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Peroral Bioassay of the Western Spruce Budworm

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Peroral Bioassay of the Western Spruce Budworm EDITOR'S~. FILE COPy United States Department of Agriculture Canada Forest Service Unfted States Pacific Northwest Forest and Range Experiment Station Spruce Budworms Research Paper PNW- 285 Program July 1981 Peroral Bioassay of N ucleopolyhedrosis Viruses in Larvae of the Western Spruce Budworm Mauro E. Martignoni and Paul J. Iwai ~.--- .- . k~ L-,-. r l //l l ~r- "T--" ,' ', . - - --_- ." ,~l Authors Mauro E. Martignoni is microbiologist and Paul J. Iwai is biological laboratory technician, Forestry Sciences Laboratory, Pacific Northwest Forest and Range Experiment Station, Corvallis, Oregon. Contents 1 Abstract 1 Summary 2 Introduction 4 Rearing and Selection of Insects for Tests 4 I. Maintenance of Choristoneura occidentalis Stocks 4 II. Selection of Fourth Instars for Bioassay 5 Peroral Bioassay 5 I. Preparation of Test Cups 6 I1. Preparation of Serial Dilutions of Virus 8 II1. Inoculation of Cups and Transfer of Test Larvae 13 IV. Evaluation 16 Results: An Example 17 Checklist of Items Needed for One Bioassay 17 Acknowledgments 18 Literature Cited 19 Appendix 19 Propagation and Processing of Experimental Quantities of Baculovirus in Larvae of Choristoneura occidentalis , Abstract Summary Martignoni, Mauro E.; Iwai, Paul J. Peroral bioassay of No information is available on the activity titers of viruses nucleopolyhedrosis viruses in larvae of western spruce pathogenic for the western spruce budworm, budworm. USDA For. Serv. Res. Pap. PNW-285. Choristoneura occidentalis Freeman. Quantification of the Portland, OR: Pac. Northwest For. and Range Exp. biological activity of these viruses is needed, both for Stn.; 1981. laboratory investigations and for field tests against this destructive defoliator. Baculoviruses isolated from nearctic The relative virulence of entomopathogenic viruses and and palearctic conifer-feeding species of Choristoneura the potency of virus preparations for control of destructive are generally cross infectious. Thus, a bioassay method is insects can be estimated reliably only by means of essential for comparing the virulence of virus isolates in a biological assay in the target species. A simple, yet screening program of pathogens of the western spruce sensitive peroral bioassay procedure is described for budworm. The method described here minimizes the diffi- preparations of nucleopolyhedrosis viruses pathogenic for culties usually encountered in bioassays and standardizes the western spruce budworm, Choristoneura occidentalis the various operations needed for measuring the activity Freeman. This procedure has been used to compare the of pathogens in a fastidious host. The procedure-- activity of Baculovirus isolates from several conifer-feeding although relatively simple--has a high degree of species of Choristoneura. reproducibility, and it does not require instrumentation other than that available in most biological laboratories. Keywords: Bioassay, Baculovirus, nucleopolyhedrosis The precision of the assays compares favorably with that virus, virus (insect), diseases (insect), quality control (virus known from other virus-host systems. preparations), biological control (forest pests), western spruce budworm, Choristoneura occidentalis. Data from three bioassays and a statistical comparison of dose-mortality regression lines illustrate an application of the peroral bioassay method in virus screening. An appendix describes simplified methods for preparing experimental quantities of baculoviruses (0.5-2 g of inclusion bodies) suitable for biological assay and biochemical studies. Future field applications of viruses against the western spruce budworm should be based on the polyhedron-to- bioactivity ratio of each pathogen rather than on polyhedron counts only. This bioassay procedure offers a method of activity titration for estimating field doses of baculovirus preparations, as generally recommended in tests with microbial control agents, and as required specifically by U.S. pesticide regulations. Introduction The western spruce budworm, Choristoneura occidentalis insecticide for control of these insects. Procedures for Freeman, is one of the most destructive defoliators in activity titration and activity standardization' of NPV are western North America. This insect occurs principally on useful not only for comparative potency assessments of true firs and Douglas-fir in the Pacific Coast States, British products used in field trials but also for comparison of virus Columbia, and the Rocky Mountain States (Carolin and strains and isolates (Martignoni and Ignoffo 1980). In Honing 1972, Furniss and Carolin 1977). Although modern addition, the US. Environmental Protection Agency (1975) forest-insect management frequently includes the use of requires that quantification of nucleopolyhedrosis and microbial insecticides, surprisingly little is known about the granulosis virus products be based on bioassays rather diseases of C. occidentalis or their role as biological control than counts of inclusion bodies. Baculoviruses found in factors. Among the viral agents, nucieopolyhedrosis virus nearctic conifer-feeding species of Choristoneura and in (NPV), Baculovirus subgroup A, has been isolated from the European C. murinana (H0bner) are generally cross wild populations of this insect in the Bitterroot Range of infectious (Stairs 1960; Cunningham and Percy 1981; Idaho, in 1967, and in the Blackfoot River drainage near M. E Martignoni, unpublished tests); methods for Missoula, Montana, in 1969 (Thomas and Poinar 1973). comparing the virulence of these diverse viruses in vivo Unfortunately, these isolates were not preserved (G. M. were needed. Thomas, Division of Entomology and Parasitology, University of California, Berkeley, California 94720, letter on file, Forestry Sciences Laboratory, Corvallis, Oregon In 1978, we started a screening and evaluation program 97331, personal communication). In 1976, NPV was for NPV pathogenic for larvae of the western spruce isolated from C. occidentalis in Canada (Cunningham et budworm. Several of these viruses are now considered al. 1978). This virus, after passage in C. fumiferana good candidates for field testing against C. occidentalis. (Clemens), was used in field tests in British Columbia to The bioassay procedure described here was developed control C. occidentalis (Cunningham et al. 1978). over the past 2 years, and it was tested repeatedly in its Populations of C. occidentalis were also treated with NPV present form. This procedure is based in part on a bioassay isolated from C~ fumiferana, with encouraging results in method for baculoviruses of the Douglas-fir tussock moth, 1978 tests (Cunningham and Percy 1981 ). In 1979, NPV Orgyia pseudotsugata (McDunnough), (Martignoni and was isolated in our laboratory from larvae of C. occidentalis Iwai 1977) and is similar in principle to that used by Ignoffo collected at White Sulphur Springs, Montana. The larvae (1966) for NPV of Heliothis zea (Boddie). The bioassay were obtained in July from G. P. Markin, Davis, California method for NPV of O. pseudotsugata was approved as part (C. G. Thompson, Forestry Sciences Laboratory, Corvallis, of the quality-control procedures of the virus product Oregon 97331, personal communication). designated "TM BioControl-l" (EPA Registration No. 27586-1, August 11, 1976). On the other hand, a vast amount of information is available on the pathology and epizootiology of nucleopolyhedrosis and other viral diseases of C. fumiferana, the spruce 1The determination of activity budworm common in eastern North America (Cunningham of a virus, or biological assay, 1978; Cunningham and Percy 1981). Since 1971, over is a process by which the 2000 ha of forest in Canada were sprayed with NPV. Aerial relation between virus dose and response in susceptible applications of this virus reduced populations of C. hosts is tested. The activity titer fumiferana, with variable success, from less than 20 or potency of a virus prepara- percent to more than 90 percent. Satisfactory foliage tion is the amount of activity per protection was achieved in some of the treatments unit weight or volume of the (Cunningham and Percy 1981 ). preparation, measured in activity units. Standardization Unfortunately, no information is available on the potency of activity is a process by which of viral preparations used in laboratory investigations or in the activity of a virus is mea- field tests against both species of spruce budworms. No sured in standard activity units. units accepted by consent as attempts to standardize the activity of spruce budworm a basis for comparison. NPV have been reported. This is most likely because (Standardization can be of propagation of spruce budworm viruses and their field use hmited import, e.g., within one have been on a modest scale only, and no current laboratory or among few commercial interest has been shown in developing a viral laboratories, or it can extend nationally and internationally as tt does for Bacillus thunngiensls Berliner preparations.) The procedure (summarized in table 1) is relatively simple, viruses pathogenic for C. occidentalis and for measure- yet sensitive; it does not require instrumentation other than ment of the potency of viral preparations for field that available in most biological laboratories. We recom- application. mend it for comparison of the activity and virulence of Table lmParameters of the bioassay procedure for Baculovirus isolates pathogenic for Choristoneura occidentalis Test insect Choristoneura occidentalis, strain COC- FS-01, initiated
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