METHOD OF CONTROLLING PLANT FEEDING MITES WITH THE FUNGUS NEOZYGITES FLORIDANA

Abstract: A method of selectively controlling plant feeding mites, particularly those of the family Tetranychidae, such as the Twospotted spider mite ( Tetranychus urticae ), is disclosed. The method comprises innoculating an agricultural field containing crop plants with a Neozygites fungus which selectively infects plant feeding mites without infecting other insects. The population of plant feeding mites is thereby reduced to a level at which they cannot substantially damage the crop plants.

Description: This invention relates to methods for controlling insect pests generally, and particularly relates to a method of controlling plant feeding mites with a fungus which selectively kills the mites. There are two serious side effects which result from the use of most modern insecticides: the development of insect resistance to the treatment, and the nonspecific and broad-ranging killing effect of many such insecticides. These two side effects can, moreover, interact with one another to exacerbate the obvious practical problems they cause. When a broadly toxic insecticide is used to control a few specific insect pests, many of the other nontargeted insects affected by the treatment can develop resistance to it even though there was no need to kill them. Later, when there is a need to control the previously nontargeted insects, they will have likely become resistant to the insecticide to which they were previously exposed, and one option for their control will have been rendered useless. A considerable amount of research has accordingly been directed, and continues to be directed, towards solving these problems. See generally L.B. Brattsten, et al., Insecticide Resistance: Challenge to Pest Management and Basic Research, 231 Science 1255 (14 March 1986). There has been some interest shown in using fungi to control insect pests. For example, Hirsutella thompsonii has been suggested as a fungus useful as a mite control agent, (or a "mycoacaricide") in citrus rust mites. See , e. g. , C.W. McCoy and T.L. Couch, 65 Florida Entomologist 116 (1982). It has been suggested that other types of fungi, such as fungi in the genus Entomophthora , might be useful as mycoacaricides because of the role they are believed to play in nature ( See U.S. Patent No. 4,021,306 to Soper; see also , U.S. Patent No. 4,530,834 to McCabe), and there has been a considerable amount of basic research on the natural effects of these fungi on insect populations--including studies of the role of the fungus Neozygites floridana in regulating the population of various plant feeding mites. Exemplary of this research are D.R. Smitley, Ph.D. Thesis, Department of Entomology, North Carolina State University (1985), R.L. Brandenburg and G.G. Kennedy, 34 Ent. Exp. and Appl. 240 (1983), R.L. Brandenburg and G.G. Kennedy, 74 Journal of Economic Entomology 428 (1981), and L.S. Boykin, W.V. Campbell and M.K. Beute, 77 Journal of Economic Entomology 969 (1984). This research has not brought forth a significant number of new mycoacaricides which can actually be applied to agricultural crops, or mycoacaricides which can confidently be said to be highly specific and selective in their action, even though there is a continuing need for such new, commercially useful, insect control agents. In an agricultural field containing crop plants, which field is infested with both insects which do not significantly harm the crop plants and undesirable plant feeding mites, we herein disclose a method of selectively reducing the population of the plant feeding mites. The method is characterized by innoculating the field with an amount of the fungus Neozygites floridana ( N. floridana ) effective to infect the plant feeding mites. As a result of this treatment, the plant feeding mites are selectively killed without substantially infecting predaceous mites and the insects which are not harmful to the crop plants. The agricultural field may be located outside, or within a greenhouse. Preferably the field is innoculated with an amount of N. floridana effective to reduce the population of plant feeding mites to a level at which they cannot substantially damage the crop plants. The plant feeding mites which can be selectively killed by our method belong to the family Tetranychidae in the Suborder Trombidiformes in the Order Parasiti. Exemplary of the genera of mites within the Tetranychidae family which can be selectively killed by our method are mites in Genus Eutetranychus , Genus Panonychus , Genus Tetranychus , Genus Oligonychus , and Genus Eotetranychus . Exemplary of particular species of mites within the family Tetranychidae which can be selectively killed by the method of the present invention are the Six-spotted spider mite ( Eotetranychus sexmaculatus ), the Texas citrus mite ( Eutetranychus banksi ), the Citrus red mite ( Panonychus citri ), the European red mite ( Panonychus ulmi ), the McDaniel mite ( Tetranychus mcdanieli ), the Pacific spider mite ( Tetranychus pacificus ), the Strawberry spider mite ( Tetranychus turkestani ), the Twospotted spider mite ( Tetranychus urticae ), the Spruce spider mite ( Oligonychus ununguis ), the Sugi Spider mite ( Oligonychus hondoensis ), and Tetranychus evansi . These plant-feeding mites cause damage to a wide variety of crops. For example, in the United States, crops which suffer significant damage from Twospotted spider mites include Citrus, Grape, Almond, Pome Fruits, Pecan, Stone Fruits, Wheat, Strawberry, Vegetable Crops, Peanut, Cotton, Corn, and ornamental and forestry crops. An important advantage of our invention is the selectivity of its killing action. We have extensively studied the effects of N. floridana on predatory insects, and have consistently found them to resist infection. Among the predatory insects we have found to resist N. floridana infection are Cecidomyiid larvae (a fly), Geocoris sp. (Big eyed bug), Orius spp. (minute pirate bug), Hemerobiid larvae (brown lacewing), Crysopa larvae (green lacewing), Coleomagilla spp. and Hippodamia spp. larvae (ladybird beetle larvae). The method of the present invention is so selective that even species of mites which are predatory, rather than plant feeding, are not infected. We have particularly observed several species of predaceous mites in the genus Neoseiulus to be unaffected by N. floridana . Further, in one hundred attempts to infect Neoseiulus fallacis with N. floridana in the laboratory, we observed no infection. Similarly, we were unsuccessful in infecting another predaceous mite, Phytoseiulus persimilis , despite 100 attempts in a laboratory study. Neozygites floridana also has been called Entomophthora floridana and Triplosporium floridana . This fungus was previously isolated by Weiser and Muma, who found it in Texas Citrus Mites ( Eutetranychus banksi ) on citrus, in Lake Alfred, Florida, U.S.A., and was identified and described in their paper entitled " Entomophthora floridana N. Sp. (Phycomycetes: Entomophthoracea), A Parasite of the Texas Citrus Mite, Eutetranychus banksi ," in The Florida Entomologist , 49 (3), 155-159 (1966). See also Selhime and Muma, Biology of Entomophthora floridana attacking Eutetranchus banksi , The Florida Entomologist , 49, (3) 161-168 (1966). The fungus can be described as it was described by Weiser and Muma: it has mycelia which are divided into short tubular or club-shaped hyphal bodies, with two to four nuclei, that grow into curved and obtuse segments which are mostly unbranched. The mycelia are found distributed throughout the host mite's body. Root-like hyphae grow through the cuticle of the host to form slightly broadened conidiophores outside the host body. The conidiophores are sometimes curved, and are single, 30 to 35µm by 6 to 8µm, and grow from root-like hyphae of the same length which are only 3 to 4µm wide. Numerous refringent granules, fat droplets or starch, are seen in the conidiophores. The hyphal protoplasm is hyaline, without granules in the septal region. The septum is formed at the periphery of the conidiophore. The conidia are pyriform, papillate, subpapillate, or epapillate type, and 13 to 18µm by 11 to 13µm, averaging 12 by 15µm with the basal end 5 to 6µm wide. The Conidia have four sperical nuclei and refringent granules of fat and starch. Weiser and Muma observed that these structures are not stained by cotton blue in Amman's solution. The conidiophores have persistent columellae, not bursting during the spore discharge. There is no gelatinous substance on the surface of conidia. Prior to discharge, both the membranes of the columella and that of the conidium are flat, rather than inflated. In fixed mounts, conidia on conidiophores are similar to the truncata type of Lakon's classification. Lakon, G., Z. Angew. Ent. 5, 161 (1919). Secondary conidia are formed from primary conidia by a single, lateral, hyphal bud. Primary and secondary conidia are of the same size and shape. Microconidia have not been observed. Anadhesive spores are produced by primary and secondary conidia at the ends of thin threads which measure 1.5µm by 50 to 60µm. These threads, which are curved adjacent to the anadhesive spore, retain a remnant of the conidial membrane at the opposite end. Mature anadhesive spores are claviform, measuring 15 to 20µm by 10 to 12µm. At the narrow end is a knob-like apex 1.5µm wide. Anadhesive spores have a brownish striated cuticle; the apex seems to be adhesive because almost all anadhesive spores become attached to host mite setae and cuticle at this end. Primary anadhesive spores produce secondary anadhesive spores at the end of a capillary tube. Resting spores, seldom seen in field collected material, are spherical or subspherical with a smooth thin three-layered wall. The upper wall of the resting spore has a rounded foramen at the former connection with the hypha. Resting spores have small refringent globules distributed throughout the protoplasm, but no oil globules or vacuoles. The resting spores are 20-23.5 by 22-26µm in diameter, with the wall only 0.5µm thick, and the foramen 4µm in diameter. Weiser and Muma, supra , accompany their description with a complete set of illustrations. This fungus has been found in other mites, in other locations, by other workers. For example, Nemoto and Aoki reported finding it attacking the Sugi Spider mite ( Oligonchus hondoensis ) in a plantation of Japanese cedar in the Kyushu District of Japan. Nemoto and Aoki, Applied Entomology and Zoology 10 ,90 (1975). Other species of Neozygities fungi known to be pathogenic on spider mites and expected to be useful in the present invention are Neozygites tetranychi and Neozygites adjarica . However, the classification of this group of fungi is not fully settled and these species may ultimately be grouped together as a single species under the name Neozygites floridana , grouped together under some other name, or further split into additional species. To describe our invention as best we are able, we have herein used the name " Neozygites floridana " for the fungus, or group of fungi, most useful in carrying it out. This term is therefore to be considered as descriptive of our invention, rather than limiting, and to encompass all equivalent fungi. The precise dosage of Neozygites floridana will depend on the type of crop and the degree of infestation, but will generally be in the range from 4.5 × 10⁷ to 4.5 × 10¹² capillaconidia per acre, or 1.1 × 10⁸ to 1.1. × 10¹³ capillaconidia per hectare. The preferred range is 4.5 × 10⁸ to 4.5 × 10¹⁰ per acre or 1.1 × 10⁹ to 1.1 × 10¹¹per hectare. The capillaconidia may suitably be applied using mite mummies as a vehicle, the mummies containing an average of 2000-4000 capillaconidia each, suitably about 3000. In the South Eastern United States, N . floridana can be found in association with populations of Tetranychus urticae . The best way to collect N . floridana is to locate dense populations of T . urticae on crop or feral host plants, and to collect several hundred mites during a period when temperatures range from 60 to 75 degrees Fahrenheit (15.5 - 23°C) and of high relative humidity or immediately following a period of prolonged rain (two to three days). The mites should be kept alive on foliage of the plants from which they were collected. After five to seven days any infected mites will die and form the mummies characteristic of N . floridana infected mites. As long as the mummies are held at a relative humidity lower than 90 percent no sporulation of the fungus will occur. Any mummies produced can be used to infect additional mites by placing them on foliage with healthy mites and holding them under conditions of l00 percent relative humidity and at a temperature of 70 degrees Fahrenheit (21.1°C) for 72 hours. We started a laboratory culture of N. floridana from infected T. urticae collected in peanut fields located in Chowan County, North Carolina. A stock culture was generated by exposing batches of spider mites raised on lima beans (cv. 'Henderson Bush') to infective spores (capillaconidia) produced from the original group of 20 infected mites. Additional infected mites were generated from the stock culture by placing one cadaver in the center of a bean leaf disc (1.5 cm in diameter) maintained on moist cotton in a Petri dish. The dish was covered, sealed in a plastic bag and held at 21.1 degrees Centigrade for 48 hours to allow the fungus to sporulate. Fifty to 100 spider mites were then added to the leaf disc. The bean leaf disc with the spider mites was held for an additional 24 hours under moist conditions before it was placed on fresh whole bean leaves maintained on moist cotton under normal laboratory conditions (25 degrees Centigrade, 30 percent relative humidity). Infected mites became brown cadavers in the lab within five days. All cadavers were stored over calcium carbonate in a four degrees Centigrade incubator or in a freezer until they were needed for experiments. Infection of live mites can be verified by placing mites in a drop of lacto-phenol cotton-blue stain on a microscope slide and covering them with a cover slip. The mites should then be squashed by pressing down on the cover slip. Infected mites will extrude fungal hyphae which stain blue. The foregoing procedures are used to collect sufficient numbers of insect mummies which have been infected and killed by the fungus, and which contain unsporulated N . floridana , to innoculate an agricultural field containing crop plants. The field can be innoculated by forming an aqueous suspension of such insect mummies, and spraying them on the crop plants. The precise number of insects which should be sprayed on the crop will vary from crop to crop, with the degree of mite infestation in the field, and according to the speed at which it is desired to cause the mite population to crash. Killed insects should preferably be held in the aqueous suspension for no longer than about three to four hours, so that they will not sporulate and become unviable. Any of a number of well-known stickers and other adjuvants may be advantageously included in the aqueous suspension of insect mummies. Exemplary of such adjuvants are Agar, "CELLOSIZE" from the Union Carbide Co., "DAEAGIN" and "FLO-GEL 1000" from the Diamond Shamrock Co., "MILLER-AIDE" and "NU-FILM 17" from the Miller Chemical and Fertilizer Corp., oils such as FC-435 from the Sun Oil Co., "ORTHO X-77" from the Chevron Chemical Co., "PYLAC" from the Hopkins Agricultural Chemical Co., and "TRITON B-1956" and "TRITON X-100" from the Rohm and Haas Co. Those skilled in the art will be aware of many other standard adjuvants which may also be used to advantage when practicing the present invention. FIELD TESTS A series of field tests were carried out to demonstrate the effect of applying N. floridana to Twospotted spider mites in corn. The results of these tests are summarized in Table 1, which shows that effective mite control was achieved by the present invention. These data were generated by applying Twospotted spider mite "mummies" in an aqueous suspension (water plus one percent agar) at the rate of 2000 mummies per 10 feet (656 per m) of corn row. This rate of application provided good control with corn, but lower rates could be used on most other row crops and many tree crops because the closed nature of the crop canopy of most crops would be expected to sustain a higher relative humidity near the leaf surface of the plants than the rather open corn canopy. Those skilled in the art will appreciate that environmental factors, such as temperatures and humidity, affect the production of primary and secondary conidia and capillaconidia in fungi such as N. floridana , and will be able to take these factors into consideration to design suitable treatments for other crops in other locations. These field tests serve to demonstrate that the fungus can survive application to an agricultural field infested by plant feeding mites, and can then be exposed to periods of sufficient length and of sufficiently high humidity for the mites to become infected by irrigating the field and thereby wetting the foliage and soil in the field (see Table 1, footnote 3).

Claims: A method of selectively reducing a population of the plant feeding mites, in an agricultural field containing crop plants, which field is infested with both insects which do not significantly harm the crop plants and with said plant feeding mites, characterized by innoculating the field with an amount of fungus Neozygites floridana effective to infect the plant feeding mites so that the plant feeding mites are selectively killed without substantially infecting predaceous mites and insects which are not harmful to the crop plants. A method according to Claim 1, wherein said plant feeding mites belong to the Family Tetranychidae. A method according to Claim 2, wherein said plant feeding mites comprise one or more Genus Eotetranychus , Genus Eutetranychus , Genus Panonychus , Genus Oligonychus and Genus Tetranychus . A method according to Claim 3, wherein said plant feeding mites are Six-spotted spider mites ( Eotetranychus sexmaculatus ). A method according to Claim 3, wherein said plant feeding mites are European red mites ( Panonychus Ulmi ). A method according to Claim 3, wherein said plant feeding mites are Strawberry spider mites ( Tetranychus turkestani ). A method according to Claim 3, wherein said plant feeding mites are Twospotted spider mites ( Tetranychus urticae ). A method according to Claim 3, wherein said plant feeding mites are Spruce spider mites ( Oligonychus ununguis ). A method according to Claim 3, wherein said plant feeding mites are Sugi spider mites ( Oligonychus hondoensis ). A method of selectively reducing a population of Twospotted spider mites, in an agricultural field containing crop plants, which field is infested with both insects which do not significantly harm the crop plants and with undesirable plant feeding mites belonging to the Family Tetranychidae, characterized by innoculating the field with an amount of fungus Neozygites floridana effective to infect the Twospotted spider mites by spraying the crop plants with an aqueous suspension of insect mummies containing the fungus, so that the Twospotted spider mites are selectively killed without substantially infecting predaceous mites and insects which are not harmful to the crop plants. A method according to any preceding claim wherein the N. floridana is applied at a dosage rate of l.l × l0⁸ to 1.1 × 10¹³ capillaconidia per hectare. A method according to Claim ll wherein the said dosage rate is 1.1. × 10⁹ to 1.1 × 10¹¹ capillaconidia per hectare. A method according to any preceding claim. wherein capillaconidia of N. floridana are applied using mite mummies as a vehicle. A method according to claim 13 wherein said mite mummies contain an average of 2000 - 4000 of said capillaconidia each.

Inventors: KENNEDY, GEORGE G., SMITLEY, DAVID R.
Applicants: NORTH CAROLINA STATE UNIVERSITY
Application year: 1987
Main classification: A01N63
IPC codes: A01N63