Title of Invention	A TERMITICIDE
Abstract	The present invention relates to a termiticide comprising an entomogenous fungus and/or a culture thereof wherein the entomogenous fungus is selected from the group of beauveria brongniarti, beauveria bassiana beauveria amorpha, metarhizium anisopliae, and verticillium lecanii.

Full Text

TERMITICIDE AND METHOD FOR TERMITE CONTROL USING THE SAME BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a termiticide containing an entomogenous fungus and/or a culture thereof, and to a method for termite control using the termiticide. Discussion of the Related Art Pesticides are usually used to control harmful insects. The use of pesticides is sometimes harmful to men and beasts. Therefore, attempts have been made to use a natural enemy for controlling harmful insects as a pesticide which is safe to men and beasts. Using a entomogenous fungus is one of such attempts. Japanese Patent Laid-Open Nos. 4-202104 and 63-258803 disclose the use of fungi to control a long-horned beetle. For termite control, organophosphate and pyrethroid pesticides have widely been used in the forms of oil solution and emulsion, which are sprinkled over the ground or lumbers, or injected into the infested structures of lumber. Recently, however, there has been increasing concern about the influence of these chemical pesticides upon humans and beasts as well as upon the natural environment. Also, termiticides are usually applied to a narrow space such as a space under the floor and therefore present operational difficulties and health hazards to the workers. SUMMARY OF THE INVENTION An object of the present invention is to provide a termiticide and a method for termite control which give no harm to humans and beasts, less environmental influences, no difficulties in handling, and eradication of termites. The present inventors made extensive studies to achieve the above object and found that fungi of certain types have a specific killing-activity against termites among arthropods including termites, cockroaches, ants, pill wood lice, sow bugs, large centipedes, and shield centipedes, and have completed the present invention. Specifically, the gist of the present invention is concerned with: (1) A termiticide comprising an entomogenous fungus and/or a culture thereof; (2) The termiticide described in (1) above, wherein the entomogenous fungus is fleauveria brongniartii; (3) The termiticide described in (1) above, wherein the entomogenous fungus is selected from the group consisting of Beauverla bassiana, Beauveria amorpha, Metarhizium anisopliae, and Veirticillium lecanii; (4) The termiticicie described in any one of (1) to (3) above, wherein the entomogenous fungus is in the state of germination or resting spore; (5) The termiticide described in any one of (1) to (4) above, wherein the entomogenous fungus and/or the culture thereof are contained in an application form selected from the group consisting of powder, granule, tablet, liquid, aerosol, band, shet, pellets, and cream; (6) The termiticide described in (5) above, wherein cell density of the entomogenous fungus in the application form is 10 to 10 cells/cm or 10 to 10 cells/g; (7) The termiticide described in any one of (1) to (6) above, wherein a target termite is selected from the group consisting of Coptotermes sp,, Retlculitermes sp., Cryptotermes sp., Xncisltermes sp., Macrotermes sp., and Odontotermes sp.; (8) The termiticide described in (7) above, wherein a target termite is selected from the group consisting of Coptotermes formosnus, Retlculitermes speratus, Retlculitermes hesperus, Retlculitermes tibialis, Retlculitermes flavilpes, Retlculitermes luclfugus, Retlculitermes santonensis, Cryptotermes domestlcus, and Incisitermes minor; and (9) A method for termite control using the termiticide described any one of (1) to (8) above. DETAILED DESCRIPTION OF THE INVENTION The type of the entomogenous fungus used in the present invention is not limited as long as the fungus can be parasitic on a termite and thereby kill the termite. Specific examples of the entomogenous fungus usable in the present invention include Beauverza brongnlartil, Beauveria bassiana, Beauveria amorpha, Metarhtzivm anlsopliae, and Verttcllllum lecantt. Among the above fungi, Beauveria brongniartii is most preferred because of its high activity against termites and quick effect. These fungi can be isolated from a body of an insect which died of the fungal parasity in the outdoors. In the present specification, "the culture of entomogenous fungus" means a culture medium where an entomogenous fungus is grown, which comprises both the fungus and the medium. The media used herein are not limited as long as the fungi as mentioned above can grow thereon. Both solid and liquid media can be used, and the components contained therein are not particularly limited. Any known components as mentioned below can be used. Examples of carbon sources include glucose, fructose, saccharose, lactose, maltose, glycerol, starch, cellulose and molasses. Examples of nitrogen sources include ammonium sulfate, ammonium chloride, and anunonium nitrate. Inorganic salts other than nitrogen sources include potassium dihydrogen phosphate, magnesium sulfate, calcium sulfate, and potassium sulfate. Natural organic substances include extracts and pulverized powder of animal tissues, such as meat extract, fish meat extract, and silkworm pupa meal; extracts and pulverized powder of vegetable tissues, such as potato exudate powder, corn steep liquor, soybean oil, malt extract, and soybean powder; microorganism cells and extracts thereof, such as dry yeast, yeast extract, and polypeptone. A medium used in the present invention can be prepared by appropriately combining the above components. In order to prepare a solid medium, an appropriate amount of agar is added in a conventional manner. In the present invention, the culture of entomogenous fungus is prepared by growing an entomogenous fungus on the above-prepared medium usually at 25°C for 3 to 7 days, but the culturing conditions are not limited to the above. The target termites for the present invention are not limited. Examples include termites of Coptotermes sp., Reticulitermes sp., Cryptotermes sp., Incisitermes sp., Macrotermes sp., and Odontotermes sp. More specific examples include termites of Coptotermes formosanus, Reticulitermes speratus, Reticulitermes hesperus, Reticulitermes tibialis, Reticulitermes flavlpes. Reticulltermes lucifungus, Reticulitermes santonensis, Cryptotermes domesticus, and incisltermes minor. The entomogenous fungus and/or the culture thereof in the present invention can be used as a termiticide without any processing. Also, because the fungus used in the present invention may be germinated fungi or resting spores, it can be processed to have an appropriate application form. Examples of the application forms include powder, granules, tablets, liquid, aerosol, band, sheet, pellets, and cream. When solid forms, such as powder, granules, tablets, band, sheet and pellets, are used, it is preferred to use dry fungi to promote conidia. The carriers used in the preparation of the above application forms are not limited, and any known carriers may be used. Examples of the carriers used in the present invention are as follows: examples of solid carriers used in the preparation of powder, granules, tablets and pellets include mineral powders (silicic acid, kaolin, active carbon, bentonite, montmorillonite, diatomaceous earth, talc, clay, calcium carbonate, alumina, acid clay, talc powder, agalmatolite powder, mica, silica sand, ammonium sulfate, etc.), vegetable powder (wood powder, soybean powder, wheat flour, tobacco powder, starch, crystalline cellulose, etc.) and clathrate compounds such as cyclodextrin; examples of carriers used in the preparation of liquid and aerosol: water; examples of carriers used in the preparation of band and sheet: synthetic fibers (polyethylene, nylon, polyvinyl chloride, polyvinylidene chloride, polyester, polypropylene, vinylone, etc.), and animal and vegetable fibers and inorganic fibers (resin, silk, cotton, wool, paper, cloth, non woven fabric, woven fabric, leather, aluminum, etc.); and examples of carriers used in the preparation of creams: hydrocarbons (liquid paraffin, vaseline, paraffin, etc.), silicones (dimethylsiloxane, colloidal silica, bentonite, etc.), alcohols (ethanol, stearyl alcohol, lauryl alcohol, etc.), polyhydric alcohols (polyethylene glycol, ethylene glycol, glycerol, etc.), carboxylic acids (lauric acid, stearic acid, etc.), and esters (beeswax, lanolin, etc.) In the present invention, medium bases prepared by mixing the above carriers and medium components are preferably used. The number of viable cells contained in each application form (cell density) is not particularly limited. It is preferably in the range of from 10 to 10" cells/cm ( 10 to 10 cells/g for solid forms), more preferably not less than 10 cells/cm (not less than 10 cells/g for solid forms). Beauveria brongniartii can exhibit a high termiticidal activity even with a small cell density (about 10 cells/cm, or 10 cells/g for solid forms), which brings about various advantages. For example, this fungus requires a shorter period of time for culturing, and makes it possible to prepare compact application forms such as thinner sheet forms. The number of viable cells per unit area, unit volume, and unit weight (cell density) can be counted by the dilution plate technique. The method for termite control of the present invention can be carried out by placing the above-mentioned termiticide of the present invention in the vicinity of a termite nest, or by directly spraying or injecting the termiticide of the present invention to the place infested with termites. The present invention provides a simple and effective termite control without causing any harm to men and beasts. EXAMPLES The present invention will be further detailed by means of the following Examples, without intending to restrict the scope of the present invention thereto. Example 1 Beauverla brongniartii was spread over the agar medium in a Petri dish and cultured at 25°C for 5 days until cell density became 10 cells/cm, the agar medium (manufactured J by Nissui Pharmaceutical K.K.) containing 0.4% by weight of potato exudate powder, 2% by weight of glucose, and 1.5% by weight of agar. On the culture medium, 10 worker termites of Coptotermes formosanus were made to walk for 1 minute. Then the termites were transferred to another Petri dish containing filter paper and water-wetted cotton. The same experiment was repeated with another 3 groups of termites. With a total of 4 groups, the death number of the termites were counted at several time points after the walking on the termiticide. The results are shown in Table 1. As an untreated control group, 10 termites were made to walk on the same medium as the above except that Beauveria brongniartii was not grown thereon, treated and kept in the same way as above, and evaluated for mortality. The results are also shown in Table 1. In Table 1, the results are expressed in cumulative mortality (%) calculated from the death number at each measurement. Beauverla brongniartii used here was isolated from the body of long-horned beetles which died from infection with this fungus in the outdoors. The fungi used in the following examples were isolated in the same way. As shown in Table 1, all the termites of Coptotermes formossnus in the treated groups in Example 1 died within 5 to 7 days. On the contrary, no death occurred before day 7 in the untreated control group. These findings demonstrate that termite Coptotermes formosanus dies from direct contact with the fungus and/or the culture thereof. This indicates that Beauver±a hrongn±art±± is usable as a termiticide- As shown in Table 2, all the termites of Coptotermes formosaiius in the treated groups in. Example 2 died within 6 to 8 days. On the contrary, no death occurred before day 8 in the untreated control group. These findings demonstrate that termite Coptotermes formosanus dies from direct contact with the fungus and/or the culture thereof. This indicates that Beauveria tiassiana is usable as a termiticide- Example 3 With Metarhizium anisopliae, the mortality of termite Coptotermes formosanus was evaluated in the same manner as in Example 1. Table 3 shows the results. As shown in Table 3, all the termites in the treated groups in Example 3 were killed within 13 to 14 days. On the contrary, no death was found before day 14 in the untreated control group. These findings demonstrate that termite Coptotermes formosanus dies from direct contact with the fungus and/or the culture thereof. This indicates that MetarTiizxum anisopliae is usable as a termiticide. As shown in Table 4, all the termites of Coptotermes formosauus in the treated groups in Example 4 died within 11 to 12 days. On the contrary, no death occurred before day 12 in the untreated control group. These findings demonstrate that termite Coptotermes formosanus dies from direct contact with the fungus and/or the culture thereof. This indicates that Verticllixmn lecanlt is usable as a termiticide. Example 5 In a Petri dish where 10 normal worker termites of CoptotetTaes formosanus were kept with filter paper and water-wetted cotton, 3 dead bodies of the termites of Coptotermes formosanus which died from infection with Beauveria brongnlartit in Example 1 were placed. Thereafter, the number of deaths in the 10 termites was evaluated at several time points. The same experiment was conducted with another 4 groups of normal termites, totaling 5 groups being evaluated for mortality at several time points after the exposure to the dead bodies. The results are shown in Table 5. A group of 10 termites were kept in the same way as the above without placing dead bodies of termites. This group was also evaluated for mortality as an untreated control group. Table 5 shows the results in cumulative mortality {%). As shown in Table 5, even if there is no direct exposure to the fungus and/or the culture thereof, normal termites can be killed by being infected with the fungus by making contact with the dead body of a termite that dies from the fungus. Example 6 On an agar culture medium where Beauveria hrongntartli was grown in the same manner as in Example 1, smoky-brown cockroach {Periplaneta fuliglnosa), German cockroach (BlatteZIa geinnanica), termite {Coptotermes formosanus), pill wood louse (Armadillidium vulgare), sow bug (Porcellio scaier), queenless ant (Pristomyrmex pungens), large centipede {Scolopendra subspinipes mutilans), and shield centipede (Thereuonema hilgendorfl) were made to have a 5-minute free walk. Each kind of the test insects was kept in a polyethylene cup with water-wetted cotton and feed, and observed for 14 days. The results of the observation are shown in Table 6. An untreated control group was prepared for each kind of the test insects by following the same procedures as the above except that an agar medium which did not grow the fungus was used. The results are shown in Table 7. As shown in Tables 6 and 7, the termiticide of the present invention was highly specific to termite CoptOtermes formosanus and did not show lethal effect to other test insects. This indicates that the termiticide of the present invention is excellent in that it gives little damages to the environment with giving no harms to other living things. Example 7 In a basal liquid medium which was prepared by adding 40 g of glucose, 10 g of peptone, and 2 g of yeast extract to 1 L of water, Beauveria brongniartii was grown at 25""C for 2 weeks. Separately, 2L of water was added to 1 kg of wheat bran and sterilized in an autoclave at 121"C for 20 minutes, to which 1 to 5 mL of the above liquid culture medium of Beauverza brongniartii. was added and cultured at 25°C for 2 weeks. Then the culture was air-dried to obtain a powder with cell density of 10 cells/g. The powder thus obtained was sprinkled in an amount of 300 g in an outdoor termite nest where not less than 5000 active termites of Coptotertnes formosanus inhabited. The area sprinkled with the powder was referred to as the treated area. Thereafter, the termites in the nest were periodically observed. The results are shown in Table 8. An untreated area was prepared in the same manner as the above except that the culture medium did not grow Beauveria brongniartii, and the termites in the nest were observed. The results are shown in Table 8. Table 8 As Shown in Table 8, it was found that all the termites in the outdoor nest can be eradicated by sprinkling the culture of the fungus (the powder) on the nest. This demonstrates the high efficacy of the present invention for termite control. Example 8 From an active nest in the outdoors where not less than 1000 termites of Coptotermes formosanntis inhabited, 300 termites (workers and soldiers) were collected. These termites were made to walk for 10 minutes on the agar culture medium of Beauveria brongniartii in the same manner as in Example 1 and returned to the nest where they were collected. The nest was referred to as "the treated area." The termites in the nest were observed periodically. The results of the observation are shown in Table 9. An untreated area was prepared by following the same procedures as the above except that termites were made to walk on the agar medium where no fungus was grown, and the termites in the nest were observed. The results are shown in Table 9. As shown in Table 9, according to the method of the present invention, treatment of only a part of termites in the nest with the culture of the fungus resulted in eradication of all the termites in the nest, indicating marked efficacy of the present invention for termite control. Example 9 In the same manner as in Example 1, 10 worker termites of Coptotermes formosannus were made to walk for 10 minutes on an agar medium where each of the fungi was grown. Then, these test termites were placed in a polyethylene cup where 50 normal termites of Coptoteimes formosarmus (workers and soldiers) were kept with wood chips and water-wetted cotton. The number of days needed before all the termites in the cup died was determined. The same experiment was conducted with another 4 groups. Involving 5 groups in total for each fungus. The results are shown in Table 10. An untreated group for each fungus was prepared by following the same procedures as the above except that an agar medium did not grow any fungus. The results in the untreated group are also shown in Table IQ. Table 10 As shown in Table 10, treatment of only one fifth the termites of Coptotermes formosannus with each of the fungi resulted in eradication of the whole group. There was no death in each untreated group. Therefore, the termiticide of the present invention can effectively control termites even if all the termites do not make direct contact with the termiticide. With Beauverla brongniartil, the number of days needed before 100% death of the termites was achieved was the shortest, indicating the highest termiticidal efficacy of the fungus. Example 10 With termite Reticulxtermes speratus, the number of deaths was evaluated in the same manner as in Example 1 where termites were treated with Beauverla brongntartii-" The results are shown in Table 11. As shown in Table 11, all the termites of Reticulitermes speratus in the treated groups died within 8 to 10 days. On the contrary, no death occurred before day 10 in the untreated group, indicating that Beauveria brongniartii can be used as a termiticide. Example 11 With termite Reticulltermes speratus, the number of death was evaluated in the same manner as in Example 2 where the termites were treated with Beauveria bassiana. The results are shown in Table 12. Table 12 fMs tiiiown in xaoie j.z, me cumuia"Cive tnortaj-itiy at day 4 was 40 to 60% in the treated groups, while no death iccurred in the untreated group. Ixample 12 With termite Reticulitermes speratus, the number of eaths was evaluated in the same manner as in Example 3 here the termites were treated with Metarhizium anisopliae. he results are shown in Table 13. As shown in Table 13, all the termites of Retlculitermes speratus in the treated groups died within 11 to 12 days. On the contrary, no death occurred before day 12 in the untreated group. These findings show that direct contact with the fungus and/or the culture thereof causes the termites to die. Therefore, though less effective than Beauveria brongnlartii in Example 10, Metarhizium anxsopliae can also be used as a termiticide. Example 13 With termite Reticulitermes speratus, the number of deaths was evaluated in the same manner as in Example 4 where the termites were treated with Verticllllum lecanil. The results are shown in Table 14. As shown in Table 14, the cumulative mortality at day 14 was 20 to 40% in the treated groups, while no death occurred in the untreated group before day 14. f Example 14 With termite Cototermes formosanus, the number of deaths was evaluated in the same manner as in Example 1 except that the termites were treated with Beauveria amorpha. The results are shown in Table 15. As Shown in Table 15, all the termites of Coptotermes formosanus in the treated group died within 11 to 12 days, while no death occurred in the untreated group before day 12. These findings show that direct contact with the fungus and/or the culture thereof causes the termites to die. Though Beauveria amorpha. is less effective in killing the termites than Beauveria brongniartii (Example 1), Beauveria amorpha can also be used as a termiticide- Example 15 Each of Beauveria brongniartii, Beauveria bassiana, and Beauveria amorpha was spread over an agar medium in a Petri dish and cultured at 25oC, the agar medium (manufactured by Nissui Pharmaceutical K.K.) containing 0.4% by weight of potato exudate powder, 2% by weight of glucose, and 1.5% by weight of agar. The duration of the culture (number of days) was controlled in order to make 3 levels of cell density, i.e. 10, 10, and 10 cells/cm. The experiment was conducted in a similar way to Example 1. The results are shown in Tables 16 to 18. Cell density was obtained by counting the number of cells per unit area using the dilution plate technique. As Shown in Tables 16 to 18, when Beauverla brongniartii was used, 100% mortality o£ termite Coptotermes formosanus was achieved even when the cell density of the fungus was low (10 cells/cm). In case of Beauverta bassiana or Beauveria amorpha, 100% mortality could not be achieved with low cell densities of not less than 10 cells/cm. Thus, since Beauverla brongniartii can achieve 100% mortality with a low cell density, this fungus is the most effective for termite control. The present invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims. WE CLAIM: 1. A termiticide comprising an entomogenous fungus and/or a culture thereof wherein the entomogenous fungus is selected from the group of Beauveria brongniarti, Beauveria bassiana, Beauveria amorpha, Metarhizium anisopliae, and Verticillium lecanii. 2. The termiticide as claimed in claim 1, wherein the entomogenous fungus is in the state of germination or resting spore. 3. The termiticide as claimed in anyone of claims 1 and 2, wherein the entomogenous fungus and/or the culture thereof are contained in an application form selected from the group consisting of powder, granule, tablet, liquid, aerosol, band, sheet, pellets, and cream. 4. The termiticide as claimed in claim 3, wherein cell density of the entomogenous fungus in the application form is 105 to 109 cells/cm2 or 105 to 109 cells/g. 5. The termiticide as claimed in anyone of claims 1 to 4, wherein a target termite is selected from the group consisting of Coptotermes sp., Reticulitermes sp., Cryptotermes sp.. Incisitermes sp., Macrotermes sp., and Odontotermes sp. 6. The termiticide as claimed in claim 5, wherein a target termite is selected from the group consisting of Coptotermes formosanus, Reticulitermes speratus, Reticulitermes hesperus, Reticulitermes tibialis, Reticulitermes flavipes, Reticulitermes lucifugus, Reticulitermes santonensis, Cryptotermes domesticus, and Incisitermes minor. 7. A method for termite control using the termiticide as claimed in anyone of claims 1 to 6.

Documents:

0268-mas-1996 others-1.pdf

0268-mas-1996 others.pdf

0268-mas-1996 abstract duplicate.pdf

0268-mas-1996 abstract.pdf

0268-mas-1996 claims duplicate.pdf

0268-mas-1996 claims.pdf

0268-mas-1996 correspondence others.pdf

0268-mas-1996 correspondence po.pdf

0268-mas-1996 description (complete) duplicate.pdf

0268-mas-1996 description (complete).pdf

0268-mas-1996 form-19.pdf

0268-mas-1996 form-2.pdf

0268-mas-1996 form-26.pdf

0268-mas-1996 form-4.pdf

0268-mas-1996 form-6.pdf

0268-mas-1996 petition.pdf