Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom

Definitions

• the present invention is generally in the field of biological control of insect pests, specifically in the area of the use of a behavioral modifier in combination with an entomopathogenic fungus for the control of termites.
• Termites cause major economic loss through destruction of wooden structures, including residences, commercial buildings, and furniture, and, by deposit of earth and clay during the infestation process. Termites also damage carpet and other floor coverings, wall coverings, plaster, siding, and dry wall materials. Infestation of a residence with termites is a particular nuisance for homeowners, especially those who are trying to sell their houses.
• Insect pathogens are a possible alternative to the common use of highly toxic chemical insecticides for the control of insect pests.
• Fungi are one promising group of insect pathogens suitable for use as biological agents for the control of termites.
• Fungi are found either as single cell organisms or as multicellular colonies. They are eukaryotic and therefore more highly differentiated than bacteria but less differentiated than higher plants. Fungi are incapable of using light as an energy source and thus are restricted to a saprophytic or parasitic existence.
• the most common mode of growth and reproduction for fungi is vegetative or asexual reproduction, which involves sporulation followed by germination of the spores. Asexual spores, or conidia, form at the tips and along the sides of hyphae, the branching filamentous structures of multicellular colonies. In the proper environment, the conidia germinate, become enlarged, and produce germ tubes. The germ tubes develop over time into hyphae that in turn form colonies.
• M. anisopliae has been administered to insect pests by a number of methods, including direct spraying, injection, and application of the fungus to the plant or soil material on or in which the insect lives or feeds. In some insect species, infection with the fungus has been shown to result in death.
• the fungi M. anisopliae as reported by Hanel and Watson, 73 Bull. Ent. Res.
• U.S. Patent Nos. 5,057,315 and 5,057,316 to Gunner et al. disclose the use of an infection chamber containing a fungal pathogen to attract and lethally infect insects with the fungus. Examples include cockroaches and flying insects.
• the infection chamber design both maintains the fungal culture and forces the insect into contact with the fungus. Insects are attracted to the chamber by anyone or more of such means as the shape of the chamber, the use of food, attractant ⁇ , flavorings, scents, or colors, depending on the insect type.
• termites are attracted into contact with a potentially lethal fungus, such as Metarhizium anisopliae or Beauveria ba ⁇ iana , using a recruitment stimulant which evokes a series of behaviors, including feeding, arresting, and trail following (referred to herein as an "attractant") , such as the fungus Gloeophyllum trabeum or its volatile products.
• a fungus is used which is naturally repellent to the termites, such as M . ani ⁇ opliae and serves to prevent termites from entering into an area bounded by the repellent fungus.
• the entomopathogenic fungus alone or in combination with an attractant fungus, is provided either in the form of an infection chamber or formulated with a nutrient composition and administered directly to inhabited galleries or corridors to the termite nest to initiate infection, transmission and colony mortality.
• An advantage of the fungi is that, once established, the fungi continue to protect a structure from termites for an indefinite period of time.
• the two most preferred entomopathogenic fungi are Metarhizium ani ⁇ opliae and Beauveria ba ⁇ iana , although other fungi can be used that are pathogenic when the termite is inoculated via the infection chamber.
• M. anisopliae is the preferred repellent fungus.
• Gloeophyllum trabeum is the preferred attractant fungus. Examples demonstrate control of termites using chambers containing M. ani ⁇ opliae and B . bassiana.
• Figure 1 is a prospective view of a termite infection chamber.
• Figures 2A and 2B are graphs of termite decline in a habit trail after exposure to fungus while foraging, graphing percent survival versus time after treatment in days.
• Figure 2A is the percent survival after exposure to M .
• ani ⁇ opliae squares
• Figure 2B is the percent survival after exposure to B . ba ⁇ iana (squares) versus control (diamonds) .
• the present invention is primarily based on two discoveries: (1) that entomopathogenic fungi can be used to infect and kill termites but that an attractant is required to induce the termite into contact with the fungus and (2) that some types of fungi, by virtue of their natural repellency to termites, can be used to repel termites from an area.
• the pathogenic fungi, alone or in combination with an attractant fungus or compounds derived therefrom, or the repellent fungus can be administered to the area to be treated or protected in a nutrient formulation or an infection chamber to initiate a fungal culture for infection or repellency of termites.
• a major problem associated with entomopathogenic fungi for termite control is that the pathogenic fungi appear to repel the termites, making it extremely difficult to achieve infection sufficiently widespread to significantly reduce the termite population. This problem has been overcome by combining a fungal or other attractant with the entomopathogenic fungus which overcomes the repellency of the entomopathogenic fungus.
• the nutrient formulation consists of the fungus on a nutrient medium such as bran or other cellulose based food product which can be administered as a dry powder, such as a cereal grain or sawdust.
• the infection chamber includes a nutrient source for the fungal culture(s) as well as contains and protects the fungi prior to establishment of the fungi at the site to be protected or treated.
• Useful fungi can be obtained as isolates from infected termites (pathogenic fungi) or from termite infested wood (attractant fungi) or as isolates from soil using conventional techniques or from stored culture collections. These fungi can be obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland (ATCC) or USDA-ARS collection of entomopathogenic fungal cultures, U.S. Plant, Soil and Water Lab, Tower Road, Ithaca, New York (ARSEF) , where they are available without restriction. Soil isolates are obtained by screening soil samples for the presence of fungi, then testing the fungi in a bioassay for their effect on insects. Pathogenicity is established when the exposed insects die.
• At least two species of entomopathogenic fungi M . anisopliae and B . bassiana , have been shown to infect and kill termites. Examples include MM. anisopliae ATCC 62176 and ARSEF 1112 which have also been shown to repel termites.
• Useful fungi can be obtained by screening soil isolates by exposing termites under laboratory conditions to the fungus, as described in the examples below.
• Useful pathogenic fungus are those that kill the termites following direct contact.
• Useful repellent fungus are those that repel termites in soil box tests as described below.
• repelent fungus includes a fungus such as Metarhizium spp. , or compounds produced by the fungus which effectively repel the termites.
• Attractants that are useful are those that attract termites, or stimulate termites to recruit other colony members to aggregate and feed.
• Preferred attractants for termites include Gloeophyllum trabeum (formerly Lenzite ⁇ trabea) and some of its metabolic products, wood decayed by certain fungi containing extractable attractant substances, and certain organic compounds which mimic trail following pheromones, as described by Watanabe and Casida, 56:3 J. Econ. Ent. 300-307 (1963), Matsumura, F. , et al., 65:2, L. Econ. Ent. 600-602 (1972), Smythe, R.V. , et al., 58:3 J___ Econ. Ent.
• Wood decayed by some fungi contains attractants to termites, as described in Matsumura, F., et al., 62:3 J. Econ. Ent. 699-603 (1969); Watanabe and Casida (1963) .
• wood decayed by the brown rot fungus G. trabeum contains (S.S.E)-3, 6, 8- dodecatriene-1-ol and other compounds known to recruit termites.
• Grace, J.K., 92 Proc. Entomol. Soc. Wash. 773-777 (1990) performed behavioral bioassays involving trail-following activity in termites in response to extracts from wood decayed by G. trabeum combined with antioxidants.
• Watanabe and Casida (1963) also found that numerous organic compounds can attract termites. It has been observed that termite attractant activity is influenced by the size of the wood, which must exceed a minimum mass to warrant recruitment.
• G. trajbeujn At least one species of fungus, G. trajbeujn, has been shown to produce substances that attract termites and mask the repellency of pathogenic fungi. Others that should be useful are fungi that decay wood. Several isolates of G. trabeum are available from the ATCC; ATCC 13021 has been tested and established to be attractive.
• Suitable culture media are known that can be used in the chamber. Examples of media known to those skilled in the art and commercially available include potato dextrose agar (PDA) , or rice agar for the growth of Metarhizium and Beauveria .
• PDA potato dextrose agar
• Rice agar consists of 1% dextrose, 1% yeast extract, 5% rice flour, 1.5% agar, and 0.5% 5x Dubois sporulation salts.
• the 5x Dubois ⁇ porulation salts consist of 15 g (NH 4 ) 2 SO 4 /1000 ml; 0.30 g MgS0 4 7H 2 O/1000 ml; 0.15 g MnSO 4 H 2 O/1000 ml; 0.0375 g CuS0 4 5H 2 O/1000 ml; 0.0375 g ZnS0 4 7H 2 /1000 ml; and 0.0038 g FeS0 4 7H 2 O/1000 ml. Each salt is completely dissolved before the next salt is added, and the solution is autoclaved.
• chamber refers to an inoculation chamber used to initiate the infection process within the resident population.
• the infection chamber (1) concentrates an extremely high concentration of pathogenic fungal inoculum in a very small space, directing entering termites into contact with the spores that infect and kill the termites; (2) contains fungal spores and/or mycelium, resulting in minimal exposure of the environment to the fungi and protecting the fungus from the environment, thereby increasing viability of the culture and minimizing contamination of the fungal culture; and (3) attracts termites to the pathogenic fungi through the presence of termite attractants, such as Gloeophyllum trabeum or its volatile products, that overcome the repellant characteristic of the pathogenic fungi, alone or in combination with sawdust or wood chips which serve as a source of food for the termites.
• termite attractants such as Gloeophyllum trabeum or its volatile products
• the chamber can be made of a non-biodegradable material, or a biodegradable material, and is designed to establish an infection locus effective against a termite infection.
• the small, lightweight infection chambers are unobtrusive and can be easily placed in locations of heavy termite infestation, increasing the efficacy of the devices.
• one fungal culture can be used as an attractant agent and a second to provide a continuous supply of infectious spores over a prolonged period of time. These spores attach to the termites and originate germ tubes that penetrate the termite cuticle, resulting in death within a short period of time.
• the attractants such as the volatile compounds produced by wood decomposition, can be provided alone or in combination with a wood decomposing fungus such as Gloeophyllum trabeum .
• the primary means of infection is by external contact, the termites can also be infected by contact with each other and by ingestion of the spores.
• the termite comes in direct contact with the entomopathogenic fungus.
• the conidia of the fungus attach to the body of the termite.
• the conidia germinate on the insect cuticle, forming germ tubes which penetrate the integument of the termite.
• the germ tubes continue their penetration until they reach the internal body cavity (hemocoel) of the termite and kill it.
• the fungal mycelia may sporulate on the body of the termite, and other termites may be infected by exposure to the conidia produced on the dead termite.
• the chamber consists of an external housing which forms the basic shape of the chamber an'd an internal core mixture which fills the housing unit.
• the chamber housing can be constructed with conventional materials including glass, metal, extrudable or moldable plastic, or wood, but is preferably constructed of a biodegradable cellulose based material such as cardboard, paperboard, wood, pressed resin-woodchip or flakeboard, expanded cellulose, papier mache or peat.
• the chamber 10 should have small openings 12 (2-5 mm in diameter) which are large enough to allow free passage of the termites or have thin walls 14 which would allow termites to easily chew through.
• the preferred embodiment is a tubular structure.
• the shape can range from circle to multipointed polygon in cross section, but a triangular tube is preferable.
• the length of the chamber should range from 10 to 30 cm, and the side width should range from 3 to 8 cm.
• the chamber tip 16 is pointed in order to facilitate placement of the chamber into the ground.
• the tip 16 can be made from the same material as the housing walls 14, and coated with resin or other materials to increase strength, or the tip can be made of a harder material such as plastic or wood.
• the top 18 of the chamber housing is covered with a paper or plastic cap after filling.
• the core of the chamber can be filled with a mixture of several components: (1) a basic food source for the termites (a cellulose base material, most inexpensively sawdust) ; (2) the attractant: a 4-8 week old culture of G. trabeum grown on sawdust, a killed culture of G. trabeum , or extracts of G. trajbeum cultures, or synthesized chemical attractants, arrestants, pheromones or kairomones; and (3) the lethal agent (M . ani ⁇ opliae or other entomopathogenic organisms growing on solid or liquid medium, formulated wet or dry with food sources and protectants, or spores) .
• a basic food source for the termites a cellulose base material, most inexpensively sawdust
• the attractant a 4-8 week old culture of G. trabeum grown on sawdust, a killed culture of G. trabeum , or extracts of G. trajbeum cultures, or synthesized chemical attract
• the core mixture should contain excess attractant in proportion to lethal fungus, at a preferred ratio of 100 g sawdust inoculated with G . trabeum : 5 g dried formulated M . anisopliae hyphae mixed with oat bran: 1 g M. anisopliae spores.
• Cultures can be applied to an inert lattice or grid which serves as a support matrix, for growing or positioning the appropriate fungal cultures or for holding nutrients within the device.
• the chamber can be appropriately placed by (1) directly forcing it into the ground if the chamber is constructed of a hardened material; (2) forcing it into a hole dug by a tool such as a trowel or core sampling device; (3) placing it into the inner sleeve of a hollow tool which would be used to insert the chambers into the ground.
• Chambers are placed in the perimeter around the area where a structure is to be protected, at a distance of not less than 3.0 meters away but preferably 5 to 10 meters away from the structure. Chambers should be concentrated in an area with termite activity or between the termite active area and the area to be protected. The chambers should be separated by a distance of 0.1 meter to 1 meter.
• An infection chamber suitable for infecting termites can be constructed by initially building a housing unit, as demonstrated in Figure 1, and then filling it with core contents.
• the housing unit can be cut from flexible flat sheets of cellulose products, folded to the desired shape and glued together. It may be more desirable to mold the sheets first from a plastic, resin/wood, papier mache or peat before cutting and folding. Molding forms the grooves and holes in the walls and folding forms the final shape. It is possible to mold the complete shape at one time or to mold sections which can also be assembled by glue, pins in holes or tabs in slots.
• the assembled housing is next filled with the core mixture.
• the core mixture is formulated in three stages.
• a cellulose based nutriment typically sawdust
• the sawdust is sterilized by autoclaving twice, in plastic bags or other containers, with a 12- 24 hour cooling period between runs.
• the sawdust is inoculated with G. trabeum mycelium harvested from a liquid culture medium such as potato dextrose broth.
• the sawdust and G. trabeum mycelium are mixed and incubated at 24-27°C for 4 to 8 weeks.
• Stage II the cellulose G . trabeum culture is mixed with formulated M . anisopliae hyphae.
• This formulation is prepared by mixing M . ani ⁇ opliae hyphae, harvested from liquid culture (1% yeast extract, 1% peptone, 4% dextrose) , with a dry food source (oat bran flour) , mineral salts and protectants. The mixture is dried and ground to form granules.
• the above preparation is mixed with M . anisopliae conidia which are harvested from rice agar growth plates or bags of inoculated rice grains after one to three weeks of growth.
• the housing is then filled by hand or by machine with the core mixture and sealed with a cap.
• a support matrix core or grid core can be inserted into the housing.
• the matrix core has pockets filled with fungal preparations as previously described: G. traJ eutn on a cellulose base and M . anisopliae on agar, or as a dried hyphal formulate or harvested spores.
• Example 1 Weed Wood Termites ⁇ Zootermopsi ⁇ angusticoll ⁇ ) Directly Exposed to Metarhizium and Beauveria to Demonstrate Efficiency of the Fungi in killing termites.
• Termites (_Reticu.Zite.rn.es flavipes) by Direct Exposure of Termites to Fungal Isolates.
• Wood containing termites was collected from a forest site in Montague, Massachusetts. The termites were separated from the wood and placed into laboratory colonies from which individuals could be removed for bioassay. Ten termites were placed for 15 minutes on the surface of a petri plate containing sporulating pathogenic fungus. After treatment, termites were transferred to a holding plate containing filter paper and observed over time. It is apparent from the results shown in Table 2 that some strains of Metarhizium and Beauveria are very effective against termites, and can be used for termite control. Since soil isolates also showed high efficacy, this demonstrates that isolates of these pathogenic fungi could be obtained from soil using conventional methods.
• Example 3 infection of Colony Members By Release of Directly Infected Termites into a Colony
• Termites Z . angu ⁇ ticolli ⁇ in groups of four were placed in a petri dish with a small piece of wood (2 cm x 0.5 cm x 0.5) coated with a mixture of cellulose powder and either M . ani ⁇ opliae or B . ba ⁇ iana conidia scraped from an agar growth plate. Termites were exposed to this source of infection for one hour, then moved to a holding plate. After 48 hours, one of the exposed termites was introduced into another group of termites which had not been exposed to the fungus. Nine days after contact, one termite from the second group, which demonstrated no signs of illness, was transferred to a third group of unexposed termites. The mortalities of each of these groups of termites were observed over time.
• Example 5 Comparison of Fungal Formulations for Application to Soil to Infect Termites from a Site Distant from a Nest
• the bottom of a plastic box (15 x 20 x 35 cm) was lined with 2 to 3 cm of soil.
• a simulated nest containing 300 termites was placed at one end of the box.
• the nest was built from two 20 x 100 mm petri dish bottoms, with four holes cut in the sides, placed open faces together, and filled with filter paper and wood fragments. Termites were allowed to adjust to the environment for 3 to 10 days.
• a hole 1.5-2 cm deep was dug in the soil, and fungal formulation was applied.
• a wood block (5 cm x 3 cm x 7 cm) was placed in the hole containing formulation, and was half buried in the soil.
• the formulations consisted of (A) a granular form (oat bran cereal and hyphae) and (B) a powder form
• the bottom of a plastic box was lined with 2 to 3 cm of soil.
• An artificial nest containing approximately 300 termites was placed at one end.
• the nest was built from two 20 x 100 mm petri dish bottoms, with four holes cut in the sides. The two petri dishes were placed open faces together, and filled with filter paper and wood fragments. Termites were transferred into the nest and allowed to adjust to the environment for three to seven days.
• a hole was dug into the soil, dried hyphal formulation was applied, a wood block (5 x 3 x 7 cm) was placed on the hole, and soil was backfilled. Wood, soil, and nest were inspected periodically for fungal activity against the termites.
• isolates which were most effective in killing termite nests were ATCC 62176 and ESC 70, a Metarhizium and a Beauveria respectively.
• Other highly effective and repellent isolates were 2080 and 1112.
• ARSEF 721 Beauveria Avoid fungus Nest alive ARSEF 1079 Beauveria Reduced activity Nest alive CONTROL Termites throughout Nest alive area
• Example 7 Tests for Termite Attractants.
• microcentrifuge tubes 1.5 ml filled with (a) potential attractants and (b) control materials were placed 15 cm apart and 25 cm away from the nest at the other side of the arena. Arenas were videotaped by a time-lapse camera and observed for 15 days.
• G . trabeum sawdust and a mixture of M. ani ⁇ opliae (conidia or hyphae) and G. trabeum sawdust were used to fill the tubes instead of G. trabeum sawdust and control sawdust. All experiments were repeated three times.
• results show that G. trabeum infected sawdust is preferred over uninfected sawdust.
• M . ani ⁇ opliae conidia or hyphae are mixed with G. trabeum , termites do not react to the presence of M . ani ⁇ opliae ; that is, they enter both the attractant tube and the attractant plus Metarhizium tube. In both cases they contacted the tubes 2-7 hours after the experiment was set up and excavated the sawdust or the G. trabeum/ M . ani ⁇ opliae sawdust mixture in the tubes. When soil was present they built soil tunnels toward the test samples. In the test comparing M . ani ⁇ opliae hyphae plus G. trabeum sawdust with G.
• Example 8 Establishment of Termite Foraging Range using Attractant Fungus.
• Arenas (122 x 366 x 30.5 cm) were set up and covered with 1 cm of soil.
• a double petri plate artificial nest containing approximately 1000 termites and filter paper, was placed at one end of the arena 61 cm away from three of the four side walls.
• G. trabeum decayed wood fragments (15 x 3 x 2 cm) were placed at distances of 122, 183 and 244 cm away from the nest in the center of the arena. These were left for one week, and were monitored on alternate days for termite activity.
• Cardboard tubes (15 cm high x 4 cm diameter) filled with sawdust which had been inoculated with G. trabeum and incubated for several weeks were placed at a distance of 122, 183 and 244 cm from the nest. These were left for one week, and were observed periodically for termite activity.
• Termites were observed building tunnels at 30 to 65 cm from the nest. Individuals were seen in areas up to 200 cm from the nest. However, no termites were observed on, in or around the wood fragments in experiment A or in the tubes in experiment B. On the other hand, as shown in experiment C, once the wood fragments or the cardboard tubes were moved closer toward the nest, termites were found in the wood pieces and the tubes overnight.
• the range of termite foraging may be more limited under laboratory conditions than in the field.
• Example 9 Attraction of Termites to Fungal Attractant Chambers.
• the test arena was 122 x 122 x 30 cm (length x width x height) , made of plexi-glass with no cover.
• the arena was filled with 5 cm of soil.
• Autoclaved wet wood fragments (15 x 3 x 2 cm) were inoculated with G. trabeum , grown for up to 8 weeks, and placed in a paper tube 4 cm in diameter and 13 cm in length.
• Autoclaved wood fragments were treated similarly but without the inoculation with G . trabeum .
• non-autoclaved wood fragments were used to fill the same kind of paper tubes.
• the termite colony was placed in the center of the arena covered with a petri dish.
• Tube C - wood Side 2 Tube D - autoclaved wood
• Tube F - G. trabeum wood Side 3 Tube G - G. trabeum wood
• Tube I - wood side 4 Tube J - autoclaved wood
• testing tubes were arranged as described above in the following order:
• the attractant was put on top of the wood block while the formulated lethal fungus (62176) was placed in the soil under the wood block.
• box A When given a choice (in box A) , termites preferred wood associated with G. trabeum over wood alone. Termites built visible tunnels over the surface of the wood block to reach G. trabeum infected wood.
• box B termites preferred the untreated wood block over the wood block placed on a M . ani ⁇ opliae formulation. These results indicate that M . ani ⁇ opliae repels termites.
• box C termites preferred the G. trabeum treated wood block over the wood block placed on a M. ani ⁇ opliae formulation. When M. anisopliae and G. trajeum were placed around the same block (box D) , the termite nest appeared to be killed faster than M . ani ⁇ opliae alone as in box B.
• G. trabeum was shown to attract and cause termites to recruit others, to build and feed.
• M . ani ⁇ opliae when encountered by termites, repels the termites. When the two fungi were combined, the termite contact with the lethal fungus was facilitated by the presence of the attractant (recruitment stimulant) . Therefore, one may conclude that combined in proper proportion and spacial configuration, the G. trabeum will overcome the repellent nature of M . ani ⁇ opliae and ultimately lead to extensive infection and demise of the colony.
• Example 12 Use of M. Anisopliae ATCC 62176 as a
• the arena was a 122 x 366 x 30.5 cm (wide x long x high) Plexi-glass arena with 15 mm depth of soil on the bottom.
• Termites were collected from Montague, Massachusetts and kept at room temperature. Prior to use, termites were removed from the wood and transferred to a petri dish filled with filter paper.
• the wood frames were constructed from stock lumber.
• Part A was made of 4 pieces of 5 x 10 x 30 cm wood with numerous 2 cm deep grooves on the surfaces. These 4 pieces of wood were nailed together and formed a 10 x 35 x 35 cm wood rectangle.
• Part B was made from 4 pieces of 5 x l0 x 30 cm wood. They were nailed together to form a 5 x 40 x 40 cm wood rectangle. Part B was nailed on top of Part A to form the wood frame.
• ATCC culture 62176 mycelium harvested from liquid culture was mixed with sterilized oat bran flour. The mixture was dried by air flow at room temperature overnight then ground and sized through a #14 sieve to form granules.
• G. trabeum wood was prepared by autoclaving wood fragments (13 x 3 x 2 cm) , inoculating .them with G. trajeum, and incubating them at room temperature for 4 to 8 weeks.
• the wood frame was placed in the center of the arena. Around and under the foundation of the wood frame, culture ATCC 62176 formulation was mixed with soil and the wood frame was set on top. In the center of the wood frame, a pile of G. ' trabeum wood was set on the soil. The wood frame was then covered with a piece of cardboard to reduce drying and keep the area dark. Into each end of the arena 120 cm away from the wood frame one termite colony (600-1000 individuals) was transferred together with a few pieces of wet filter paper. The colony was then covered with a petri dish which had a few openings on the side to form termite nests. Of the two termite colonies used, one had about 1000 termites while the other had about 600 termites.
• Each arena was then examined once or twice a week for fungal growth as well as termite activities in the wood frame and in the G. trabeum wood pile, and sprayed with water periodically to keep soil moist.
• Another arena was set up with similar materials except that a control formulation (oat formulation without 62176 mycelium) and two termite colonies, each containing 1000 termites, were used.
• the termite nest and soil in the arena as well as the "wood frame" were taken apart and examined thoroughly. Less than 10% of the termite population in one nest (the one where dead termites were found) were alive, i.e., more than 90% of the termites in this nest were killed. In the other nest, no dead termites were found and the number of termites recovered from the nest and soil approximated the original termite number. More importantly, the wooden structure had no visible damage from termite attack. In a control experiment where the formulation used at the structure contained no M . ani ⁇ opliae , termites were found at the wood structure after one week. Subsequently, they tunneled up, around, and into the wood. Several hundred termites, including secondary reproductives and eggs, were found living inside the wood frame.
• Chambers Paper tubes (3 cm diameter x 5 cm high) were filled with a uniform mixture of G. trabeum sawdust, M. ani ⁇ opliae ATCC 62176 Formulation and 62176 conidia in the ratio 100:5:1.
• Blank Control Chambers Paper tubes filled with G. trajbeum sawdust and granular at a ratio of 100:5.
• the arena (122 x 122 x 30) and termites were the same as in Example 9.
• Each petri dish contained about 1000 termites and was treated as a colony. Arenas were filled with soil to a depth of 5 cm.

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