Biological ovicide for control of lepidopterous insects

An agricultural formulation of a virulent isolate of Beauveria bassiana, which has the characteristics of B. bassiana ATCC 74040, can be used to effectively control lepidopterous insects. This fungal strain has been found to be active against the egg stage of lepidopterans. Activity against the larval stages of lepidopterans is also shown.

FIELD OF THE INVENTION 
The invention is directed to a method of controlling pests, in particular 
insect pests belonging to the Order Lepidoptera, using a biopesticidal 
composition containing a highly virulent entomopathogenic fungus of the 
species Beauveria bassiana. 
BACKGROUND OF THE INVENTION 
Larvae of lepidopterous insects cause millions of dollars of damage each 
year to agricultural crops grown for food and fiber production. While 
chemical insecticides such as chlorinated hydrocarbons, organophosphorus 
compounds, carbamates, and synthetic pyrethroids are conventionally used 
to control these insects, most species are broadly and highly resistant to 
chemical insecticides due to the continued reliance upon such substances 
by producers in order to prevent crop damage and consequent economic 
losses. 
Application of these largely ineffective chemicals have significant impact 
upon the environment in which they are placed. In addition to 
environmental pollution, use of chemical insecticides create potential 
health hazards for agricultural workers and consumers. The problem of 
residues on consumer products and water quality present major drawbacks. 
Detrimental effects of these chemical insecticides on nontarget species 
often result in secondary pest outbreaks. Moreover, such chemicals are 
often phytotoxic to exposed plants. 
Because of the problems associated with the use of chemical pesticides, 
safer and more effective methods of controlling insect pests are essential 
for continued agricultural production. While biological control agents are 
a reasonable alternative to chemical pesticides, none have been identified 
as being commercially feasible for controlling lepidopterous pests by 
direct activity against the egg stage. Bacillus thuringenis, for example, 
is an biological control agent designed specifically as an insecticide for 
control of certain leaf-eating caterpillars (Lepidoptera). The active 
ingredient is a stomach poison and, as such, must be eaten by the larvae 
to be effective. Larvae of lepidopterous insects must, therefore, be 
actively feeding on treated exposed plant parts. 
Recently the development of mycoinsecticides have been directed towards 
specific insects such as Bemisia tabaci. No mycoinsecticide has, however, 
been heretofore identified as having ovicidal activity. 
SUMMARY OF THE INVENTION 
The invention is directed to the use of a highly virulent strain of an 
entomopathogenic fungus to control lepidopterous insects which attack and 
damage plants. It has been discovered that a strain of Beauveria bassiana 
having the characteristics of Beauveria bassiana ATCC 74040, shows 
virulence against the egg stage as well as the larval stages of 
lepidopterous insects. The entomopathogenic fungus used in the practice of 
the invention is a B. bassiana culture having the characteristics of B. 
bassiana ATCC 74040, and mutants thereof which substantially retain the 
virulence of the parent strain. The use of this fungus as a 
mycoinsecticide does not produce the hazards associated with conventional 
chemical control agents. The fungus can be applied directly to the eggs or 
larvae of insects or to the plants which are to be protected. 
It is an object of the invention to provide a method of controlling 
lepidopterous insect pests. The method comprises applying a pesticidal 
composition containing a fungus having the identifying characteristics of 
B. bassiana ATCC 74040 to lepidopterous insects or to the foliage of 
plants to be protected. 
B. bassiana having the identifying characteristics of B. bassiana ATCC 
74040 has been found to be virulent against the egg stages of 
lepidopterous species of insects as well as to the larvae thereof. The 
method of the invention has been found to be particularly advantageous 
when the pesticidal composition is applied to lepidopterous insects in the 
egg stage. 
DETAILED DESCRIPTION OF THE INVENTION 
Chemical insecticides and some biological insecticides are currently 
employed in the control of insect pests. These have associated problems 
and are not completely effective. Because of the problems associated with 
the use of chemical pesticides, safer and more effective methods of 
controlling insect pests are needed. There is a need for alternative 
materials that can be used in a complementary fashion with existing 
controls and which can replace existing control agents that may lose 
efficacy due to resistance or other factors. 
The development of a broad spectrum of pesticides would reduce the need for 
many of the petrochemically based pesticides. While biological control 
agents have been tried, their availability, limited host range, cost and 
reliability have reduced the potential for implementing the use of these 
biological control agents. By using fungi to control insect pests, the 
potential for resistance development is minimized, which, in turn, will 
stabilize many pest management programs. 
At least six species of Beauveria are recognized based on morphological and 
biochemical characteristics: B. alba, B. amorpha, B. bassiana, B. 
brongniartii, B. velata, and B. Vermiconia (Mugnai et al., 1989, A 
chemotaxonomic evaluation of the genus Beauveria. Mycol. Res., 
92:199-209). Not only do significant differences exist between species of 
Beauveria, but significant intraspecies variability exist as well. 
Different strains of B. bassiana are known to exhibit different 
insecticidal effects. As disclosed by Peczynska-Czoch et al. (Formation of 
beauvericin by selected strains of Beauveria bassiana, 1991, Archivum 
Immunologiae et Therapiae Experimentalis, 39:175-179), significant 
intraspecies variability of B. bassiana isolates exist. Ferron (Pest 
control by the Fungi Beauveria and Metarhizium, In: Microbial Control of 
Pest and Plant Diseases, 1970-1980, Burges, Ed., 1981, Academic Press, pp. 
465-4820) not only discloses that it is known that entomopathogenic fungi 
have certain specificity, but also discloses that within the same species 
of fungus different strains can have different activity spectra. Reference 
is also made to Ferron, Biological Control of Insect Pests by Entomogenous 
Fungi, 1978, Ann. Rev. Entomol., 23:409-442, which also discloses that 
different fungal strains have different activity spectrum. 
It has been discovered that a strain of Beauveria bassiana having the 
characteristics of Beauveria bassiana ATCC 74040, shows virulence against 
the egg stage as well as the larval stages of lepidopterous insects. The 
entomopathogenic fungus used in the practice of the invention is a B. 
bassiana culture having the characteristics of B. bassiana ATCC 74040, and 
mutants thereof which substantially retain the virulence of the parent 
strain. A biologically pure culture of an isolate of Beauveria bassiana 
was deposited under Accession No. ATCC 74040 in the American Type Culture 
Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, on Mar. 11, 
1991. This isolate has also been deposited in the USDA--ARS Collection of 
Entomopathogenic Fungal Culture under Accession No. ARSEF 3097. This 
strain, which was isolated from a boll weevil cadaver in the lower Rio 
Grande Valley of Texas, has been confirmed to be a B. bassiana (Balsamo) 
Vuillemin. 
The taxonomic description of Beauveria bassiana ATCC 74040 is the same as 
that for other members of the species B. bassiana. B. bassiana is an 
imperfect fungus (Fungi Imperfect) of the subdivision Deuteromycotonia. 
The genus Beauveria Vuill is within the Class Deuteromycetes and is 
distinguished from other genera by having conidia that are borne singly, 
not catenulate. The fertile portion of the conidiophore is zigzag in shape 
and drawn out at the tip. The species B. bassiana has spherical, not 
ellipsoid, conidia measuring 2 to 3 micrometers by 2 to 2.5 micrometers 
and with conidiophores forming dense bunches. 
Like most entomogenous fungi, B. bassiana initiates infection by a 
germinating spore (conidium) attaching to and subsequently penetrating the 
cuticle of the insect host. B. bassiana ATCC 74040 attaches very securely 
to the cuticle of the targeted insect pest and is typically not removed by 
the grooming activities thereof. While not wishing to be bound by any 
particular theory, this may account somewhat for the high virulence of the 
fungus. As the fungus penetrates the target pest cuticle, the invasive 
hyphae begin to enter the host tissues and ramify through the hemocoel. 
Hyphal bodies or segments of the hyphae distribute throughout the 
hemocoel, filling the dying insect with mycelium. Emergent hyphae grow out 
through the insects integument and produce spores on the external surface 
of the host. These spores, or conidia, are dispersed and capable of 
infecting new host insect pests. Although the mode of infection of 
Beauveria bassiana fungi is generally by cuticular penetration by the germ 
tube of the fungal conidia and may also occur through the respiratory or 
alimentary tract (such as mouth parts). Ingestive fungal spores voided in 
the feces may provide another source of contact with the cuticle of the 
targeted insect pest. Death of the host may occur either by release of 
fungal toxins or by tissue destruction. 
While B. bassiana ATCC 74040 is taxonomically the same as other members of 
the species B. bassiana, this isolate differs from other members of its 
species metabolically and biochemically. B. bassiana having the 
characteristics of B. bassiana ATCC 74040 has been heretofore found to be 
highly virulent against Anthonomus grandis (boll weevils), Bemisia tabaci 
(sweet potato whiteflies) and Pseudatomoscellis seriatus (cotton 
fleahoppers). Reference is made to copending application Ser. No. 
08/058,795, the disclosure of which is incorporated herein by reference. 
B. bassiana ATCC 74040 has also been observed by the inventor to have 
mycobiological activity against citrus mealybug, Planococcus citri; 
thrips, Frankliniella spp.; onion thrips, Thrips tabaci; armyworms, 
Spodoptera spp.; Colorado potato beetle, Leptinotarsa decemlineata; two 
spotted spider mite, Tetranychus urticae; gypsy moth, Lymantria dispar; 
pepper weevil, Anthonomus eugenii; webworms, Pyralididae; corn rootworms, 
Diabrotica app.; flies, Muscidae; chinch bugs, Blissus spp.; corn borers, 
Diatraea spp.; and other pests. 
It has now been discovered that this isolate is highly virulent to the eggs 
of a number of important lepidopterous pests. Such insect pests include 
Heliothis virescens, Heliocoverpa zea, Spodoptera frugiperda, Trichoplusie 
ni and Plutella xylostella. The use of this fungus as a mycoinsecticide 
does not produce the hazards associated with conventional chemical control 
agents. The fungus can be applied directly to the eggs or larvae of 
insects or to the plants which are to be protected. 
Prior to applicant's discovery of Beauveria bassiana ATCC 74040, a 
Beauveria bassiana species having effectiveness required for commercial 
use was not known. Although the potential for commercial exploitation of 
entomopathogenic fungi as an insecticide has been explored, various 
factors such as the virulence of the strain, the susceptibility of the 
host insect and the mode of infectivity (i.e., not only through the gut 
but through spiracles and, in particular, through integument) have 
deterred the formulation of a commercially effective product. For example, 
all possible ways of infection are not necessarily exploited by a given 
fungus for a given insect. In addition, conditions encountered in the 
field, such as humidity and temperature, must be considered. 
The inventor was the first to produce a commercially useful biopesticide 
containing B. bassiana, specifically B. bassiana ATCC 74040, as required 
for use in the claimed invention. Reference is made to the product 
(NATURALIS.RTM.) described by Knauf and Wright ("Fermone Exp 7744: A 
Biorational Insecticide for Whitefly Control. A Review of Research and 
Cooperator Trials in Florida and Texas Greenhouses," 1992, and "A Summary 
of Research and Cooperator Studies of Naturalis.RTM.-L and Fermone Exp 
7744 Bioinsecticides for Control of Sweet Potato Whitefly," 1992). 
The fungus used to practice the method of the invention can be successfully 
grown on several different media including potato dextrose agar (PDA), 
Subraund dextrose agar (SDA), oatmeal agar, and mixed bran agar. Based on 
the diameter of the colonies, spore production, and the cost and 
availability of the agars, SDA provides an excellent medium for growing 
the fungus. The B. bassiana used in the practice of the invention may be 
cultured and mass produced by methods used to culture Beauveria spp. See 
for example, U.S. Pat. No. 4,925,663; Microbial Control of Pest and Plant 
Diseases 1970-1980, published by Academic Press, pp. 471-473 (1981; edited 
by H. D. Burges); and Feng et al., J. Invertebrate Pathology, Vol. 46, no. 
3, November 1985, page 260, the disclosures of which are incorporated 
herein by reference. The fungal growth range is between 40.degree. and 
95.degree. F. in a wide range of humidity with high humidity necessary to 
germinate spores and to increase spore production. The concentration of B. 
bassiana used in the composition is readily determinable of skilled 
practitioners depending, for example, on the extent and degree of 
infestation, time, weather conditions, life cycle stage of the pest, and 
concurrent usage of other insecticides. Generally 2.times.10.sup.5 to 
2.times.10.sup.14 spores per milliliter, preferably, at least about 
2.times.10.sup.8 spores per milliliter, is sufficient to control 
lepidopterous insect pests. 
The composition used to practice the invention may consist of B. bassiana 
alone, or may comprise B. bassiana in combination with a conventional 
agriculturally acceptable carrier. Solid and liquid formulations may be 
used. Additional expedients used in the art, such as emulsifiers, 
thickeners, foaming agents, etc., may be used. The composition may also 
include other chemical or biological control agents. Particularly 
advantageous is the use of a formulation comprising B. bassiana having the 
identical characteristics of ATCC 74040 and B. thuringenis. Compositions 
may also be applied, either simultaneously or sequentially, with other 
chemical or biological control agents. Application of conventional 
chemical insecticides, at a reduced rate, combined with fungal 
compositions has been found by the inventor to impact damaging insect 
populations at a faster rate than when the fungus is applied alone. 
Application of the fungal composition may be accomplished using standard 
operating equipment used in the agricultural industry by conventional 
ground spreaders or sprayers, or aerially. 
Field evaluations in Mississippi, Louisiana and Texas have confirmed the 
biological activity of B. bassiana ATCC 74040 against the egg stage of 
lepidopterous pests. When lepidopterous eggs are exposed to B. bassiana 
ATCC 74040 the eggs are killed when the fungus colonizes the egg. Timing 
of application is important in relationship to the age of the egg, as 
newly oviposited eggs are more susceptible to the virulent activity of B. 
bassiana ATCC 74040. The fungus, however, also will colonize larvae when 
exposed directly or when newly hatched larvae feed on the egg shell from 
which it emerged. This activity gives immediate and long term control of 
the pests. 
The following Examples demonstrate the activity of B. bassiana ATCC 74040 
against the eggs of important lepidopterous species and also related 
activity against larvae. The data reported in the following Tables is the 
average of three trials. One hundred insects (eggs or larva) being treated 
in each trial.

EXAMPLE 1 
Eggs of Heliothis virescens were treated with different concentrations of 
B. bassiana spores. The percent mortality is shown in Table 1. 
TABLE 1 
______________________________________ 
Concentration 
(no. of spores/ml) 
% of eggs colonized 
______________________________________ 
2.5 .times. 10.sup.5 
58 
2.5 .times. 10.sup.6 
56 
2.5 .times. 10.sup.7 
70 
1.0 .times. 10.sup.8 
90 
______________________________________ 
EXAMPLE 2 
Eggs of Heliocoverpa zea were treated with different concentrations of B. 
bassiana spores. The percent mortality is shown in Table 2. 
TABLE 2 
______________________________________ 
Concentration 
(no. of spores/ml) 
% of eggs colonized 
______________________________________ 
2.5 .times. 10.sup.5 
39 
2.5 .times. 10.sup.6 
38 
2.5 .times. 10.sup.7 
36 
1.0 .times. 10.sup.8 
59 
______________________________________ 
EXAMPLE 3 
Eggs of Spodoptera frugiperda were treated with different concentrations of 
B. bassiana spores. The percent mortality is shown in Table 3. 
TABLE 3 
______________________________________ 
Concentration 
(no. of spores/ml) 
% of eggs colonized 
______________________________________ 
2.5 .times. 10.sup.5 
73 
2.5 .times. 10.sup.6 
65 
2.5 .times. 10.sup.7 
80 
1.0 .times. 10.sup.8 
88 
______________________________________ 
EXAMPLE 4 
Eggs of Trichoplusia ni were treated with different concentrations of B. 
bassiana spores. The percent mortality is shown in Table 4. 
TABLE 4 
______________________________________ 
Concentration 
(no. of spores/ml) 
% of eggs colonized 
______________________________________ 
2.5 .times. 10.sup.5 
18 
2.3 .times. 10.sup.6 
21 
2.5 .times. 10.sup.7 
16 
1.0 .times. 10.sup.8 
44 
______________________________________ 
EXAMPLE 5 
Eggs of Plutella xystella were treated with different concentrations of B. 
bassiana spores. The percent mortality is shown in Table 5. 
TABLE 5 
______________________________________ 
Concentration 
(no. of spores/ml) 
% of eggs colonized 
______________________________________ 
5.0 .times. 10.sup.7 
56 
1.0 .times. 10.sup.8 
50 
______________________________________ 
EXAMPLE 6 
Larvae, first and second instar of Heliothis virescens were treated with 
different concentrations of B. bassiana. The percent mortality is shown in 
Table 6. 
TABLE 6 
______________________________________ 
Concentration 
(no. of spores/ml) 
% of larvae colonized 
______________________________________ 
2.5 .times. 10.sup.6 
13 
2.5 .times. 10.sup.7 
13 
1.0 .times. 10.sup.8 
19 
______________________________________ 
EXAMPLE 7 
Larvae, third and fourth instar of Heliothis virescens were treated with 
different concentrations of B. bassiana. The percent mortality is shown in 
Table 7. 
TABLE 7 
______________________________________ 
Concentration 
(no. of spores/ml) 
% of larvae colonized 
______________________________________ 
8.4 .times. 10.sup.8 
75 
1.2 .times. 10.sup.9 
76 
1.5 .times. 10.sup.9 
76 
1.9 .times. 10.sup.9 
78 
______________________________________ 
EXAMPLE 8 
Larvae, first and second instar of Heliocoverpa zea were treated with 
different concentrations of B. bassiana. The percent mortality is shown in 
Table 8. 
TABLE 8 
______________________________________ 
Concentration 
(no. of spores/ml) 
% of eggs colonized 
______________________________________ 
2.4 .times. 10.sup.7 
42 
1.2 .times. 10.sup.8 
64 
2.4 .times. 10.sup.9 
48 
______________________________________ 
It will be understood that the foregoing examples are for purposes of 
illustration only and are not meant to limit the scope of the claimed 
invention. Various changes in the details, materials and arrangement of 
parts which have been described and illustrated herein in order to explain 
the nature of the invention may be made by those skilled in the art within 
the principle and scope of the invention as expressed in the appended 
claims.