Novel virus composition to protect agricultural commodities from insects

A potent, stable virus composition useful for protecting agricultural commodities from insects and a simple, efficient, economical and labor-saving method to produce and formulate large quantities of the virus composition are described. The method is particularly useful to prepare nuclear polyhedrosis virus or granulosis virus compositions for control of postharvest pests such as the Indianmeal moth.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention relates to a novel virus composition useful for protecting 
agricultural commodities from insect pests and preparation thereof. 
2. Description of the Art 
Baculoviruses, in particular nuclear polyhedrosis viruses (NPVs) and 
granulosis viruses (GVs), have many characteristics which make them 
particularly suitable for control of insects. NPVs and GVs are specific in 
their pathogenicity to the class Insecta and many are highly virulent to 
their hosts (Baculoviruses for Insect Pest Control: Safety Considerations, 
Ed. M. Summers et al., American Society for Microbiology, Washington, 
D.C., p. 7, 1975). Such viruses can be used to control insects which 
infest agricultural commodities such as the Indianmeal moth, the raisin 
moth, the almond moth, the tobacco moth and the like. A major problem in 
the use of baculoviruses to control insects is the lack of methods to 
readily produce large quantities of virus at low cost. 
The problem of protecting agricultural commodities from insects is 
illustrated with reference to the Indianmeal moth, Plodia interpunctella 
(Hubner) (Lepidoptera: Pyralidae), hereinafter IMM. This insect is 
recognized as a major insect pest that infests grains, dried fruits and 
nuts, and other stored commodities worldwide. Presently, there is no 
protection for commodities such as dried fruits and nuts from IMM invasion 
or damage after the commodities have left the processing plant, and it has 
been estimated that over 90% of the losses caused by insects to dried 
fruits and nuts after processing are due to IMM. One problem in 
controlling IMM is that it has shown resistence to certain chemical 
insecticides, thus other possible control agents are being investigated. 
Granulosis virus (GV) isolated from IMM is a highly virulent insect 
specific virus; it has been shown to be an effective protectant against 
IMM for stored dried nuts (Hunter et al., Journal of Invertebrate 
Pathology 22: 481 (1973) and Journal of Economic Entomology 70: 493-494 
(1977)), raisins (Hunter et al., Journal of Stored Products Research 15: 
65-69 (1979)), and wheat and corn (McGaughey, Journal of Economic 
Entomology 68: 346-348 (1975)). Because GV is unique to insects and does 
not infect man, it offers potential for use as a protectant against IMM on 
food commodities. 
Until 1979, formulations of the IMM granulosis virus (IMMGV) were produced 
by rearing IMM larvae infected with GV on a diet and removing the infected 
larvae by hand from the diet. After removal, a given number of infected 
larvae were homogenized in distilled water to free the virus from the 
insect bodies, and the virus was used immediately. Alternatively, 
hand-collected larvae were freeze-dried and then homogenized and suspended 
in water just prior to use. The primary deficiency of this method was that 
hand-removal of the larvae from the rearing diet was tedious and time 
consuming and made this method of control of IMM on commodities 
economically unfeasible. Further, these wet formulations had the problems 
that loss of activity of the labile nonoccluded infectious components 
occurred, and the preparation process had to be repeated whenever inoculum 
was required. 
Cowan et al., Journal of Economic Entomology 79: 1085-1090 (1986) described 
a freeze-dried formulation of IMMGV. This formulation was prepared by 
rearing GV-infected IMM larvae on a diet, and homogenizing the larvae in 
the diet to obtain a composition containing GV-infected larvae plus the 
diet components. The composition was freeze-dried, milled, and formulated 
with a carrier, and the formulation tested for control of IMM on almonds 
and raisins. The diet components used to rear the IMM larvae consisted of 
wheat bran, honey, water, glycerol, a vitamin mixture, and 
fungicides/fungistats. The problems associated with this composition 
(GV-infected larvae plus diet components) are that the presence of honey 
and glycerol caused the formulations after freeze-drying to be sticky and 
difficult to mill as well as hygroscopic. Attempts to minimize these 
problems included washing the formulation several times prior to 
homogenizing and freeze-drying. However, even with several washings, not 
all the glycerol and honey was washed out. In addition to adding 
additional labor with attendant increased costs, the washing procedure 
resulted in significant losses in potency of the formulation. 
SUMMARY OF THE INVENTION 
The present invention provides a simple, efficient, and labor-saving method 
to produce and formulate large quantites of virus for use as an 
agricultural commodity protectant at less than one tenth the cost of the 
wet formulations and at significantly less than the cost of the Cowan et 
al. formulation, while eliminating the problems associated with the prior 
art methods. 
In the method of the invention, host larvae are reared on a diet devoid of 
honey and glycerol, components which provide sources of readily-utilizable 
sugars, vitamins, and trace minerals. Heretofore it was thought that honey 
and glycerol were nutritional components essential for larvae growth and 
development. Surprisingly, however, removal of these "critical" components 
from the diet did not deter larval growth or consequent virus production, 
and further, resulted in a 20-30 fold increase in activity over the Cowan 
et al. formulations, while eliminating the washing steps of Cowan et al. 
With regard to the earlier formulations wherein the infected larvae were 
hand-removed from the diet, the instant method provides a labor-saving 
method to produce and formulate large quantities of virus at less than one 
tenth previous costs. The dry formulation of the invention is also much 
easier to assay and store, and eliminates the need of making wet 
formulations for laboratory or commercial use. 
In accordance with this discovery, it is an object of the invention to 
provide a method for preparing a virus composition useful to protect 
agricultural commodities against insects. 
A further object of the invention is to provide virus compositions devoid 
of honey and glycerol which are useful as commercial insecticides. 
Another object of the invention is the provision of a simple, cost-saving, 
efficient method to obtain large quantities of virus for use to control 
insects in raw agricultural commodities, in stored commodities after 
harvesting and processing, in packaged commodities, and in warehouses or 
processing plants. The invention provides a way to protect commodities 
from insects through marketing channels to consumption. 
Other objects and advantages of the invention will become readily apparent 
from the ensuing description. 
DETAILED DESCRIPTION OF THE INVENTION 
The invention comprises preparation of a virus composition for use in 
protecting agricultural commodities from insects while eliminating honey 
and glycerol from the larvae-virus production diet and eliminating the 
hand-removal of larvae. 
Preparation of the Virus composition of the Invention. 
Step 1. Preparation of the Diet. 
A diet is prepared which contains a substrate which functions 
simultaneously as a nutritional component for the host larvae (larvae 
which are to serve as hosts for production of the virus), a substrate to 
absorb excess moisture in the diet, a bulking agent, and a carrier for 
other components. Examples of such a substrate are wheat bran and rice 
bran. The preferred substrate is wheat bran. Other components in the diet 
are water added in an amount sufficient to disperse the diet components on 
the substrate; yeast, preferably Brewers yeast, in an amount sufficient to 
provide vitamin complexes necessary for growth; and vitamins. Optional 
ingredients include fungicides or fungistats. It is a critical feature of 
the invention that honey and glycerol be omitted from the diet. 
Step 2. Infestation and First Incubation. 
The diet described in step 1 is infested with sufficient insect eggs or 
larvae to adequately utilize the diet without adversely affecting virus 
production, and incubated for a time and at a temperature to develope the 
larvae to the middle stages of larval development, e.g., until they are 
about half-grown. In the case of the IMM, for example, suitable 
development generally is obtained in about 10 days when the temperature is 
27.+-.1.degree. C. 
Step 3. Inoculation of the Infested Diet and Second Incubation. 
An inoculum of virus is prepared by homogenizing virus-infected larvae in 
water by any standard method, for example, as described by Cowan et al., 
supra. The diet plus infested larvae from step 2 is inoculated with the 
virus inoculum and incubated for a second incubation for a time and at a 
temperature sufficient to cause the larvae to become infected with the 
virus. In the case of production of IMMGV, for example, at least 90% of 
the larvae will become highly infected, that is will have patent infection 
or be moribund, in about 10 days at 27.+-.1.degree. C. 
Step 4. Homogenization. 
Next, sufficient water is added to homogenize the diet plus virus-infected 
larvae obtained in step 3, and the mixture is homogenized to have the 
consistency of a slurry. Homogenization must be carried out at 
temperatures which do not adversely affect the activity of the virus. 
Temperatures below about 27.degree. C. are preferred. 
Step 5. Recovery of the Homogenate. 
Next, the homogenate is dried by a method which removes water without 
adversely affecting virus activity, for example, freeze-drying, and the 
dried material is milled to a fine powder at temperatures which do not 
adversely affect the activity of the virus in the composition. 
The virus composition can be used immediately or it can be stored 
refrigerated or frozen for future use. 
Application of the Virus Composition. 
The virus composition prepared as described in steps 1-5 is applied by any 
method known to those in the art for microbial agents used for insect 
control. For example, it may be formulated with water to provide an 
aqueous suspension for spraying on commodities. To prepare the suspension, 
the virus composition is mixed with sufficient water to provide coverage 
of the commodity with the virus composition without raising the water 
content of the commodity such that mold or other adverse problems occur. 
Generally about 0.5-1% (w/w, water/commodity) is suitable. Alternately, 
the virus composition is added to a carrier, for example, milled wheat 
bran, to obtain a dust formulation for dusting on the commodity to be 
protected. In all cases, the amount of virus in the application 
formulation must be a pesticidally effective amount, that is, an amount 
which will result in a significant mortality rate of a treated group as 
compared to an untreated group. The actual effective amount may vary with 
the species of pest, type of virus, and other related factors. For most 
applications, it is preferred to achieve an LC.sub.95 (concentration that 
kills 95% or more of exposed insects) dose/gram of commodity based on 
quality control bioassays. 
It is preferred that the virus composition comprise baculovirus, 
particularly nuclear polyhedrosis virus or granulosis virus, as these 
viruses are restricted in their pathogenicity to the class Insecta, are 
often genus or species specific, and are often highly virulent to their 
hosts. Further, they can be used in insect control programs without harm 
to beneficial arthropod species. 
Uses. 
The virus composition of the invention is useful as a protectant for 
commodities after harvest and particularly as a protectant for commodities 
after processing. However, it can be used on raw agricultural commodities 
as well. The composition may also be used in warehouses, processing 
plants, and other areas in sanitation programs to reduce risk of 
infestation of the commodities. It is also useful for topically treating 
packaged commodities to kill larvae prior to entering the packages. 
Another use of the formulation is in combination with an insect attractant 
to attract and contaminate insect adults and thus disseminate the virus 
into the environment of the target insect. 
The virus composition finds particular use in controlling postharvest pests 
such as the IMM, the raisin moth, the tobacco moth, the almond moth and 
the like. For example, the IMMGV composition is useful as a microbial 
control agent for IMM infesting dried fruits and nuts and grains; it 
provides excellent control of IMM at a cost similar to that of the 
presently used pesticides. 
It was unexpected that a diet devoid of honey and glycerol would be useful 
for larvae growth, as it was thought that these components provided 
nutrients essential for growth and development of the larvae. 
Surprisingly, however, not only did the larvae grow on the diet, removal 
of these components resulted in a 20-30 fold increase in activity of the 
resulting virus composition as compared to the Cowan et al. preparation. 
Further, the need for washing these components out of the diet was 
eliminated.

EXAMPLES 
The following examples are intended only to further illustrate the 
invention and are not intended to limit the scope of the invention which 
is defined by the claims. 
EXAMPLE 1 
IMM eggs and neonate larvae were obtained from stock colonies reared at the 
U.S. Department of Agriculture Agricultural Research Service Horticultural 
Crops Research Laboratory, Fresno, Calif. Eggs were surface-sterilized in 
10% formalin for 20 minutes and washed with sterile, distilled water for 
30 minutes. 
A diet was prepared which contained 3 gallons of autoclaved wheat bran; 400 
ml deionized water; 100 gram Brewers yeast, and 0.1 gm Vanderzants vitamin 
mixture in 10 ml water. Sorbic acid and methyl-p-hydroxybenzoate (4,000 
ppm each) were mixed into the diet as fungistats. After preparation, 150 
grams of the diet was placed in 0.95 liter wide-mouth canning type jars, 
the metal lids of which were replaced by fine-mesh brass screen discs and 
filter paper circles (9 cm diam). The jars were infested with 100 mg of 
IMM eggs, closed, and incubated at 26.7.+-.1.degree. C. for 10 days. Next, 
the jars were inoculated with an aqueous spray containing approximately 93 
mg of homogenized, GV-diseased IMM larvae in 25 ml of sterile distilled 
water. The inoculum was lightly stirred into the diet-larvae mixture. The 
diseased IMM larvae-diet mixture was then incubated for an additional 10 
days at 26.7.+-.1.degree. C. 
Next, 200 ml of cold, sterile, distilled water was added to the larvae-diet 
mixture, and the mixture was homogenized into a fine slurry and 
freeze-dried. Care was taken to maintain the temperature of the slurry 
below about 27.degree. C. The preparation was then milled into a fine 
powder, with care taken to avoid inactivation temperatures. After milling, 
the preparation was passed through a fine sieve to eliminate any coarse 
particles that may have remained and also to prevent clogging of nozzles 
if an aqueous spray is used. 
Bioassay Procedure. To determine viral activity, the GV composition was 
diluted in sterile distilled water so that the addition of 4 ml of the 
dilutions per 20 g of maintenance diet (Cowan et al., supra) provided 
concentrations between 1.0 mg/g and 0.001 microgram/g depending on the 
potency of the formulation. After thorough mixing, each of two cups (236 
ml each) per concentration were infested with 50 newly hatched IMM larvae 
and were covered with a plastic lid and incubated to adult emergence. 
Mortality was based on the number of moths emerging from each 
concentration. Controls were prepared identical to the samples except that 
GV composition was not added. 
The results are shown in Table 1. As can be seen from the data, the GV 
composition showed LC.sub.50 values in the range of 0.22 (0.19-0.26) 
microgram/gm of larval diet for formulations resulting from the above 
preparation. This preparation maintained its activity after storage for 22 
months at -20.degree. C. (Table 1). 
For comparison purposes, the above preparation was carried out except that 
honey and glycerol were included in the diet for virus production, and 
subsequently the honey and glycerol were washed out of the formulation 
(Cowan et al., supra). Bioassays of the resulting preparation showed 
LC.sub.50 values in the range of 5.6 (2.2-10.7) micrograms/gm of larval 
diet. This amounts to approximately a 25-fold difference in potency 
between the formulation of the invention and the formulation of Cowan et 
al. 
TABLE 1 
__________________________________________________________________________ 
Potency (micrograms GV per gram diet) 
GV Composition 
(95% CL) 
Sample 
Bioassay date(s) 
LC.sub.50 
LC.sub.95 
LC.sub.99 
__________________________________________________________________________ 
F-12-12-20 
3/86-10/86 
0.22 3.64 11.61 
(0.19-0.26) 
(2.72-4.87) 
(7.89-17.10) 
F-12-12-20.sup.1 
1/88 0.40 3.87 9.93 
(0.23-0.62) 
(2.09-11.63) 
(4.41-46.10) 
__________________________________________________________________________ 
.sup.1 Stored for 22 months at -20.degree. C. 
Use as Commodity Protectant. The GV composition prepared as described above 
was tested for control of IMM on raisins as follows. The GV composition 
was added to water to provide an aqueous spray that would treat the 
raisins at 10 mg or 100 mg GV composition per kg of raisins. The spray was 
applied to the raisins at a rate of 0.75% (w/w, water/raisins). Next, the 
raisins were artificially infested with IMM eggs and incubated until adult 
emergence was complete. Percent survival of IMM adults and percent 
visually damaged raisins were measured. The results are shown in Table 2. 
As can be seen from the data, damage to the raisins and survival of IMM 
adults were significantly reduced by treatment with the GV composition. 
TABLE 2 
______________________________________ 
GV dose.sup.1 % raisins 
% survival 
(mg/kg) damaged to adult 
______________________________________ 
0 (control) 86 2.3 
10 30 1.1 
100 3 0.1 
______________________________________ 
.sup.1 Composition F12-12-20. 
It is understood that the foregoing detailed description is given merely by 
way of illustration and that modification and variations may be made 
therein without departing from the spirit and scope of the invention.