NOVEL TOLYPOCLADIUM ALBUM STRAIN

Abstract

A novel fungal strain of Tolypocladium album (synonym Chaunopycnis alba) is disclosed that has enhanced toxicity against insects and mites. This novel Tolypocladium album strain is capable of producing terpendoles A, C, I, J, K, N, O and P through fermentation and does not produce nalanthalide.

Claims

1. A biologically pure fungal strain of Tolypocladium album, having all the identifying characteristics of Tolypocladium album HL-105-64-AC11 having ATCC accession number PTA-124560.

2. A biologically pure fungal culture of a Tolypocladium album, HL-105-64-AC11.

3. The fungal strain of claim 1, wherein the strain produces terpendoles A, C, I, J, K, N, O and P.

4. The fungal strain of claim 1, wherein the strain does not produce nalanthalide.

5. A pesticidal composition comprising a mixture of an effective amount of the fungal strain of claim 1 and a suitable carrier.

6. A pesticidal composition comprising a mixture of an effective amount of a fermentate of the fungal strain of claim 1 and a suitable carrier.

7. A method of controlling a pest comprising applying an effective amount of the fungal strain of claim 1 to a pest or the pest's environment.

8. A method of controlling a pest comprising applying an effective amount of a fermentate produced from the fungal strain of claim 1 to a pest or the pest's environment.

9. The method of claim 8, wherein the pest is an insect.

10. The method of claim 8, wherein the pest is an aphid.

11. The method of claim 8, wherein the pest is a lepidopteran.

12. The method of claim 8, wherein the pest is a thrips.

13. The method of claim 8, where the pest is a mite.

14. The method of claim 8, wherein the pest is a whitefly.

15. The method of claim 10, wherein the aphid is selected from the group consisting of cotton aphid (Aphis gossypii), foxglove aphid (Aulacorthum solani), cabbage aphid (Brevicoryne brassicae), birdcherry-oat aphid (Rhopalosiphum padi) and green peach aphid (Myzus persicae).

16. The method of claim 11, wherein the lepidopteran is selected from the group consisting of diamondback moth (Plutella xylostella) and cabbage looper (Trichoplusia ni).

17. The method of claim 12, wherein the thrips is western flower thrips (Frankliniella occidentalis).

18. The method of claim 13, wherein the mite is a twospotted spider mite (Tetranychus urticae).

19. The method of claim 14, wherein the whitefly is a silverleaf whitefly (Bemesia tabaci).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] FIG. 1. Internal transcribed spacer (ITS) sequence (SEQ ID NO: 1) of Tolypocladium album strain HL-105-64-AC11.

[0014] FIG. 2. A. Morphology of colonies of T. album HL-105-64-AC11; B. Morphology of, mycelia and spores of T. album HL-105-64-AC11.

[0015] FIG. 3. Correlation coefficient of terpendoles isolated from T. album HL-105-64-AC11

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention is generally directed to a novel, biologically pure strain of T. album, HL-105-64-AC11. This strain was deposited with the American Type Culture Collection in Manassas, Va. on Nov. 17, 2017 having accession number PTA-124560. HL-105-64-AC11 exhibits improved pesticidal activity as compared to other T. album strains. Specifically, HL-105-64-AC11 produces terpendole C when fermented. Further, unlike other T. album strains, HL-105-64-AC11 does not produce the toxic compound, nalanthalide when fermented.

[0017] The phrase biologically pure fungal strain as used herein means a strain essentially free from biological contamination and having a genetic uniformity such that different substrains taken therefrom will display substantially identical genotypes and phenotypes.

[0018] DNA sequencing including random amplification of polymorphic DNA (RAPD), may serve to characterize the genetic architecture of a strain and thus serve as a further identifier of the fungal strain of the invention.

[0019] Genetic identification was performed by sequencing of an ITS region of ribosomal DNA and comparison of obtained results with the ones published in genetic databases for genetic identification of this species.

[0020] The new fungal strain was identified as Tolypocladium album with 100% match with a Tolypocladium album isolate in a gene bank. Tolypocladium album can also be referred to Chaunopycnis alba as they are synonyms.

[0021] The results of genetic analysis of the ITS sequence from the strain Tolypocladium album strain HL-105-64-AC11 defined in this invention, are shown in FIG. 1 as SEQ ID NO:1. The strain is characterized by the lack of nalanthalide production as described in Example 1.

[0022] HL-105-64-AC11 strain of T. album may be produced by methods disclosed in the present application.

[0023] In one embodiment, the present invention is directed to a pesticidal composition comprising a mixture of an effective amount of HL-105-64-AC11 or a fermentate produced by HL-105-64-AC11 and a suitable carrier.

[0024] Compositions of the present invention may contain from about 0.1% by weight to about 99% by weight, preferably from about 0.1% by weight to about 95% by weight of HL-105-64-AC11 or a fermentate of HL-105-64-AC11 and from about 1% to about 99.9% by weight of an acceptable solid or liquid inert carrier.

[0025] As used herein, the term fermentate refers to the resulting product of the breakdown of a carbon source by HL-105-64-AC11 strain of Tolypocladium album. The fermentate may contain alcohols, fatty alcohols, organic acids, salts and other metabolites such as terpendole A, C, I, J, K, N, O and P. The fermentate may be differentiated from other T. album strains by its lack of production of nalanthalide. See Example 1, below.

[0026] As used herein, all numerical values relating to amounts, weight percentages and the like are defined as about or approximately each particular value, namely, plus or minus 10%. For example, the phrase at least 5% by weight is to be understood as at least 4.5% to 5.5% by weight. Therefore, amounts within 10% of the claimed values are encompassed by the scope of the claims.

[0027] The term effective amount means the amount of the formulation that will kill the target pest. The effective amount will vary depending on the mixture concentration, the type of pest(s) being treated, the severity of the pest infestation, the result desired, and the life stage of the pest during treatment, among other factors. Thus, it is not always possible to specify an exact effective amount. However, an appropriate effective amount in any individual case may be determined by one of ordinary skill in the art.

[0028] After production of the HL-105-64-AC11 strain of T. album according to the above methodology, large-scale fermentation may be carried out using media and fermentation techniques which are often optimized for improved yield as commonly practiced in the fermentation industry. The fermentation broth as a whole, or an extract from the HL-105-64-AC11 fermentate may then be concentrated, lyophilized, spray-dried and/or formulated in any of a number of well-known ways, including as a liquid concentrate, dry or wettable powder or suspension for spraying on or under foliage, and a granular preparation for application to soil or foliage. Alternatively, the fermentation broth may be formulated directly without extraction or other processing.

[0029] The phrase acceptable carrier as used herein means an otherwise inert filler or excipient which confers upon the composition desirable storability, material handling and application characteristics; commonly-used carriers may include fillers, binders, surfactants, dispersants, adhesion agents and the like.

[0030] The pesticidal compositions comprising HL-105-64-AC11 or a fermentate of HL-105-64-AC11 may be in the form of, for example, a suspension, a dispersion, an aqueous emulsion, a dusting powder, a dispersible powder, an emulsifiable concentrate, an aerosol or micro or microencapsulated granules or any other formulation that gives controlled release of T. album.

[0031] In another embodiment, the present invention is directed to a method of controlling a pest comprising applying an effective amount of HL-105-64-AC11 or a fermentate produced by HL-105-64-AC11 to a pest or the pest's environment.

[0032] As used herein, controlling a pest refers to decreasing the negative impact of the pest on plants or animals to a level that is desirable to the grower or animal.

[0033] As used herein, pest's environment refers to any area where the pest is present during any life stage. One environment likely to be treated by the methods of the present invention includes the plants that the pest is living on/in and the surrounding soil. The pest's environment may also include harvested plants, gardens, fields, greenhouses, or other buildings, and various indoor surfaces and structures, such as furniture including beds, and furnishings including books, clothing, etc.

[0034] The articles a, an and the are intended to include the plural as well as the singular, unless the context clearly indicates otherwise. For example, the methods of the present invention are directed to controlling pest but this can include control of a multiple pests (such as a more than one insect or more than one insect species or more than one mite or more than one mite species).

[0035] As used herein terpendole A refers to the following structure

##STR00001##

with CAS number 156967-65-5.

[0036] As used herein terpendole C refers to the following structure

##STR00002##

with CAS number 156967-64-6.

[0037] As used herein terpendole I refers to the following structure

##STR00003##

with CAS number 167612-17-1.

[0038] As used herein terpendole J refers to the following structure

##STR00004##

with CAS number 167427-26-1.

[0039] As used herein terpendole K refers to the following structure

##STR00005##

with CAS number 167427-27-2.

[0040] As used herein terpendole N refers to the following structure

##STR00006##

[0041] As used herein terpendole O refers to the following structure

##STR00007##

[0042] As used herein terpendole P refers to the following structure

##STR00008##

[0043] As used herein nalanthalide refers to the following structure

##STR00009##

having CAS number 145603-76-5.

[0044] The pesticidal compositions of the invention can be applied directly to the plant by, for example, spraying or dusting an effective amount of the HL-105-64-AC11 or the HL-105-64-AC11 fermentate at the time or after the pest has begun to appear on the plant or before the appearance of pests as a protective measure. Plants to be protected within the scope of the present invention include, but are not limited to, cereals (wheat, barley, rye, oats, rice, sorghum and related crops), beet (sugar beet and fodder beet), drupes, pomes and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, and blackberries), leguminous plants (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoa beans, groundnuts), cucumber plants (cucumber, marrows, melons), fiber plants (cotton, flax, hemp, jute), citrus fruit (oranges, lemons, grapefruit, mandarins), vegetables (spinach, lettuce, asparagus, cabbages and other brassicae, carrots, onions, paprika), lauraceae (avocados, cinnamon, camphor), solanaceae (potato, tomato, eggplant, chili peppers, bell peppers, ground cherries, tomatillos, cape gooseberry, wolfberry, Chinese lantern, petunia, browallia, hyacinth, mandrake, deadly nightshade, tobacco), deciduous trees and conifers (linden-trees, yew-trees, oak-trees, alders, poplars, birch-trees, firs, larches, pines), or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines hops, bananas and natural rubber plants, as well as ornamentals. The preferred mode of application is by foliar spraying. It is generally important to obtain good control of pests in the early stages of plant growth as this is the time when the plant can be most severely damaged. The spray or dust can conveniently contain another insecticide or pesticide, e.g., fungicide, grass herbicide or fertilizer, if this is thought necessary. In a preferred embodiment, the composition of the invention is applied directly to the plant.

[0045] The compositions of the present invention are effective against arthropods. As used herein arthropod refers to pests that belong with Phylum Arthropoda. In a preferred embodiment, the pests are insects. As used herein, insect refers to pests that belong to the Class Insecta. In another preferred embodiment, the pests are mites. As used herein, mite refers to pests that belong to the Subclass Acari of the Class Arachnida.

[0046] In another preferred embodiment, the pest is an aphid. As used herein, aphid refers to pests that belong to the Family Aphididae. Exemplary aphids include cotton aphid (Aphis gossypii), foxglove aphid (Aulacorthum solani), cabbage aphid (Brevicoryne brassicae), birdcherry-oat aphid (Rhopalosiphum padi) and green peach aphid (Myzus persicae).

[0047] In another preferred embodiment, the pest is a lepidopteran. As used herein, lepidopteran refers to pests that belong to the Order Lepidoptera including butterflies, moths and their larval stages. Exemplary lepidopterans include diamondback moth (Plutella xylostella) and cabbage looper (Trichoplusia ni).

[0048] In another preferred embodiment, the pest is thrips. As used herein, thrips refers to pests that belong to the Family Thripidae. Exemplary thrips include western flower thrips (Frankliniella occidentalis).

[0049] In another preferred embodiment, the pest is a whitefly. As used herein, whitefly refers to pests that belong to the Family Aleyrodidae. Exemplary whiteflies include tobacco whitefly (Bemisia tabaci).

[0050] In another preferred embodiment, the pest is a planthopper. As used herein, planthopper, refers to pests that belong to the Infraorder Fulgoromorpha of the Order Hemiptera. Exemplary planthoppers include brown rice planthopper (Nilaparvata lugens).

[0051] In another preferred embodiment, the pest belongs to the infraclass Neoptera of the Class Insecta, the Subfamily Aphidinae of the Family Aphididae or the Parvorder Heteroneura of the Family Lepidoptera.

[0052] The following examples are presented by way of illustration and are not intended to limit the invention in any way.

EXAMPLES

Example 1. Phenotypic Characteristics of T. album HL-105-64-AC11

[0053] Tolypocladium album strain HL-105-64-AC11 was grown at 26 C. on a potato dextrose agar plate or in potato dextrose broth for 2-3 days. On plates, it forms larger, white, fluffy colonies. In liquid media, it produces both mycelia and spores. The liquid grown biomass can be used as an inoculum for fermentation studies. Its mycelia and spores in the liquid media can be observed under a microscope. A picture showing its typical colony morphology and microscopic observation of mycelia and spores is shown in FIG. 2.

Example 2. Terpendole and Nalanthalide Production of Various Tolypocladium album Strains

[0054] Various T. album strains including HL-105-64-AC11 were grown in various media to determine terpendole and nalanthalide production. Specifically, a 500-milliliter shake flask containing 100 milliliters of fermentation medium was inoculated. The inoculated flask was incubated at 26 C. on a shaker at 250 revolutions per minute for 7-8 days. The fermentation broth was then analyzed for terpendoles and nalanthalide by high performance liquid chromatography.

[0055] The basic fermentation medium contained 2% w/v mannitol, 0.3% w/v yeast extract, 1.5% w/v soy flour, 0.1% w/v magnesium sulfate and 0.1% w/v monopotassium phosphate in water. Media 1-4 and 35 in Table 1, below, were modified from the basic fermentation medium composition for better yield of terpendoles. Results of this assay can be seen in Table 1, below.

TABLE-US-00001 TABLE 1 Total Fermentation Terpendole C Terpendole Nalanthalide Medium (mg/L) (mg/L) (mg/L) Tolypocladium Medium 1 15.9 97.6 454 pustulatum Medium 2 0 4.21 0 (HL-90-POR-P01) Medium 3 3.69 68.9 121 Medium 4 0 37.1 179 T. album Medium 1 14 148.9 20.7 (HL-89-CL01-Q10) Medium 2 8.14 155.8 31.9 Medium 3 28.9 202.6 43.8 Medium 4 5.64 69.7 0 T. album Medium 1 33.5 198.7 26 (HL-103-22-R03) Medium 2 11.3 161.8 27.1 Medium 3 20.2 229.4 41.7 Medium 4 16.4 156.6 89.4 T. album Medium 1 106 268 97.3 (HL-105-03-AD02) Medium 2 23.3 302.1 79.7 Medium 3 150 323 122 Medium 4 44.9 256.4 188 T. album Medium 1 75.6 426 0 (HL-105-64-AC11) Medium 2 84.2 326 0 Medium 3 24.9 200 0 Medium 4 34.7 164.9 0 T. album Medium 35 0.00 0.00 0.00 (Chaunopycnis alba ATCC 201787)

[0056] As seen in Table 1, above, T. album strain HL-105-64-AC11 produced the most terpendole C and total terpendoles. Further, HL-105-64-AC11 is the only Tolypocladium spp. strain assayed that did not produce the toxic compound nalanthalide in any of the media tested.

Example 3. Insecticidal Activity of Tolypocladium album Strain HL-105-64-AC11

[0057] An HL-105-64-AC11 fermentation broth was produced as in Example 2. The HL-105-64-AC11 fermentation broth was then diluted with water to 50%, 25% and 12.5% v/v and tested for control of diamondback moth (Plutella xylostella) and cabbage looper (Trichoplusia ni). Results of this assay can be seen in Table 2, below.

TABLE-US-00002 TABLE 2 Dilution 12.5% 25% 50% % Efficacy 24 hours 48 hours 24 hours 48 hours 24 hours 48 hours Diamondback 30 50 57 70 70 92 moth Cabbage 22 64 62 94 90 100 looper

[0058] As can be seen in Table 2, HL-105-64-AC11 fermentation broth was effective at controlling both diamondback moth and cabbage looper at all three dilutions.

Example 4. Miticidal Activity of Tolypocladium album Strain HL-105-64-AC11

[0059] An HL-105-64-AC11 fermentation broth was produced in shake flasks. The HL-105-64-AC11 fermentation broth was then diluted with water to 1.39% and 13.9% v/v and tested for control of twospotted spider mites (Tetranychus urticae) and green peach aphids (Myzus persicae). The results of these assays are summarized in Tables 3 and 4.

TABLE-US-00003 TABLE 3 Dilution 1.39% 13.9% % Efficacy 24 hours 48 hours 24 hours 48 hours Twospotted 49.4 51.5 86.1 88.4 spider mite

[0060] As can be seen in Table 3, HL-105-64-AC11 fermentation broth was effective at controlling twospotted spider mites at both dilutions.

TABLE-US-00004 TABLE 4 Dilution 1.04% 3.125% 6.25% 12.5% % 24 48 24 48 24 48 24 48 Efficacy hours hours hours hours hours hours hours hours Green 48 67 92 98 90 100 97 100 peach aphid

[0061] As can be seen in Table 4, HL-105-64-AC11 fermentation broth was effective at controlling green peach aphids at four dilutions.