PROCEDES ET COMPOSITIONS INSECTICIDES ELIMINANT LES HETERORHABDITIS BACTERIOPHORA

HETERORHABDITIS BACTERIOPHORA INSECTICIDE COMPOSITIONS AND RELATED PROCESSES

Abrégé français

L'invention se rapporte à une composition insecticide comprenant une quantité efficace d'une souche hybride d'Heterorhabditis bacteriophora Hb (NJ) entomopathogène, insecticide, se présentant à l'état de formes juvéniles infectantes, ou de mutants ou de variants de ceux-ci, et un support biocompatible. Les compositions insecticides de l'invention s'avèrent améliorer l'activité insecticide par comparaison avec des compositions insecticides connues renfermant un nématode H. bacteriophora connu HBHP88. L'invention se rapporte également à un procédé visant à éliminer la population d'insectes se développant dans les sols, ce procédé consistant à appliquer sur le lieu infecté par ces insectes la composition insecticide décrite ci-dessus. L'invention se rapporte de plus à la souche hybride de nématodes Hb (NJ).

Abrégé anglais

Provided is an insecticidal composition comprising an effective amount of a
juvenile infective-stage of insecticidal entomopathogenic Heterorhabditis
bacteriophora hybrid strain Hb (NJ), or mutants or variants thereof and a
biocompatible carrier therefor. The insecticidal compositions provided have
improved insecticidal activity compared to known insecticidal compositions
containing a known H. bacteriophora nematode HBHP88. Also disclosed is a
process for reducing the population of soil-inhabiting insects in soil by
applying to a soil locus infected with such insects the above-described
insecticidal composition. Additionally, the Hb (NJ) hybrid nematode strain is
provided.

Revendications

What is Claimed is:
1. An insecticidal composition useful in reducing the population of one or
more soil-inhabiting insects comprising an effective amount of a juvenile
infective-stage of an insecticidal entomopathogenic nematode having
insecticidal properties of Heterorhabditis bacteriophora hybrid strain
Hb(NJ) or effective variants or mutants thereof and a biocompatible carrier
therefor.
2. A composition of Claim 1 wherein the nematode is Heterorhabditis
bacteriophora hybrid strain Hb(NJ).
3. A process for reducing the population of soil-inhabiting white grubs or otherinsects comprising applying to a soil locus infested with said insects an
insecticidal composition comprising an effective amount of an
infective-stage of an insecticidal entomopathogenic nematode having insecticidalproperties of Heterorhabditis bacteriophora hybrid strain Hb(NJ)) or
variants or mutants thereof.
4. A process of Claim 3 wherein the nematodes are the infective-stage of
Heterorhabditis bacteriophora hybrid strain Hb(NJ) nematodes.

5. A nematode of Heterorhabditis bacteriophora hybrid strain HB (NJ)
capable in the juvenile infective-stage of reducing the population of one or
more soil-inhabiting insects when applied to the infested soil locus.
6. A juvenile infective-stage of the nematode of Claim 5.

Description

CA 022113~9 1997-07-24
WO 96/23871 PCT/US96/02603
Heterorhabditis Bact~riophora Insecticide Compositions
and Related Processes
FIELD OF INVENTION
This invention relates to novel insecticide compositions comprising
infective-stage heterorhabditid hybrid nematodes with enhanced insecticidal
effectiveness, novel nematodes having said characteristics, and process for
reducing soil-inhabiting insect pest populations by applying an effective
amount of said insecticide composition to the infected locus.
BACKGROUND O~ INVENTION
Insecticide compositions having as an ingredient a biological insecticide
or entomopathogenic nematodes offer an attractive field of biological control
agents for use against soil-inhabiting insect pests. They offer many important
advantages over available chemical insecticides. Advantages of insecticide
nematodes include high virulence, broad host range, safety, exemption from
government registration and ease of mass production.
Nematodes of the family Heterorhabditidae form a unique symbiotic
association with Photorhabd2/s bacteria. They are pathogenic to a diverse
group of insects. Heterorhabditids are hermaphroditic. In general, infective
juveniles penetrate into the hemocoel of their host insects such as white grubs,usually through the gut or spiracles. Heterorhabditids have a dorsal tooth that
might also facilitate penetration in other areas (e.g., intersegmental
membranes). Bacteria from the digestive system of the nematodes are released
within the host, and the host dies from septicemia, typically within 48 h. The
nematodes feed, develop, mate and reproduce within the host cadaver, and,
often after multiple generations, infective juveniles are again produced, emerge,
and seek new host insects.

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It is desired to provide insecticide compositions comprising insecticide
nçm~todes which provide improved effectiveness against soil-inhabiting insect
pests, novel nematodes providing the improved effectiveness, and the process
for controlling soil-inhabiting insect pests by use of such improved insecticidenematodes, and process and device for producing said improved insecticide
1 5 nematodes.
SUMMARY OF INVENTION
Provided by this invention are novel insecticide compositions and an
improved process for controlling soil-inhabiting insect pests by applying said
novel insecticide composition having an effective amount of infective-stage
entomopathogenic nematodes of this invention to the infested locus. The
insecticidal compositions of this invention comprise an effective amount of a
juvenile infective-stage of an insecticidal entomopathogenic nematode having
insecticidal properties of Heterorhabdi~is bacleriophora hybrid strain HB (NJ)
and effective variants, mutants or hybrids thereof and a biocompatible carrier
therefor. Also, provided by this invention are the novel entomopathogenic
nematodes of the insecticidal compositions of this invention, which have high
effectiveness in controlling soil-inhabiting insects including an increased
pathogenicity to diverse insect hosts and an enhanced fecundity.

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DETAILED DESCRIPTION O~ THE INVENTION
The hybrid strain of Heterorhabditis bacteriophora HB (NJ) was
attained by hybridizing a large series of wild-type isolates of H. bacteriophoraobtained from soil samples collected from four dirrerent habitats. The habitats
were turf, turf with scattered trees, weedy field and weedy field with scatteredtrees. The habitats represented many collection sites.
The enhanced properties of the hybrid strain include enhanced patho-
genicity or infectivity to diverse insect hosts and increased fecundity.
Infectivity is deterrnined by the following test. The standard test of
infectivity used is to apply 50 infective juveniles to the top of a sand column (5
cm diameter x 5 cm high) with a target insect (the wax moth, Galleria
mellonella) at the bottom. The test is typically run at 25~C and the sand
contains 10% moisture by weight. The nematodes are allowed 24 h to find the
target insect and at that point the insect is removed, rinsed in distilled water and
incubated for a further 24 h at 25~C to allow the nematodes time to develop in
the host. The host is then dissected and the number of nematodes that
successfully established in the host is counted. In two tests of 20 replicates
each, HB (NJ) had a mean infectivity rate of 10.45 and 11.1 nematodes per host
(SE = 1.053 and 1.095 respectively) with all hosts having been infected and
killed during the experiment. In comparable tests, the HB (NJ) strain showed
results much superior to results of two known H. bacteriophora strains (HB~C
and HBHP88). They killed only 50% and 70% of the hosts, respectively, and
had mean infectivity rates of only 0.08 and 1.20 nematodes per host,
respectively. Infectivity rates against other insects indicate similar superiority
of the HB (NJ) strain.

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Fecundity is determined by the following test. The standard test of
fecundity is to quantify the number of new infective juveniles produced from
single hosts (Galleria nle110nella) of a standard weight (0.24-0.26 gms) and
with a standard inoculation of infective juveniles (n = 20) at 25~C. For 30 d
replicates with HB (NJ), a mean fecundity level of 383,046.7 (SE = 15212.9)
was obtained. Indications are that the fecundity of HB (NJ) is superior.
The Heterorhabditis bac1eriophora hybrid strain HB (NJ) provided by
this invention has a deposit number of ATCC
The insecticidal compositions can be in the folm of an aqueous spray
composition, in the form of gel pellets or granules made using calcium alginate
or other suitable carriers, in the form of a solid sheet, and the like.
The carriers, which immobilize the nematodes or partially desiccate
them, reduce their metabolism and improve their tolerance to temperature
extremes. Nematodes are also commercially available on a moist substrate
such as sponge, vermiculite, and peat, although extended storage at room tem-
perature is generally poor because nematode viability declines rapidly. The
Heterorhabditid compositions of this invention are suitably stored at 10~C-
15~C and the org~nism.c should be maintained in the presence of oxygen.
Nematodes can be applied with common agrichemical equipment
including small pressurized sprayers, mist blowers, electrostatic sprayers, fan
sprayers, and helicopters. Entomopathogenic nematodes can withstand appli-
cation pressures of 300 lb/in' and can be delivered with all common nozzle type
sprayers (e.g., "01" nozzles) with openings as small as 50 microns in diameter.
However, some types of pumping equipment produce a considerable amount of
heat, and should the temperature in the sprayer plumbing rise above 32~C, the
nematodes could be adversely affected. Fortunately, this rarely occurs and is

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usually only a problem when the spray tank is almost empty and liffle water is
available as a heat sink.
Field concentrations exceeding 2.5 billion nematodes/ha (l billion/acre)
are usually applied to ensure that a sufficient nematode population will come incontact with the target insects to provide control. A high concentration is
needed to overcome the negative impacts of the abiotic and biotic soil environ-
ment. High nematode concentrations are also needed against certain insects
which only remain in soil for a few days before tunneling into roots, such as
cabbage maggots (D. radiC21M?), and against insects which are not highly sus-
ceptible to nematode infection because of their small size and active movement.
For example, high nematode concenbrations are desirable against early
imm~ re stages of mole crickets (Scap~erisc2~s spp.), root maggots (Delia
spp.). and corn rootwor~ns (Diabrotica spp.)
An extensive discussion of formulation and application applicable to
providing the insecticidal compositions of this invention and the processes of
this invention is found in the breatise Entomopathogenic Nematodes in Biologi-
cal Control, editors Randy Gaugler, Ph.D. and Harry K. Kaya. Ph.D., Chapter
9. Forml~l~ion and Application Technology, by Ramon Georgis, CRC Press
(Boca Raton, Ann Arbor, Boston), l990.
The Galleria mellonella recognized test for evaluating insecticidal
activity of nematodes is carried out as follows~ n No. 1 filter paper is
placed in 35 mm pebi dishes. A Galleria mellonella host is placed on the filter
paper. In 500 microliters of water, lOO juvenile nematodes are suspended. The
aqueous suspension is placed on the filter paper by wetting the filter paper in
several locations. If the Galleria mellonella host has turned red at the end of
the test period, this is a showing that the Galleria n7ellonella host is dead.

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In the Galleria mellonella bait method for isolating nematodes, the
Galleria mellonel1a infected with Heterorhabditis bacferiophora normally die
and turn a red color. The soil samples taken are 2 cm in diameter and lS cm in
length. In providing the He~erorhabditis bacteriophora hybrid strain HB (NJ)
of this invention, about lS0 Heterorhabditis bacteriophora isolates were
utilized.
The crossbreeding used to provide the HB (NJ) strain involve
conventional procedures.
Changes and modifications of the invention provided will be apparent to
those skilled in the art and to the extent they are within the spirit of this
invention, are intended to be within the scope thereof.