) cause damage to turf
and pastures around the world. In
the USA, the Japanese beetle can be effectively controlled with H.
bacteriophora and S. glaseri but other important white grubs species
are far less susceptible to these nematodes.
The major reason for the limited use of nematodes in the USA is the
availability of cheaper or more effective insecticides.
In countries where insecticides are not available or too expensive, Hb
(Germany) or Sg (Japan) are successfully marketed for white grub control.
Mole crickets (Orthoptera: Gryllotalpidae) cause severe damage to turf
and pastures in the southern USA. The
mole cricket-specific nematode S. scapterisci can provide effective
control of adult mole crickets but not of nymphs and thus has to be applied
preventively. Because various insecticides are available in turf that are
cheaper (curatives) or more effective (preventives), Ss has to be
marketed for ‘sensitive’ areas and with an emphasis on long-term suppression
in areas with some tolerance for damage. In
pastures, Ss can be marketed as an inoculative control agent because
insecticides are too expensive. Billbugs
(Coleoptera: Curculionidae) can be effectively controlled by S. carpocapsae.
In the USA, nematode use is limited by the availability of cheaper
(curatives) or more effective (preventives) insecticides.
In Japan, Sc is the primary means of billbug control on golf
courses because no effective insecticides are available.
Black cutworm (Lepidoptera: Noctuidae) is a cosmopolitan pest on golf
course greens. It is highly
susceptible to Sc. But the
extremely low tolerance for damage on greens combined with the availability of
even more effective and reliable insecticides limits the use of Sc.
Glasshouse and musrooms
Marek
TOMALAK, Department
of Biological Pest Control and
Quarantine, Institute of Plant
Protection, Miczurina 2060-318
Poznan, Poland
No
abstract
Field efficacy of EPNs in
nursery and tree applications
R.W.H.M.
VAN TOL1 and M.J. RAUPP2, 1Plant Research
International, P.O. Box 16, 6700 AA Wageningen, the Netherlands, 2University
of Maryland, Central Maryland Research and Education Center, 11975 Homewood
Road, Ellicott City, MD 21042, USA
Although
EPNs have become an increasing successful mean to control several soil borne
pests in ornamental tree production, results in the field are still variable. In
contrast to the reliable and only limited varying efficacy of agrochemicals,
this variation in EPN efficacy is an important limiting factor for the
large-scale acceptance and use in pest control. To determine what factors
influence this variation we analysed a large number of field data where EPNs are
applied in ornamental trees and shrubs as well as information available about
growers’ perception to EPN use and economic factors. The results reveal that
several management and economic aspects like protocols for monitoring and total
pest control, qualified support by extension service, labour costs for pest
monitoring and visibility of control as well as reliability of the commercial
EPN products are important limiting factors. Important factors causing variable
field efficacy by the EPN products are quality variation of the products,
limited persistence of activity after application, EPN species/strains used in
the products, plant species, application timing (autumn vs. spring), pot or
field application and trial set-up. The field results indicate that the
tritrophic interaction between plant species, insect and EPN species/strain used
is more important for control than assumed before. Many of these and other field
factors need more research to understand their influence on efficacy and improve
the product reliability. Finally, unrealistic set-up of trials (e.g.
inoculation with larvae prior to EPN application) often leads to an
overestimation of the efficacy of EPNs. This may finally result in disappointing
control when applied in the field. Standard protocols for field testing of EPN
products would be a great help to avoid unnecessary contrasting results.
Field-testing should more closely resemble the natural situation of the pest at
the time of the season when growers apply the EPNs.
Veterinary and livestock
pests
Michael
SAMISH, Kimron Veterinary Institute, P.O. Box 12, Bet Degan, 50250, Isreal
No abstract
Vegetable
and tuber crops
Guy
BÉLAIR1, Denis J. WRIGHT2 AND Giovanna CURTO3,
1Horticultural Research and Development Centre,
Agriculture and Agri-Food Canada,
St-Jean-sur-Richelieu, Quebec, Canada J3B 3E6; 2Department
of Biological Sciences, Imperial College London, Silwood Park campus, Ascot,
Berkshire SL5 7PY, UK; 3 Servizio
Fitosanitario Regione emilia-Romagna, Bologna, Italy
Steinernematid
and heterorhabditid nematodes have been shown to infect and kill over 200 insect
hosts in the laboratory. From this list, a limited number of insects are
reported to feed on vegetable and tuber crops of economic importance. These are
mainly representative of the order Coleoptera, Diptera and Lepidoptera. For many
of these agricultural pests, the exclusive reliance on chemical control, and the
poor level of control obtained in the 1970s, stimulated research on alternative
control methods, including the use of biological control agents such as
entomopathogenic nematodes (EPN). In vegetable crops, the implementation of
integrated pest management practices, including scouting and monitoring of
insect pests, has help to reduce pesticide use but has also helped to provide
information on the biology and ecology of the pests needed for the introduction
of biological control agent. Vegetables are high value crops and the economics
have been favourable for the introduction of these costly practices. Yet,
success with EPN has not been achieved in the field despite these economics and
promising laboratory or field trials. Our objective is to review the work
performed so far at the field level and provide some directions for the future
of EPN on these crops. We will be reporting on those pest species that have
received the most attention within each of the following vegetable group: 1) roots
and bulbs 2)
tuber roots and industrial crops and 3/ leafy and other above-ground
vegetables. Although EPN have shown some promise for controlling pests on such
crops, they are not yet a viable management strategy mainly because they cannot
compete against the current management tactics. Improvements
in production technology, distribution, and application will be the key to
reducing nematode costs and insuring quality. For most crops, the organic
vegetable market, where the number of management tools is limited and the return
is high, has been identified as the most probable niche for EPN. The rapid loss
of nematode efficacy in the field suggests that improved formulation and
enhanced longevity will be necessary to acquire a wider potential in field
vegetable crops. New species and strains with higher virulence are also needed
against many pest insects. Against foliar pests, the use of EPN will require
optimization of formulations, application technology and spray regimes. This is
most likely to be attainable in humid conditions for protected crops and in the
humid tropics and sub-tropics on high value crops where the high relative cost
of nematodes compared with chemical insecticides is a less significant factor.
The withdrawal of approvals for agrochemicals on many horticultural food crops
within Europe, North America and elsewhere is likely to represent an increasing
market opportunity for biopesticide products, including nematodes.
Nematodes against forest pests
Peter TORR1, Mike WILSON1 and
Stuart HERITAGE2, 1School of Biological Sciences, University of Aberdeen,
Cruickshank Building, St Machar Dr., Aberdeenshire, AB24 3UU, UK, 2Entomology
Branch, Forest Research, Northern Research Station, Roslin, Midlothian, EH25
9SY, UK
EMC:
A new Xenorhabdus isolate of some unusual phenotype
Andrea
Máthé1, András Fodor1,
Mária Hevesi3, Tibor Ersek4 Judit Marokházi2,5,
Richard ffrench-Constant5, Betsy Anderson6, Michael
G. Klein6 and Charles Krause6,1,2
Eötvös University, Departments of Genetics1
and Biochemistry2;
H-1117 Pázmány P. setány 1/C, Budapest, Hungary; 3Quaranteen Laboratory, Szent István University, Faculty of Horticulture,
Budapest, 4Institute of Plant Protection, Hungarian Academy of
Sciences, H-1525, Hermann O. u. 15, Budapest, Hungary;
5Department
of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK; 6USDA-ARS,
Wooster, Ohio, USA.
The
Xenorhabdus symbiont of a South American Steinernema strain was isolated directly from the
infective dauer juvenile (IJ). The colonies of the new bacterium strain (EMC)
are dark brown color in LB and blue on LBTA agar media. They swarm more
intensively on the agar surface than we have observed with any other Xenorhabdus
strain before. The swarming behavior can be influenced by supplying salt in the
medium. After a few days, colorful crystals of strange form and of fractal
structure could be observed under and around the EMC colonies in the agar. When
growing on media with higher concentration of sugar, the crystals could also be
observed on the surface of the colonies as well. The size of the crystals
increased with to time, but stopped growing when the colonies were removed. The
crytals were isolated by laying adouble-layer of cellophane on the surface of
the agar and pipetted a drop of overnight-grown EMC suspension were pipetted
(localized with a sterile rubber ring) on the surface of the upper layer and let
it growing. When EMC were grown on a bacterium filter (of 0.2 µm pore size) the
crystals also appeared under the filter after a couple of days. Before the
crystals appeared, a diffusion zone of a strong purple color appeared around the
bacterium colony on the filter. We present both light and EM pictures of
different magnification of the crystals. The shapes and colors are shocking and
spectacular. By ThermoNORAN EM-image analysis (Dr. Krause and his
associates) we found that there is no nitrogen content in the crystals. Thery
are built of C, O, H and some metal, surprisingly B. For further chemical
analysis we isolated about a 40 mg of material. It is extremely hydrophobic and
pratcically insoluable in ethanol. By using acetonitril and chloroform, a
coloured compond having absorption maximum in the UV range can be separated from
a colorless, insoluable material. EMC is an excellent producer of antimicrobial
compounds, but not of per os toxins. Their effect of its antibiotics is
cytotoxic (not cytostatic) on Erwinia amylovora (Hevesi),
E. carotivora, E. coli (Fodor and Klein). The antibiotics activity
inhibits of the growth of B.subtilis, B.cereus all the Phytophtora
species tested, as well as that of Fusarium sp. (Ersek). The 16S
rDNA sequence has been determined. The oral toxicity tests on Manduca sexta
have already been accomplished (Marokházi and ffrench-Constant).
Antibiotic
substances produced by new Xenorhabus isolates: an option to control Erwinia
amylovora, the bacterium pathogen causing
fire blight
Andras
Fodor1, Attila Szentirmai2, Ferenc Sztaricskai3,
Maria Hevesi,4, Andrea Mathe1, Lajos Foldes5,8,
Aranka Kormany6,8, Szilvia Pekar4, Michael G. Klein7
and Ferenc Inantsy8, Department of Genetics, Eötvös University,
H-1117 Budapest, Pázmány Peter sétány 1/C, Hungary
Entomopathogenic
bacteria belonging to Xenorhabdus and Photorhabdus genera are
producing natural compounds of biological activity which can be used in
biological plant protection. We have continuously screening the antibiotics
activity of more than 100 EPB strains of the EPB Collection at the Eotvos
University in Budapest, Hungary (Mathe, Fodor). Our results demonstrate,
that compounds of antimicrobial activity of some strains are active against
several agricultural microbial pests such as Erwinia amylovora (Hevesi,
Pekar), Phytophtora spp. and
Fusarium spp (Ersek, see Poster of Mathe et al). We have
got the best results with our new Xenorhabdus isolates EMA and EMC. (The
origin of the strains are not presented, because of patenting considerations).
In Hungary, large volume of EMA fermentation „soup” (Szentirmai) was
studied in phytotrone, greenhouse in (Foldes, Kormany) and field (Inantsy
et al.) tests against fire blight and provided promising results. In the last
year we have established and operated a fermentation facility to grew both EMA
and EMC in the USDA Japanese Beetle Laboratory in Wooster Ohio (Klein, Fodor)
last year. Later on (in Debrecen, Hungary) we have elaborated the technology of
isolation and purification of the compounds of antimicrobial activity (Sztaricskai,
Szentirmai). Some details are presented. We have separated several fractions
of extreme antimicrobial activity. Although it was expected, we did not fint
nematophine (only triptamyne)
amongst the compounds of extracelluar substances The chemically synthetised
nematophin (Sztaricskai) showed no antimicrobial activity in our tests (Fodor,
Hevesi, Szentirmai).
The
recognition of bacterial symbionts Photorhabdus and Xenorhabdus
spp. by entomopathogenic nematodes (Heterorhabditis and Steinernema
spp.)
Narayan Prasad Pokharel1, Olaf Strauch2, Ralf-Udo Ehlers2,
1Tribhuvan University, Kathmandu, Nepal, 2Dept. f.
Biotechnology and Biological Control, Inst. f. Phytopathology, Christian-Albrechts-University
Kiel, 24223 Raisdorf, Germany
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.
The
Yin and Yang of Photorhabdus temperata: Insect pathogen and nematode
symbiont.
Todd A Ciche and Jerald C. Ensign, Department of Bacteriology; University of
Wisconsin-Madison, Wisconsin,
USA
The
gamma-proteobacterium Photorhabdus
temperata is the causative agent of a vector-borne disease of insects.
It is mutually symbiotic with this vector, a nematode Heterhabditis
bacteriophora. The dauer
juvenile (DJ) nematode transmits a monoculture of the bacteria to the hemocoel
of an insect larva where it requires the bacteria for pathogenicity and as a
substrate for growth and reproduction. We employed a mini-Tn5 transposon
mutagenesis and Green Fluorescent Protein (GFP) marker to illuminate the
mutualistic interactions between the bacterium and nematode host. A
phosphopantetheinyl transferase (PPTase) homolog was found to be essential for
the bacterium to support nematode growth and reproduction and to produce
siderophore and antibiotic activities. The PPTases are required to
activate carrier proteins involved in the biosynthesis of fatty acids and a
great diversity of secondary metabolites. One of these, a novel catechol
siderophore, photobactin, was identified from P.
temperata, but was not required for the bacterium to support nematode growth
and reproduction. The labeling of P. temperata allowed the bacteria to be observed in situ in the
nematode and insect hosts. We were able to determine that the juveniles
regurgitate the bacteria when immersed in arthropod hemolymph. It should
be possible to adapt genetic techniques from Ceanorhabditis elegans to H.
bacteriophora and to use Drosophila
melanogaster as an insect host. This will provide great potential for
P. temperata and further promote the study of the molecular basis for mutualism
and pathogenesis.
Poster
Code: A014
Primary
/ secondary phenotypes of and gnotobiological analysis in Photorhabdus
Katalin Lengyel, Erzsébet Böszörményi, Jeanette Steenroos-Ek, Viktor Vancsó, Antonia Völgyi, András Fodor and Steven Forst, 1Dept. Genetics, Eötvös University, H-1117 Budapest, Pázmány Peter sétány, Hungary, 2Biology Department, University of Wisconsin-Milwaukee, Milwaukee, WI, USA.
All
but the human pathogen Photorhabdus species of the genus are natural
symbionts the Heterorhabditis
EPN genus. The EPN/EPB symbiosis is developmentally regulated from both sides
and strictly taxon-specific. Considering that in connection with the primary(10)
/secondary(20) variation
as well as with the taxon specificity there „more legend than experimental
facts in the literature” (ffrench-Constant, unpublished), in our
systematic gnotobiological analysis we attempted to answer two fundamental
questions: (i) Is it true that intimate symbiosis (including retention) can only
be established between primary forms od the EPB (Photorhabdus) and the IJ
of the infective (Heterorhabditis) dauer juvenile? (ii) Does the „taxon
specificity” mean species –specificity? By other terms: can we really talk
about co-speciation? In our study the the 10 and 20 forms
of strains belonging to P. luminescens ssp. luminescens (Hm,
Hb), and laumondii (RH), P. temperata (WX6, WX8, NC19), P.
temperata ssp. temperata (HSH) were compared phenotypically in
details. The crystal protein mutants NC19 (A+B- and A-B+) two mutants of Jerry Ensign
as well as the intermediate form of the WX8 (Barna Fodor) were also
included. Axenic J1 as well as IJ of the natural EPN were transferred onto TSY
plates with bacterial lawn of the primary and secondary forms to see, if they
survive, molt, develop, produce progeny, produce pathogenic IJ progeny. In case
the WX strains NC19 nematodes were used. No nematode grew on Hm1 or Hm2. We
found that no but Wx6 let nematodes grow and develop normally. Neither of the
crystal protein mutants supported growth of nematodes and was not retained. As
for the phenotypic analysis we compared the cell-, and colony morphology,
hemolysis, antibiotics production, presence or absence of crystal protein
inclusion, septic toxicity, exoenzyme production (lipase, lecitinase, proteases,
outer membrane protein) and the
only surprise was that we could not find crystal proteins neither of variants
the P. temperata ssp. temperata strain we studied so far. As for the
exoproteases, the only really non-secreting and non-producing variant was Hm2.
As for gnotobiological analysis of taxonomic nature we found that the EPB
symbionts of H. bacteriophora strains carrying P. luminescens
ssp. laumondii (HP88, RH1, Brecon, AZ36 etc.) and P. boemari
(AZ29, HU1, HU2, MOL) could mutually be exchanged, but it was not true for NC19.
The symbionts of American NWE H. megidis could not relly be exchanged,
while H. marelatus could grow and develop on the symbiont of H. megidis
OHI. The symbionts of different H. indica strains (H. indica, H.
hawaiense, IS5, EG2) could not be replaced each other, altough all belong to
P. luminescens ssp. akhursti. Also, the symbnionts of H. downesii
from Ireland (K122) and Central Europe (EU 349) cannot freely be exchanged.
Neither those of EUR349 and H.Jun, inspite the latter carry conspecific EPB
symbionts. These data do not confirm the theory of cospeciation. We have started
a genetic screen for getting antibiotics-nonproducer and „host range”
mutants by using a pLOF (Kan) mutagenesis. We isolated a 9,000 mutants so far.
The mutagenesis protocol as well as our preliminary results are also presented.
We have 8 antibiotics non-producing mutants which might be used in RNAi work
with H. downesii K122.
Poster
Code: A015
Genetic
diversity among strains of Photorhabdus and Xenorhabdus
Heather
L. Smith,1 Jeff B. Jones,2 Frank J. Louws,3
and Byron J. Adams4, Department of Entomology and Nematology,1
Department of Plant Pathology,2 University of Florida, Gainesville,
FL 32611-0680, Department of Plant Pathology,3 North Carolina State
University, Raleigh, NC 27695-7616, Department of Microbiology and Molecular
Biology,4 Brigham Young University, Provo, UT 84602-5253, USA
Entomopathogenic
nematodes and their bacterial endosymbionts are important biological control
agents against a broad range of soil inhabiting insect species. In the past,
much study has been devoted to the insecticidal qualities of the bacterial
endosymbionts, yet recently, the diversity, co-evolution, and symbiotic
properties of the bacteria are beginning to emerge. For the present study,
relationships among 54 strains of Photorhabdus and 52 strains of Xenorhabdus
bacteria were analyzed by generating genomic fingerprints based on the
amplification of repetitive DNA (BOX element, repetitive extragenic palindromic
[REP], and the enterobacterial repetitive intergenic consensus [ERIC]) sequences
distributed throughout the chromosome (rep-PCR).
The rep-PCR products were analyzed by agarose gel electrophoresis,
revealing strain-specific patterns. Analysis
of the combined BOX, REP, and ERIC fingerprints showed the formation of 3
distinct clusters for both genera that correlated with the species of nematode
from which the bacteria were isolated. However,
some strains that were isolated from Steinernema glaseri are dispersed
paraphyletically throughout the dendrogram, while a few other strains formed
unique, independent lineages. This study demonstrated that BOX-, REP-, and
ERIC-like DNA sequences are commonly distributed in Photorhabdus and Xenorhabdus
strains, and therefore, rep-PCR may provide an efficient and sensitive
diagnostic tool for identifying and characterizing the bacterial endosymbionts
of entomopathogenic nematodes.
Poster
Code: B010
Age-related
effects on infection behavior of three entomopathogenic nematode species
Poster
Code: B011
Trehalose
accumulation at sub-lethal temperatures by entomopathogenic nematodes and their
survival at environmental extremes
We
tested whether the nematodes will accumulate trehalose during acclimation at
sub-lethal warm and cold temperatures and whether the accumulated trahalose
correlates with enhanced desiccation, heat, and freezing tolerance.
Three species, Steinernema carpocapsae, S. feltiae, and S.
riobrave were acclimated at 35 and 5
o
C for 1 and 4 days, respectively and
their trehalose contents were measured. Survival
of acclimated- and non-acclimated nematodes at –20 (freezing) and 40
o
C (heat), and in 25% glycerol
(osmotic desiccation) was compared. Steinernema
riobrave and S. carpocapsae accumulated high amounts of trehalose at
35
o
C, and S. feltiae at 5
o
C.
Heat tolerance in warm- and cold-acclimated S. carpocapsae and S.
feltiae increased, but was unaffected in acclimated S. riobrave.
Freezing tolerance increased in acclimated S. carpocapsae and S.
riobrave but not in S. feltiae.
Warm acclimated S. carpocapsae and cold acclimated S. riobrave
showed the highest desiccation survival at 5
o
C.
Overall, the trehalose accumulation appears to enhance desiccation,
freezing and heat tolerance of entomopathogenic nematodes.
Poster
Code: B011
Genetics
of infective juvenile longevity and stress tolerance in the entomopathogenic
nematode Heterorhabditis bacteriophora
Parwinder
S. Grewal and Nethi Somasekhar, Department of Entomology, Ohio State University,
OARDC, Wooster, OH 44691
Quantitative
and qualitative genetic analysis of life span in experimental model animals
(rat, Drosophila, Caenorhabditis elegans) predicts that resistance
to stress and longevity are positively correlated.
In a recent study we found a strong positive correlation between
infective juvenile longevity and heat tolerance in field populations of the
entomopathogenic nematode Heterorhabditis bacteriophora.
In this study we report on the genetic variability in the infective
juvenile longevity, and tolerance to heat, UV, hypoxia, and desiccation in
inbred lines of two strains of H. bacteriophora: GPS11 and HP88.
We found large genetic variability for the infective juvenile longevity
and tolerance to heat, UV, hypoxia, and desiccation in the inbred lines of both
strains. We also found that the
infective juvenile longevity is positively correlated with heat, UV, and hypoxia
tolerance, but not with desiccation tolerance.
Poster
Code: B012
A
trade-off between infective juvenile longevity and early infectivity in Heterorahbditis
bacteriophora
Parwinder
S. Grewal and Xiaodong Wang, Department of Entomology, Ohio State University,
Wooster, OH 44691, USA
Life
history theory predicts that senescence results from the detrimental effects of
aging genes that have positive effects early in life, thus increased life span
might be associated with reduced early fitness. A trade-off between enhanced longevity and decreased early
infectivity was discovered in natural populations of the entomopathogenic
nematode, Heterorhabditis bacteriophora. H. bacteriophora is a lethal endoparasite capable of
infecting a broad range of insects. Dauer
(enduring) juveniles disperse from depleted host cadavers to seek new hosts.
Because they are the only free-living stage, the dauer juveniles are
highly resistant to environmental stresses, but they must immediately search for
new hosts upon emergence because they have limited stores of energy. The
longevity of dauer juveniles from 15 populations of H. bacteriophora
collected from around the world varied from 6 to 16 weeks at 25oC
(longevity was defined as weeks to 90% mortality). Dauer juvenile infectivity against the wax moth (Galleria
mellonella) was negatively correlated with longevity one week after
emergence from host cadavers. Hence,
increased longevity of dauer juveniles was associated with decreased early
infectivity. This result is
consistent with the pleiotropy theory of aging which predicts that genetic
alterations that increase life span also reduce early fitness. Genetic selection for 24 generations of a long-lived
population of H. bacteriophora resulted in increased infectivity against G.
mellonella, but longevity declined. These
results provide strong empirical evidence for the existence of a life-history
trade-off between early infectivity and longevity.
Although, a trade-off between virulence and transmissibility of parasites
is often observed, to our knowledge, this is the first demonstration of a
relationship between lifespan and infectivity of a parasite.
Poster
Code: B013
Spodoptera
frugiperda turns tables on endophyte-mediated plant defense and virulence of an
entomopathogenic nematode
Douglas
S. Richmond1, Brian A. Kunkel2, Nethi Somasekhar3,
and Parwinder S. Grewal1, 1Department of Entomology, The
Ohio State University, Ohio Agricultural Research and Development Center, U.S.A,
2Department of Nematology, Sugarcane Breeding Institute (ICAR),
Coimbatore, 641 007, India
The
fungus Neotyphodium lolii forms a symbiotic relationship with its grass
host Lolium perenne (perennial ryegrass).
The fungus benefits from access to plant nutrients and photosynthate,
whereas the plant benefits from acquired chemical defence against herbivory.
This study examined the potential for endophyte-mediated plant defenses to
influence interactions between fall armyworm Spodoptera frugiperda, and
the entomopathogenic nematode Steinernema carpocapsae and clarified
biological mechanisms underlying our observations. In laboratory and greenhouse
experiments, S. frugiperda larvae were fed endophytic or non-endophytic L.
perenne, then exposed to S. carpocapsae or injected with the
nematodes’ symbiotic bacteria Xenorhabdus nematophila. In all
instances, S. frugiperda larvae fed endophyte-infected grass suffered
significantly lower mortality than those fed non-endophytic plants.
Although larvae fed endophyte-infected grass often had significantly
lower biomass than those fed uninfected grass, these differences did not account
for altered susceptibility to the nematode.
Endophyte-mediated reductions in herbivore susceptibility to the nematode
pathogen represent a herbivore adaptation that effectively turns the tables on
both the plant and the natural enemy by reducing the virulence of the
nematodes’ symbiotic bacteria and expanding the window of herbivory.
HU86:
A Heterorhabditis strain highly pathogenic for several scarabs, including
Melolontha melolontha
András
Fodor1, Andrea Máthe1 and Michael G. Klein2, 1Eötvös
Loránd University, Faculty of Natural Sciences, Department of Genetics,
Budapest, Hungary, 2USDA, Agricultural Research Service,
Horticultural Insect Research, Wooster, Ohio, USA
Within
the frame-work of a long-term US - Hungarian international cooperation we have
been involved in this subproject to elaborate a laboratory technique by which a
bunch of EPN strains could be tested, and all ineffective strains could
unambiguously be excluded. With the permission of the USDA/ARS, Japanese Beetle
Laboratory, the standard protocol, elaborated by us is available for researchers
interested in using it. More than 30 EPN strains (including steinernematids of
the long dauer phenotype) have been tested and those which were unable to
efficiently infect scarabs under optimum condition were excluded. The bulk of
the experiments were carried out at the USDA/ARS Japanese Beetle Laboratory, in
Wooster, Ohio on local pests (northern masked chafer [Cyclocephala borealis),
European chafer (Rhizotrogus majalis) and Japanese beetle (Popillia
japonica)] and continued in Hungary on Maybeetle (Melolontha melolontha)
grubs. The amount, the humidity, the viscosity and the aeration of the soil, as
well as the temperature of the experimental chamber, were standardized. The
number of the infective dauer juveniles (IJ) was carefully determined before
being mixed evenly with the soil. IJs were provided in great excess, since we
were interested only in the biological potential of the EPN strains. The
effective strains from these initial tests are to be studied with reproducible
parameters of practical application. This study includes data from different
application techniques of EPN strains. Although we found significant differences
between the susceptibilities of the different scarab species to EPN strains in
general, we could made an order of EPN strains concerning their biological
activities against grubs. From the more than 30 strains we tested, we have kept
only 9, as potential candidates of controlling Maybeetle.
These are to be produced on a large scale in a bioreactor at Újfehértó,
Hungary. We found that Heterorhabditis strain in general were more
efficient than any Steinernema strain we studied in these tests. Strains of the
same species (such as H. bacteriophora) proved rather different. We have
finally chosen HU86 as a best candidate to control M. melolontha. IJs
emerging from the cadavers of Northern Masked Chafer (NMC) and Japanese Beetle (JB)
were brought back to Hungary and used to infect more than 800 M. melolontha
grubs of different age in the same test system. We found a surprisingly high
(> 80%) mortality independently of the size of the grubs. By a selection
procedure (which is not as easy as it had been thought) we could improve the
efficiency further. There are two problems which should be solved. (i) It is not
too easy to grow HU86 on its own symbiont in vitro; since the symbiont we
isolated is extremely toxic even for its nematode symbiont partner; (ii) we have
to use an IJ dose much higher than used in general (500-1000 IJ/g soil of 14.1%
humidity).
Poster
Code: C011
New
strains of the entomopathogenic nematode, Steinernema riobrave:
are they better for biological control of the citrus root weevil, Diaprepes
abbreviatus?
Robin
J. Stuart1, David Shapiro-Ilan2, Rosalind James3,
Khuong Nguyen4, and Clayton W. McCoy1
1Citrus
Research and Education Center, IFAS, University of Florida, Lake Alfred, FL
33850, 2USDA-ARS, Southeast Fruit and Tree Nut Research Lab, Byron,
GA 31008, 3USDA ARS NPA,
Pollinating Insect-Biol., Mgmt. Syst., Utah State Univ., Natural Resources
Biology Bldg., Logan UT 84322, 4Department of Entomology and
Nematology, IFAS, University of Florida, PO Box 110620, Gainesville, FL 32611,
USA
The
entomopathogenic nematode Steinernema riobrave has proven
effective against Diaprepes abbreviatus in certain Florida soils.
However, this species is known from only a single strain, and it is
possible that other strains of this species might be much more effective. Therefore,
we recently returned to the lower Rio Grande Valley in Texas and Mexico where
the original strain was isolated and took a series of soil samples in an effort
to find new strains. These samples
yielded ten new isolates of S. riobrave as well as a new species
of entomopathogenic nematode in the genus Heterorhabditis.
Laboratory assays comparing the new S. riobrave strains,
the old strain, and a mixed strain (formed by pooling all ten new strains)
against D. abbreviatus found significant differences in virulence.
Under our experimental conditions (24şC, 70% RH, Candler sand at 8%
moisture in 25 dram snap-cap vials and 200 infective juveniles), and after 7
replicates of 20-30 vials per strain per replicate, the old strain produced the
lowest average mortality level (57.7%) whereas the mixed strain produced the
highest average mortality level (84.1%). Thus,
the best and the worst strains differed by 26.4%, with the best strain killing
45.7% more than the worst strain. These
data indicate that some of the new strains are considerably more virulent than
the older strain and justify further testing of the new strains for
characteristics important to the biological control of D. abbreviatus
in Florida citrus.
Differences
in the susceptibility of white grub species (Coleoptera: Scarabaeidae) to
entomopathogenic nematodes
P.
S. Grewal*, S. K. Grewal*, and M. G. Klein**, *Department of Entomology, OARDC,
Ohio State University, Wooster, OH 44691, USA and **USDA, Agricultural Research
Service, Horticultural Insects Research Laboratory, Wooster, OH 44691, USA
Invasive,
non-native, white grubs (Coleoptera: Scarabaeidae) cause significant damage in
urban landscapes. Although the lack
of natural enemies in their new home is often suggested as an important factor
in the establishment and spread of invasive species, the potential of incumbent
generalist parasites and pathogens to delay their establishment and spread has
not been explored. We compared the
susceptibility of the introduced Popillia japonica and the native Cyclocephala
borealis to 16 species and strains of entomopathogenic nematodes isolated
from within or outside the geographic ranges of the two scarabs. We found large variation in the virulence of the
species/strains of nematodes with over 50% mortality of P. japonica
produced by Heterorhabditis zealandica strain X1 and H.
bacteriophora strain GPS11 and of C. borealis by H. zealandica
and H. bacteriophora strains KMD10 and NC1.
Heterorhabditis indica and H. marelatus caused less than
20% mortality of both scarab species. When
considered as a group, the nematode species and strains from within and outside
the geographic ranges of either P. japonica or C. borealis did not
differ in virulence towards either scarab species.
Dose response studies with selected nematode species and strains against P.
japonica and two additional non-native species Anomala (Exomala)
orientalis and Rhizotrogus majalis and the native C. borealis
indicated that R. majalis was the least susceptible and P. japonica
and A. orientalis were as susceptible as the native C. borealis.
Heterorhabditis zealandica was significantly more virulent than
any other species or strain against P. japonica with a LC50 of
272 infective juveniles/grub. The
LC30 and LC50 values for H. zealandica were also
the lowest among the four nematode species/strains tested against A.
orientalis and C. borealis. The
LC50 values for H. zealandica and H. megidis (UK
strain) were signficantly lower for the native C. borealis than the
introduced A. orientalis. Heterorhabditis
zealandica also showed the highest penetration efficiency and the lowest
encapsulation in P. japonica and C. borealis grubs.
Results suggest that the introduction of the exotic H. zealandica
into the front-line states with respect to the movement of P. japonica
and A. orientalis should be explored as a tactic to delay their
establishment and spread. The
results also suggest that the manipulation of the indigenous H. bacteriophora
populations may help in delaying spread and mitigating losses caused by the
invasive grub species.
Poster
Code: C013
Enhanced
consistency in biological control of white grubs with new strain of
entomopathogenic nematodes.
P.
S. Grewala,
K. T. Powera,
S. K. Grewala, A. Suggarsb,
and S. Hauprichtb, aDepartment
of Entomology, OARDC, Ohio State University, 1680 Madison Avenue, Wooster, OH
44691, USA bTrueGreen
Technical Center, TrueGreen ChemLawn, 135 Winter Road, Delaware, OH 43015, USA
Achieving
predictability (i.e. consistency of control) is a key challenge in the use of
entomopathogenic nematodes for the control of white grubs.
We evaluated, Heterorhabditis
zealandica X1, Heterorhabditis
bacteriophora GPS11 and HP88, Steinernema glaseri NJ and MB, and S. kraussei UK strains in a series of field trials against the
second and third instar white grubs Popillia
japonica and Cyclocephala borealis
at different locations over a 2-year period. A combined analysis of the results from all 8 trials
containing 46 treatments indicates that the control of P.
japonica and C. borealis by the
new nematode strains applied at 2.5 x 109
infective juveniles/ha is equal to or better than the curative application of
trichlorfon. Overall, H.
zealandica X1 and H. bacteriophora GPS11 strains were the most
effective nematodes resulting in 73-98% and 34-97% control of P. japonica
and 72-96% and 47-83% control of C. borealis, respectively. Nematode efficacy was however, strongly influenced the
post-application irrigation plus rainfall.
Under optimal amount of post-application irrigation plus rainfall, the
control of both grub species was 80-96% by Hb-GPS11 and 96-98% by Hz-X1 strain.
Trichlorfon applied as a curative treatment in September provided 29-92%
control of P. japonica, 49% control of C. borealis, and 0-77%
control of mixed populations of the two grub species.
Halofenozide and imidacloprid applied as preventative treatments in July
provided 97 and 98% control, respectively of P. japonica.
Imidacloprid applied as a curative treatment in September provided 47%
control of C. borealis but 0% control of P. japonica. We
conclude that the new strains of nematodes, Hb-GPS11 and Hz-X1 can provide
better or as good curative control of white grubs as the most widely used
insecticide, trichlorfon.
Poster
Code: C014
Comparison
of efficacy of entomopathogenic nematode species against fungus gnat, Bradysia
coprophila, in floriculture
Efficacies
of Steinernema carpocapsae, S. feltiae, Heterorhabditis
bacteriophora GPS11, H. indica, H. marelatus and H. zealandica
were compared against the fungus gnat, Bradysia coprophila infesting
poinsettia (Euphorbia pulcherrima) grown in two potting media (Ball-mix
and Pro-mix) in growth chamber at 22 ± 1
o
C.
Nematodes were applied at the rate of 1.25 X 10
5
infective juveniles/m
2
in a single or split application.
Efficacy of nematodes against fungus gnats differed with potting media
and species. In the Ball-mix single
application of H. bacteriophora significantly reduced fungus gnat
emergence relative to the control 3, 7 and 14 days after treatment (DAT).
Steinernema feltiae significantly reduced fungus gnat emergence
relative to the control 3 and 7 DAT and S. carpocapsae 3 DAT.
Heterorhabditis indica and H. zealandica significantly
reduced fungus gnat emergence relative to the control 7 DAT.
In the Pro-mix, H. bacteriophora, H. marelatus and H.
zealandica significantly reduced fungus gnat emergence relative to the
control only 3 DAT. However, H.
indica significantly reduced fungus gnat emergence relative to the control
from 28 DAT until the end of the experiment.
When nematodes were applied at the rate of 1.25 X 10
5
infective juveniles/m
2
in four split dosages at weekly
interval, only S. feltiae was significantly effective in reducing fungus
gnat emergence throughout the experiment. Since
nematodes species, H. bacteriophora, H. indica, and H. zealandica
were as effective as S. feltiae in reducing fungus gnat emergence at 22
± 1
o
C, further research is needed to
explore the effectiveness of these warm-adapted nematodes to manage fungus gnats
in the greenhouses.
Poster
Code: C015
Improvement
of persistence and infectivity of entomopathogenic nematodes using formulation
adjuvants for foliar application against Plutella xylostella
Sibylle
Schroer & Ralf-Udo Ehlers, Institute
of Phytopathology, Dept. Biotechnology & Biol. Control, Christian-Albrechts-University
Kiel, Klausdorferstr. 28-36, 24223 Raisdorf, Germany
In
the past decades the Diamondback moth (DBM), Plutella xylostella,
developed resistance against every insecticide applied on Brassica crops
world-wide. In 2001 an EU funded project (DIABOLO) started with the objective to
manage resistance in DBM populations and to support natural antagonists. Novel
integrative biological strategies will be tested in China and Indonesia. One
particular subject of DIABOLO is the substitution of chemical insecticides with
entomopathogenic nematodes (EPN) against last instars of DBM. To achieve
satisfying control results EPN persistence and infectivity on the foliage must
be increased significantly. Screenings of different EPN species confirmed best
pathogenicity of Steinernema carpocapsae (Sc) against 3rd instar
DBM. Penetration of Sc into DBM larvae was more effective when applied on
filter paper than on cabbage leaf, indicating an active invasion of infective
juveniles (IJ) rather than an ingestion by DBM. Formulating EPN with 0.3%
surfactant based on castor oil and 0.5% xanthan gum improved the efficacy
compared to water. The infective dose (ID)50 is lowered from 20.3 to
6.7 Sc/larva. Using 100 Sc/larva the infective time (IT)50 is 8.7h in
water and only 1.4h in the formulation. Without formulation adjuvants ~70% of
the EPN applied on cabbage foliage run
off the leaves. The ability to prolong EPN persistence on the leaf was evaluated
using the polymer-surfactant-formulation enriched with 0.25% cross-linked
polyacrylamide (PA) or 0.25% alginate gel. Ten hours after application the
decrease of EPN infectivity is 50% using the polymer-surfactant-formulation,
with or without PA. However, using the alginate gel infectivity was decreased by
only 10%.
Poster
Code: C016
Control
of fungus gnat infection by Steinernema
feltiae in a commercial greenhouse
Karen
Ferdinand and Susan Bornstein-Forst, Marian College, Marian, Wisconsin
Fungus
gnats are among the more pervasive insect pests found in commercial greenhouses
and can rapidly destroy large flats of a wide-variety of plants.
Entomopathogenic nematode (EPN) Steinernema feltiae has
demonstrated significant ability to prevent plant destruction and is available
for agricultural applications through a variety of commercial sources.
This study compared the efficacy of S. feltiae spray applications
from both research and commercial sources on fungus gnat infestation of
impatiens plants. Three trials
using doses of up to 10,000 EPN/plant were applied to sets of 40 impatiens
plants in 4” pots. Control plant
sets were not subject to nematode sprays. Both
experimental and control plants were infected naturally through the resident
fungus gnat population found in the greenhouse and plants were evaluated for
health by measuring water uptake, growth of new foliage, damage by fungus gnats,
and height. The fungus gnats were
collected and counted using a sticky trap bioassay.
Significant protection from insect infection was observed in every
category measured. Furthermore, it
was also observed that cross-protection from spider mite infestation was notable
in nematode treated plants. A cost
analysis indicates that EPN sprays are much less costly than chemical
counterparts, which are potentially dangerous to human health and can select for
resistant mutations in insects. This
commercial study is a model report for typical greenhouses across the USA.
Poster
Code: C017
Managing
late instar Diaprepes root weevil with entomopathogenic nematodes and an
insecticide
C. Mannion and H. Glenn, Tropical Research and
Education Center University of Florida, IFAS
Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae), an introduced pest, has spread over a large area of central and southern Florida where it is damaging citrus, ornamental plants, sugar cane and numerous other crops. In addition to the damage caused by this pest, there are regulatory concerns of spreading Diaprepes to non-infested areas. Previous research has demonstrated that bifenthrin (Talstar) is efficacious against neonates and young larvae and that some entomopathogenic nematodes are efficacious against various stages of larvae. Bifenthrin is currently recommended as a drench or incorporated into the potting media at a rate of 25 ppm based on the bulk density of the media. Tests were conducted to evaluate bifenthrin and entomopathogenic nematodes, alone and in combination, for control of older larvae (> fifth instar) in container ornamentals. In all cases, the combination treatment of bifenthrin and the entomopathogenic nematodes provided the best control suggesting a synergy or additive effect between treatments. The results of a field test did not mirror the results from the tests with containerized plants, however, there was a trend of increased control in the combination treatments.
Poster
Code: C018
Use
of steinernematid nematodes for post harvest control of navel orangeworm (Lepidoptera:
Pyralidae, Amyelois transitella)
in fallen pistachios and almonds
J.P. Siegel1, L A. Lacey2, R. Fritts, Jr.3,
B. S. Higbee4, and P. Noble1, 1USDA/ARS,
San Joaquin Valley Agricultural Sciences Center, 9611 South Riverbend Avenue,
Parlier, CA 93648
2USDA/ARS,
Yakima Agricultural Research Laboratory, 5230 Konnowac Pass Road, Wapato, WA
98951, 3Certis USA, 9145 Guilford Road, Suite 175, Columbia, MD
21046, 4Paramount Farming Company, 33141 E. Lerdo Highway,
Bakersfield, CA 93308, USA
Four
trials employing 1-m2plots were conducted between November 2002 and
April 2003 in Madera County, CA, to evaluate the ability of two species of
nematodes, Steinernema carpocapsae (Weiser)
and Steinernema feltiae (Filipjev) to
control navel orangeworm, Amyelois transitella
(Walker), in fallen infested pistachios and almonds.
The plots were located in two 16.2 ha blocks of pistachio trees and the
soil type was sandy loam. Nematodes were applied with a backpack sprayer at
concentrations ranging from 5-100 infective juveniles (IJs)/cm2 (10
IJs/cm2 was assessed in all four trials) in 374ml/m2
water. A total of 4,300 larvae were
recovered from 17,593 laboratory-infested pistachios (24.4% average infestation)
and 1,827 larvae were recovered from 6,425 naturally infested almonds (28.4%
average infestation). S.
carpocapsae was more effective than the formulations tested of S.
feltiae in pistachios and produced > 72% mortality at a concentration of
10 IJs/cm2 when nighttime temperatures were above freezing. S. carpocapsae was equally effective in bare and leaf-covered
plots and persisted longer in sandier soil (8 weeks) than did S.
feltiae. S. carpocapsae has
the potential to multiply in the field; 51.3% of the cadavers examined 21 d
after application contained nematodes (n=226).
Our trials demonstrated that S.
carpocapsae can play a role in the post harvest control of navel orangeworm
and that the formulation tested produced greater mortality than formulations of S.
feltiae applied at the same concentration.
Poster
Code: C019
The
efficacy of the bacteria Xenorhabdus nematophilus and X. bovienii
and their toxic metabolites against the Black vine weevil, Otiorhynchus
sulcatus
A.N.Mahar,
S.R.Gowen, N.G.M.Hague, S.A. Elawad and M.Munir, Department of Agriculture,
University of Reading, P.O.Box. 236, Earley Gate, Reading, RG6 6AT, UK.
Broth suspensions containing cells of Xenorhabdus nematophilus and X. bovienii mixed into compost were effective against larvae of the black vine weevil Otiorhynchus sulcatus when applied at 15 and 20 °C. Bacterial cells were always recovered from dead larvae showing that the bacteria were motile and able to penetrate into the larvae. Treatments with cell-free solutions from the bacteria were also affective against O.sulcutus larvae indicating that it is the toxin in the bacterial metabolites which are responsible for the lethal affects observed. Application of cells and cell-free metabolites of both species of bacteria to strawberry plants infected with larvae of O. sulcatus were effective. Preliminary results indicate that cells and the toxins in cell-free solutions of both species bacteria retain their effectiveness when stored in compost at 20 and 25 °C.
Poster
Code: D010
Persistence
and seasonal population dynamics of entomopathogenic nematodes Heterorhabditis
bacteriophora and Steinernema feltiae
Susurluk, I. A. & Ehlers, R. U., Institute for Phytopathology, Department for Biotechnology and Biological Control, Christian Albrechts University Kiel, 24223 Raisdorf, Germany
The establishment and
persistence of entomopathogenic nematodes (Heterorhabditis bacteriophora and
Steinernema feltiae) was investigated in organic agriculture. Nematodes
were sprayed at a dose of 5x105 infective juveniles/m˛. S.
feltiae was applied on red clover in October 2001 on an area of 18 x 500 m.
In spring 2002 oats were sown. H. bacteriophora was applied on oil seed
rape in October 2001. White clover was sown in autumn 2002. In June 2002 H.
bacteriophora was again applied on field beans which were followed by winter
wheat. The latter nematode was always applied on an area of 9 x 500 m. To
monitor the natural nematode population before and immediately after spraying,
100 soil samples per field of approximately 35 g (soil core of 2 cm diameter and
10 cm depth) were collected. Later samplings took 50 samples per field. The
samples were transferred to 25°C and 2 last instars of Galleria mellonella
were added for 3 days. Trapping of nematodes was replicated twice and the
percentage of samples with nematodes was recorded. After the application in
October 2001 the population of H. bacteriophora decreased to 50% and that
of S. feltiae to 25% of the released amount. Both nematode populations
applied in October 2001 disappeared during the winter and nematodes were again
recorded during the summer until September and were than again detected at 2%
positive samples in February 2003. A natural population of H. bacteriophora
was never detected, but the released population invaded the neighbouring
controls probably due to anthropological influence. As the occurrence of S.
feltiae in the H. bacteriophora field did not differ from that in the
field that was applied with S. feltiae we consider that the natural
population was not suppressed by the introduction of H. bacteriophora.
The establishment of H. bacteriophora in June 2002 in beans seemed to be
more successful as over 50% of the samples were positive for H. bacteriophora
in the following months. The population was quite frequently detected also
during the winter. The beans suffered from an attack of Sitona lineatus
which could have supported the succesful establishment. It cannot be concluded
about an effect on the natural population of S. feltiae in this field
because this species was not detected neither in the treated nor in the
untreated areas.
Poster
Code: D011
Naturally
occuring insect infections by entomopathogenic nematodes. Interactive database
in the Internet.
Arne
Peters, e-nema GmbH, Klausdorfer Str. 28-36, 24223 Raisdorf, Germany
Inspired
by an OECD-meeting on the safety of introduced entomopathogenic nematode species
to ecosystems in 1996 the database was created containing published and
non-published observations on naturally occurring insect infections with Steinernema,
Neosteinernema and Heterorhabditis species. All this information
is incorporated into the Ecological Database on the World Insect Pathogens (EDWIP).
The original database, however, has now been updated and is available from the
website
www.e‑nema.de under the name
NINFINS (Nematode Infections of Insects). The database provides information on
the nematode species, the insect species, the habitat and the location as well
as the circumstances of finding the infected insect. If available, the
percentage of insects infected is also included. It was put on the Internet not
only for convenience of possible users but also to encourage researchers to
enter their observations in the formsheet included. Your observation is
anxiously desired by the scientific community! The observation will be checked
and included into the Excel‑based database. New entries will be regularly
sent to the University of Illinois to be included into the EDWIP.
Poster
Code: D012
Influence
of host interactions on the regulation of endemic and exotic strains of the
entomopathogenic nematode, Heterorhabditis bacteriophora
Janet
Lawrence, Casey W. Hoy and Parwinder S. Grewal, Department of Entomology, Ohio
Agricultural Research and Development Center, Ohio State University. Wooster,
Ohio 44691, USA
An
ultimate aim of our research is to develop strategies to conserve populations of
entomopathogenic nematodes within vegetable production systems. Research
investigations were conducted to determine the impact of host supply on the
persistence of an endemic and an exotic strain of Heterorhabditis
bacteriophora. A spatially
explicit stochastic model, developed to simulate the population dynamics of H.
bacteriophora in 100 patches each 1m2 for length of a cropping
season (120 days), predicted an interaction between mortality rate and host
supply within the range of mortality rates observed in our field experiments.
Consistent with these model predictions, higher levels of persistence were
attained in treatments in which crops were manipulated to ensure a continuous
supply of hosts than those treatments in which host supply was sporadic.
Poster
Code: D013
Effect
of turfgrass management practices on endemic entomopathogenic nematode
populations
A.
Alumai and Parwinder S. Grewal, Department of Entomology, Ohio State University,
OARDC, Wooster, OH 44691, USA
Entomopathogenic
nematodes are effective biocontrol agents that are used commercially to control
insect pests of turfgrass. However,
little information is available regarding the effects of routine turfgrass
management practices on endemic nematode populations.
We investigated the effect of the intensity of management practices on
three golf course surfaces (putting greens, fairways, and roughs) on the natural
occurrence and survival of entomopathogenic nematodes.
Of the positive sites, the nematodes were recovered from 67% of rough
areas and 33% of fairways, with no recovery from putting greens.
Steinernema carpocapsae was recovered from 4, S. glaseri
from 1, and H. bacteriophora from 4 of the positive sites.
In a replicated field study, we tested the effect of 6 insecticides on
endemic populations of H. bacteriophora on a golf course rough area.
We found that only Fipronil (Chipco Choice 80 WDG) significantly reduced
the persistence of H. bacteriophora.
In a related study, we compared the viability and pathogenicity of H.
bacteriophora HP88 strain and S. carpocapsae All strain exposed to 8
products at three dilutions. We
found no significant variation in viability of S. carpocapsae or H.
bacteriophora. Trichlorfon (Dylox
80) significantly reduced pathogenicity of both H. bacteriophora and S.
carpocapsae, whereas aluminum tris (Aliette), chlorpyrifos (Dursban Pro),
and carbaryl (Sevin) had negative effects only on S. carpocapsae
pathogenicity. The insecticides
imidacloprid (Merit 75) and carbaryl (Sevin) had synergistic effects on H.
bacteriophora pathogenicity at low concentrations.
These results suggest that nematodes are more likely to occur in less
intensely managed areas that receive fewer to no chemical insecticides than the
more intensely managed fairways and putting greens.
Persistence of
endemic entomopathogenic nematodes in heterogeneous landscapes in Northeast Ohio
Janet Lawrence,
Casey Hoy and Parwinder Grewal, Department of Entomology, Ohio State University,
OARDC, Wooster, Ohio 44691, USA
Understanding
abiotic and biotic factors which affect the persistence of endemic
entomopathogenic nematodes is critical for sustaining populations in
agroecosystems. Surveys conducted in vegetable cropping areas of Northeast Ohio
during 2000-2001 revealed that entomopathogenic nematodes were absent within
cultivated areas but present within adjacent grassy banks. To identify factors
responsible for these observations, a model was developed to simulate the
dynamics of nematodes across 100 sites over a cropping season. Model parameters
included mortality rate (environmental and agronomic stresses) and birth rate
(function of host quality and quantity). Simulations indicated that at mortality
rates ≥ 0.1 nematodes would be undetectable within habitats regardless of
host quantity or quality and at rates of ≤ 0.5, host factors greatly
influenced the maintenance of population at detectable levels. The model also
predicted that sites with detectable populations would more likely remain
detectable over time than sites without detectable populations. Sites where
nematodes were not detectable could occasionally increase to detectable levels.
A revisit to sites sampled in the grassy banks, showed that 40% of sites
at which nematodes were previously detected remained positive and 10% of
negative sites became positive. An additional survey of undisturbed forests,
undisturbed shrub lands, grassy banks, and cultivated areas resulted in nematode
detection only along grassy banks. Data confirm model predictions of temporal
variation of populations and suggest that differential detection among habitats
may be due to differences in mortality rates caused by agronomic or
environmental stresses.
Ecological
consequences of inundative biological control: impact of entomopathogenic
nematodes on the nematode community in turfgrass
N.
Somasekhar1, 2 P.S. Grewal, 1, E. A.B. De Nardo1,
3, and B. R. Stinner1, 1Department of Entomology,
Ohio State University, OARDC, Wooster, Ohio 44691, USA, 2Permanent
Address: Sugarcane Breeding Institute (ICAR), Coimbatore 641 007, India, 3Permanent
Address: Embrapa Meio Ambiente, Jaguariuna, 13000.820, Brazil
Biological
control is generally considered an ecologically safe pest management approach.
This notion is often based on its relative safety to humans and above
ground flora and fauna. However,
there is a paucity of data to support that the large-scale releases of
biocontrol agents do not have an impact on below ground food webs.
We addressed this issue using native and non-native entomopathogenic
nematodes and soil nematode community in a turfgrass ecosystem as a model.
Application of Heterorhabditis bacteriophora strain GPS11 (native
to Ohio), H. bacteriophora strain HP88 (non-native to Ohio), and H.
indica (non-native to US) significantly reduced the abundance, species
richness, diversity, and maturity of the nematode community by reducing the
number of genera and abundance of plant parasitic but not free-living nematodes.
In contrast to the entomopathogenic nematode treatments, trichlorfon, a
commonly used insecticide in turfgrass, reduced the number of genera, abundance
and diversity of nematode community by adversely affecting both plant parasitic
and free-living nematodes. The
decreases in diversity and species richness of soil nematode community were
significantly greater in non-native as compared to the native entomopathogenic
nematode treatment and untreated control 30 days after treatment.
This indicates rapid response of soil nematode community to application
of non-native entomopathogenic nematodes. Sixty
days after treatment, non-native nematodes recorded significantly lower
diversity and species richness relative to the control, but no significant
differences were observed in native and non-native nematode treatments.
The reduction in abundance and diversity of plant parasitic nematodes
without any adverse effect on free-living nematodes that play a role in nutrient
cycling may be a beneficial non-target effect of entomopathogenic nematodes.
Inundative application of
entomopathogenic nematodes poses no threat to the soil microbial activity and
nutrient cycling
E.
A. B. De Nardo1, 2, P. S. Grewal, 1 D. McCartney1
and B. R. Stinner1, 1Department of Entomology, Ohio State
University, OARDC, Wooster, Ohio 44691, USA, 2Permanent Address:
Sugarcane Breeding Institute (ICAR), Coimbatore 641 007, India, 3Permanent
Address: Embrapa Meio Ambiente, Jaguariuna, 13000.820, Brazil
Entomopathogenic
nematodes (EPNs) and their associated symbiotic bacteria have been considered as
a safer approach to pest control than the chemical pesticides.
They have been proved to be safe to the humans and several other above
and below ground vertebrates and invertebrates. However, some recent studies have indicated that EPNs have
the potential to affect the diversity of native fauna in soil ecosystems even
though they do not have any direct parasite/host or predator/prey relationship.
EPNs are applied often as inundative strategy and repeated applications
of these nematodes to control recurring pest populations may sustain the impact. Metabolic products of symbiotic bacteria of EPNs are reported
to possess a broad spectrum of biological activities and fundamental questions
arise about their impact on soil fauna and flora and consequently affecting soil
processes. The impact of an
inundative release of Steinernema carpocapsae and the insecticide
Trichlorfon (Dylox 80) in the presence or absence of Galleria mellonella larvae,
on the soil microbial respiration; microbial biomass (total nitrogen), and
mineral nitrogen (NH4-N, NO3-N) were evaluated in a
microcosm study. The results from
the first trial indicated that treatment with S. carpocapsae with or
without G. mellonella larvae do not cause detrimental affect on the soil
processes measured. In fact, the
EPNs increased the amount of NH4-N and N-NO3,
significantly, compared to the pesticide and control treatment, at least until
15 days. Microbial biomass was not affect by the treatments.
Poster
Code: E012
The safety of the
slug-parasitic nematode Phasmarhabditis hermaphrodita (Rhabditida) to the
earthworms Lumbricus terrestris and Eisenia fetida under
laboratory conditions
E. A.
B. De Nardo1, S. K. Grewal1, A.
Sindermann2 and P. S.Grewal1, 1Department of
Entomology, Ohio State University, OARDC, Wooster, Ohio 44691, USA, 2Wildlife
International Ltd, Ecotoxicology & Analytical Testing Service, Easton, MD,
USA
Phasmarhabditis hermaphrodita is a lethal nematode parasite of slugs and in
Europe has successfully been developed as a biological control agent NemaslugTM.
Recent research in the USA showed
the efficacy of this nematode for managing pest slug population present there,
and nematode producers have expressed interest to import P. hermaphrodita, since
it has not been found occurring naturally in the USA. To bring it in, we need to demonstrate it’s safe to
non-target organisms. Recent claims
that a nematode similar to P. hermaphrodita preys on earthworms have cast
some doubt on the safety of this slug-control agent. In our study, intact and
injured adults of L. terrestris
and E. fetida were exposed to the commercial formulation of P.
hermaphrodita DMG 0001 strain “Nemaslug”in muck soil and artificial soil
for 14 days respectively. Each
treatment had four replicate, each containing ten worms. The average body
weight, burrowing behavior, mortality and other clinical signals of the
earthworms were evaluated. No statistically significant differences in those
parameters were found at any concentration tested when compared to the control
groups and also no nematodes were found in the dissected earthworms that died
during the experiment.
Poster
Code: E013
Effects
of entomopathogenic nematodes and their symbiotic bacteria on plant-parasitic
nematodes in turf grass in sandy soil
Ganpati
B. Jagdale, M. S. Nahar, Parwinder S. Grewal, and Robin. M. Giblin-Davis1,
Department of Entomology, Ohio State University, OARDC, Wooster, OH 44691-4096,
USA, 1University of Florida, Ft. Lauderdale, FL 33314-7799, USA
Effects
of Steinernema carpocapsae All strain and Heterorhabditis
bacteriophora GPS11 strain, and their symbiotic bacteria Xenorhabdus
nematophilus and Photorhabdus luminescens, respectively were
evaluated against plant-parasitic nematodes associated with turfgrass in Ohio
during 2002 and 2003. The site was
a golf course and turf consisted of a 50:50 Kentucky bluegrass and perennial
ryegrass blend and the soil was sandy. Total
plant parasitic nematode number did not differ significantly in any of the
treatments in 2003, but there were some differences in 2002.
Infective juveniles of H. bacteriophora and both the species of
bacteria caused 32-57% reduction in total populations of plant-parasitic
nematodes relative to the control only 30 days after treatment (DAT) in 2002.
Only four plant-parasitic nematode genera, Mesocriconema, Helicotylenchus,
Trichodorus and Tylenchorynchus were significantly influenced by
the application of the entomopathogenic nematodes and bacteria.
The populations of Mesocriconema were
significantly reduced in S. carpocapsae (65%), H. bacteriophora
(80%) and P. luminescens (76%) treatments relative to the control
60 DAT in 2002 but not in 2003. The
populations of Helicotylenchus were significantly reduced (57-65%) in the
plots treated with both bacteria 60 DAT only in 2002.
The population of Trichodorus was significantly reduced (64%) in S.
carpocapsae treatment relative to the control 30 DAT in 2003 but not in
2002. The population of Tylenchorynchus was significantly reduced in H.
bacteriophora (40%) and P. luminescens (40%) treatments
relative to the control 15 DAT and in P. luminescens (67%) 30 DAT
in only 2002. The populations of moss feeder nematode, Tylenchus spp.also
reduced only in H. bacteriophora treatment 15 DAT relative to the control
in 2002 and in all the treatments 30 DAT in 2003.
The non-stylet bearing nematodes were not affected by the application of
either entomopathogenic nematodes or bacteria in both years.
Poster
Code: E014
The
effect of bacteria from three species of entomopathogenic nematodes against
root-knot nematode (Meloidogyne javanica)
M. A. Tabil, S. R. Gowen & N. G. M. Hague, Department
of Agriculture, University of Reading, P.
O. Box 236, Reading RG6 6AT, UK
Broth
suspensions of cells of Xenorhabdus nematophila [isolated from Steinernema
carpocapsae (ALL)], X. bovienii (isolated from a Norwegian isolate of
S. feltiae) and a bacterium isolated from an unidentified species of Steinernema
(United Arab Emirates) prevented the hatching of second stage juveniles (J2)
from eggs of Meloidogyne javanica and immobilised the J2s.
Cell-free solutions containing toxic metabolites obtained from these
species of bacteria also immobilised the J2 and inhibited egg hatch.
A
survey of entomopathogenic nematodes (EPNs) in Meghalaya, India
Arun
K. Yadav1, Lalramliana1, Parwinder Grewal2 and
A. N. Shylesha3, 1Department of Zoology, North-Eastern
Hill University, Shillong 793 022, INDIA, 2Department of Entomology
Ohio State University/OARDC, Wooster, Ohio 44691, USA, 3Division of
Entomology Indian Council of Agricultural Research, ICAR Research Complex, Umroi,
Shillong, INDIA
A
one year survey of entomopathogenic nematodes in the Ri-bhoi District of
Meghalaya, India showed the presence of Steinernema
spp. in some soil samples. The greater wax moth larvae, Galleria
melonella were used as baiting agents. The soil samples were collected
randomly at regular time-intervals from different sites in the district, namely
dry land, wet land, forest and jhum lands. Samples withdrawn from wet lands and
forests with mostly the loamy and lacerite soil showed a comparatively higher
prevalence of EPNs. This is the first report of occurrence of EPNs, in general,
and Steinernema spp, in particular
in Meghalaya, India.
Evaluation
of optimal temperature and concentration of infective juveniles for the
bioefficacy test of indigenously isolated entomopathogenic nematodes in
Meghalaya, India
Lalramliana1,
Arun K. Yadav1, Parwinder Grewal2 and A. N. Shylesha3,
1Department of Zoology, North-Eastern Hill University, Shillong 793 022, INDIA, 2Department
of Entomology
Ohio State University/OARDC, Wooster, Ohio 44691, USA, 3Division of
Entomology Indian Council of Agricultural Research, ICAR Research Complex, Umroi,
Shillong, INDIA
Knowledge of
optimum environmental conditions is an essential pre-requisite to undertake any
field efficacy related test of an indigenous EPN species. Laboratory studies
were undertaken to understand the role of optimum temperature and concentration
of infective juveniles (ijs) on the rate of mortality of an indigenously
isolated Steinernema spp. The greater
wax moth larvae i.e. Galleria melonella served
as experimental insect host. Three different temperatures viz. 100 C,
250 C, 300 C and four different concentrations of
infective juveniles/larva viz. 25 ijs/l, 50 ijs/l, 75 ijs/l, and 100 ijs/l were
selected. Both, at 300 C and 250 C with 75 ijs/l and 100
ijs/l, a 100 % mortality of insect larvae occurred within 48 hrs. Again at the
same temperatures with 25 ijs/l and 50 ijs/l, 100% mortality of larvae could
complete within 120 hours and 72 hours, post inoculation, respectively. At 100
C with 75 ijs/l and 100 ijs/l, the larval mortality was delayed, as only
10% and 20% mortality of larvae occurred within 120 hours, post inoculation,
respectively.
Liquid
culture production of biocontrol nematodes
Ralf-Udo Ehlers and Arne Peters*, Institut for Phytopathologie, Dept. for
Biotechnology and Biological Control, Christian-Albrechts-University Kiel, 24223
Raisdorf. * E-Nema GmbH, 24223 Raisdorf, Germany
Insect pathogenic
nematodes of the genera Steinernema and Heterorhabditis are used
to control insect pests in soil environments. Several SMEs in Europe produce
nematodes commercially. Typical for rhabditid nematodes is the dauer juvenile,
an enduring third stage, which is adapted for long term survival in the soil
environment, which can be stored and formulated into a pest control product and
applied with conventional spraying equipment. Unique for Steinernema spp.
and Heterorhabditis spp. is their close association with symbiotic
bacteria of the genera Xenorhabdus and Photorhabdus, respectively.
For mass production nematodes are produced in liquid media pre-incubated with
the symbiotic bacterium before the dauer juveniles are inoculated. Nematode
yields and the process duration can vary significantly. For example, H.
megidis dauer juvenile yields can reach 100,000/ml, however, the mean of
several processes is usually not high than 40,000/ml. The process time is either
15 days (one-generation) or 24 days (two generation process). Major reason for
process instability is a variation of the nematode population dynamics. Process
conditions which are able to increase the development of the nematode inoculum
are presented. Different types of bioreactors have been tested. Internal loop
bioreactors equipped with a marine propeller can provide the necessary oxygen at
low shear forces. The system has been proven to be superior to airlift and
conventionally stirred bioreactors and has been successfully scaled up to 7,500
l.
Factors
influencing dauer juvenile recovery of Heterorhabditis
bacteriophora in liquid culture
Ehlers, R.-U., Aumann, J.; Strauch, O., Institute for Phytopathology, Christian-Albrechts-University
Kiel, Department for Biotechnology & Biological Control, 24223 Raisdorf,
Germany
The production
of entomopathogenic nematodes for commercial uses is mainly done in large scale
bioreactors filled with liquid media (Ehlers 2001). Liquid cultures of the
nematodes are pre-inoculated with their symbiotic bacteria and the DJs harvested
from a previous culture are used as nematode inoculum (Ehlers et
al., 1998). One bottle neck in the production of entomopathogenic nematodes
is the recovery of the inoculated DJ from the developmentally arrested dauer
stage. The development of the DJ to a propagative hermaphrodite is stimulated by
unknown chemical signals (food signals) which occur in a living insect and which
are produced by the symbiotic bacteria (Strauch et
al. 1998, Jessen et al. 2000,
Aumann & Ehlers, 2001). In contrast to the nematode development in the
insect, the percentage of recovering DJs in artificial liquid cultures is highly
variable (1-96% after 4 days, Strauch, 1997). Consequently, the starting
conditions in the liquid culture process are highly variable as well. The
recovery defines the number of hermaphrodites developing from the nematode
inoculum and the hermaphrodite density in the culture defines the final nematode
yield of the culture (see poster “Liquid culture production of biocontrol
nematodes”, Ehlers & Peters). The factors causing the high variability of
the recovery in liquid cultures are under investigation and the results of these
investigations are summarized in this presentation.
Requirements
for nutritional prototrophy and iron-acquisition during X. nematophila colonization of nematodes
Eric C.
Martens, Frances Russell,
Kurt Heungens and Heidi Goodrich-Blair, Department of Bacteriology, University
of Wisconsin-Madison, Madison, Wisconsin, USA
Xenorhabdus
nematophila is a
mutualist of the entomopathogenic nematode Steinernema
carpocapsae and colonizes a specialized intestinal organ, the vesicle, of
the infective juvenile (IJ) stage of S.
carpocapsae. Previously, we
determined that transposon insertions in the serC,
aroA and lrp genes of X. nematophila disrupt its ability to fully colonize IJ nematodes.
Here, we clarify the partial colonization phenotypes of these mutants and
investigate their molecular causes. We
constructed serC, aroA and lrp mutant
strains labeled with green-fluorescent protein (GFP) and followed the
colonization proficiency of these labeled strains in a visual colonization assay
using fluorescence microscopy. Comparison
of these visual assays with another assay to determine the average number of X.
nematophila/IJ in a population (gross assay), reveal that these mutants
colonize a minority of IJs in a population to near wild-type levels and that a
majority of IJs are not productively colonized.
Furthermore, some IJs that are not productively colonized by mutant
bacteria retain GFP-specific signal in their vesicles and clear this signal over
time, suggesting that these mutants are most often cleared from the intestinal
vesicle before productive colonization is established.
Using a defined medium and an assay for production of iron-scavenging
siderophores, we determined that the serC
and aroA mutants are auxotrophs for serine and aromatic amino acids,
respectively; and, that all three mutants show defects in siderophore
production. To understand which of
these phenotypes (nutrient acquisition or siderophore production) is essential
for normal IJ colonization, we are constructing new mutants with individual
defects in each of these phenotypes.
Isolation and
characterization of Photorhabdus temperata
motility mutants
Brandye Day, Erik Janicki, Ryan Jackobek, and Louis S. Tisa*, Department of
Microbiology, University of New Hampshire, Durham, NH 03824-2617
Photorhabdus temperata is a bacterium
that lives in symbiotic association with entomopathogenic nematodes of the Heterorhabditidae
family. In symbiotic and pathogenic bacteria, motility plays an important
role in the successful infection of a host. Photorhabdus
temperata was motile both by swimming (movement in liquid) and swarming
(movement on surfaces) under the appropriate conditions. Swimming motility
always required additional NaCl, MgCl2 or KCl, and was optimum with
86-176 mM NaCl. Oxygen inhibited swimming by the secondary-phase cells, but had
no effect on primary-form cells. Transmission electron microscopy revealed the
presence of peritrichous flagella on the primary-form cells under both
conditions, while the secondary-form cells produced flagella only under anoxic
conditions. However, both phase variants swarmed on solid media under anoxic and
oxic conditions and produced lateral flagella. From a bank of 10,000 transposon
mutants, we have begun to isolate mutants that are altered in motility. From our
preliminary screens, four classes of motility mutants were identified: 1.
hyperswimming mutants that swam at an accelerated rate, 2. hyperswarming mutants
that swarm at an accelerated rate, 3. swimmers that did not require additional
salt, 4. non-motile mutants, and 5. mutants with aberrant motility. For
the non-motile mutant, the disrupted gene was identified as an flgE
homolog, responsible for the hook protein of flagella. The physiological and
genetic properties of these mutants are being further determined. These studies
are directed toward understanding the role of motility and signal transduction
in the life cycle of the bacterium as both a pathogen and a symbiont.
An analysis of
the role of polar (phospholipid) membranes, polyunsaturated fatty acids and
sterols in the thermo adaptation of entomopathogenic nematode (Steinernema)
species
Erika Virágh1,
Elfridea Fodor1, Tibor Farkas1, András Fodor2,
Ghazala Furgani2, David J. Chitwood3, Michael G. Klein3,
1Biological Research Centre of the Hungarian Academy of Sciences,
Szeged, Hungary, 2Department of Genetics, Eötvös University,
Budapest, Hungary, 3Nematology Unit, USDA ARS, Beltsville, MD, USA, 4Japanese
Beetle Laboratory, USDA-ARS, Wooster, Ohio, USA
As in other
organisms, the survival of entomopathogenic nematodes depends on the ability of
membrane fluidity to adjust appropriately to changes in environmental
temperature. Therefore, we
performed a series of investigations examining the chemical composition and
specific biophysical parameters of the phospholipids from numerous Steinernema isolates from different geographical origins.
In some cases, the nematodes were cultured at cold (18 şC) and warm (25
şC) temperatures. Interestingly,
the phospholipids of steinernematids of the long dauer phenotype contained
higher proportions of saturated fatty acids and lower proportions of
polyunsaturated acids than did phospholipids from S. carpocapsae and S. feltiae.
The differences were reflected in the saturated/unsaturated acid ratios
between the isolates. When four nematode strains of different geographic origins
belonging to S. feltiae and S. carpocapsae were
propagated at low and warm temperatures, the warm climate strains increased the
proportion of the eicosapentaenoic acid (EPA) more than the boreal ones. This
result may indicate that strains inhabiting colder regions do not require as
much EPA to maintain structural and functional integrity of their membranes as
do strains from warmer areas when grown at lower temperature.
In two different measures of membrane fluidity—Fourier transform
infrared spectroscopy and fluorescence depolarization—membranes prepared from
phospholipids form the boreal nematode S.
feltiae VIJE (from Norway) were more fluid than those from S.
feltiae IS6 (from Israel), regardless of the culture temperature of the
nematodes. Membrane fluidity in Steinernema may be further modified by the observed differences in
membrane cholesterol and other sterols.
Race to death:
the encapsulation response by insect hemocytes is mediated by the surface coat
proteins and cuticular proteins of Heterorhabditis
bacteriophora.
D.L. Cox-Foster, Xinyi Li, Abid Kazi, and Kristine Miller, Department of
Entomology, Penn State University, University Park, PA, USA.
Entomopathogenic
nematodes, like Heterorhabditis
bacteriophora and Steinernema glaseri,
have evolved a lifecycle in which survival depends upon overcoming insect
immunity for survival and reproduction. After the nematode invades the insect
hemocoel and before an extensive cellular immune response by the insect, the
symbiotic bacterium must be released from the nematode gut and established for
nematode reproduction. The underlying mechanisms used by the nematode to
evade the insect immune response and the countermeasures used by the insect
present a unique system for study of host/pathogen coevolution and for discovery
of key regulators of cellular immunity. Evidence is presented that the initial,
immediate insect immune response is critical and that the nematode itself is
contributing to overcoming this defense. The interactions of the nematodes
and the hemocytes from a series of resistant and susceptible hosts (Manduca
sexta, Galleria mellonella, Popilla
japonica, and Acheta domesticus)
were visualized by light microscopy in sterile, in vitro cultures, and captured
with time-lapse computer-generated movies (available through a web-site access).
In addition, the cellular interactions were examined using scanning electron
microscopy. Initial recognition of the nematode by the hemocytes
determines success of nematode. For H.
bacteriophora (Oswego) In the resistant host M. sexta, hemocytes rapidly recognize the nematode ends and
encapsulate the entire nematode, while producing reactive oxygen species.
In a semi-permissive host (P. japonica),
recognition is also rapid but directed first at the middle of the nematode and
then the ends, permitting release of the bacteria. In the susceptible host
G. mellonella, hemocyte recognition is
weak, allowing release of the bacteria and survival of the nematode. Preliminary
data suggest that less than 15 major proteins are present in the surface coat
proteins, and that these can disrupt melanization and coagulation by Manduca
hemocytes. The nematode/bacterium produce factors eliminating reactive
oxygen species that underlie the killing of invaders by insect hemocytes.
In a semi-permissive host like the Japanese beetle, these factors may permit the
nematode to survive until the bacterium can act. Thus, a triad of
interactions governs the fate of the nematode/bacterium versus insect.
Evasive behavior of white grub species against
entomopathogenic nematodes
Corrie A. Yoder and Parwinder S. Grewal, Department of
Entomology, Ohio State University, OARDC, Wooster, OH 44691, USA
Emphasis on
biological alternatives to pesticides has increased in agriculture due to
concern about environmental pollution. Entomopathogenic
nematodes (EPNs) are used as biological control agents for soil dwelling insects
with varying success. Some grub species, for example, have been shown to vary in
susceptibility to EPNs. We
hypothesized that differences in the evasive behaviors of grub species at least
partially account for variation in the susceptibility of grubs to nematodes.
In this study, we evaluated the evasive behavior of Rhizotrogus
majalis (EC), Popillia
japonica (JAB), Exomala orientalis (OB), Macrodactylus
subspinosa (RC), Maladera castanea
(AGB), and Phyllophaga sp. (JUB)
against Heterorhabditis zealandica in
glass chambers containing field soil. Grub
movement was tracked after the inoculation of 2000 infective juveniles (IJs) in
close proximity to the grub for 2 hours. Water
was inoculated as a control treatment. Mean
distance traveled per 20-min increment, mean distance traveled over 2 h, and
percent grub mortality was quantified for each treatment and species.
Of the species studied, OB had the strongest evasive behavior against H.
zealandica as indicated by the total distance traveled and in distances
traveled per time interval. RC
moved the least overall.
Prospects
of a genome-wide analysis of EPN for
dauser
regulatory and other genes
using tools of C. ELEGANS molecular
genetics
András Fodor1, Parwinder
Grewal2 and Michael G. Klein3, 1Eötvös
University, Budapest, Hungary; 2Ohio State University, Wooster, Ohio;
USA, 3USDA-ARS, Wooster, Ohio, USA
The
efforts of elaborating an animal model system, which can be used to approach the
genetic regulation and molecular basis of almost all biological phenomena, has
resulted in the nematode Caenorhabditis
elegans having a completely described cell lineage and genomic sequence.
Considering the entomopathogenic nematodes (EPN) are also nematodes, our
community is in a privileged position to benefit the most from the information
gathered by the C. elegans research
community.
The
powerful tools of molecular genetics and functional genomics elaborated by the C.
elegans community which might be adapted to EPN research are the following:
1.
Classical genetic analysis, methods of cytogenetics and molecular
genetics aimed at determine the degree of syntheny between C
elegans and EPN chromosomes, as well as to distinguish between related and
unrelated sequences. We are focusing on two sublines: (i) to elaborate and
optimize the methodology of producing transgenic EPN for research and economic
use and (ii) to discuss the options of adopting the methods of functional
genomics, particularly the RNAi.
2. We have found the optimum
promoter and the optimal co-transformation markers: hsp-2
and yeast tps-1, respectively.
3. The possible ways of
difficulties of adopting RNAi are also discussed.
We are
suggesting an international co-operation in the field between EPN / EPB
researchers and the C. elegans
research communities.
Bacteria-mediated
RNA interference in Steinernema
carpocapsae
Kevin Drace and Creg Darby, University of Alabama in Birmingham, Microbiology Department, Birmingham, Alabama, USA
S.
carpocapsae is an
entomopathogenic nematode that together with its bacterial symbiont, Xenorhabdus
nematophila, parasitizes a variety of insect larvae. During the S.
carpocapsae life cycle, a developmental stage known as the infective
juvenile can infect insect larvae. Once inside the insect, S.
carpocapsae releases X. nematophila that
has been growing in a “vesicle” inside its gut. X.
nematophila then rapidly reproduces and secretes factors to kill and digest
the insect and factors that enhance S.
carpocapsae reproduction. S.
carpocapsae reproduces, producing large numbers of infective juveniles per
insect, each colonized with X. nematophila.
The current
focus in our lab is to use RNA interference (RNAi) to identify S. carpocapsae genes essential for this benign colonization. RNAi
was first discovered in the nematode Caenorhabditis
elegans. C. elegans has been well
characterized over the years and may become a useful genetic tool to study other
nematodes. Since, there has been little genetic work in S.
carpocapsae, RNAi could prove to be an informative and quick way to analyze
genes involved in this process without having to know the genome or have
specific genes in mind to analyze.
Both C. elegans and S. carpocapsae eat bacteria, and this has led to a useful strategy for introducing double-stranded RNA (dsRNA) for RNAi. Double-stranded RNA is delivered to the worms when they eat bacteria transformed with a vector that has the ability to produce dsRNA. We began by using 2 genes that are highly conserved among nematodes. One gene, unc-22, when knocked out causes a severe twitching phenotype. The other, unc-54, causes paralysis. When these constructs are fed to C. elegans, a phenotype corresponding to a knockout of the genes can be observed. We are currently implementing this strategy to assess the possibility that RNAi can also work in S. carpocapsae.