FIELD OF THE INVENTION

The invention relates to the treatment of plants by a composition containing B. subtilis, B. cereus, or bacillus BPOl (ATCC 55675) in the form of cell, spores, or suspensions.

BACKGROUND OF THE INVENTION

Agricultural chemical manufacturers are always looking for ways to improve the efficacy of active ingredients used on plants. This is particularly true where the applied material is a plant growth regulator or systemic agent (e.g., insecticide, fungicide, or herbicide). Transport mechanisms into the plant and translocation among the various plant tissues is important and, in some instances, may be the primary factor determining the efficacy of the applied ingredient. For some active ingredients, an improvement in the transport mechanism could translate into improved performance at existing application rates, the need for less active ingredient, or the ability to treat new species that were previously resistant to the active ingredient.

One area where this sentiment is particularly true is herbicides. Many herbicides could also use a boost in activity without an increase in the amount of applied herbicide. Farmers and herbicide manufacturers are often faced with a need to control weeds and noxious plants without exceeding the application levels of proven herbicides, if the plants can be controlled at all. Some plants, like Florida Pusley, Bull Grass, Bermuda grass, Dog Fennel, and Primrose are all highly resistant to herbicides proven to be effective. It would be useful to have a means for increasing the uptake of agriculturally active ingredients, such as herbicides, into plants.

Mepiquat chloride is another active ingredient where plant uptake and transport is important. Mepiquat chloride (N,N-di emylpiperidinium chloride) is used annually as the active ingredient for stunting vegetative plant

growth and increasing fruit retention on millions of acres of cotton. Mepiquat chloride also has some uses on potatoes, sweet potatoes, peanuts, grapes, corn, wheat, citrus, tomatoes, and onions. Mepiquat chloride has the effect on cotton plants of stunting vegetative growth thereby forcing the plant to redirect its energies into fruit (cotton boll) production. With appropriate application of mepiquat chloride to plants that are beginning to exhibit excessive vegetative growth, cotton plant yields can be maintained or increased without harm to the plant. The growth stunting effects are particularly desired when the crop is grown in fertile soil or after weather conditions that favor plant growth rather than fruit production.

Cotton plants have a predictable life cycle and growth period. Cotton plants emerge 7-10 days after the seeds are planted in a furrow. The cotton plant exhibits growth of a root system and the extension of plant height through stem and branch growth in a pattern referred to as "vegetative growth" until about the 4th - 8th node. Thereafter, the plant produces a reproductive branch (the "1st fruiting site"), and all subsequent branches are reproductive. Cotton growers attempt to control the growth of the plant to ensure that the ratio of vegetative growth to reproductive growth (boll production) favors the desired range of reproductive growth. Cotton growers generally prefer to see about 2 inches (5 cm) between main stem nodes. This ratio represents a balance between too much reproductive growth (boll production) which can cause the plant growth to outpace the rate of vegetative growth and terminate before the yield is maximized, and too much vegetative growth which reduces the number of mature bolls.

Cotton plants that have directed the majority of the available plant energy to vegetative growth are referred to as "rank" cotton and produce less bolls which mature later and are vulnerable to weather extremes for longer

periods of time. Cotton that exhibits signs of going rank are readily visible by abnormal plant height relative to the boll loads and number of reproductive main stem nodes. Mepiquat chloride is used to stop cotton from going rank by modifying the cotton plant's growth characteristics. The branches off the main stem generally always extend from alternating sides of the stem. Each branch site is called a "node" with 5-7 nodes being formed above the cotyledon leaves before the first fruit bearing branch with true leaves is formed. Node counting starts at the bottom of the plant and extends up the main stem. The "internode length" is the distance between branch sites with a new node being formed roughly every three days. For purposes of measurement and comparison, the number of nodes and internode length above node 8 are generally used to eliminate interplant fruiting node variations because fruit bearing branches will necessarily have been formed by node 8. The counting of fruiting nodes thus conventionally starts from the first reproductive node, usually no. 7 or no. 8.

Fruiting sites in cotton are referred to as "squares. " Each fruit bearing branch will form 1-6, normally about 3, fruiting sites ("squares") with approximately six days between square formations on each branch. New squares and the beg ning of reproductive growth in cotton plants are referred to as "pinhead" squares due to their barely visible size. After about 5-10 days, the square has grown to about the size of a match head and is a period in the plant cycle referred to as a "match head square. " The match head square continues to grow to about the size of an average adult fingernail before blooming ("early bloom"). Three days later, a boll has formed beneath the bloom. Roughly tiiirty days after early bloom, the product boll is fully mature and ready for harvest. Overall, about 80% of the total cotton yield is set within the first 3 weeks after early bloom and 95% of the total yield is set within 5 weeks of early bloom.

Generally, mepiquat chloride is applied to cotton plants in one of two ways. The method used until about 1986 was a single application of 8-16 ounces per acre of a 4.2 wt% solution at early bloom. This type of single treatments did control plant height although it was noticed that plant yields were occasionally reduced particularly if the plant was stressed during or after the application.

Since 1987, the trend has been to apply mepiquat chloride in a series of applications each having a lower dose than the single dose application. The first treatment occurs at match head square with a second treatment 7-14 days thereafter. Both treatments are made at a rate within the range from about 0-8 ounces of 4.2 wt% solution per acre with the specific application rate depending on whether the cotton plant was exhibiting signs of being stressed (no application), moderate growth (about 2 ounces of solution per acre), or vigorous growth (about 4 ounces of solution per acre). Thereafter, two additional treatments at 7-14 day intervals may be used with application rates extending up to about 8 ounces of 4.2 wt% mepiquat chloride solution with the specific application rate dependent on the amount of vegetative growth in the field. Further experimentation by individual growers has resulted in a wide variety of multiple application rates. It would be desirable if the use of mepiquat chloride could be integrated into a system of treatment that would increase plant tissue mass in the roots, stems, and leaves to provide higher levels of nutrient transfer while, at the same time, restricting vegetative growth to enhance fruit production. SUMMARY OF THE INVENTION It is an objective of the present invention to provide a composition and method of use therefore to increase the uptake of agriculmrally effective active ingredients, such as herbicides and mepiquat chloride, as well as the intraplant transport thereof.

It is a further objective of the invention to provide a composition and method for its use on fruit-producing plants and seeds that increases the number of fruiting sites on treated plants with the goal of providing increased yields of fruit. It is another objective of the invention to provide a composition and method for its use in which treated plants grow in a more healthy condition.

In accordance with these and other objectives of the invention that will become apparent from the description herein, a composition according to the invention comprises: (a) an agriculmrally effective active ingredient, and (b) a transport enhancer consisting essentially of B. subtilis, B. cereus, or a bacillus strain exhibiting characteristics of both B. subtilis and B. cereus, like ATCC 55675, in the form of cells, cultures, or suspensions and in an amount sufficient to enhance the effectiveness of said active ingredient. Preferably, the transport enhancer is free of plant growth hormones when used in combination with plant growth regulating agents, like mepiquat chloride, that suppress plant growth hormones.

Compositions according to the present invention improve the efficacy of the applied agriculturally active ingredient. The same amount of active material that is conventionally applied will be more effective. Lower levels of active ingredient can be used to achieve the same effect as the higher conventional application rate. In addition, plants that have treated with compositions according to the invention are healthier with the attendant benefit of being more resistant to disease or other stress as well as exhibiting higher numbers of fruiting sites and increased yields. DETAILED DESCRIPTION

The invention provides a method for treating fruit-producing plants with a composition containing a transport enhancer consisting essentially of B. subtilis, B. cereus, or a bacillus exhibiting characteristics of both B. subtilis

and B. cereus, like ATCC 55675, in an amount sufficient to enhance the effectiveness of an agriculmrally effective active ingredient applied simultaneously, before, or after application of the transport enhancer. The increased effectiveness can be used to reduce the amount of applied agriculmrally effective active ingredient or, when the active ingredient is applied at the same rate, the bacillus increases the effectiveness of the applied agriculturally effective active ingredient. Such increased effectiveness is useful for controlling weeds that are otherwise difficult to control with regular herbicides. Bacillus subtilis and B. cereus are naturally occurring soil saprophytes found throughout the world. The bacillus deposited as ATCC 55675 exhibits characteristics of both B. subtilis and B. cereus. For compositions according to the invention, gram positive bacillus strains can be used in the form of cells, spores, cultures, or suspensions thereof and added to a spray tank or distribution reservoir as a stable, aqueous concentrate solution exhibiting an equivalent spore concentration within the range from about 300,000 colony forming units per milliliter (CFU/ml) to about 1.5 million CFU/ml, preferably about 1 million to about 1.2 million CFU/ml. Optionally and in a preferred embodiment, the spray tank will also contain at least one agriculmrally effective active ingredient that is made from one or more plant growth regulating agents or systemically acting agents (e.g., fungicides, insecticides, or herbicides).

The specific dilution and application rate will depend on the method by which the solution is to be applied to the plant surfaces. For example, aerial spraying will employ a different dilution rate and application quantity than boom spraying or the use of manual sprayers. Preferably, the concentrated bacillus solution is applied to plant foliage at a rate within the range from about 0.1 X 10 ¹⁰ CFU/acre to about 10 X 10 ¹⁰ CFU/acre, preferably within the range

from about 0.5 X 10 ¹⁰ CFU/acre (0.5 fl. oz./acre of concentrate) to about 8 X 10 ¹⁰ CFU/acre (2 fl. oz./acre of concentrate). Conventional equipment can be used for the application. If desired, the bacillus can be mixed with other treatments and applied simultaneously or can be applied in a discrete treatment step. Foliar application is the preferred method for increasing the number of fruiting sites on fruit-producing plants.

The concentrate can also be used to formulate a ready-to-use, packaged mixture of growth regulating agents and bacillus. So prepared, the bacillus suspension is diluted to an amount within the range from about 150,000 CFU/ml to about 600,000 CFU/ml and stored at a pH of less than 7. If necessary, any of the conventional acidifying agents or buffers (preferably food grade or those classified as "Generally Regarded As Safe" by the U.S. Environmental Protection Agency) may be used to maintain a suitable acidic pH to ensure storage stability. Under such acidic conditions, the spores remain stable and exhibit good storage stability. When diluted for use and following application, the pH of the solution will raise to greater than 7 thereby causing the bacillus suspensions to become live, vegetative colonies. The bacillus will thereby reproduce on the treated plant surfaces and facilitate transport or translocation of the co-applied plant growth regulating agent. For the invention, virtually any strain of B. subtilis, B. cereus, or a bacillus exhibiting the characteristics of these strains (like ATCC 55675) that promotes the formation of fruiting sites in fruit producing plants can be used in accordance with the present invention. In the 1992 edition of the American Type Culture Collection, 182 different strains of B. subtilis are listed and incorporated herein by reference. A preferred B. subtilis strain for use in the present invention includes GB03. A preferred bacillus strain for use in the present invention includes strain ATCC No. 55675 which exhibits characteristics of both B. subtilis and B. cereus.

Previously, B. subtilis GBO3 was recognized as a biological fungicide and commercially used as a seed treatment under the names KODIAK™ HB or GUS 2000™ by Gustafson, Inc., Piano, Texas 75093 (EPA Reg. No. 7501- 146). This product is available as a 2.75% powder formulation containing not less than 5.5 X 10 ¹⁰ viable spores per gram and is to be applied at a rate ranging from 2-4 ounces per 100 pounds of seed. The use directions indicate that the product is to be used for treatment of crop seeds only. The bacillus is said to colonize the developing root systems and compete with disease organisms that would attack the roots. Foliar application is not listed. Plants that can be treated by the present invention include virtually any plant that produces fruiting sites from which fruit will grow. Such plants preferably include any of the raw agriculmral commodity and especially cotton, soybeans, peanuts, grapes, apples, citrus (e.g., lemons, limes, oranges, grapefruit), berries (e.g., strawberries, blackberries, raspberries), tubers (e.g., potatoes, sweet potatoes), corn, cereal grains (e.g., wheat, rice, rye), tomatoes, onions, cucurbits (e.g., watermelon, cucumbers, and cantaloupes). The bacillus can be applied as a discrete treatment or simultaneously with a variety of other agriculmrally effective active ingredients. Useful agriculmrally effective active ingredients include plant growth regulating agents, systemic insecticides, systemic fungicides, plant growth smnting agents and herbicides. Preferably, the composition is a combination of either a plant growth smnting agent or herbicide and a transportation enhancer of B. subtilis, B. cereus, or ATCC 55675 (BPOl) in an amount of at least 0.1 X 10 ¹⁰ CFU/acre. Plant growth regulators include plant growth enhancing agents as well as plant growth smnting agents. Suitable plant growth enhancing agents for the present invention include plant growth hormones such as at least one of the 84 identified gibberillins with GA ₃ , GA ₄ , GA ₅ , GA ₇ and GAo being preferred;

cytokinins (e.g., zeatin, kinetin, benzyladenine, dihydrozeatin, and isopentenyl adenine); auxins (e.g., indolacetic acid (IAA), indolebutyric acid (IB A), and naphthalenacetic acid (NAA)); sodium ortho-nitrophenolate; sodium para- nitrophenolate; sodium 5-nitro-guaicolate; and polyhydroxycarboxylic acids of 2, 4, 5, and 6 carbon structures; ethephon; and fertilizers.

Suitable plant growth smnting agents useful in the invention include chlormequat chloride, mepiquat chloride, as well as maleic hydrazide and its esters. Such plant growth regulators affect and alter plant metabolic processes to enhance or retard plant growth. All such agents can be used according to the application rates and tuning specified by the manufacturer on the product label.

Suitable systemic agents that will benefit from enhanced plant uptake, transport, and process assimilation include the systemic pesticides and systemic fungicides. Systemic agents for plants that benefit from the present invention include, inter alia, the insecticides aldicarb, acephate, carbofuran, dimethoate, phorate, and terbufos. Systemic fungicides that will benefit from the mixtures of the invention include tridemorph, metalaxyl, iprodione, fosetyl-aluminum, thiophanate, benomyl, triadimefon, carboxin, oxycarboxin, carbendazim, thiabendazole, thiophanate, ethirimol, bupirimate, and dimethirimol. Systemic herbicides the triazines (e.g., atrazine), the ureas, glyphosate, sulfosate, glyfosinate, and sethoxydim.

In a particularly preferred embodiment, a gibberellin-free transport enhancer containing the bacillus is applied to the foliage of cotton plants at the same time the plants are treated with mepiquat chloride. An aqueous tank mixture containing bacillus (preferably ATCC 55675) and mepiquat chloride is a convenient method for simultaneously applying the components.

It should be noted that formulations according to the present invention should not include combinations of materials that attempt to act in a

contradictory fashion on the plant metabolism. For example, mepiquat chloride is commonly used on cotton foliage to suppress plant growth hormones and stunt the vegetative growth of the plant. A formulation would not be prepared that included plant growth hormones because the effects of the mepiquat chloride and the growth hormones would place inconsistent demands on the plant metabolism, reduce the efficacy of the mepiquat chloride, and lead to inconsistent results. With the present invention, however, the combination of mepiquat chloride and ATCC 55675 consistently produces treated plants that have higher yield, more healthy growth, and a higher resistance to disease. While not wishing to be bound by any particular theory of operation and with respect to the combined use of bacillus and mepiquat chloride on cotton, the bacillus appears to be specifically affecting the plant growth mechanism to increase the retention of bolls on fruiting sites 1 and 2 and increases the number of bolls overall by producing and retaining fruiting sites on normally vegetative branches.

For the present invention, mepiquat chloride is used at the application rates and during the conventional stages of cotton plant growth. Conventionally applied rates of mepiquat chloride are up to about 60 g/acre (25 g/acre) or about 1-16 ounces per acre with individual application rates falling within the range from about 2.5 g/acre (1.0 g/hectare) for a 2 ounce/acre application of 4.2 wt% solution to 10 g/acre (4.1 g/hectare) for an 8 ounce/acre application of the same 4.2 wt% solution. If mepiquat chloride of higher or lower purity and/or activity is used, the specific application rate should be adjusted up or down according to the change in conventional mepiquat chloride activity. EXAMPLES

Example 1

An aqueous mixture of 4.2 wt% mepiquat chloride and 560,000 CFU/ml B. cereus (lab sample BPOl, ATCC 55675) was prepared in a 1:1 volumetric ratio. This solution was applied by conventional sprayer in four applications to the foliage of growing cotton plants in test areas of fields A (10 of 50 acres in Mississippi) and B (8 of 60 acres in Tennessee). Field A was treated with one dose at the rate of 4 fluid ounces per acre followed in five weeks by a treatment at the rate of 8 fluid ounces per acre.

Field B received a different treatment regiment. The first three applications in Field B were at the rate of 4 fluid ounces per acre. (The first and second were 24 days apart. The second and third were at 6 days apart). The final application in Field B was at the rate of 8 fluid ounces per acre 11 days later. In all cases, the control fields were treated with the same rate of only mepiquat chloride.

After treatment, sampling in Fields A and B was conducted by hand of representative rows. All known biases were placed in favor of the control treatment with only mepiquat chloride.

In Fields A and B, beginning and final plant heights of the treated cotton plants was measured. The total plant heights of the control fields was comparable to those treated according to the invention. See, Table 1.

Table 1

Field Beginning Height (in.) Final height (in.)

A - Control 293 601

A - Treated 291 597

B - Control 338 580

B - Treated 334 636

During the growing season, the differences between the control and treated plants were readily observed. When the young plants contained only squares in the first month of fruiting, an unusually high number and size of additional fruiting branches were seen where the primary fruiting branch exits the main stem. Many were as large or almost as large as the primary fruiting branch and contained fruit prior to first bloom. Extra fruit was also observed at the intersection of the fruiting branches and main stem.

As the bloom extended up the plant and only bolls or missing sites were found below the bloom, an unexpected amount of back fruiting was also observed. Double fruiting was observed in squares, blooms, small bolls, and "now open bolls" (rare). Although double fruiting occurred in both the control and treated plants, the instances were higher in the treated plants.

About 3-4 weeks before the end of the growing period, the plants were inspected for the number of fruiting sites, the number of bolls on nodes 1 and 2, as well as the type of boll. Table 2 reports the results of that inspection.

Table 2

Field Sites Bolls % Open Open + Other Bolls Green Bolls

A - Control 240 115 48 73 270 25 A - Treated 292 155 53 173 359 103

B - Control 344 207 60 81

B - Treated 366 234 64 — __ 163

In Field B and at the end of the test period, 20 plants were inspected. The plants were at cut-out. There were no open bolls in the control group and only 7 in the treated group. The younger fruit, however, were larger and older for the treated plants compared to the control plants. This indicates that the treated plants were growing at a faster and more favorable rate. The treated plants had a total of 265 bolls compared to 238 bolls for the control group, an improvement of 11 % .

The treated plants also exhibited an increase in the number of fruit on the vegetative branches in the ratio of 88:51 , an increase of about 73 % . The treated plants also produced an increase in other fruit, i.e., those on fruiting branch positions 3 and wider, at the ratio of 86:50 (72% increase). The estimated weight (seed cotton) of the fruit from the 20 plants was also higher in the treated plants, 1796 g. v. 1456 g. (23% increase). The extra fruit on the plants brought an expectation that the additional plant parts and young fruit would be a drain on the plant's system. Bolls would open sooner, but would adversely affect yield. This expectation was not realized. The treated plants grew at a faster rate and in a more healthy condition than the control. To reduce bias, all lint was then harvested by hand from the test fields including unfluffed lint from partially opened bolls. Such unfluffed lint are usually from bolls that never opened correctly and are low on the plant or from bolls just opening that are high on the plant. Table 3 reports the weight of seed cotton and the number of green bolls per 10 foot of row in the treated and control fields.

Table 3

Field Seed cotton (g.) Number of Green Bolls

A - Control 5322 97

A - Treated 6287 0

B - Control 4532 175

B - Treated 5058 42

The test results show that the combination of mepiquat chloride applied simultaneously with a transport enhancer containing B. subtilis according to the invention produces higher cotton yields and healthier plants than use of mepiquat chloride alone. Example 2

Bacillus cereus strain BPOl (ATCC 55675) was used in combination with a widely used herbicide, atrazine, to determine whether the BPOl would affect control over weeds that are recognized as difficult to kill with triazines. Atrazine is used to provide season-long control in corn, sorghum, and other crops at a suggested rate of 2 pounds active ingredient per acre. At sufficiently high rates, such as those used in this example, atrazine is recognized for its ability to provide nonselective weed control. In sandy soil, three replicates of each test were performed in 12 ft. X

25 ft. plots using a randomized complete block pattern. The weeds were 2-6 inches (5-15 cm) in height at the time of treatment. Atrazine was applied from aqueous solution at rates equivalent to either 1 or 2 pounds of active ingredient per acre. BPOl concentrate was added to the atrazine in an amount equivalent to either 0.5 or 1 fluid ounce per acre (0.5-1 X 10 ¹⁰ CFU per acre). For comparison, crop oil concentrate (about 85% paraffinic oil and about 17% surfactant) was used as is conventional with triazine herbicides to increase their

efficacy. Tables 4-8 report the degree of control for Florida Pusley (Table 4), Bull Grass (Table 5), Bermuda grass (Table 6), Dog Fennel (Table 7), and Primrose (Table 8).

Table 4 - Florida Pusley

% Control After Treatment

Treatment 4 days 9 days 16 days 23 days

Control 0 0 0 0

Atrazine (1 lb.) 3 33 42 45

Atrazine (2 lb.) 17 57 72 83

Atrazine (1 lb) + crop oil cone. (1 gal.) 28 62 68 68

Atrazine (1 lb) + 0.5 oz. BPOl 27 43 60 60

Atrazine (1 lb) + 1 oz. BPOl 22 53 65 67

Table 5 - Bull Grass

% Control After Treatment

Treatment 4 days 9 days 16 days 23 days

Control 0 0 0 0

Atrazine (1 lb.) 10 40 40 55

Atrazine (2 lb.) 25 70 80 90

Atrazine (1 lb) + crop oil cone. (1 gal.) 25 40 65 60

Atrazine (1 lb) + 0.5 oz. BPOl 35 40 55 63

Atrazine (1 lb) + 1 oz. BPOl 30 60 80 90

Table 6 - Bermuda grass

% Control After Treatment

Treatment 4 days 9 days 16 days 23 days

Control 0 0 0 0

Atrazine (1 lb.) 0 10 15 15

Atrazine (2 lb.) 8 15 22 27

Atrazine (1 lb) + crop oil cone. (1 gal.) 25 27 30 43

Atrazine (1 lb) + 0.5 oz. BPOl 7 20 37 68

Atrazine (1 lb) + 1 oz. BPOl 10 23 53 63

Table 7 - Dog Fennel

% Control After Treatment

Treatment 4 days 9 days 16 days 23 days

Control 0 0 0 0

Atrazine (1 lb.) 15 40 55 60

Atrazine (2 lb.) 17 55 70 95

Atrazine (1 lb) + crop oil cone. (1 gal.) 25 75 93 98

Atrazine (1 lb) + 0.5 oz. BPOl 33 88 96 99

Atrazine (1 lb) + 1 oz. BPOl 30 90 97 100

Table 8 - Primrose

% Control After Treatment

Treatment 4 days 9 days 16 days 23 days

Control 0 0 0 0

Atrazine (1 lb.) 10 40 50 60

Atrazine (2 lb.) 30 65 70 80

Atrazine (I lb) + crop oil cone. (1 gal.) 20 83 88 93

Atrazine (1 lb) + 0.5 oz. BPOl 20 60 72 88

Atrazine (1 lb) + 1 oz. BPOl 23 70 65 80

From Tables 4-8, it can be seen that BPOl generally improved the effectiveness of the atrazine at 23 days after treatment. The control rate at 1 lb. of atrazine with the bacillus was better than the control rate of 2 lb. atrazine for Bermuda grass, Dog Fennel, and Primrose, and the two treatments had the same control rate for Bull Grass. Only with Florida Pusley and 1 lb/acre of atrazine with BPOl was the control rate reduced relative to the 2 lb/acre treatment with atrazine..

Similarly, the BPOl also improved the control rate of atrazine relative to a mixmre of atrazine and crop oil concentrate in all weeds except for Florida Pusley and Primrose. Such an improvement suggests that the bacillus is not acting as a surfactant, but is enhancing effectiveness by either or both of the metabolic activity or translocation characteristics of the co-applied agent.

The preceding are intended solely for purposes of illustrating the invention and are not intended to act as limitations on the scope of the appended claims.