CH702874A2 - Entomopathogenic virus preparation. - Google Patents

Abstract

A new composition is proposed for effective control of the peach winder. The new composition comprises at least one granulosis virus isolate of the new type CpGV-AND and, in its action against the larvae of the codling moth Cydia pomonella, show no appreciable reductions, but at the same time an activity increased by a factor of 65.6 compared to the known isolate of the CpCV-M type against the larvae of the peach winder Grapholita molesta.

Description

Technical area

The present invention relates to an entomopathogenic composition which is suitable for the biological control of the larvae of the peach moth Grapholita molesta and biological pesticides which contain these virus preparations or replicates of these preparations, as well as its use in the control of noxious insects.

[0002] The novel composition comprises a novel previously unisolated or identified viral preparation based on insect specific granulosis viruses.

Technological background and state of the art

The peach winder Grapholita molesta (also Cydia molesta, Pfirsichtriebbohrer or Pfirnaufriebwickler) has a worldwide distribution and is the most important pest in peach cultivation, Also in nectarines, apricots and apples cause the larvae of the peach winder considerable crop losses (Rothschild & Vickers, 1991 ). The control of the pest with synthetically produced insecticides is difficult, in particular because of the problem of residues in pome fruit. In addition, the peach winder has already developed resistance to various synthetic insecticides (Kanga et al., 1997).

As an alternative to the chemical insecticides biological plant protection products have been proven for years in both biological and integrated fruit growing. They allow a residue-free pest control and thus have no harmful effects on humans and the environment. One of the important biological control methods of butterfly pests in orchards is the use of baculoviruses, and in particular granulosis viruses (GV), also called granuloviruses, a subgroup of baculoviruses. These naturally occurring viruses infect certain types of host insects and lead to the death of the host within a few days. Thus, for example, to control the codling moth (Cydia pomonella) various biological preparations are available on the market, which contain the codermaker granulovirus CpCV. Most of these are the so-called CpCV-M ("Mexican Strain"), a virus which had been isolated from infected codling moth larvae in Mexico in 1964. If codling moth larvae nourish the virus in sufficient quantities after application of the preparation they die within a few days, causing no more damage.

The larvicidal activity of a particular granulovirus is usually limited to a well-defined butterfly group (e.g., CpCV on codling moth larvae). Although some effect may be present on other (even closely related) host insects, it is usually too small for efficient control. It is described in the prior Techink that the known CpGVs have a larvicidal action not only on the codling moth larvae but also on the peach worm larvae (Lacey et al., 2005). However, this activity of known CpCV against peach wrappers is 557 to 589 times lower than codling mothers. For a person skilled in the art, the larvicidal action of the currently available CpGV preparations and the CpCV strains and isolates described in the literature is clearly too low for a commercial use against the peach winder.

Object of the invention

It is therefore an object of the present invention to provide a biological preparation for controlling the peach winder.

These and other objects are achieved by a composition according to the invention according to the independent claim. Further advantageous embodiments are given in the dependent claims.

Presentation of the invention

For effective control of the peach winder, a new composition is proposed which comprises at least one granulovirus strain of the known CpCV can be extended to the peach winder.

In advantageous embodiments of the composition according to the invention, this comprises an isolate or an isolate mixture of the new type CpCV-AND, which has identifiable characteristic differences with the known isolate CpCV-M and a significantly extended spectrum of activity in its construction with relatively large correspondence. It is advantageous that these new compositions show no significant reductions in their action against the moth worm larvae, but at the same time have an activity against the larvae of the peach worm, which is increased by a factor of 65.6 compared to the known CpCV-M.

The inventive composition comprising the novel virus isolate can be used as an active ingredient in a formulated, sprayable pesticide for controlling the peach winder, the codling moth or other butterfly pests on which the inventive virus isolate has larvicidal activity. Optionally, further additives can be added to the composition.

Characterization of the isolates

A baculovirus isolate must always be considered as a mixture of different genotypes. These genotypes can have numerous differences, which have come about through insertions or deletions of DNA sequences. Depending on the number of different genotypes that are present in an isolate, the isolate can be described as very homogeneous or rather heterogeneous. In any case, it can be assumed that the genotype composition of an isolate decisively influences the virulence and the host spectrum of the isolate.

Baculovirus isolates are normally analyzed and characterized by restriction enzyme analysis (OECD, 2002). The DNA is cut by restriction enzymes in certain recognition sequences and there is a characteristic pattern with DNA fragments of different lengths. Comparing the restriction fragment patterns of two isolates, even small differences in genotype composition can be detected.

By means of restriction enzyme analysis, the virus DNA of the isolate according to the invention was compared with a known CpGV-M isolate (reference isolate). For this, the viruses in the new isolate were purified by centrifugation. From this purified virus suspension and the reference isolate, the virus DNA was subsequently isolated. The viral DNA was then digested with the four restriction enzymes BamHI, EcoRI, Sali and EcoRV. These restriction enzymes have been found to be very useful in previous studies of CpCV isolates.

For the restriction enzyme analysis 17 .mu.l of the isolated virus DNA were incubated with 2 .mu.l of buffer and 1 .mu.l of enzyme solution at 37 ° C for 3 h. One tenth of each sample was used to test if the digestion process was successful and to compare the DNA content. Subsequently, 6 to 14 μl of sample material were loaded onto an electrophoresis gel (0.8% agarose) and the DNA fragments were separated overnight with a TAE buffer system according to their length.

Fig. 1 shows a schematic representation of gel electrophoresis (0.8% agarose) with purified virus DNA from the (1) reference isolate CpCV-M, and (2) inventive virus isolate type CpGV-AND. The letters designate the restriction fragments in increasing fragment length. The columns on the far left and on the right show reference bands with a known fragment length (given numbers).

The resulting restriction fragment patterns of the new isolate of the type CpCV-AND and the reference isolate are shown in FIG. For the four restriction enzymes used, the following results were obtained:

Digestion with BamHI:

All restriction fragments of the new isolate are consistent with those of the reference isolate. The new isolate shows no missing or additional bands.

Digestion with EcoRI:

All bands of Referenzisoltas found also in the new isolate at the corresponding positions. However, the new isolate has an additional band at 5.8 kb (marked with an arrow in FIG. 1).

Digest with EcoRV:

All bands of Referenzisoltas found also in the new isolate at the corresponding positions. However, the new isolate has two additional bands at 13 kb and 6.5 kb (marked with arrows in Fig. 1).

Digestion with sali:

All restriction fragments of the new isolate are consistent with those of the reference isolate. The new isolate shows no missing or additional bands.

The restriction fragment pattern of the new virus isolate agrees to a large extent with that of the reference isolate. However, the three characteristic additional bands in the new isolate after digestion with the enzymes EcoRI and EcoRV show that DNA of an additional genotype is present in the new isolate virus compared to the reference isolate. The new isolate, hereinafter referred to as CpGV-AND, can be reliably characterized by the restriction fragment pattern and has a significantly higher activity against peach worms.

Activity of the isolate in the bioassay

The activity of a baculovirus isolate or its virulence against the larvae of a particular butterfly pest can be measured by how many viruses must be taken up by the larvae (i.e., eaten) to achieve a particular effect. For example, the LD50 indicates the median lethal dose at which 50% of the animals die. The lower this lethal dose (i.e., the viral dose administered), the higher the virulence of the virus isolate compared to a reference isolate.

In laboratory experiments (bioassays) with peach worm larvae from the company's own laboratory breeding the Applicant LD50 was determined for new virus isolates of the type CpGV-AND and compared it with that for a known CpGV isolate (CpCV-M). It could be clearly demonstrated that the new isolate of the type CpGV-AND has a significantly higher activity against peach worm larvae than the previously known CpCV-M.

Preparing the bioassay:

In the bioassay an artificially prepared nutrient medium was used (Stonefly Heliothis Diet, Item No. 38V0600, available from Wards Natural Science, NY). The dry nutrient medium must be mixed with water. In each case, 12.5 g of dry nutrient medium were placed in a small plastic bag. Subsequently, a defined amount of aqueous virus solution was added and the nutrient medium in the bag by hand well kneaded, so that a homogeneous, moist mass is formed.

For each virus isolate (new isolate CpGV-AND and reference isolate CpGV-M) five different virus concentrations were tested; for the new isolate, from 648 to 52,480 viruses per gram of nutrient medium and for the reference isolate of 19,753 to 16,000,000 viruses per gram of nutrient medium (Table 1). The amount of aqueous virus solution with known virus concentration was chosen so that the desired virus concentration could be achieved in the finished nutrient medium (Table 1). In addition to the treatments with virus solution, Biotest breeding units without viruses were also used as control (only water in the nutrient medium). A Biotest breeding unit consisted of a plastic box (140 × 70 × 20 mm, PS crystal clear, consisting of bottom, lid and grid insert 50-fold).

Preparation of the bioassay:

The bioassays were performed under sterile conditions. The contents of a kneaded plastic bag were distributed evenly on the bottom of the Biotest breeding unit. Thereafter, the grid was pressed by the nutrient medium on the bottom of the shell, so that 50 equal-sized compartments with the same amount of medium emerged. The freshly hatched larvae (1st larval stage, also designated L1) were then carefully transferred to the surface of the nutrient medium using a fine brush. One compartment was used per compartment. Once all compartments were occupied, the grid was covered with cling film and weighted with the inverted lid. The lid was fastened with rubber bands (Fig. 2). Subsequently, the Biotest breeding units were incubated in a climatic chamber (25 ° C, rLf 60-80%).

Fig. 2 shows the finished closed Biotest breeding unit.

Evaluation of the bioassay:

After 14 days of incubation, the survival rate of the animals was determined. For this purpose, the breeding units were opened, each compartment examined closely and the number of living (i.e., moving) larvae recorded. The results are shown in Table 1.

Using the probit analysis (Finney, 1971), the dose-effect curve (FIG. 3) and the LD50 (Table 2) were calculated for the new virus isolate and the reference isolate. Table 2 also shows the activity of the new virus isolate on peach worm larvae in relation to the effect achieved by the reference isolate CpCV-M on these larvae.

Table 1. Overview of the structure and the results of the bioassay with the new virus isolate and the reference isolate. <Tb> <sep> ID <sep> Quantity dry nutrient medium <sep> virus concentrate, in aqueous virus solution [OB / ml] <sep> Quantity used Virus solution <sep> Amount added Water <sep> Virus / g Broth <sep> Number living Larvae after 14 days <tb> Reference isolate CpGV-M <sep> Conc. 1 <sep> 12.5 g <sep> 4.00E + 07 <sep> 0.025 ml <sep> 37.5 g <sep> 19 753 <sep> 43 <Tb> <sep> Conc. 2 <sep> 12.5 g <sep> 4.00E + 07 <sep> 0.074 ml <sep> 37.4 g <sep> 59 259 <sep> 30 <Tb> <sep> Conc. 3 <sep> 12.5 g <sep> 4.00E + 07 <sep> 0.222 ml <sep> 37.3 g <sep> 177 778 <sep> 21 <Tb> <sep> Conc. 4 <sep> 12.5 g <sep> 4.00E + 07 <sep> 0.667 ml <sep> 36.8 g <sep> 533 333 <sep> 6 <Tb> <sep> Conc. 5 <sep> 12.5 g <sep> 4.00E + 07 <sep> 2,000 ml <sep> 35.5 g <sep> 1 600 000 <sep> 0 <tb> New Isolate <sep> Conc. 1 <sep> 12.5 g <sep> 1.31E + 06 <sep> 0.025 ml <sep> 37.5 g <sep> 648 <sep> 35 <Tb> <sep> Conc. 2 <sep> 12.5 g <sep> 1.31E + 06 <sep> 0.074 ml <sep> 37.4 g <sep> 1 944 <sep> 25 <Tb> <sep> Conc. 3 <sep> 12.5 g <sep> 1.31E + 06 <sep> 0.222 ml <sep> 37.3 g <sep> 5 831 <sep> 6 <Tb> <sep> Conc. 4 <sep> 12.5 g <sep> 1.31E + 06 <sep> 0.667 ml <sep> 36.8 g <sep> 17 493 <sep> 3 <Tb> <sep> Conc. 5 <sep> 12.5 g <sep> 1.31 E + 06 <sep> 2,000 ml <sep> 35.5 g <sep> 52 480 <sep> 0

Table 2. LD50 values for the new virus isolate CpGV-AND and the reference isolate CpGV-M with lower and upper limit of the 95% confidence interval. The relative activity of the reference isolate was set to 1. <tb> <sep> LD 50 (v / g) <sep> 95% <sep> 95% <sep> Relative Activity <sep> 95% (lower Limit) <sep> 95% (top Limit) <Tb> CPCV-M <sep> 121 600 <sep> 91080 <sep> 160 250 <sep> 1.0 <sep> 0.7 <sep> 1.5 <Tb> News Virus isolate <sep> 1852 <sep> 1372 <sep> 2454 <sep> 65.6 <sep> 44.2 <sep> 98.1

Fig. 3 shows linearized dose-effect curves (transformation of the probit units) for the new virus isolate CpGV-AND and the reference isolate CpGV-M. The dashed lines show the 95% confidence limits for each mean effective dose.

The new virus isolate type CpGV-AND has an LD50 of 1852 viruses per gram of nutrient medium 65.6 times higher activity against codling moth larvae than the reference isolate CpGV-M. Such an activity difference is far beyond the usual blur range of such bioassays and has never before been achieved with a baculovirus on peach worm larvae. The LD50 obtained by the new virus isolate here on peach worm larvae corresponds more or less closely to an LD50 as commonly observed with CpGV-M on the target organism codling moth. This means that the peach winder with the new virus isolate CpGV-AND in the field can be fought just as efficiently as the codler with the CpGV-M isolate.

This new virus isolate CpGV-AND with significant increase in activity compared to the known virus isolates of the type CpGV-M could be isolated by targeted multistage virus selection, without the use of genetic engineering methods.

Quoted literature

Finney D. 1, 1971. Probit Analysis, 3rd edition, Cambridge University Press, Cambridge.

Lacey LA, Arthurs S.P., Headrick H., 2005. Comparative activity of the codling moth granulovirus against Grapholita molesta and Cydia pomonella (Lepidoptera: Tortricidae). J. Entomol. Soc. Brit. Columbia 102: 79-80.

[0037] OECD 2002. Consensus Document on Information Used in the Assessment of Environmental Applications Involving Baculovirus (Monograph). Series on Harmonization of Regulatory Oversight in Biotechnology, No. 20, OECD Environment, Health and Safety Publications.

Rothschild C.H.L, Vickers RA, 1991. Biology, Ecology and Control of the Oriental fruit moth. In: van der Geest L.P.S., Evenhuis H.H., editors. Tortricid pests, their biology, natural enemies, and control. Amsterdam, The Netherlands: Elsevier Science Publishers. pp. 389-412.

Kanga L.H.B., Pree DJ., Van Lier IL, Whitty K.J., 1997. Mechanisms of resistance to organophosphorous and carbamate insecticides in Oriental fruit moth populations, Grapholita molesta (Busck). Pesticide Biochemistry and Physiology. 59: 11-23.

Claims (6)

Composition which has activity against noxious insects, characterized in that the composition comprises a granulosis virus isolate of the CpGV-AND type, the type CpGV-AND being used in the restriction enzyme analysis with the four restriction enzymes BamHI, EcoRI, SalI and EcoRV of a when digested with EcoRI all the bands of the reference isolate and an additional band at 5.8kb and when digesting with EcoRV all bands of the reference isolate and additionally two additional bands at 6.5 kb and at 5.8 kb. 2. Composition according to claim 1, characterized in that it is suitable for the effective control of the larvae of the peach winder Grapholita molesta. 3. Composition according to claim 1 or 2, characterized in that it is suitable for the effective control of the larvae of codling Cydia pomonella or other butterfly pests. Formulated, sprayable plant protection product for controlling the peach winder, codling moth or other butterfly pests, characterized in that it comprises as active ingredient a composition according to one of claims 1 to 3 which is effective for controlling the pests. 5. A method for controlling the larvae of the peach winder, codling moth or other butterfly pests, characterized in that the pests to be controlled are exposed to an effective dose of the composition according to any one of claims 1 to 3. 6. A process for the preparation of a composition according to claims 1 to 3, characterized in that a virus isolate of the type CpGV-AND is isolated by targeted multistage virus selection and provided for the preparation of the composition.