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(Orthoptera: Gryllidae), Using the Fungal Pathogen

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(Orthoptera: Gryllidae), Using the Fungal Pathogen Plant Protection Quarterly Vol.ll(1) 1996 9 There is no published information on Biological control of the black field cricket, natural enemies of black field cricket other than pathogens. As part of a pro- Teleogyllus commodus (Walker) (Orthoptera: gramme evaluating pathogens for control of crickets, Reinganum et al. (1981) sur- Gryllidae), using the fungal pathogen Metarhizium veyed diseases in Victoria. Live crickets anisopliae (Metsch.) Sorokin (Deuteromycotina: were sampled from 232 sites in 1979 and pathogens either apparent at the time of Hyphomycetes) sampling, or which appeared within two weeks of collection, were identified. Richard J. Milnefi, Lisa MilkerB,George G. LuttonAand Felice DeriveF Cricket paralysis virus was found most ACSIRO, Division of Entomology, GPO Box 1700, Canberra, ACT 2601, commonly (42.7% of sites) and Australia. Metarhiziurn anisopliae (Metsch.) Sorokin BAgricultureVictoria, Colac, Victoria 3250, Australia. (Deuteromycotina: Hyphomycetes), the only other pathogen found, infrequently (5.2% of sites). Neither pathogen was suf- ficiently abundant to have any influence Summary when applied as an oil-based on population density. A number of iso- The hyphomycete fungus, Metarhizium mycoinsecticide. lates of M. anisopliae were obtained dur- anisopliae, is a natural pathogen of ing the survey and many of these were black field cricket, Teleogyllus Introduction evaluated in the present study (Table 1). commodus, in Victoria. Comparison of The black field cricket, Trleogryllus Reinganum et al. (1981) concluded that 12 isolates, using Random Amplified cornrnodus (Walker) (Orthoptera: the fungus, since it was pathogenic and Polymorphic DNA (RAPDs), from this Gryllidae), is a serious pest of pastures in easily grown on inexpensive media such insect collected in Victoria showed that parts of Victoria and South Australia as rice, was promising for development as they were all very similar genetically causing over $8m damage in some years a mycoinsecticide, however no field trial but quite distinct from the group 3 (Anon. 1989). It is also a serious pest in results have been published. M. flavoviride isolates from grasshop- parts of the north island of New Zealand Recently, it has been shown that certain pers and locusts. Screening bioassays (Blank 1982). Populations are particularly isolates known as M.flavoviride Gams and showed that isolates from crickets were high in western Victoria where cracking Rozsypal group 3 (Prior 1995) comprise a more virulent for this pest than isolates clay soils provide essential shelter during genetically uniform and distinct group, from other sources. One of these cricket hot dry weather. T. cornmodus is and are virulent for a range of grasshop- isolates F11099 was selected for use in univoltine: over-wintering diapause eggs pers and locusts. Interestingly, natural in- field trials. An oil formulation of are laid in the soil in autumn and hatch in fections of M. flavoviride group 3 have conidia of FI1099 was tested either as a early summer (December); the nymphs only been found on grasshoppers and lo- ultra-low volume (ULV) spray or in a develop through five instars before be- custs which may suggest that they have high volume oillwater emulsion at two coming adults usually in the autumn co-evolved. Furthermore, it has been sites in Victoria. At Warrambeen, a dose (March). Populations decline with the on- found that by formulating the conidia of of 2 x loi3 conidia per hectare gave a set of cooler weather in April and few, if M. flavoviride in oil, the fungus can con- 30-40% reduction in the cricket popula- any, adults over-winter. Pasture defolia- trol locusts and grasshoppers when ap- tion after 21 days compared with 80% tion can be severe in late summer and au- plied using ultra-low volume (ULV) tech- control in the malathion plots. At tumn (February-April). The only method niques or in an oil-emulsion using high Turkeith, the higher dose of 4 x 10'j of control available to farmers is the ap- volume boom sprayers (Baker et al. 1994). conidia per hectare gave 60-70% control plication of chemical insecticides using The oil not only prevents evaporation of while the malathion again gave about malathion cereal baits (Stahle et al. 1983). water during ULV spraying but also 80% control. These results are promising Many thousands of hectares are treated aids attachment and penetration of the and show that M. anisopliae has the po- each year, often using aerial application target host by the fmgus. This enables tential to control black field cricket methods. the conidia to germinate under field Table 1. List of isolates and the sources of Metarhizium spp. used in this study. Isolate Species name Original host Source Place F1610 M. anisopliae termite mound CSIRO Brown Mt., New South Wales FI 985 M. flavoviride Austracris guttulosa ARSEF 324 Rockhampton, Queensland FI 1030 M. anisopliae T. cornmodus IMI 351 830 Victoria FI 1037 M. anisopliae T. cornrnodus ARSEF 448 Victoria FI 1066 M. anisopliae soil IIBC Ben 4 Benin FI 1067 M.flavoviride acridid IlBC 192-701 Benin FI 1090 M. anisopliae T. cornrnodus ARSEF 435 Warrnambool, Victoria FI 1091 M. anisopliae T. cornrnodus ARSEF 437 Victoria FI 1092 M. anisopliae T. cornrnodus ARSEF 438 Victoria FI 1093 M. anisopliae T. cornrnodus ARSEF 445 Carapook, Victoria FI 1094 M. anisopliae T. cornmodus ARSEF 440 Warrnambool, Victoria FI 1095 M. anisopliae T. cornrnodus ARSEF 441 Warrnambool, Victoria F11096 M. anisopliae T. cornrnodus ARSEF 442 Hawkesdale, Victoria FI 1097 M. anisopliae T. cornrnodus ARSEF 443 Camperdown, Victoria FI1098 M. anisopliae T. cornrnodus ARSEF 444 Byaduck, Victoria FI 1099 M. anisopliae T. cornrnodus ARSEF 445 Carapook, Victoria FI 1100 M. anisopliae T. cornrnodus ARSEF 446 Mortlake, Victoria 10 Plant Protection Quarterly Vol.ll(1) 1996 conditions of low humidity which nor- was washed twice with 500 pL of 7O0/0 experiment, the crickets were T. mally prevent or seriously delay the in- ethanol, then dried in a vacuum and cornrnodus while in the second the closely fection process (Bateman et al. 1993, resuspended in 400 pL of lOmM Tris HC1 related species T. oceanicus was used as Milner et al. 1995). Crickets are also at pH 8.0, ImM EDTA and 10 pg/mL they were more abundant in the labora- orthopteroid insects, and the present RNase A. The DNA was incubated at tory colonies. In previous laboratory ex- studywas undertaken to determine if the 37" for 30 minutes and then extracted periments, it had been found that these grasshopper control strategy could be ex- with 1 volume phenol : CHC1,: isoamyl species are similar in their susceptibility tended to crickets. alcohol (50:48:2), followed by CHC1,: to Metarhiziurn spp. (Milner unpublished For genetic fingerprinting, randomly isoamyl alcohol (24:l) until the interface data). amplified polymorphic DNA (RAPD) was clean. The DNA was precipitated by markers have been used to generate mo- adding 40 pL 3M sodium acetate pH 5.4 Field trials lecular markers (Welsh et al. 1991, and two volumes ethanol. The DNA was Two field trials were set up in February Williams et al. 1991) and to analvse recovered by brief centrifugation in a 1995. The first was a replicated small plot genomes and populations in a variety of microfuge, washed twice with 70% trial established at the Alcoa Landcare organisms including fungi (Fegan et al. ethanol, then dried in a vacuum. Pellets Demonstration Farm, "Warrambeen" 1993, Bidochka et al. 1994), humans and were resuspended in 5mM Tris HC1, near Colac, Victoria. Each plot was 25 x plants (Williams et al. 1991), and insects 0.5mM EDTA. 25 m and separated by an untreated (review by Haymer 1994). These DNA RAPD-PCR (polymerase chain reac- buffer of 20 m. There were four treat- polymorphisrns may arise from repetitive tion) amplifications were performed in a ments: untreated control, baited with and unique regions and indicate the de- total volume 25 pL. Each reaction con- malathion at a rate of 125 mL a.i. to 10 kg gree of relatedness between isolates. tained 2.5 pL of 10X Bresatec reaction oats ha-', sprayed with a highvolume oil/ The aims of the study were to use mo- buffer (16.6mM (NH,),SO,, 67mM Tris water emulsion of M. anisopliae at 2 x 1013 lecular fingerprinting techniques to assess HC1 at pH 8.8, 0.45% Triton X100, 200 pg conidia ha-' in 130 L ha-', and the same the genetic status of isolates from T. mL-' gelatin) 3.5mM MgCl,, 200pM each number of conidia applied using a commodus, select the most virulent isolate dATP, dTTP, dCTP, dGTP, 30pM primer, MicroUlva Plus ULV sprayer at 2 L ha-'. for this host and to undertake initial field 10-15 ng DNA and 2.4 U Taq polymerase The plots were arranged in a randomized tests of the efficacy of the selected isolate (Bresatec). The reactions were covered by block with four replicate plots per treat- in oil-based sprays. a drop of light mineral oil. Primers were ment. The treatments were applied late in those used by Fegan et al. (1993) from the evening on February 15 as the very Materials and methods Random Primer kits H and F (Operon hot, windy, day-time conditions were un- Technologies, Alameda, California). Re- suitable for spraying. Isolates actions were placed in a thermocycler The other trial was set up at "Turkeith", A list of isolates used in this study are (Corbett Research, Australia) and DNA a property located about 20 km south of given in Table 1. The isolates from T. was amplified using the following tem- Warrambeen and with a history of seri- commodus were all from the ARSEF col- perature cycles: one cycle at 94" for five ous cricket infestations. There were two lection (USDA Insect Pathogenic Fungi minutes, 40°C for t-wo minutes, 72" for unreplicated plots each 50 x 50 m sepa- Culture Collection at Ithaca, New York) three minutes, then 39 cycles at 94°C for rated by a 20 m untreated buffer.
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