Nematode biopesticide

ABSTRACT

A composition and method for controlling scarabs, especially lawn scarabs, utilising certain strains of the entomopathogenic nematode species, are disclosed. The nematode strains used in the composition and method generally have an LD50 value of less than 300 IJ as measured by pot assays against final instar scarab larvae.

INFORMATION

DETAILED DESCRIPTION OF THE INVENTION

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

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

FIG. 1 shows a RAPD gel conducted on DNA from and other comparative strains. The primer used was OP-A04: 5′-AATCGGGCTG-3′ (SEQ ID NO:1) (Operon Technologies Inc.). (Key: 1. 100 bp DNA Ladder; 2(Great Keppel A); 3(Great Keppel B); 4(Great Keppel C); 5(Windsor); 6(NZH3); 7(WA Het); 8. Heterorhabditis sp. (JB6); 9(JB3D); 10(JBX1); 11. Heterorhabditis sp. (HT390); 12(Microbio); 13(NJ)).

FIG. 2 shows a RAPD gel conducted on DNA from and other comparative strains. The primer used was OP-F03: 5′-CCTGATCACC-3′ (SEQ ID NO:2) (Operon Technologies Inc.). (Key: 1. 100 bp DiNA Ladder; 2(Great Keppel A); 3(Great Keppel B); 4(Great Keppel C); 5(Windsor); 6(NZH3); 7(WA Het); 8. Heterorhabditis sp. (JB6); 9(JB3D); 10(JBX1); 11. Heterorhabditis sp. (HT390); 12(Microbio); 13. Heterorhabditis sp. (M145); 14(NJ)).

FIG. 3 shows a RAPD gel conducted on DNA from and other comparative strains. The primer used was OP-X11: 5′-GGAGCCTCAG-3′(SEQ ID NO3) (Operon Technologies Inc.). (Key: 1. 100 bp DNA Ladder; 2(Great Keppel A); 3(Great Keppel B); 4(Great Keppel C); 5(Windsor); 6(NZH3); 7(WA Het); 8. Heterorhabditis sp. (JB6); 9(JB3D); 10(JBX1); 11. Heterorhabditis sp. (HT390)).

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

FIG. 5 provides graphical results of pot assays conducted to assess the mortality of achieved by strains JB1/X1 and NZH3strain NC34 and HP88.

FIG. 6 provides graphical results of pot assays conducted to assess the mortality of achieved by strains JB1/X1 (Per 100, 330 and 100 nematodes) and NZH3, and strain NC34.

FIG. 7 provides graphical results of pot assays conducted to assess the mortality of achieved by strain JB1/X1 and strain NC34.

FIG. 8 provides graphical results to assess the mortality of adult achieved with strains JB1/X1, NZH3 and Botany, strain NC34and BW.

FIG. 9 provides graphical results of studies conducted to deterimne LD50s of strains in pot assays against final instar

FIG. 10 provides graphical results in field trials of X1 against African black beetle scarab.

EXAMPLE 1

Isolation and Characterisation of 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 2). Eight of the isolated nematodes were assigned to the species

Samples of three of the strains, namely JB1/X1, GKB and JB3D were deposited under the Budapest Treaty with the Australian Government Analytical Laboratories (AGAL), P.O. Box 385, Pymble, New South Wales 2073, Australia. These deposits have been accorded the Accession Nos. 10726, 10727 and 10728, respectively.

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

EXAMPLE 2

Control of Scarabs with Nematodes

A variety of trials were conducted with several strains of 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 (see LD50 Trials).

Comparison Trials

1. Dermolepida

Pot assays using the method of Bedding, Molyneux and Akhurst (1983) (see LD50 Trials), were conducted using final instar larvae of Dermolepida dosed with 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 NZH3 and NC34 at the same dose (see FIG. 4) showed that JB1/X1 achieved superior kill (over 90% kill after 3 weeks with JB1/X1 versus 57% after 3 weeks with 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 larvae with the NC34 strain of HP88 and the NZH3 and JB1/X1 strains of (at 500 Ijs/pot). The results obtained (see FIG. 5) showed that JB1/X1 achieved superior kill. Subsequent assays comparing JB1/X1 with a range of other strains of (namely, JB3/D, Botany, GKA, GKB, GKC and JB3/F) at a dose of 200 IJs/pot showed JB1/X1 to equal (JB3/D, Botany, GKB and GKC) or superior (B3/F and GKA) in killing power. In addition, pot assays comparing JB1/X1 to a range of field collected material at 200 IJs/pot (niamiely, JB4/A to JB4/E, Qld CS9, and JB4/H, all of which are ), JB1/X1 was again found to achieve superior kill.

3

Pot assays were conducted on final instar comparing strains JB1/X1 and NZH3 and strain NC34. The results (see FIG. 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 lower dose (500 instead of 1000) resulted in all of strain MicroBio strain HP88 and strains JB1/X1 and NZH3 achieving a very poor level of kill.

4

Pot assays were conducted on final instar comparing strain JB1/X1 and strain NC34. The results (see FIG. 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

Pot assays were conducted on adult (African Black Beetle) comparing strains JB1/X1, NC34, NZH3 and Botany, , and BW at 1000 IJs/pot. The results (see FIG. 8) showed that BW achieved some control while the others were ineffective.

6

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

7. Anoplognathus sp.

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

LD50 Trials with

Assays to determine the LD50s of the strains Windsor, JB1/X1, GKB, NZH3, GKA, JB6, JB3/D and HT390 (H. sp) against freshly collected final instar were conducted in accordance with the following method:

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=1.3). 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 removed and if dead were dissected and microscopically examined for neatode infection in the insect Ringers solution. The LD50 values were computed using the probit analysis of Finney 1971.

The results are shown in FIG. . In particular, FIG. 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 as follows in Table 3:

LD50 Trials with Other Scarab Species

LD50s of strain JB1/X1 against other species of scarabs (i.e. the Japanese beetle, ) 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 eveii moisture content of 7% (Pf=1.3). A larval scarab is placed at the bottom of each specimen jar, sand lightly packed over it and the top screwed on tigltly.

2. Nematodes are introduced in 1 ml of water into a centrally placed well (0.5 cm 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 40 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.

5. 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 of against various scarab species using the above method.

LD50 and LD90 and confidence limits were calculated using the probit analysis and Fieller procedures in Genstat 5.

EXAMPLE 3

Effectiveness of 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 , larvae. Four turfed area of 10 m2 were treated with JB1/X1 at an amount of 250,000 IJ/m2 as part of a random 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 FIG. ), whereas there was negligible neinatode 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 bird feeding damage in the treated area but significant damage nearby.

2. In a second trial, 500,000 IJ/m2 were sprayed over 100 m2 of a Canberra soccer field (Australian Capital Territory) heavily infested with Argentine scarab, . After eight days, 33% of the nemiatodes were dead, after 23 days 52%, and after thirty days 61% were dead over six sample areas but only 3% were dead in ain area of dry soil. After 68 days no scarabs were found alive and 16 dead in thirteen 200 cm2 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 were treated with at one million IJ/m2. 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 (91%) 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 at 500,000 IJ/m2. After two weeks there was a little nematode death, after three weeks all average of 48% nematode death over three plots, after 4 weeks 81%, and after six weeks 90% nematode death.

The results above indicate that certain strains of may be used as biological control agents for scarabs. Since 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.

References

1. BEDDING, R. A. (1981). Low cost in vitro mass production of Neoaplectana and Heterorhabditis species (Nematoda) for field control of insect pests. 27: 109-14.

2. BEDDING, R. A. (1984). Large scale production, storage and transport of the insect parasitic nematodes Neoaplectana spp. and Heterorhabditis spp. . 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. 21: 109-10.

4. BEDDING, R. A., MOLYNEUX, A. S. AND AKHURST, R. J. (1983). Heterorhabditis spp., Neoaplectana spp. and : Interspecific and intraspecific differences in infectivity for insects. 55: 249-57.

5. BEDDING R. A., M. S. STANFIELD, AND G. W. CROMPTON (1991). Apparatus and Method for Rearing Nematodes, Fungi, Tissue Cultures and The Like, and For Harvesting Nematodes. International Patent Application No PCT/AU91/00136.

6. HASHMI, G., GLAZER, I. & GAUGLER, R. (1996). Molecular comparisons of entomnopathogenic nematodes using Random amplified Polymorphic DNA (RAPD) markers. ., 18:55-61.

CLAIMS

1. A composition comprising an amount of an entomopathogenic nematode selected from the group of H. zealandica strains consisting of strains JB1/X1, GKB and JB3D, optionally in admixture with a suitable agricultural and/or horticultural carrier.

2. The composition of claim 1, wherein the amount of the entomopathogenic nematode is an amount in the range of about 50 to 10,000 nematodes/ml of composition.

3. The composition of claim 2, wherein the amount of the entomopathogenic nematode is an amount in the range of about 500 to 10,000 nematodes/ml of composition.

4. 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 claim 1, thereby the population of the larval and/or pupal scarabs is controlled.

5. The method of claim 4, wherein the composition is applied to the affected area so as to provide a dose of 50,000 to 1,000,000 IJ/m2.

6. The method of claim 5, wherein the composition is applied to the affected area so as to provide a dose of 100,000 to 500,000 IJ/m2.

7. The method of claim 4, wherein the composition is applied to the affected area at dusk.

8. The method of claim 4, 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. pruinosis, 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 horticola, Haplididia etrusca, Maladea matrida, Costelytra zealandica, Amphimallon solstatialis and Ligyrus subtropicus.

9. The method of claim 8, wherein the method is for controlling a population of larval and/or pupal scarabs of the species of C. signaticollis.

10. The method of claim 8, wherein the method is for controlling a population of larval and/or pupal scarabs of the species of P. japonica.

11. An isolated nematode selected from the H. zealandica strains designated JB1/X1, GKB and JB3D.

12. A process for producing a composition for controlling larval and/or pupal scarabs comprising: (i) subjecting one or more candidate entomopathogenic nematodes of the species Heterorhabditis zealandica to a 14-day pot assay against final instar Cyclocephala signaticallis larvae and/or final instar Popilla japonica larvae; (ii) selecting an entomopathogenic nematode which has an LD50 value of less than 300 IJ as measured by the 14-day pot assay; and (iii) admixing the entomopathogenic nematode of step (ii) with a suitable agricultural or horticultural carrier.

13. The process of claim 12 wherein the entomopathogenic nematode is a strain of H. zealandica which has an LD50 value of less than 175 IJ as measured by the 14-day pot assay against final instar C. signiticollis larvae and/or final instar P. japonica larvae.

14. The process of claim 12 wherein the entomopathogenic nematode is selected from the group of H. zealandica strains consisting of strains JB1/X1, GKB and JB3D.

15. The process of claim 12 wherein the amount of the entomopathogenic nematode admixed with the carrier is in the range of about 50 to 10,000 nematodes/ml.

16. 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 produced in accordance with claim 12, thereby the population of the larval and/or pupal scarabs is controlled.

17. The method of claim 16 wherein the composition is applied to the affected area so as to provide a dose of 50,000 to 1,000,000 IJ/m2.

18. The method of claim 16 wherein the composition is applied to the affected area at dusk.

19. The method of claim 16 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 morbilliosis, Anagplognathus porosus, Ataenius imparalis, Sericesthis geminant, S. pruinosis, S. noigrolineata, 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, Phyllophertha horticola, Haplididia etrusca, Maledea matrida, Costelytra zealandica, Amphimallon solstatialis, and Lygyrus subtropocus.

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