CA2001794A1 - Biological control of turf disease - Google Patents

Abstract

ABSTRACT OF THE INVENTION
A biocontrol preparation for control or prevention of turf diseases comprising microorganisms, preferably fungi, formed into prill appropriate for application with a broad cast or drop spreader. The preparation has a broad spectrum, is easy to apply, and is shelf-stable.

Description

2~)0~7~4 BACKGROUND OF THE INVENTION
This invention relates generally to the control and prevention of turf diseases and to the enhancement of plant health through the use of biological agents. More specifically, an effective and stable dry biomass delivery svstem for these agents is discl~sed. Biocontrol formulations of this invention contain sporulated microbial biomass which, when added to turf, allows growth of the biocontrol agent through the soil to reduce infestation of plant pathogens and the diseases these pathogens cause to growing turf.
The use of biological agents for control of plant diseases is not itself a new idea, nor is the controlled release of such agents from a matrix. For example, Lewis et al., "Formulation and Delivery Systems for siocontrol Agents E~fective Against Soilborne Plant Pathogenic Fungi," Proceed. Intern. Symp. Control. Rel. Bioact.
Mater., Vol. 12, pp. 341-3 (1985), describes the use of alginate pelle'ts containing propagules of potential fungal and bacterial biocontrol isolates to reduce the incidence of damping-off diseases in cotton, sugar beets and radish seedlings. Similarly, Fravel et al., "Encapsulation of Potential Biocontrol Agents in an Alginate-Clay Matrix,"
Phytopathology, Vol. 75, pp. 772-7 (1985), discloses a method for the encapsulation in alginate pellets of microorganisms that have potential to control plant diseases.
Backman et al., "A System for the Growth and Delivery of siological Control Agents to the Soil," Phytopathology, Vol. 65, pp. 819-21 ~1975~ describes a diatomaceous earth granule impregnated with molasses, disclosed as suitable for growth and delivery of Trichoderma harzianum.

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Paulitz et al., "Effect of Peat:Vexmiculite Mixes Containing Trichoderma harzianum on Increased Growth Response of Radish," J. Amer. _oc. Hort. Sci., Vol. 111, pp. 810-16 (1986) describes addi-tion of T. harzianum to mixes of Canadian sphagnum peat and vermiculite for increased growth of radishes, independent of detectable plant pathogens. European Patent Application No.
86309438.9 ~Agracetus), discloses a fungal culture product in which a medium of ground vermiculite, a nutrient and water is prepared and inoculated with a microbial culture.
The above uses of biocontrol agents were all directed at control of diseases of seeds or seedlings, not established plants. It is unexpected that a biocontrol agent will work on an established plant. Additionally, these previous uses of biocontrol agents were for localized applications (i.e., seed coating, application at seeding, planting, or directly to plant roots). Never before have biocontrol agents been applied to wide areas in a broad cast'manner, such as treatment of an entire lawn or field.
For purposes of this discussion, the term "mycofungicides" shall mean microbes which act in the control of fungal diseases in plants. Soilborne diseases, caused by about fifty genera of fungi, and a few bacteria and viruses, cause about 50% of the losses due to plant diseases. It is conservatively estimated that, in the United States alone, soilborne diseases cause at least $5 billion losses on crops annually. A
major obstacle to the implementation of biological control is a lack of technology in developing and formulating effective and stable preparations whlch can be produced commercially and which will be accepted in 3~7 agricultural and horticultural systems.
While the composition of the invention is similar to others, the use and effectivity of biocontrol agents on established turf had not been previously demonstrated.
The term "turf" refers to any grass area that is mowed, including established lawns, golf greens, sod farms, athletic fields, lawns undergoing renovation, and newly seeded or sodded lawns.
The term "biomass" refers to biological microorganisms having biocontrol properties on turf. The definition of biomass includes mycelia, hyphae, juvenile and mature spores.

SUMMARY OF THE INVENTION
The dry delivery system of this invention is very effective for enhancing plant health in consumer, horticultural, ornamental or recreational applications.
The biocontrol composition comprises a microorganism effective for control of turf disease, preferably a sporulated fungi. Preferably, the sporulated fungi is rich in chlamydospores.
It is a primary purpose of this invention to provide an economical delivery system for the application of microorganisms, including mycofungicides or other biological agents to soils in consumer, horticultural, ornamental or recreational settings for prevention of turf diseases and for the enhancement of plant health via soilborne biological activity. It is a related objective that the delivery system be easy to handle and apply in these settings.
Another important object is to provide a biomass delivery system which can be formulated and packaged 2~0 ~7g4 primarily under nonsterile conditions without detrimental bacterial contamination. The delivery system of this invention is easily stored and shipped. It can be stored in compact containers and can be stored for extended periods. Drying the biocontrol preparation of this invention reduces or eliminates bacterial proliferation.
An additional object of the invention is to provide a dried materi-al which comprises a temporarily inactive form of a biological agent. It is intended that the biological agent can be maintained until the time of application under conditions which would otherwise result in cell death. Upon application to the turf, and with wetting, the inactive agent may be activated, or reconverted into biologically active form.

DETAILED DESCRIPTION OF THE INVENTION
The invention disclosed herein is a delivery system for applying biological microorganisms, including mycofungicides or other biologically active agents to turf to control or prevent turf diseases (such as dollar spot, brown patch, downy mildew, fusarium patch, and typhula blight) or to provide some beneficial result to the turf.
A fungicidally effective amount of the biocontrol preparation is used. The biological turf control composition of this invention comprises a microorganism biomass, preferably a fungal biomass formed into prill.
The prill of the invention has a diameter of 0.5-5 millimeters and would include powders. The microorganisms are selected from species which are effective for con~rol of soilborne diseases or maintenance of plant health. The preferred biological control preparation is prepared for delivery by: (l) growing the selected fungus and allowing 2q~ 794 it to sporulate under conditions favoring production of resistant spores or other survival structures, (2) forming spherical prill, and (3) drying the prill. This dried -prill is applied to the turf to be protected with a broad cast or drop spreader and wetted to activate the biomass.
The spores germinate to produce vegetative cells which act to control the target turf pathogens or to otherwise enhance the health of the turf.
Fungi may reproduce through the formation of sexually or asexually produced spores. Spores produced under unfavorable conditions (e.g., chlamydospores, ascospores, etc.) are quite resistant to hostile environmental conditions, such as heat, dryness, unfavorable pH or nutrient conditions, etc. When fa~orable conditions return, the spores germinate. Under continued favorable conditions, large numbers of asexually derived spores may be produced. This causes rapid spread of the fungus. 3 In order for fungal biocontrol to work for turf diseases such as those described above, living, actively metabolizing fungi must be present in the vicinity of the plant pathogen. Biocontrol of these soilborne plant pathogens requires the presence of the living antagonist fungi where the pathogens exist and/or where they attack the plant.
The delivery system described herein meets these requirements. The biomass composition is versatile enough to be applied in agricultural, horticultural and consumer settings in such a manner that the biocontrol microorganism permeates the infested soil. The dried product may be applied on the turf. Alternatively, the product may be mixed with water and applied as a simple aqueous solution. In any event, upon wetting of the soil 21~ 17~4 and biocontrol product, the spores begin to germinate in the soil, thereafter acting as biocontrol agen~s against the target pathogens.
The dried prill is applied to the turf about two weeks before a particular disease is expected to appear as damage to the turf. For best results, it is important that the prill be added before the disease has a chance to spread. The prill will control both foliar disease, such as dollar spot, as well as root rots, such as brown patch, if the inoculant is established before the disease takes hold. Multiple applications can also help to ensure disease control if the onset of disease is unknown.
Biologically active fungi are selected and prepared according to the method described herein. A number of fungi are known to control diseases caused by plant pathogenic fungi. Many more have the potential to be effective in reducing the incidence of turf disease.
Biocontrol fungi which can be used to control turf diseases include:
Trichoderma viride, Trichoderma hamatum, Trichoderma harzianum, Talaromyces flavus, Gliocladium virens, Gliocladium roseum, Paecilomyces fumosoroseus, Penicillium oxalicum, and Laetisaria arvalis. ~
For preparation of the product of this invention, the desired biocontrol agent is selected~ is grown in culture and is caused to sporulate. Although this description Z~13~31794 refers to preparation of a single fungus, it will be appreciated that a mixture of genera or species may be desirable in some applications. Cultures of common species of fungi, such as those examples listed above, are readily available from sources such as the Agricultural Research Service Culture Collection at the Northern ~egional Research Laboratory (NRRL), 1815 N. University Street, Peoria, Illinois 61604 and the American Type Culture Collection ~ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, as well as from private, university and government sources.
The fungal culture is maintained on a medium suitable for sustaining growth (i.e., containing carbon and nitrogen sources) under appropriate culture conditions for the species selected. The ability to grow the biological agents used in this invention is within the knowledge and ability of one of ordinary skill in the art. Conventional large-scale liquid fermentors can be used.
Fermentat;on is continued until sporulation occurs.
Sporulation may be induced by continuing to culture to depletion of the nutrients required by the selected biological agent. Alternative methods for induction of sporulation, such as heat or temperature shock, may be used as well. With such biocontrol fungi as Trichoderma and Gliocladium it is preferred to obtain biomass rich in chlamydospores because these spores, rather than the conidia, are the resistant survival structures of the biocontrol fungi. With other fungi, such as Laetisaria, specialized mycelium may form as the resistant structure, and with Talaromyces, ascospores or conidia may form. In other species, blastospores or oospores would be the resistant survival structure.

21~ 94 g Liquid fermentation media substrates such as brewer's yeast, molasses, corn steep liquor, corn sugar, etc. are suitable culture media and are readily available.
Fermentation is continued until large quantities of resistant spores are produced, that is, at least about 106 CF~T per gram dry weight of biomass. The sporulated cell mass is then harvested.
Preferably, the cell mass is concentrated prior to further processing. Concentration eliminates the need for handling the culture medium along with the cell mass, thus streamlining processing requirements as well as concentrating the biocontrol agent. The biomass may be separated from the spent medium by filtration, centrifugation or any other convenient method.
Advantageously, this invention allows for formulation directly from the wet biomass, without the need for a mid-stage drying step. Alternatively, the biomass may be dried and powdered, for example by milling or grinding to particles preferably sized to pass through a 40-mesh screen.
The biomass, whether wet or dry, is blended with a nutrient base. Wheat bran is a particularly suitable base. Alternatively, chitin or cellulose-based substances such as corn cobs, peanut hulls and the like, or a mixture thereof, may be used. The nutrient base is first heated (for example, at 80-90C for 48 hours) or otherwise treated to reduce growth of contaminating microorganisms.
The addition of nutrient base to the biomass ensures the survival of the biomass upon ultimate reconstitution.
An encapsulating substance is blended with biomass either simultaneously with, or subsequent to, addition of nutrient base. Alginate is a particularly suitable 21~C.~1794 encapsulating substance. Alternatively, starch-borate, starch-xanthate, or carrageenan gels, or mixtures thereof, may be used.
The biomass/nutrient base/encapsulating substance mixture is added dropwise into a gellant solution to form spherical biocontrol prill. The preferred gellant solution is CaCl2. The preferred size of the biocontrol prill is about 2mm. The biocontrol prill is dried under vacuum at a low temperature (~30C). The dried biocontrol prill preferably has a diameter of about 0.5-5 mm and a density greater than 0.3 g/ml. The most preferable diameter is about 2 mm. This size and density is particularly good for use in a broad cast or drop spreader. A smaller particle could be dust-like and float away during administration, while a larger particle might clog the spreader.
This biocontrol prill is slowly dried to a total volatiles content of less than about 10.0%, most preferably less than about 2.0%. At low total volatiles levels, both bacterial and fungal growth is minimized or eliminated, allowing storage of the biocontrol prill. It is preferred that the prill be as dry as possible to decrease bacterial and fungal growth at this stage. The dried biocontrol prill is shelf-stable and may be stored for up to six weeks or longer.
The dried biocontrol prill should contain ~ungal spores in sufficient quantity to have a viable spore count of at least about 10 colony forming units ~CFU) per gram of the dried biocontrol prill. A colony forming unit is a measure of the viability of the spore preparation. A
useful range is about 103 to about 10l, preferably about 104 to about 106, CFU per gram. It should be appreciated Zt~ 794 that the preferred spore count of the drled biocontrol prill will depend on the anticipated dosage used on the turf. A dosage of about 1.0 to about 10,00~ grams per square meter of turf is suggested. A preferred dosage is about 10 to 100 grams per square meter. The biocontrol prill is applied to turf in a suitable manner such as with a broad cast spreader at a desired rate.
The dried biocontrol prill may be packaged into any container which is suitable for storage and shipment.
Dense packing is possible with the material of this invention. Packaging materials and ~design should be adequate to maintain the low moisture content of the packaged product for its anticipated shelf life.
As mentioned above, the dried biocontrol prill may be 1~ applied to the turf to be protected. Alternatively, the dried prill may be suspended in water or other liquid and applied to the turf as a drench.
The present invention allows for activation of the fungal spores ~hat, when added to turf, permits growth and development of the biocontrol agent under natural conditions. After addition to turf, the effectiveness of the formulation is ascertained by reduction in pathogen inoculum density and in prevention or reduction of turf disease. Use of the biocontrol prill of this invention can reduce or prevent turf diseases caused by Rhizoctonia solani, Sclerotinia homoeocarpa, Pythium ultimum, Sclerotium rolfsii, Verticillium dahliae, and other species of turf pathogenic fungi.
The examples which follow are given for illustrative purposes and are not meant to limit the invention described herein. The following abbreviations have been used throughout in describing the invention:
C - degree(s) Centigrade CFU - colony forming unit(s) cc - cubic centimeter cm - centimeter g - gram(s) " - inch 1 - liter M - molar mg - milligram(s) ml - milliliter(s) N - normal ~ - percent rpm - rotations per minute sq. ft. - square feet TV - total volatiles wt. - weight Example 1 Preparation and Application of Biocontrol Products Soil inoculant prill were prepared by transferring a culture of Gliocladium virens GL-21 from agar slant or frozen culture to a sterile 2 liter shake flask containing 1 liter of medium consisting of 30 g/l molasses and 5 g/l brewer's yeast. The solution pH was adjusted to 6.0 and held at 26C for 40-48 hrs. The shake flask was then use~d;
to inoculate a 20 liter fermentor containing 15 liter of medium consisting of 15 g/l pharmamedia (Trader Protein, Ft. Worth, Texas), 10 g/l cornsteep liquor and 60 g/l glucose. The fermentor was aerated (0.5 to 1 VVM), agitated (1000 rpm) and glucose concentration is monitored and maintained at 5 to 30 g/l by periodic addition (of a zi~C~1794 - 700 g/l glucose solution). The pH of the fermentation was held at pH 6 during the growth phase (6 N NH40~ used for control) and after 48 hours the fungus begins to sporulate until the 120th to 160th hour.
After the fermentation was fully sporulated the cell mass was harvested by centrifugation.
The sporulated cell mass was then blended to make up an alginate solution of l~t sodium alginate, 20~ bran and 10% cell concentrate. Prill are then formed using a pr`illing column to make spherical prill gelled by dropping into a solution of O.lM CaC12. The ~elled prill are then filtered off and air dried.

Example 2 Control of Dollar S~ot on Burmudagrass with G. virens In an effort to determine whether the presence of Gliocladium virens would reduce the incidence of the fungal disease ,dollar spot on hybrid bermudagrass, plots treated with the biocontrol agent (G. virens) were compared'to plots treated with both standard and experimental fungicides, and untreated plots.
Applications of G. virens were initiated early with anticipation that this fungus would impede sclerotial ~
germination of Sclerotinia homoeocarpa, the causal agent of dollar spot. t The biocontrol agent (formulated and applied as described in Example 1) and the standard or experimental fungicides were applied to 4 different 25 sq. ft. plots in a randomized manner. Treatment schedules are provided in Table 1.

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- Table 1 Rates ànd Times of Applications of G. virens ~ to Hybrid sermudagrclss Treatment Rate Application Date(s) 1. Control 2. G. virens 77 g/25 sq ft 14 day intervals ~ 3/17~6/05 3. Daconil 2787 3 oz/1,000 sq ft 14 day inter~als ~ 75WP 3/17-6/05 4. G. virens plus Same as 2 and 3 Same as 2 and 3 Daconil 2787 5. Banner 2E 2 oz/1000 sq ft 5/17 and 6/02 6. Chipco 26019 1 oz/1000 sq ft 5/17 and 6/02 7. CGA 455 ~ oz/1,000 sq ft 5/17 and 6/02 8. CGA 455 ~ oz/1,000 sq ft 5/17 Ratings of each treatment were based on the number of "hits" per plo,t. "Hits" are defined as damage spots in the burmudagrass ranging in size from % to 2" in diameter.
Ratings were taken June 5, June 16, and June 28, 1989 and statistically analyzed by the computer program ANOVA
(analysis of varience) ~Humàn Systems Dynamics, _ California). Results are provided in Table 2.

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Table 2 Disease Incidence Ratings of Dollar Spot on Hybrid Bermudagrass -Treatment 6/05/89 6/26/89 6/28/89 1 30.3 51.7 85.5 2 9.3 2~.0 46.5 3 2.8 4.3 22.0 4 1.3 0.8 15.8 0.0 0.3 3.3 6 0.8 6.0 15.0 7 1.0 ` 0.8 14.0 8 5.0 20.0 49.3 LSD 0.05 4.6 9.1 18.3 .

Results of this experiment show that G. virens did reduce the incidence of dollar spot. Fungicides, such as Daconil 2787 and Banner, controlled the disease better than G. virens in this experiment, but there is every indication that with optimization of dosage and timing, G. virens will be comparable or better than fungicides in treating dollar spot. The biocontrol agent also has the added advantage that it is not nearly as toxic to the environment. :

Example 3 Control of Brown Patch on Centipedegrass with G. virens G. virens was tested on centipedegrass to determine its effectiveness for controlling brown patch (caused by 2~ 79 R. solani AG2T2). Six-month-old centipedeyrass grown in 10 cm pots was placed in a growth chamber where temperatures in the 14 hour light cycle ranged from 28 to 30GC and temperatures of the dark cycle ranged from 19 to 22C on day one of the experiment. Each treatment was applied to 4 different pots in a completely randomized design. For Treatment 1, on day 1, G. virens, prepared as in Example 1, was applied at a rate of .35 g per pot to four pots. The G. virens particles were suspended in water for ease of application. On day 14, R. solani was inoculated to the pots at the rate of 1 g of infested fescue seed per pot. For Treatment 2, four pots were infested with R. solani only on day 14, at the specified rate to serve as a positive control for infestation. For Treatment 3, four pots were infested with both R. solani and G. virens on day 14 at the rates specified above. For Treatment 4, four pots were infested with G. virens only on day 14. Plants without any fungi added served as negative controls (Treatment 5). Treatments were rated by % sheath rot after 30 days. The results shown in Table 3 indicate that G. virens provides significant control of brown patch on centipedegrass.
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Tabl~ 3 Treatment and Ratings (Based on Percent Sheath Rot) of srown Patch on Centipedegrass Treatment % Sheath Rot 1. G. virens 5/25 plus R. solanl 6/7 22.5 2. R. solani 6/7 72.5 3. G. virens plus R. solani 6/727.5 4. G. virens 6/7 0.0 5. Control 0.0 I.SD (0.05) 11.5 Example 4 Control of Yellow Patch on Bentgrass with G. virens The experimental design of Example 2 was used to test the ability of G. virens to control yellow patch (caused by Rhizoctonia cerealis) on bentgrass. No symptoms developed on any plots 6 months after treatment. Due to lack of symptom development, it was inconclusive whether G. virens would have any controlling effect on yellow patch, but the G. virens did not impair the color or growth of the bentgrass.
The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the invention.

Claims (16)

1. WHAT IS CLAIMED IS:
   
   l. A biocontrol preparation for control or prevention of turf diseases comprising biomass of one or more species of microorganism effective for control of turf diseases, formed into prill appropriate for application with a broad cast or drop spreader.
2. 2. The biocontrol preparation of Claim 1, wherein said microorganism is a fungus.
3. 3. The biocontrol preparation of Claim 2, wherein said fungus is Gliocladium virens strain GL-21.
4. 4. The biocontrol preparation of Claim 1 further comprising a nutrient base.
5. 5. The biocontrol preparation of Claim 2 which comprises a fungicidally effective amount of said fungus for controlling soilborne fungal pathogens.
6. 6. The biocontrol preparation of Claim 5 which has a viable spore count of at least 103 colony forming units of said fungus per gram (dry weight).
7. 7. The biocontrol prill of Claim 1 which have a diameter between 0.5 and 5 millimeters and a density greater than 0.3 grams per milliliter.
8. 8. The biocontrol prill of Claim 7 which has a total volatiles content of less than about 10.0%.
9. 9. The biocontrol prill of Claim 8 which has a total volatiles content of less than about 2.0%.
10. 10. The biocontrol preparation of Claim 4 in which said nutrient base is selected from the group consisting of wheat bran, chitin, corn cobs, peanut hulls or a mixture thereof.
11. 11. The biocontrol preparation of Claim 2 which is prepared by:
    (a) fermenting said fungus in a culture medium until sporulation occurs and resistant spores are produced, (b) harvesting the sporulated biomass, (c) mixing biomass with a nutrient base and encapsulating substance, (d) forming prill from the biomass/nutrient base/encapsulating substance mixture, and (e) drying the prill.
12. 12. The biocontrol preparation of Claim 11 in which the fermentation of step (a) is continued until sufficient quantities of resistant spores are produced to yield at least about 106 colony forming units per gram dry weight of biomass.
13. 13. A method of enhancing plant health by applying to turf a biocontrol preparation for control or prevention of turf diseases, said biocontrol preparation comprising biomass of one or more species of microorganism effective for control of turf disease.
14. 14. The method of Claim 13 wherein said microorganism is a fungus.
15. 15. The method of Claim 14, wherein said fungus is Gliocladium virens strain GL-21.
16. 16. The method of Claim 13 in which said biocontrol preparation is applied to the soil or growth medium at a rate of between about 1.0 and about 10,000 grams per square meter.