AU779650B2 - Nematode biopesticide - Google Patents

Description

WO 00/38510 PCT/AU99/01152 1 NEMATODE BIOPESTICIDE Field of the Invention: The present invention relates to a composition and method for controlling scarabs, especially lawn scarabs, utilising certain strains of the entomopathogenic nematode species Heterorhabditis zealandica.

Background of the Invention: The family of beetles known as Scarabaeidae includes a number of species that are known as significant agricultural and horticultural pests.

Larvae of lawn scarabs (such as Cyclocephala signaticollis, Heteronychus arator, Adoiyphorus couloni, Antitrogus morbillosus, Anoplognathus porosus, Ataenius imparalis, Sericesthis geminata, S. pruinosus, S. nigrolineata, Scityla sericans, Saulostomus villosus, Aphodius tasmaniae, Heteronyx spp, Rhopoea magnicornis, Popillia japonica, Cyclocephala borealis, C. hirta, C. parallela, Melolontha melolontha, Anomala aenea, Phyllophaga phyllophaga, P.

hirticula, Phyllopertha horlicola, Haplididia etrusca, Maladea matrida, Costelytra zealandica, Amphimallon solstatialis, and Ligyrus subtropicus) feed on the roots of grasses thereby causing considerable damage to pastures, lawns and amenity turf. Control treatments typically involve the use of chemical pesticide sprays, however these have a number of disadvantages including low efficacy (particularly against final instar larvae), low specificity and public concern regarding pesticide residues. Consequently, there is a need for a viable alternative to the control of lawn scarabs by chemical pesticide spraying. In this regard, the present inventors have identified certain strains of the entomopathogenic nematode species H.

zealandica that are suitable for use as biological control agents for lawn scarabs.

Disclosure of the Invention: Thus, in a first aspect, the present invention provides a composition for controlling a populainon of lanral and/or nunal scarahs. comprising an A A.

amount of an entomopathogenic nematode optionally in admixture with a suitable agricultural and/or horticultural carrier, wherein said entomopathogenic nematode belongs to the species Heterorhabditis zealandica and has an LD50 value of less than 300 infective juveniles (IJ) as measured by 14-day pot assays against final instar Cyclocephela signaticollis larvae and/or Popillia japonica larvae.

More preferably, the entomopathogenic nematode belongs to a strain of H. zealandica which has an LD50 value of less than 175 IJ as measured by 14-day pot assays against final instar C. signaticollis larvae and/or final instar P. japonica larvae. Especially preferred are the strains designated JB1/X1, GKB and JB3D.

Compositions will include an amount of the entomopathogenic nematode which is, typically, about 50 to 10,000, more preferably about 500 to 1000, IJ/ml of composition.

Compositions will also typically include a suitable agricultural and/or horticultural carrier. Where the composition is desired to be in the form of an aqueous spray, the carrier may be selected from, for example, water or *i 15 solutions in water of polyethylene glycol or glycerol or small quantities of wetting agent or various substances to stimulate nematode activity such as citric acid, insect blood or low concentrations of chemical pesticide. Where the composition is desired to be in a solid form, the carrier may be selected from, for example, calcium alginate and polyacrylamide (as would be suitable 20 for gelled pellets), attapulgite or vermiculite (as would be suitable for solid granules), or other moist substrates such as peat, sponge, sawdust or cellulose. Compositions in solid form may be dispersed into an aqueous carrier (such as those mentioned above) for use as an aqueous spray.

Compositions are preferably stored at low temperature 2 to 10 oC) under aerobic conditions, and at a water activity of about A, 0.97.

In a second aspect, the present invention provides a method for controlling a population of larval and/or pupal scarabs in an affected area, said method comprising applying to said area a composition in accordance with the first aspect.

For compositions in the form of aqueous sprays, application may be carried out with typical agricultural and/or horticultural spraying equipment including pressurised, fan sprayers venturi sprays and boom sprayers. For compositions to be applied in a solid form, application may be carried out with typical agricultural and/or horticultural scattering equipment such as those used for spreading fertilisers on lawn.

The composition will typically be applied to an affected area which has been subjected to heavy watering in amounts sufficient to provide 50,000 to 1 million IJ/m 2 more preferably 100,000 to 500,000 IJ/n 2 Following application, it is also preferable to submit the affected area once again to heavy watering in order to soak the composition into the root zone where the larval scarabs feed. Application of the composition is preferably conducted at dusk.

The composition and method of the invention may be used for the control of lawn scarabs (such as those mentioned above) and other pest scarabs sugar cane scarabs, blueberry scarabs, etc.) In a further aspect, the present invention provides a nematode, in a 0 substantially purified form, selected from the H. zealandica strains designated JB1/X1, GKB and JB3D.

The term "controlling" as used herein in relation to a population of 15 larval and/or pupal scarabs, is intended to refer to both maintaining (i.e.

preventing increases) and reducing said population.

The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature or group of steps, components or features with or 20 without the inclusion of a further step, component or feature or group of steps, components or features.

0 Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

The invention will hereinafter be described with reference to the following non-limiting examples and accompanying figures.

Brief description of the accompanying figures: Figure 1 shows a RAPD gel conducted on DNA from H. zealandica and other comparative strains. The primer used was OP-A04: 3' (Operon Technologies Inc.). (Key: 1. 100 bp DNA Ladder; 2. H. zealandica (Great Keppel 3. H. zealandica (Great Keppel 4. H. zealandica (Great Keppel 5. H. zealandica (Windsor); 6. H. zealandica (NZH3); 7. H.

zeoiandica (WA Het); 8. Heterorlzabditis sp. (JB6); 9. H. zealandica (JB3D); H. zealandicci (JBX1); 11. Heterorizabditis sp. (I-Ff'390); 12. H. megidis (Microbjo); 13. H. bacteriopliera 0SOO S S 0O S 5.55 S *5

SO

S 0 0@ 0 6* Oe S. S

S.

S@

6 055005 0 0 0000 *5 0O 6 *005 0006 0O 05 S

S

005505 a WO 00/38510 PCT/AU99/01152 4 Figure 2 shows a RAPD gel conducted on DNA from H. zealandica and other comparative strains. The primer used was OP-F3: 5'-CCTGATCACC-3' (Operon Technologies Inc.). (Key: 1. 100 bp DNA Ladder; 2. H. zealandica (Great Keppel 3. H. zealandica (Great Keppel 4. H. zealandica (Great Keppel 5. H. zealandica (Windsor); 6. H. zealandica (NZH3); 7. H.

zealandica (WA Het); 8. Heterorhabditis sp. (JB6); 9. H. zealandica (JB3D); H. zealandica (JBX1); 11. Heterorhabditis sp. (HT390); 12. H. megidis (Mvicrobio); 13. Heterorhabditis sp. (M145); 14. H. bacteriophera Figure 3 shows a RAPD gel conducted on DNA from H. zealandica and other comparative strains. The primer used was OP-X11: 3' (Operon Technologies Inc.). (Key:1. 100 bp DNA Ladder; 2. H. zealandica (Great Keppel 3. H. zealandica (Great Keppel 4. H. zealandica (Great Keppel 5. H. zealcuandica (Windsor); 6. H. zealandica (NZH3); 7. H.

zealandica (WA Het); 8. Heterorhabditis sp. (JB6); 9. H. zealandica (JB3D); 10 H. zealandica (JBX1); 11. Heterorhabditis sp. (HT390)).

Figure 4 provides graphical results of pot assays conducted to assess the mortality of Dermolepida achieved by H. zealandica strains JB1/X1, JB1/Q and NZH3 and Steinernenia glaseri strain NC34.

Figure 5 provides graphical results of pot assays conducted to assess the mortality of C. signaticollis achieved by H. zealandica strains JB1/X1 and NZH3, S. glasei strain NC34 and H. bacteriophora HP88.

Figure 6 provides graphical results of pot assays conducted to assess the miortality of A. parvulus achieved by H. zealandica strains JB1/X1 (Per 100, 330 and 100 nematodes) and NZH3, and S. glaseri strain NC34.

Figure 7 provides graphical results of pot assays conducted to assess the mortality of L. negatoria achieved by H. zealandica strain JB1/X1 and S.

glaseri strain NC34.

Figure 8 provides graphical results to assess the mortality of adultH.

arator achieved with H. zealandica strains JB1/X1, NZH3 and Botany, S.

glaseri strain NC34, S. feltiae and S. carpocapsae BW.

Figure 9 provides graphical results of studies conducted to determine of H. zealandica strains in pot assays agrinst final inqtar C.

signaticollis.

WO 00/38510 PCT/AU99/01152 Example 1: Isolation and characterisation ofH. zealandica nematodes.

Soil samples infested with nematodes were collected from a number of field sites throughout Australia. Using the method of Bedding and Akhurst (1975), nematodes were isolated and subsequently assigned to a species on the basis of morphological characterisation (see Wouts (1979)) and DNA analysis (see Tables 1 and Eight of the isolated nematodes were assigned to the species H. zealandica.

Samples of three of the H. zealandica strains, namely JB1/X1, GKB and JB3D were deposited under the Budapest Treaty with the Australian Government Analytical Laboratories (AGAL), Pymble, New South Wales, Australia. These deposits have been accorded the Accession Nos 10726, 10727 and 10728 respectively.

Random Amplification of Polymorphic DNA (RAPD) studies were conducted on DNA from the H. zealandica nematodes in accordance with the method of Hashmi, Glazer and Gangler (1996). The results which are presented in Figures 1 3, indicate that various of the strains may be seperated on the basis of their RAPD patterns strains JB1/X1 and JB3D can be distinguished on the basis of their RAPD pattern obtained with the primer OP-X11).

Table 1: Percentage 0 Similarity of Domain 3 DNA Sequences of Heterorhabditis Strains. (299 bp Region) NZH3 Windsor JBX1 JBQ1 WA Net Irish M145 HT390 Microbjo NJ C1 NZH3 100 Windsor 100 100 JBX1 100 100 100 J13Q1 100 100 100 100 WA Het 1010 100 100 100 100 Irish M145 98.67 98.67 98.67 98.67 98.67 100 IT390 98.67 98.67 98.67 98.67 98.67 100 100 Microbio 98.33 98.33 98.33 98.33 98.33 99.67 99.67 100 NJ 94.98 94.98 94.98 94.98 94.98 96.32 96.32 95.99 100 C1 94.65 94.65 94.65 94.65 94.65 95.99 95.99 94.98 99.00 100 Known comparative strains: NZH3 zealandica), Microbio megidis), Irish M145 ("Irish Megidis"), NJ C1 bacteriophora). Deletions have been included as base pair changes.

0 Table 2: Percentage Similarity of Internal Transcribed Spacer 1 DNA Sequences of Heterorhabditis Strains. (805 bp Region) NZH3 Windsor JBX1 JBQ1 WA Het Irish M145 HT390 H. hepialus H. marelatus Microbio NZH3 100 Windsor 100 100 JBX1 99.88 99.88 100 JBQ1 99.88 99.88 100 100 WA Het 99.88 99.88 100 100 100 Irish M145 89.07 89.07 89.19 89.19 89.19 100 HT390 88.57 88.57 88.70 88.70 88.70 94.78 100 H. hepialus 81.80 81.80 81.80 81.80 81.80 91.24 99.31 100 H. marelatus 81.57 81.57 81.57 81.57 81.57 91.01 99.08 99.77 100 Microbio 84.97 84.97 85.09 85.09 85.09 93.66 89.69 87.56 87.33 100 Known comparative strains: NZH3 zealandica), Microbio megidis), Irish M145 ("Irish Megidis"). H. marelatus and H. hepialus sequences were extracted from Genbank and their comparisons are based on a 434 bp overlap of the 805 bp ITS1 region. Deletions have been included as base pair changes.

WO 00/38510 PCT/AU99/01152 8 Example 2: Control of scarabs with H. zealandica nematodes.

A variety of trials were conducted with several strains ofH. zealandica and other nematodes against scarabs. Laboratory based trials included a comparison of strains by dosing a homogeneous selection of larvae with an equivalent number of nematodes of each strain and assessing the number of larvae killed (see Comparison Trials), and the determination of LD50s from pot assays against final instar Cyclocephala signaticollis (see LD50 Trials).

Comparison Trials.

1. Dermolepida Pot assays using the method of Bedding, Molyneux and Akhurst (1983) (see Trials), were conducted using final instar larvae of Dermolepida dosed with IH. zealandica strains JB1/X1 and JB1/Q. JB1/X1 achieved 70% kill while JB1/Q had little effect (at 1000 IJs/pot). Subsequent experiments with the above strains as well as H. zealandica NZH3 and S. glaseri NC34 at the same dose (see Figure 4) showed that JB1/X1 achieved superior kill (over 90% kill after 3 weeks with JB1/X1 versus 57% after 3 weeks with S. glaseri NC34, being the best of the others). Control mortality was too high in tests done on first instar larvae to give a valid result.

2. Cyclocephala Pot assays were conducted on final instar C. signaticollis larvae with the NC34 strain of S. glaseri, H. bacteriophora HP88 and the NZH3 and JB1/X1 strains of IH. zealandica (at 500 Ijs/pot). The results obtained (see Figure showed that JB1/X1 achieved superior kill. Subsequent assays comparing JB1/X1 with a range of other strains of H. zealandica (namely, JB3/D, Botany, GKA, GKB, GKC and JB3/F) at a dose of 200IJs/pot showed JB1/X1 to equal (JB3/D, Botany, GKB and GKC) or superior (JB3/F and GKA) in killing power.

In addition, pot assays comparing JB1/X1 to a range of field collected material at 200 IJs/pot (namely, JB4/A to JB4/E, Qld CS9, and JB4/H, all of which are H.

zealandica), JB1/X1 was again found to achieve superior kill.

3. Antitrogus paivulus Pot assays were conducted on final instar Antitrogus parvulus comparing H.

zealandica strains JB1/X1 and NZH3 and S. glaseri strain NC34. The results (see Figure 6) showed that NZH3 achieved the best kill particularly in the short term. Further, NZH3 achieved close to its maximum level of control in one week whereas JB1/X1 required two weeks. Subsequent assays using a WO 00/38510 PCT/AU99/01152 9 lower dose (500 instead of 1000) resulted in all ofH. megidis strain MicroBio H. bacteriophora strain HP88 and H. zealladica strains JB1/X1 and NZH3 achieving a very poor level of kill.

4. Lepidiota negatoria Pot assays were conducted on final instar Lepidiota negatoria comparing H.

zealciadica strain JB1/X1 and S. glaseri strain NC34. The results (see Figure 7) showed that JB1/X1 achieved over 80% kill with 100 nematodes per larvae after 2 weeks, whereas NC34 achieved only 35% kill with 1000 nematodes after the same period.

5. Heteronych us arator Pot assays were conducted on adult Heteronychus arator (African Black Beetle) comparing H. zealandica strains JB1/X1, NC34, NZH3 and Botany, S.

feltiae, and S. caipocapsae BW at 1000 IJs/pot. The results (see Figure 8) showed that BW achieved some control while the others were ineffective.

6. Xylotrupes gideon Preliminary studies have showed that the H. zealancdica strain JB1/X1 at a dose of 1000 IJs/pot, is capable of killing Xylotrupes gideon larvae.

7. Anoplognathus sp.

Preliminary studies have showed that the H. zealandica strain JB1/X1 at a dose of 1000 IJs/pot, is capable of killing anAnoplognathus species.

Trials with C. signaticollis Assays to determine the LD50s of the H. zealandica strains Windsor, JB1/X1, GKB, NZH3, GKA, JB6, JB3/D and HT390 sp) against freshly collected final instar C. signaticollis were conducted in accordance with the following method: C. signaticollis larvae (collected from a playing field at the Australian National University, Canberra, Australian Capital Territory) were exposed individually to nematodes within plastic screw cap specimen jars (diameter 4.2 cm, height 6 cm) filled to within 1 cm of the top with approximately 80 g of fine sand, moisture content to about 7% (pF= The larvae were placed at the bottom of the jars. Nematodes were introduced in 1 ml of water into a centrally placed well at the top 0.5 cm diameter, 2 cm deep), which was then filled with sand. Numbers of nematodes were estimated by dilution counts. There were 20 applications of each dosage for each nematode strain.

After 14 days incubation at a temperature of 23 degrees C. larvae were WO 00/38510 PCT/AU99/01152 removed and if dead were dissected and microscopically examined for nematode infection in the insect Ringers solution. The LD 50 values were computed using the probit analysis of Finney 1971.

The results are shown in Figure 9. In particular, Figure 9 shows the level of kill achieved by each strain at a range of doses (corrected for control mortality).

These results allowed the calculation of LD50s against C. signaticollis as follows in Table 3: 00 Table 3: of various strains of Heterorhabditis zealacndica against final instar Cyclocephala signaticollis Strain LD50 95% Limits Strain LD90 950% Limits JB3ID JB 1/X 1

WINDSOR

GKB

NZH3

GKA

J136 HT390 110 121.

148 23CI 5 96 562 3170 4831 57 65 83 109 340 286 1194 1064 222 203 250 444 1052 1094 47424 2E+08 JB3/D JB1/X1

WINDSOR

GKB

NZH3

GKA

JB6 1H7390 739.7 848.7 10004 3755 4339 6041 122652 65842 315.7 430.8 506.4 1473 2062 2427 15096 9024 14430 3708 4384 28650 22781 63009 199788676 1.29E+20 and LD9COs and confidence limits calculated using the probit analysis and Fieller procedures in Genstat WO 00/38510 PCT/AU99/01152 12 Trials with other scarab species of H. zealcuadica strain JB1/X1 against other species of scarabs the Japanese beetle, P. japonica) may be determined by the following method.

1. Scarab larvae are exposed individually to nematodes within plastic screw cap specimen jars (diameter 4.2 cm, height 6.0 cm) filled to within one cm of the top with 80 g of clean, fine sand carefully mixed with water to achieve an even moisture content of 7% (Pf A larval scarab is placed at the bottom of each specimen jar, sand lightly packed over it and the top screwed on tightly.

2. Nematodes are introduced in 1 ml of water into a centrally placed well cmi diameter. 2 cm deep), which is then filled with sand.

3. Five dosages of IJs are each applied to 20 scarab larvae with a further larvae as controls (total of 140 larvae). The dosages used are 10, 33, 100, 330, and 1000 IJ/ml.

4. Larvae are examined after one week and two weeks at 23 degrees C.

with live and dead recorded on both occasions.

LD50s are calculated using the probit analysis and Fieller procedures within the software package, Genstat 5 (Genstat 5, Release 4.1, Third Edition, Lawes Agicultural Trust, (IACR-Rothamsted), 1997).

Tables 4 and 5 provides the results of LD50s ofH. zealandica against various scarab species using the above method.

WO 00/38510 WO 0038510PCT/AU99/01 152 T1able 4: and LD9Os of various scarabs HI. zealwidica JB1/XI Scarab Species LD50 Adoryphorus couloiii 128 Lep-idiota negatoria 177 An titrogus parvulus 186 Cyclocepzala sign aticollis 121 Popillia japonica 115 .6* after 2 weeks exposure to 95% Limits 228 317 449 203 181 Adoivlioru11s couloni Lepidiota n egatoria Antitrogus parvulus Cyclocepliala sign aticollis Popillia japonica 1396 964 3402 848.7 1174 95% Limits 627 438 1009 430.8 871 6486 6810 227541 3708 5742 froin pooled data and LD90 and confidence limits were calculated using the probit analysis and Fieller procedures in Genstat WO 00/38510 WO 0038510PCT/AU99/Ol 152 14 'rable results from tests on Popillia japonica Rep 1 LD50 Lower 95% Upper After 1 week 5236 681.1 3.94E +18 After 2 week 182.3 95.42 410.9 Rep 2 LD50 Lower 95% Upper After 1 week 147.9 86.81 265.9 After 2 week 76.38 41.01 134.9 Pooled reps LD50 Lower 95% Upper After 1 week 412.7 233.2 993.7 After 2 week 115.6 74.81 180.8 Lower 95% Upper After 2 weeks 1174 871 5742 Example 3: Effectiveniess of H. zealandica nematodes against various scarabs in the field.

Small scale field trials were conducted by treating small turfed areas, followed by periodic "digging uip" to count live and dead scarabs and larvae.

1. One trial was made during February 1999 at the Peninsula Golf Club in Victoria (Australia) where there was a heavy infestation of black beetle H.

aratoi', larvae. Four turfed area of 10 1n, were treated with H. zeal an dica JBI/XI at an amount of 250,000 IJ/in 2 as part of a randomn block design including various other treatments. Observations revealed that 50% of larvae in the treated area were killed after two weeks and 80% after three weeks (see Figure 10), whereas there was negligible nematode death in the control plots.

Separate from this trial, the golf club superintendent treated larger areas of turf (about 2 hectares) with only 100,000 IJ/m2' and found no hird, feedIi ngc damage in the treated area but significant damage nearby.

2. In a second trial, 500,000 IU/rn were sprayed over 100 in' of a Canberra soccer field (Australian Capital Territory) heavily infested with Argentine scarab, C. sign aticollis. After eight days, 33% of the nematodes were dead, WO 00/38510 PCT/AU99/01152 after 23 days 52%, and after thirty days 61% were dead over six sample areas but only 3% were dead in an area of dry soil. After 68 days no scarabs were found alive and 16 dead in thirteen 200 cm 2 samples of treated area whereas 18 live larvae were found in untreated areas.

3. Small areas in two Canberra back garden lawns (Australian Capital Territory) with very lush grass and severe infestations of C. signaticollis were treated with H. zealandica at one million IJ/m 2 In the first garden, after 11 days, there were 30% nematodes dead in one plot and after 35 days, 74% nematode death in one plot with 90% death in another (based on only 19 and 11 larvae respectively). In the second garden, no dead scarabs could be found after 20 days, but after 40 days a total of 42 dead and 4 live scarabs were found in samples of three plots.

4. In late February 1999, 4 areas of turf within a quadrangle at the Australian National University (Australian Capital Territory) were treated with H. zealandica at 500,000 IJ/n 2 After two weeks there was a little nematode death, after three weeks an average of 48% nematode death over three plots, after 4 weeks 81%, and after six weeks 90% nematode death.

WO 00/38510 PCT/AU99/01152 16 The results above indicate that certain strains of H. zealandica may be used as biological control agents for scarabs. Since H. zealandica nematodes may be readily and cost-effectively reared using solid culture as described by Bedding (1981, 1984); their use in liquid or solid compositions would appear to offer a viable alternative to lawn scarab control by chemical pesticide spraying.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

WO 00/38510 WO 0038510PCT/AU99/01 152 17 References: 1. BEDDING, R.A. (1981). Low cost in vitro mass production of Neoaplectana and Heter-orliabditis species (Neinatoda) for field control of insect pests. Nemcztologica 27: 109-14.

2. BEDDING, R.A. (1984). Large scale production, storage and transport of the inisect parasitic nemnatodes Neoaplectana spp. and Heterorhiabditis spp. Ann. appl.Bio] 104: 117-120.

3. BEDDING, R.A. AND AKHURST, R.J. (1975). A simple technique for the detection of insect parasitic rhabditid nematodes in soil.

Neinatologica 21: 109-10.

4. BEDDING, MOLYNEUX, A.S. AND AKHURST, R.J. (1983).

I-et erorhabditis spp., Neoaplectania spp. and Stein ernema kraussei: Interspecific and intraspecific differences in infectivity for insects.

Expeimental Parasitology 55: 249-57.

5. BEDDING M.S. STANFIELD, AND G.W. CROMPTON 1991).

Apparatus and Method for Rearing Nematodes, Fungi, Tissue Cultures and The Like, and For H-arvesting Nematodes. Intern ati on a] Patent Application No PCT/AU91/00136 6. HASHMI1, GLAZER, 1. GAUGLER, R. (1996). Molecular comparisons of entoinopathiogenic nemiatodes using Random amplified Polymorphic DNA (RAPD) markers. Fundam. app]. Nematol., 18:55-61

Claims (11)

1. A composition for controlling a population of larval and/or pupal scarabs, comprising an amount of a substantially purified entomopathogenic nematode optionally in admixture with a suitable agricultural and/or horticultural carrier, wherein said entomopathogenic nematode belongs to the species Heterorhabditis zealandica and has an LD50 value of less than 300 IJ as measured by 14-day pot assays against final instar Cyclocephala signaticollis larvae and/or Popilliajaponica larvae.

2. The composition of claim 1, wherein the entomopathogenic nematode belongs to a strain of H. zealandica which has an LD50 value of less than 175 U as measured by 14-day pot assays against final instar C. signaticollis larvae and/or final instar P. japonica larvae. 15 3. The composition of claim 1, wherein the entomopathogenic nematode is selected from the group of H. zealandica strains consisting of the strains JBl/X1, GKB and JB3D. 9

4. The composition of any one of the preceding claims, wherein the amount of the entomopathogenic nematode is an amount in the range of about 50 to 10,000 nematodes/rnm of composition.

5. The composition of claim 4, wherein the amount of the entomopathogenic nematode is an amount in the range of about 500 to 1,000 nematodes/ml of composition.

6. A method for controlling a population of larval and/or pupal scarabs in an affected area, said method comprising applying to said area a composition in accordance with any one of the preceding claims.

7. The method of claim 6, wherein the composition is applied to the affected area so as to provide a dose of 50,000 to 1,000,000 IJ/m 2

8. The method of claim 7, wherein the composition is applied to the affected area 50 as tou provide a dose of 100,000 to 500,000 IJ/m 2 COMS ID No: SBM1-01038093 Received by IP Australia: Time 11:30 Date 2004-12-14 14/12 '94 11:29 FAX 613 9863 3099 F.B. RICE Co. 013/015 19

9. The method of any one of claims 6 to 8, wherein the composition is applied to the affected area at dusk The method of any one of claims 6 to 9, wherein the method is for controlling a population of larval and/or pupal scarabs selected from the group of scarab species consisting of Cyclocephala signaticollis, Heteronychus arator, Adoryphorus couloni, Antitrogus morbillosus, Anoplognathus porosus, Ataenius imparalis, Sericesthis geminata, S. pruinosus, S. nigrolineata, Scityla sericans, Saulostomus villosus, Aphodius tasmaniae, leteronyx spp, Rhopoea magnicornis, Popilliajaponica, Cyclocephala borealis, C. hirta, C. parallela, Melolontha melolontha, Anomala aenea, Phyllophagaphyllophaga, P. hirticula, Phyllopertha horticola, Haplididia errusca, Maladea matrida, Costelytra zealandica, Amphimallon solstatialis, and Ligyrus subtropicus. 1: 15 11. The method of claim 10, wherein the method is for controlling a population of larval and/or pupal scarabs of the species C. signaticollis.

12. The method of claim 10, wherein the method is for controlling a population of larval and/or pupal scarabs of the species P. japonica.

13. A nematode, in a substantially purified form, selected from the H. zealandica strains designated JB 1/X1, GKB and JB3D. 0@

14. A composition according to claim 1 substantially as described herein. *oe Dated this fourteenth day of December 2004 Commonwealth Scientific and Industrial Research Organisation Patent Attorneys for the Applicant: F B RICE CO COMS ID No: SBMI-01038093 Received by IP Australia: Time 11:30 Date 2004-12-14