WO1990006056A1 - Bioherbicide for purple nutsedge - Google Patents

Abstract

The subject invention concerns a novel bioherbicide and its use to control a major weed found in many fields in the Southern United States and in the Caribbean. Specifically, a novel Colletotrichum isolate in an agricultural composition can be used to effectively control purple nutsedge without adversely affecting field crops. Further, the novel isolate in a mixture with other biocontrol agents can be used to control nutsedge and other undesired vegetation, such as Florida beggarweed, sicklepod, showy crotalaria and coffee senna.

Description

DESCRIPTION

BIOHERBICIDE FOR PURPLE NUTSEDGE

Background of the Invention

Weeds are a tremendous problem for farmers throughout the United States and the Caribbean region. Weeds cause a 10-12% loss of value for agricultural products in the United States, the most recent estimate being $20 billion annually (McWhorter, C.G. [1984] Weed Sci. 32:850-855). In Florida vegetable production alone, losses due to weeds are estimated to be over $100 million. According to the most recent estimates, 41% of the cost of plant protection was for the control of weeds. Herbicides are applied to more acres than fungicides and insecticides combined. Weeds act as alternate hosts for insects, fungi, bacteria, and viruses. They affect man, not only by competing with crop plants, but by poisoning range animals, interfering with right-of-ways and roadways, decreasing forest production, and marring landscapes.

Nutsedges (Cyperus spp.) comprise a group of commonly occurring weeds that are among the most difficult to control. In the Caribbean Basin there are three species which are of most consequence, namely C. rotundus (purple nutsedge), C. exculentus (yellow nutsedge), and C. iria (rice flatsedge). Purple nutsedge has been called the world's worst weed because of its distribution worldwide and its resistance to control measures (Holm, L.G., D.L. Plucknett, J.V. Pancho, and J.P. Herberger [1977] The World's Worst Weeds: Distribution and Biology. University Press of Hawaii, Honolulu). It is a problem in 52 crops in more than 90 tropical and subtropical countries. It is considered a serious weed in the United States and a principal weed in Puerto Rico. Yield reduction due to nutsedge varies. In agronomic crops, yield reduction can be as high as 79%, as was seen in maize. In horticultural crops, yield reductions can reach 80%, as has been seen in beans. Yield reduction in tomato, a very valuable crop in the Caribbean region, can be as high as 53%. Nutsedges cannot compete with crops if a dense crop canopy is established, making early season control of these weeds essential.

In Florida, purple and yellow nutsedge, and rice flatsedge are problems in virtually every crop grown in the state. In Puerto Rico, the major problem is with purple nutsedge, although the other species are also found. Vegetables, particularly tomato, pumpkin, pepper, and onion, are affected. In the Virgin Islands the major species is also purple nutsedge which causes the greatest problems in vegetables. Purple nutsedge is also widely distributed in other crops throughout the Caribbean Basin.

Many herbicides have been tested for control of purple and yellow nutsedge. Pereira et al. recently reviewed this research (Pereira, W. , G. Crabtree, and R.D. William [1987] Weed Technology 1:92-98). Herbicides based on virtually every mode of action have been studied. Examples are: 2,4- D ("control erratic"), atrazine ("control inconsistent"), linuron ("marginal control"), paraquat ("inconsistent"), alachlor and metolachlor ("control temporary"). Some herbicides that have been used successfully include glyphosate, dichlobenil, EPTC, arsenicals, and soil fumigants. Success or failure of a herbicide treatment depends on such factors as nutsedge growth stage at application, soil moisture and temperature, and addition of adjuvants to the spray mixture.

Chemical weed control programs are seriously inadequate for control of this weed. Frequently the weed germinates below the treated zone and avoids herbicide injury. Although many herbicides have been developed and tested in the last three decades, farmers still rely heavily on dinoseb, a herbicide developed in the 1950's. Dinoseb is a contact herbicide that causes injury to some field crops. Alternative approaches include monosodium methanearsonate (MSMA), paraquat, toxaphene, and triazine herbicides, but these chemicals are not registered for use on some crops for reasons of toxicology and/or crop safety.

The use of chemical pesticides in agriculture is currently a major concern in the U.S. Nowhere is this concern more obvious than in the San Joaquin Valley of California, where pesticides are being blamed for an epidemic of cancer in children and young adults (Weisskopf, M. [1988] The Washington Post Weekly Edition 5(47):10-11, Washington, D.C.). New technologies in detection methods are enabling researchers to find pesticides in the environment that were previously thought to be totally degraded. Perhaps the major public concern of the 1980's is protection of groundwater. The Environmental Protection Agency (EPA) estimates that 100,000 of the nation's 1.3 million wells are contaminated with pesticides (Fleming, M.H. [1987] Amer. J. Alternative Agriculture 2:124-130). This has alarmed the general public since 50% of all Americans depend on groundwater wells for their fresh water supplies. Because herbicides are so widely used in agriculture, and because they are often applied directly to the soil, the potential for movement into groundwater by leaching is perhaps greater than any other pesticide. Other inadequacies of chemical controls include lack of residual control, injury to non-target organisms, undesirable residues in harvested products, and carryover in subsequent crops.

There are no selective herbicides which can be used to control these sedges in all crops. Indeed, those herbicides which are available do not always give acceptable control of these weeds. An alternative is offered by the use of biological herbicides based on plant pathogens (mycoherbicides) which are specific for the problem weeds, and do not infect desirable plants.

Phatak et al. (Phatak, S.C., M.B. Callaway, and C.S. Vavrina [1987] Weed Technology 1:84-91) have reviewed biological control of purple and yellow nutsedge. Cost effective procedures for the use of insects to control these species have not been developed. Phatak has reported that manipulation of a rust pathogen (Puccinia canaliculata) of yellow nutsedge has reduced stand, tuber formation, and completely inhibited flower formation of this weed. Phatak points out that little research has been directed toward integrating biological and chemical control of nutsedge. It was shown that rust-paraquat (1,1'-dimethyl-4,4'- bipyridinium ion) combinations were much more effective than either treatment alone. Other work indicates that sequential applications of the rust and other herbicides, such as bentazon [3-(1-methylethyl)-(1H)-2,1,3- benzothiadiazin-4-(3H)-one 2,2-dioxide] provided significantly better control than either applied alone. Although this system is promising, there are many aspects that must be investigated, such as spore production and maintenance in storage. Phatak also states that other pathogens should be sought to complement the rust system.

Therefore, the use of bioherbicides is becoming an increasing important alternative to chemical herbicides. This importance is exemplified by several patents which have issued for bioherbicides and their use. Some of these patents, by way of illustration, are as follows: U.S. Patent No. 3,849,104 (control of northern jointvetch with Colletotrichum gloeosporioides Penz. aeschynomene); U.S. Patent No. 3,999,973 (control of prickly sida [teaweed] and other weeds with Colletotrichum malvarum); U.S. Patent No. 4, 162, 912 (control of milkweed vine with Araujia mosaic virus); U.S. Patent No. 4,263,036 (control of Hydrilla verticillata with Fusarium roseum Culmorum); U.S. Patent No. 4,390,360 (control of sicklepod, showy crotalaria, and coffee senna with Alternaria cassiae); and U.S. Patent No. 4,419,120 (control of prickly sida, velvetleaf, and spurred anoda with fungal pathogens).

Despite the successful identification of several effective biocontrol agents, the process by which these control agents can be obtained is by no means straightforward or predictable. Often, the first step in the process is to identify weeds which appear to have been injured by a fungal pathogen. Unfortunately, weeds may be injured by a number of environmental factors, and it may not be readily apparent whether a given injury was caused by a fungus, or by insects, worms, dry conditions, physical damage from machinery or people, chemical herbicides, excess or inappropriate fertilizer, or a combination of these factors.

Once a fungus is correlated with a plant lesion, the work is just beginning. Among other things, the investigator must determine whether the fungus actually caused the lesion, whether the fungus would be capable of killing or controlling healthy plants, whether the fungus can selectively control weeds without adversely affecting desirable vegetation, what concentration of the fungus is needed to achieve control, what environmental conditions are needed for the fungus to exert its effects, and whether the fungal pathogen can be grown efficiently in quantities sufficient to produce the necessary amount for use as a biocontrol agent. The researcher must also determine the precise conditions under which to grow the fungus in order to retain and, possibly, to enhance the virulence of the control agent.

There is a range of important cultural and pathogenicity requirements that must be investigated: inoculum rates, moisture/temperature requirements, susceptible stage of weed growth, and preemergence vs. postemergence applications. Research on the various interactions with commonly used agricultural chemicals is also of great importance if a mycoherbicide is to be integrated into a total plant protection system. Research needs to be conducted to show that a mycoherbicide can be integrated with conventional weed control practices to provide weed control over a wide range of conditions.

Unfortunately, these determinations are largely empirical and must be repeated for each and every damaged plant that is encountered and each and every potential biocontrol agent identified. Thus, it is not surprising that, after years of research and the countless hours spent searching for useful biocontrol agents, there is still less than a 4% success rate in identifying biocontrol agents. Evidence of the unpredictability of these procedures is found in the enormous number of weeds which are still problematic to farmers around the world. Brief Summary of the Invention

The subject invention concerns a novel bioherbicide which is effective, when used according to the process disclosed herein, in controlling purple nutsedge without adversely affecting the growth and yield of the desired field crop. Preferably, the subject invention relates to the use of a composition comprising spores from a novel Colletotrichum in association with an agricultural carrier wherein said spores are in a concentration of from about 1 x 10⁴ spores/ml of carrier to about 1 x 10⁷ spores/ml of carrier. The concentrated spore formulation can be adapted for distribution over geographical locales or situs where the spores germinate and infect purple nutsedge. Detailed Description of the Invention

The novel Colletotrichum described here is the first fungal pathogen that has been used to successfully control purple nutsedge.

A subculture of the Colletotrichum has been deposited in the permanent collection of the American Type Culture

Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland

20852 USA on November 30, 1988. The culture was assigned the following accession number by the repository: Culture Accession number Deposit date

Colletotrichum CR488D ATCC 20912 November 29, 198

The subject culture has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of the deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject culture deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it.

The organism of the subject invention was cultured in petri dishes containing standard potato dextrose agar (PDA). The spores were washed from the dishes with 10 ml of sterile distilled water, diluted with sterile distilled water, mixed with TWEEN™20 at 0.5% v/v, and applied to purple nutsedge plants raised from tubers in a greenhouse. The concentration of the suspension was 1.41 x 10⁵ spores/ml as determined with a hemacytometer in the first experiment. The concentration was 4.5 x 10⁶ spores/ml in the second experiment. The treated plants were placed in a dew chamber at 26°C for 16 hours, then returned to the greenhouse for a two week incubation period. Control plants were treated with sterile distilled water containing 0.5% TWEEN™20 v/v, and given the same dew period and incubation conditions. Isolations were made from tissue showing lesions of any kind from both treatments. The tissue was surface sterilized by soaking in a 2.6% solution of sodium hypochlorite for 30 seconds. The tissue was subsequently rinsed in sterile distilled water for 30 seconds. The isolates were cultured on PDA. The fungi thus isolated were compared microscopically to the original isolate, and the results were compared with a comparison of binomials (Snedecor, G.W., and W.G. Cochran [1980] Statistical Methods. Iowa State Press, Ames, pp. 124-125). The following tables give the results of these tests. The results shown in Tables 1 through 3 indicate that this isolate is a pathogen of purple nutsedge.

Colletotrichum can be grown on solid or in liquid media. Solid media that can be used include water agar, potato dextrose agar, V-8 agar, and string bean agar (strained extract of macerated string beans solidified in agar). Spores are produced on solid V-8 medium exposed to fluorescent light. Specifically, solid media can be, for example, (1) water agar, (2) potato dextrose agar (Difco), (3) lima bean agar (Difco), (4) corn meal agar (Difco), (5) potato-carrot agar (Tuite 19), and (6) Desmodium agar (blend 10 g Desmodium plant parts or plant extracts in 1000 ml water and solidify with 20 g agar).

For large scale production in fermentation tanks, liquid media is used, for example: Formula 1 - Modified Richard's Solution - V-8*

Sucrose 50 gm

Potassium nitrate 10 gm

Potassium phosphate, monobasic 5.0 gm

Magnesium sulfate«7H₂O 2.50 gm

Ferric chloride 0.02 gm

V-8 juice 15 ml

Distilled water to make 1000 ml *Trademark, The Campbell Soup Company for mixed vegetable juices.

Formula II - Modified Richard's Solution - Distillers Solubles - Same as Formula I but substitute 15 gm Distillers solubles for V-8 juice.

Formula III - Modified Richard's Solution - Brewers yeast - Same as Formula I above but substitute 15 gm brewers yeast for V-8 juice.

Formula IV - Modified Richard's Solution - Torula Yeast - Same as Formula I above but substitute 16 gm torula yeast for V-8 juice.

Formula V - Oatmeal solution - 4%+2% sugar - 40 gm oatmeal

20 gm sucrose

1000 ml distilled water

The preparation of spores is commenced in preseed liter flasks containing about 300 ml of liquid medium which have been inoculated with spores. The medium is incubated for 1-3 days with agitation at a temperature of about 26°C to about 30°C.

The preseed is then transferred aseptically to 20 liter seed tanks with additional sterile medium as described above. The tanks are provided with sterile air and agitation. The cycle is continued at a temperature of about 26°C to about 30°C for 1 to 3 days.

Larger fermentors (250 liter) are aseptically inoculated with the seed tanks (entire contents), described above. Additional sterile medium, as used above, is added and the pH adjusted to about 6.0. The fermentor is supplied with sterile air and agitation, and is maintained at a temperature of about 26°C to about 30°C for from 1 to 3 days. The fermentor is then harvested by filtering the contents to remove insoluble solids and mycelia growth. The filtered beer is then centrifuged, the supernatant is discarded, and the remaining spore concentrate is collected, placed in plastic bags, and stored in ice. The concentrate so stored maintains an 80% germination for up to 21 days.

The spore concentrate is mixed with an agriculturally acceptable diluent or carrier for application to the undesired host vegetation or a situs. By the term "situs" is meant those areas infested with the undesired vegetation or potential infestation sites.

The preferred carrier is water, and the spore concentrate is dispersed to make a concentration of from about 2 x 10⁴ to 2 x 10⁷ spores/ml. The formulation is then sprayed on the undesired vegetation or situs by conventional spraying equipment in an amount of from about 50 to 500 liters per hectare.

The potential exists to combine pathogens from these isolates into a mycoherbicide for nutsedge control. The potential also exists for combining these isolates with the isolates of rust being evaluated for control of yellow nutsedge. Spores of the novel Colletotrichum herbicide can be mixed with spores of other bioherbicides to enlarge the scope of control of undesired vegetation. For example, a mixture of the novel Colletotrichum with Alternaria cassiae can be used to control both purple nutsedge and sicklepod (Cassia obtusifolia). Further, spores of the novel Colletotrichum can be mixed with those of A. cassiae to control purple nutsedge and showy crotalaria or coffee senna. The use of A. cassiae to control sicklepod, showy crotalaria and coffee senna is disclosed in U.S. Patent No. 4,390,360, which is incorporated herein by reference thereto. The culture, means of growing, and application to these weeds disclosed in U.S. Patent No. 4,390,360 can be used herein. Mixtures of Colletotrichum and A. cassiae, for example, A. cassiae NRRL 12533, can be made by methods well known in the art, utilizing the disclosure of U.S. Patent No. 4,390,360, and that contained herein.

The novel Colletotrichum of the subject invention can also be mixed, in an agricultural carrier, with the rust pathogen Puccinia canaliculata. P. canaliculata has been reported to control yellow nutsedge and the mixture can be used to control both of these nutsedges. The novel Colletotrichum described here can also be mixed with C. truncatum (Schw.) Andrus & Moore, having the identifying characteristics of NRRL 15933, and is used to control

Florida beggarweed as described in U.S. Patent No.

4,693,756, issued to Cardina et al. This mixture could be used to control both purple nutsedge and Florida beggarweed.

The effectiveness of the novel Colletotrichum may also be enhanced by mixing it with chemical herbicides such as 2,4-D, atrazine, linuron, paraquat, alachlor, metolachlor, glyphosate, dichlobenil, EPTC, and arsenicals.

Table 1 provides a list of other groups of herbicides which could be used in conjunction with the novel fungus of the subject invention.

Though spores are the preferred form of the fungi, the fungi can also be used in their vegetative form. For example, fragmented mucelia can be formulated and applied to purple nutsedge in much the same manner as described above for the spore form.

American Type Culture Collection

12301 Parklawn Drive• Rockville, MD 20852 USA• Telephone: (301)881-2600 Telex: 898-055 ATCCNOR TH

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7.3

AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10.2

To: (Name and Address of Depositor or Attorney)

Thomas A. Bewick, Ph.D.

Vegetable Crops Department

University of Florida

1255 Fifield Hail

Gainesville, Florida 32611

Deposited on Behalf of: Thomas A. Bewick, Ph.D.

Identification Reference by Depositor ATCC Designation

Colletotrichutisp., CR488D 20912

The deposit was accompanied by:_a scientific description X a proposed taxonomic description indicated above.

The deposit was received November 29.1988 by this lnternational Depository Authority and has been accepted.

AT YOUR REQUEST:

X We will infora you of requests for the strain for 30 years.

_ We will not informyou of requeste for the strain.

_ The strain is available to the scientific public upon request as of

The strain will be made available if a patent office signatory to the Budapest Treaty certifies one's right to receive, or if a U.S. Patent is issued citing the strain.

lf the culture should die or be destroyed during the effective term of the deposit, it shall be your responsibility to replace it with a living culture of the sane.

The strain will be maintained for a period of at least 30 years after the date of deposit, and for a period of at least five years after the most recent request for a sample. The United States and many other countries are signatory to the Budapest Treaty.

The viability of the culture cited above was tested December 14, 1988. On that date, the culture was viable.

International Depository Authority: American Type Culture Collection, Rockville, MD 20852 USA

Signature of person having authority to represent ATCC:

Date: December 14, 1988

cc: Roman Saliwanchik, Esq.

Form BP 4/9

Claims

culture deposit ATCC 20912 , together with Colletotrichum truncatum (Schw.) Andrus & Moore, in association with an inert agricultural carrier. 6. A composition for agricultural application for controlling purple nutsedge, said composition comprising a mixture of an isolate of the fungus Colletotrichum having the identifying characteristics of culture deposit ATCC 20912, together with a suitable chemical herbicide. 7. A composition, according to claim 6, wherein said chemical herbicide is selected from the group consisting of 2,4-D, atrazine, linuron, paraquat, alachlor, metolachlor, glyphosate, dichlobenil, EPTC, and bentazon. 8. A biologically pure culture of the novel isolate of Colletotrichum having the identifying characteristics of culture deposit ATCC 20912. 9. A process for controlling purple nutsedge, said process comprising the application of an agricultural composition comprising an isolate of the fungus Colletotrichum having the identifying characteristics of ATCC 20912, onto said purple nutsedge or onto the situs of said purple nutsedge. 10. A process, according to claim 9, wherein said novel fungus is in combination with an inert biological carrier. 11. A process, according to claim 9, wherein said fungus is in the spore form at a concentration of from about 1 x 10⁴ spores/ml of carrier to about 1 x 10⁷ spores/ml of carrier.

12. A process for controlling purple nutsedge and yellow nutsedge, said process comprising the application of an agricultural composition comprising a mixture of an isolate of the fungus Colletotrichum having the identifying characteristics of culture deposit ATCC 20912, together with the rust pathogen Puccinia canaliculata in association with an inert agricultural carrier. 13. A process for controlling purple nutsedge and sicklepod, said process comprising the application of an agricultural composition comprising a mixture of an isolate of the fungus Colletotrichum having the identifying characteristics of culture deposit ATCC 20912, together with Alternaria cassiae, in association with an inert agricultural carrier. 14. A process for controlling purple nutsedge and Florida beggarweed, said process comprising the application of an agricultural composition comprising a mixture of an isolate of the fungus Colletotrichum having the identifying characteristics of culture deposit ATCC 20912, together with Colletotrichum truncatum ( Schw. ) Andrus & Moore, in association with an inert agricultural carrier. 15. A process for controlling purple nutsedge, said process comprising the application of an agricultural composition comprising a mixture of an isolate of the fungus Colletotrichum having the identifying characteristics of culture deposit ATCC 20912, together with a suitable chemical herbicide. 16. A process, according to claim 15, wherein said chemical herbicide is selected from the group consisting of 2,4-D, atrazine, linuron, paraquat, alachlor, metolachlor, glyphosate, dichlobenil, EPTC, and bentazon.