AU738993B2 - Agricultural compositions containing bacteria - Google Patents

Description

WO 98/23157 PCT/US96/19116 AGRICULTURAL COMPOSITIONS CONTAINING BACTERIA FIELD OF THE INVENTION -The invention relates-to the treatment of plants by a composition containing an agriculturally effective active ingredient and an enhancer additive containing a substantially pure culture of bacteria selected from the genus Bacillus or a soil bacteria. The added culture may be in the form of cell, spores, or suspensions.

BACKGROUND OF THE INVENTION Agricultiural 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 (growth stunting or growth enhancing), -herbicide, or systemic agent insecticideor Sfungicide). 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.

Mepiquat chloride is an active ingredient where plant uptake and transport is important. Mepiquat chloride (N,N-dimethylpiperidinium chloride) is used annually as the active ingredient for stunting vegetative cotton 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 therebyforcingjhe. plant to redirect its energies into fruit (cotton boll) production. With appropriate application of mepiquat chloride to plants that WO 98/23157 PCT/US96/19116 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 lessbolls 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 WO 98/23157 PCT/US96/19116 3 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 iiternode 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. 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 beginning 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. Roiighly thirty days after early bloom, theproduct 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 t-he application. WO 98/23157 PCT/US96/19116 4 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), mioderate 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 tissuemiass 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.

The technology of plant herbicides has a continuing desire for enhanced efficacy without a corresponding increase in the application-rate. 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 efficacy of agriculturally-active ingredients, such as herbicides, without increasing the amount of the applied active ingredient.

CD01 145003.0 It will be understood that the term "comprises" or its grammatical variants as used in this specification and claims is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

SUMMARY OF THE INVENTION The present invention relates to a composition and method of use therefore to increase the efficacy of agriculturally effective active ingredients.

The invention further relates to 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.

The invention is also relates to a composition and method for its use in which treated plants grow in a more healthy condition.

oo° ~The invention described herein therefore provides a composition according to the i invention comprising: an agriculturally effective active ingredient selected from the group consisting of a plant growth regulating agent, herbicide, systemic fungicide, and a systemic insecticide; and an enhancer of spores, cultures, or suspensions of a suitable Bacillus or soil bacteria at a pH sufficiently less than 7 to maintain said enhancer in spore form, provided that the composition does not contain plant growth hormones when said plant growth regulating agent is a plant growth stunting agent. Preferably, the enhancer is free of plant growth hormones when used in combination with plant growth stunting agents, like mepiquat chloride, that suppress plant growth hormones in the treated plant.

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 been 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.

WO 98/23157 PCT/US96/19116 6 The invention provides a method for treating plants with a composition containing an agriculturally effective active ingredient and an enhancer containing a culture of a bacteria selected from the genus Bacillusor a soil bacteria in an amount sufficient to enhance the effectiveness of an agriculturally effective active ingredient applied simultaneously, before, or after application of the enhancer. The increased effectiveness attributable to the bacteria-containing enhancer component can be used to reduce the amount of applied agriculturally effective active ingredient or, when the active ingredient is applied at the same rate, the bacillus increasesthe effectiveness ofthe applied agriculturally effective active ingredient. Such increased effectiveness is useful for controlling weeds that are otherwise difficult to control with regular herbicides.

THE BACTERIA-CONTAINING ENHANCER The enhancer component contains spores, cultures, and suspensions of a bacteria from the genus Bacillus or a soil bacteria. Preferably, the bacteria for the enhancer component is in the form of spores as a result of a suitable adjustment in temperature, pH, salinity, etc.

Suitable bacteria for use in the present invention include those bacteria that exhibit an ability to increase the effectiveness of an agriculturally effectiveactive ingredient by any mechanism. Methods for screening bacterial strains for bioactivity and therefore their capacity to enhance the effectiveness of a plant growth regulator, a herbicide, a systemic fungicide, or a systemic insecticide will be apparent to one of ordinary skill in the art in.view of the disclosure and examples set forth herein. For-example, a candidate bacterial strain, such as a Bacillus cereus, can be cultured and maintained under standard laboratory conditions. (See Sambrook et alr"Molecular Cloning:, A Laboratory Manual" Cold Spring Harbor Laboratory press (1989)). Specificplants can be chosen for their susceptibility or resistance-to a particular WO 98/23157 PCT/US96/19116 7 agriculturally effective compound such as a herbicide or a plant growth regulator. For example, and as set forth in the Example section contained herein, cotton can be used to evaluate the bioactivity of a bacterial strain in combination with a plant growth regulator as reflected in the number of fruiting sites and/or the number of bolls produced and compared to treating similar plants with only the plant growth regulator or the bacteria.

Alternatively, plants susceptible to certain fungal diseases, such as tomato leaf blight, potato scab, wheat stem rust, corn smut, or leaf rust of various plant s- peciS-, can be grown under controlled conditions and treated either with an 1- appropriate fungicide, a fungicide with a candidate bacterial species, or the bacterial species alone, either before, concurrently, or subsequent to controlled inoculation of the plants With the-disease causal organism, to evaluate the capacity of the bacterial species to enhance the activity of the fungicide. These types of experiments can be carried out in the field, under semi-controlled conditions such as a greenhouse, or under relatively controlled conditions such as within a growth chamber. These experiments involve routine screening of organisms for their enhancement effect with compounds oi-compositions on known plant species, and the parameters used to evaluate the effects are likewise known and routinely measured.

These similar'types of screens can be utilized with bacterial strains described herein to provide an enhancement effect to a particular agriculturally effective active ingredient, the Bacillus cereus strain deposited with the American Type Culture Collection as ATCC 55675 providing an enhancement effect to the plant growth regulator mepiquat chloride and the herbicideatrizine, to screen the bacterial species for potential enhancement with other agriculturally effective active ingredients, such as systemic insecticides and systemic fungicides.

WO 98/23157 PCT/US96/19116 8 Particularly preferred are cultures, spores, and suspensions of soil bacteria encountered between and among the roots of thriving growths of the plant type in the local soil. Foliar application of the local soil bacteria at an application rate within the range from about 0.1 X 10 CFU/acre to about X 1010 CFU/acre enhances the natural mechanisms associated with plant growth and propagation to a level sufficient that the agriculturally active ingredient exhibits enhanced activity on or within the treated plant.

A wide variety of bacterial species within the genus Bacillus and within the known species of soil bacteria are useful within the present invention. The following is a list of species for the present invention: Bacteria from the genus Bacillus Bacillus acidocaldarius Bacillus acidoterrestris Bacillus alcalophilus Bacillus alvei Bacillus aminoglucosidicus Bacillus aminovorans Bacillus amyloliquefaciens Bacillus amylolyticus Bacillus anthracis Bacillus aneurinolyticus Bacillus apiarius Bacillus azotofixans Bacillus brevis Bacillus badius Bacillus capitovalis Bacillus cereus Bacillus. circulans WO 98/23157 -PCT/US96/19116 9 Bacillus cirroflagellosus Bacillus coagulans Bacillus colofoetidus Bacillus cycloheptanicus Bacillus epiphytus- Bacillus fastidiosus Bacillus filicolonicus Bacillus firmus Bacillu'skfeudenreidii Bacillus fructosus Bacillus globigii Bacillus globisporus Bacillus. insolitus Bacillus-laevolacticus Bacillus larvae- Bacillus laterosporus -Bacillus lautus Bacillus lentimorbus Bacillus lentus Bacillus lichenformis Bacillus macerans Bacillus macquari.ensis Bacillus maroccanus Bacillus macroides Bacillus medusa Bacillus megaterium -Bacillus mycoides Bacillus nato U WO 98/23157 WO 9/23157PCT[US96/19116-- -Bacillus nigrifcans Bacillus pabuli Bacillus pacificus Bacill us 7pantothenticus Bacillus parabrevis Bacillus pasteurfi Bacillus polymyxa Bacillus popilliae- Bacillus pulvifa Bacillus pulvifaciens Bacillus pumilus Bacillus prodigiosus Bacillus psychrophilus Bacillus psychrosaccharolyticus Bacillus racemilacticus Bacillus sphaericus Bacillus stearothermophilus Bacillus subtilis Bacillus therrnodenitrifi cans Bacillus thiaminolyticus *Bacillus thuringiensis Bacillus uniflagellatus Bacillus validus Soil Bacteria Achromobacterpesifer Alcaligenes eutrophus -Alcaligenes latus WO 98/23157 WO 9823157PCTIUS96/191 16 A mycolata autotrophica Archangium gephyra Arthrobacter viscosus Azotobacter chroococcum Bacillus acidovorans Bacillus brevis Bacillus cereus, Bacillus circulans Bacillus insolitus Bacillus laterosporus Bacillus megaterium Bacillus moj ave nsis Bacillus mycoides Bacillus pasteurii Bacillus poty~nyxa.

Bacillus psychrosaccharalyticus -Bacillus sphaericus Bacillus subtilis Bacillus viscosus Chainia hygroatrocyanea Clostridium absonum Cystobacterfuscus CJhPh0agajohnsonae Ens {fer adhaerens HIyphomicrobiumfacilis Micrococcus luteus Micromonospora chalcea Micromonospora coerulea Micromonospora grisea- WO 98/23157 PCT/US96/191 16 12 Micromonospora polytrota Microtetraspora glazuca Mycobacterium agri Mycobacterium aichiense Mycobacterium aurum- Mycobacterium chitae Mycobacterium ciwbuense Mycobacterium diernhoferi Mycobacteriumfortuitum Mycobacterium necaurum Mycobacterium parafortuitum Mycobacterium terrae Mycobacterium,.thermoresisti bile Myxococcus coralloides Myxococcusfulvus Myxococcus macrosporus.

-Myxococcus-xanthus Nannocystis exedens Ntrosolobus multiformis.

Nocardia brasiliensis Nocardio ides albus Ochrobactrum anthropi Polyangium cellulosum -Pseudomonas-aeruginosa Pseudomononasflutorescens Pseudomonas glathei Rahnella aquatilis Saccharobacterium acuminatum -Saccharomonospora viridis WO 98/23151^- WO 9823157PCTIUS96/191-16 Serratia marcescens Streptomyces anandii Streptomyces aureofaiciens Streptomyces chatreusis Streptomyces cyaneus -8-Stpeptomyces cymaro genes Streptomyces diastatochromo genes -Streptomycesflavogriseus -Streptomyces gelaticus Streptomyces hygroscopicus Streptomyces indigocolor Streptomyces katrae-- Streptomyces lipmanii Streptomyces ion gisporus Streptomyces massasporeus Streptomyces nobilis Streptomyce s odorifer Str eptomyces omiyaensis Streptomyces parvulus Streptomyces phaeochromo genes Streptomyces pseudo griseolus Streptomyces roseoflavus Streptomyces rubiginosohelvolus Streptomyces rut gersensis Streptonyces scierogranulatus Streptomyces toxytricini Streptomyces violaceoruber Streptomyces viola~ceus Streptomyces violarius WO 98/23157 WO 9823157PCTJUS96/191 16 14 Thermoactinomyces vulgaris Thiobacillus denitrtfi cans Thiobacillus (hi oparus non-fluorescent Pseudomonas Rhizobium Agrobacterium, Corynebacterium ureafaciens.

Arthrobacter ureafaciens Pseudomonas aeruginosa Bacillus fastidosus Micrococcus dentrifcans Mycobacterium phlei A erobacter aiero genes Fusarium moniliforme .Histoplasma cadpsulata Penicillinum chrysogenum Particularly useful are species of B. subtilis, B. cereus, and B.

Megaterium. Bacillus subtilis and B. cereus are naturally occurring soil saprophytes found throughout the-world. In the 1992 edition. of the American Type Culture Collection;- 182 different strains of B. subtilis are listed and i ncorporated herein by reference. The following is a l ist of B. subtilis that would be useful in the present invention: Subtilis ATCC 10783 ATCC 15818 ATCC 15819 ATCC 15245 (Bacillus natto).

WO 98/23 157 PCTJUS96/19116 ATCC 15134 (Bacillus uniflagellatus) ATCC 13542 ATCC 13472 ATCC 15575_ ATCC 27505 ATCC 21697 (Achromobacter nitriloclastes) ATCC 15811 ATCC 4925 (Bacillus nigrificans) ATCC 27370 ATCC 605 1a ATCC_7003, ATCC 11838 ATCC 15563 7ATCC 33234 ATCC 25369 ATCC 27689 ATCC 55033- ATCC 13933 ATCC 15244 ATCC 27328 ATCC 12695 ATCC 12100 ATCC 21554 -ATCC 21555 ATCC 15561 ATCC 15562 ATCC 9799 ATCC 12711 WO 98/231.57 PCTIUS96/191-16 16 ATCC 14593 ATCC 4944 ATCC 31002 ATCC 31004 ATCC 9943 -ATCC 13407 ATCC 7067 ATCC 29056 ATCC 31524 ATCC 31526 ATCC 21359 ATCC 21360 ATCC 13954 ATCC 13955.7 ATCC 15044 ATCC 33677- ATCC 3 1003 ATCC 31522 ATCC 465 ATCC 12432 ATCC 43223 ATCC*13952 ATCC 13953 ATCC 14662- ATCC 15039 ATCC 15040 ATCC 15041 ATCC 15042 WO 98/23157 WO 9823157PCTIUS96/191 16 ATCC 15043 ATCC 15181.

ATCC 15182 ATCC 15183 ATCC 15184 ATCC 21183 ATCC 21336 ATCC 49343 ATCC 6537 ATCC 21394 ATCC 8473 ATCC 31523 ATCC 31525 ATCC 31527 ATCC 29233 ATCC 14660 ATCC 14661 ATCC 31268 ATCC 4925.

ATCC 55405 ATCC 9524.

ATCC 15476 ATCC 23858 ATCC. 23859.

ATCC_7060 AT.CC 7058 ATCC 7059 ATCC 7480MkBaillus endoparaitcus).

WO098/23157 .PCTIUS96/19116 18 ATCC 21584 ATCC 31022 ATCC 21331 ATCC 21332_ ATCC 21777 ATCC 21778 ATCC 6598 (Bacillus licheniformis) ATCC 49822 ATCC 2-3857 ATCC 19221 ATCC 9858 ATCC 21742 ATCC 4529 ATCC 35148 ATCC 33608 ATCC 19549 ATCC 19550 ATCC 21556 ATCC 31340 ATCC 49760 ATCC 53325 ATCC 14807 ATCC 21228 -ATCC 15512 ATCC 15841 ATCC 10774 ATCC 31091 ATCC 31092 WO 98/23157 PCTIUS96/19116 19 ATCC 31094 ATCC 31096 ATCC 31097 ATCC 35946 ATCC 39374 ATCC 11774 ATCC 15116 ATCC 35021 ATCC 31954 ATCC 19062 ATCC 23059.

ATCC-53115- ATCC 15115 ATCC 13956 ATCC 21952 ATCC 82 ATCC 21603 ATCC 31785 ATCC 21697 ~ATCC 15477 ATCC31098 ATCC 19162 ATCC 14617 AT-CC 14618 .ATCC 33713 ATCC 33-714 ATCC 55422 ATCC 6461 WO 98/23157 PCT/US96/19116 ATCC 21007 ATCC 21770 ATCC 6984 ATCC 19163 ATCC 21663 ATCC 19217 ATCC 19219 ATCC 19220 ATCC 21005 ATCC 21006 A- preferred B. subtilis strain for use in the present invention includes GB03. Previously, B. subtilis GBO3 was recognized as a biological fungicide and commercially used as a seed treatment under the names KODIAKT HB or- GUS 2000T 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 10'0 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. This strain is said to colonize the developing root systems and. compete with disease organisms that would attack the roots. Foliar application is not listed.

The following is a list ofB. Cereus that would be useful in the present invention.

B. Cereus ATCC 55675 (BP01) ATCC 13824 NCIB 2600 (Bacillus cereus var. luorescens) ATCC 14603 ATCC 15816 WO 98/23157 WO 982315 PC-TIUS96/191 16-.

21 ATCC 15817 ATCC 13472 ATCC 14737 ATCC 9592 (Bacillus metiens).

ATCC 27877 ATCC 19637 ATCC 11950 ATCC 23261- -ATCC 6464 ATCC 11773 (Erwinia atroseptica).

ATCC 10876 ATCCl10987- ATCC 4342 (Bacillus lacticola).

ATCC 21182 ATCC 7004 (Bacillus albolactis).

ATCC 10702 ATCC 12480 ATCC 490 63 ATCC 2 (Bacillus agri) ATCC'19265 (Bacillus cereus subsp. alesti) ATCC 21634 ATCC 12826 ATCC 21768 ATCC 21769 ATCC 21771 ATCC 21772 ATCC 9139 ATCC 21928 WO-98/23157,-- PCTIUS96/19116 22 ATCC 27522 ATCC 31430 ATCC 43881 ATCC 246 (Bacillus lactimorbus).

ATCC 2177Q (Bacillus cereus).

ATCC 21929 (Bacillus cereus).

ATCC 13367- ATCC 31429 ATCC 31293 -ATCC 21366 (Bacillus coagulans) ATCC 25621 (Bacillus medusa) ATCC 7039 (Bacillus metiens) ATCC 14893 (Bacillus subtilis).

ATCC 9818 (Bacillus lactis) ATCC 33018 ATCC 33019 ATCC 55055 ATCC 53522 ATCC 1f3366 ATCC 7064 (Bacillus siamensis) ATCC-1 1949 ATCC 10792 (Bacillus cereus) ATCC 27348 -ATCC 23260 (Bacil1lus endorhythmos) ATCC 13061 ATCC 13062 (Bacillus megaterium) -ATCC 25972 ATCC .14579 WO 98/23157 PCT/US96/19116 23 ATCC 19646 ATCC 49064 ATCC 11778 ATCC 39152 ATCC 19146 FDA strain PCI 818 (Pseudomonas sp.) A preferred B. cereus strain for use in the present invention includes strain ATCC No. 55675. In another embodiment, the B. cereus strain for use in the present invention has the characteristics as ATCC No. 55675. In yet another embodiment, the B. substilis strain for use in the present invention has the 1o characteristics as ATCC No. 55675.

The,following: is-a4ist of B. megaierium that would be useful in the present inventions: B. Megaterium ATCC 33166 ATCC 33167 ATCC 33168 ATCC 33169 SATCC 12872 ATCC 33164 ATCC 11478 ATCC 33165 ATCC 11561 ATCC 11561a ATCC 11561b ATCC 11561c ATCC 11561d ATCC 11561e ATCC 6458 WO 98/23157 WO 98/3 157PCTfUS96/19116 24 ATCC 6459 ATCC 14946 ATCC 27327 ATCC 33729 ATCC 9885 ATCC 13639 ATCC 1363.2 ATCC 15374 ATCC 49099 ATCC 49096 ATCC 43725 A-TCC 19136 ATCC 25848 ATCC 4531 ATCC 13402 ATCC 15046 ATCC 15047 ATCC 25300 ATCC 19380 ATCC 13368 ATCC 21 181- ATCC'35985 ATCC 39383 ATCC 11562 _ATCC 15127 ATCC 15128 ATCC 7703 ATCC 14945.

WO 98/23157 WO 9823157PCTIUS96/191 16 ATCC 15177 -ATCC 19218-.

A-TCC 25833 ATCC- 15450 ATCC 19135 ATCC 19137 ATCC 21916- ATCC 8245_ ATCC 15781 ATCC 31294 ATCC 15117 ATCC 15118 ATCC 19160 ATCC 19161 ATCC 89 ATCC 21209 ATCC 35076 ATCC 49098.

ATCC 39118 ATCC 7051 ATCC- 7052 ATCC..10778 ATCC 8011 ~ATCC 7056.-.- ATCC 14581 ":--ATCC 13062 ATCC 49097 ATCC 19213 WO 98/23157 PCT/US96/19116 26 ATCC 71 ATCC 35075 ATCC 21603 ATCC 21738 ATCC 72 ATCC 21737 ATCC 49095 ATCC 15451 THE ACTIVE INGREDIENTS- Agriculturally effective active ingredients used in the present invention can be selected from awide. variety of materials that act on and through the metabolism of the treated plants. For premixed materials, it is desirable to select active ingredients that do not degrade or otherwise become impaired by extended storage at the conditions used to maintain the bacteria-containing enhancer component in a spore form.

The general term "plant growth regulating agent" encompasses a number-of active ingredients that affect a plant in different ways. Generally speaking, plant growth regulators encompass plant growth stunting agents, plant growth enhancing agents, and herbicides.

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 GA 9 being preferred; cytokinins zeatin, kinetin, benzyladenine, dihydrozeatin, and isopentenyl adenine); auxins indolacetic acid (IAA), indolebutyric acid (IBA), and naphthalenacetic acid sodium ortho-nitrophenoiate; sodium para-nitrophenolate; sodium nitro-guaicolate; and polyhydroxycarboxylic acids of 2, 4, 5, and 6 carbon structures; ethephon; and a variety of nitrogen or phosphorous-containing fertilizers.

WO 98/23157 PCT/US96/19116 27 Suitable plant growth stunting 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 timing specified by the manufacturer on the product label.

Herbicides include the triazines atrazine), the ureas, glyphosate, sulfosate, glyfosinate, and sethoxydim.

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,ltnter 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.

Plants that can be treated by the present invention include virtually any plant grown in soil and that is affected by an agriculturally effective active ingredient. Exemplary plants include commodity grain crops corn, wheat, and soybeans), sorghum, desired and undesired grasses, weeds, herbs,etc.

The invention is well suited to increased production of fruit in plants that produce fruiting sites from which fruit will grow. Such plants preferably include any of the raw agricultural commodity and especially cotton, soybeans, peanuts, grapes, apples, citrus lemons, limes, oranges, grapefruit), berries strawberries, blackberries, raspberries), tubers potatoes, WO 98/23157 PCT/US96/19116 28 sweet potatoes), corn, cereal grains wheat, rice, rye), tomatoes, onions, cucurbits watermelon, cucumbers, and cantaloupes).

METHOD OF USE The compositions of the present invention may take the physical form of a liquid, emulsion, suspension, solid granule, aggregate, or composite granule active ingredient solids carried on.an inert carrier particle).

Application of each physical form to plant foliage will generally proceed with conventional techniques.

Gram positive bacteria strains can be used in the enhancer component in the form of cells, spores, cultures, or suspensions thereof. In a liquid or dispersible solid form, the enhancer is added to a spray tank or other form of liquid 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 to make a composition that is applied to plant foliage at a rate within the range from about 0.1 X 1010 CFU/acre to about 100 X 10' 0 CFU/acre, preferably at a rate within the range from about 0.1 X 10'0 CFU/acre to about 10 X 1010 CFU/acre, and most preferably within the range from about 0.5 X 100 CFU/acre (0.5 fl. oz./acre of concentrate) to about 8 X 1010 CFU/acre (2 fl. oz./acre of concentrate).

Optionally and in a preferred embodiment, the spray tank will also contain the agriculturally effective active ingredient component for simultaneous application of both components.

Solid forms of the components can be dry mixed or formed into aggregates before broadcast. One or more of the conventional adjuvants may be used to enhance dispersion;,breakdown, adhesion to foliage, etc.

The specific application rate can vary somewhat depending on the method by which the solution is to be applied to the plant surfaces. For WO 98/23157 PCT/US96/19116 29 example, aerial spraying will employ a different dilution rate and application quantity than boom spraying, manual sprayers, or broadcast of granules.

Conventional equipment caibe used for the application. If desired, the enhancer component 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. So prepared, the enhancer is diluted to an amount in.the package container that is within the range from about 150,000 CFU/ml to about 600,000 CFU/ml and with conditions adequate to ensure that the bacterial component rtemaiis in a spore form but will become vegetative after application.

For many bacteria, use of a pH of less than 7 acidic) will maintain the bacteria in a spore form. If necessary, any oTthe 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 bacteria in the enhancer to become live, vegetative colonies. The bacteria will thereby reproduce on the treated plant surfaces and facilitate or translocation of the agriculturally effective active ingredient.

The bacteria-containing enhancer component can be applied as a discrete treatment or simultaneously with a variety of other agriculturally effective active ingredients. Useful agriculturally effective active ingredients include plant growth enhancing agents, plant growth stunting -agents, WO 98/23157 PCT/US96/19116 herbicides, systemic insecticides, and systemic fungicides. Preferably, the composition is a combination of either a plant growth stunting agent or herbicide and an enhancer containing a substantially pure strain of B. suibtilis, B. cereus, or ATCC 55675 (BP01) applied at the rate of at least 0.1 X 1010 CFU/acre.

In a particularly preferred embodiment, a gibberellin-free 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 the bacteria-containing enhancer (preferably ATCC_55675) and mepiquat chloride is a convenient method for simultaneously applying the components. If premixed, the mepiquat achoride and bacteria-containing enhancercan be stored readily at a pH within the range from about 4-6.5.,-most preferably within the range of about 5-6.5.

It should be noted that formulations according to the present invention desirably do 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 preferably 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 the bacteria-containing enhancer and mepiquat chloride on cotton, the bacteria appears to be affecting the plant WO 98/23157 PCT/US96/19116 31 growth mechanism to increase the retention of bolls on fruiting sites 1 and-2 and increasing the number of bolls overall by producing and retaining fruitingsites on normally vegetativebranches.

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 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 Sapplication 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 aiid 560,000 CFU/ml B. cereus (lab sample BP01, ATCC 55675) was prepared in a 1:1 volumetric ratio. This solution was applied by conventional sprayerinfour applications to the foliage of growing cotton.plantsin test areas of fields A 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 fluidounces per acre 11 days later--In all cases, the control fields were treated with the same rate ofonly mepiquat chloride.

WO 98/23157 PCT/UJS96/191-16 32 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 mepiqua-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 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 onlysquares 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.

S- Table 2 WO 98/23157 PCT/US96/19116 Field Sites Bolls Open Bolls Open Other Bolls Green A- Control 240 115 48- 73 270 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, 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 S the treated plants, 1796g. 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 ata 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 frombolls that never opened correctly and are low on the plant or from WO 98/23157 PCT/US96/19116 34 bolls just opening that are high on the plant. Table 3 reports the weight ofseed cotton and the number of green bolls per 10 foot of row in the treated and control fields.

Table 3 Field Seed cotton 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 an enhancer containing ATCC 55675 according to the invention produces higher cotton yields and healthier plants than use of mepiquat chloride alone.

Example 2 Bacillus cereus strain BP01 (ATCC 55675) was used in combination with a widely used herbicide, atrazine, to determine whether the BPO1 would affect control over weeds that are recognized as difficult to kill with triazines.

Atrazine is used to provide season-long control iln-orn, 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 ft. plots using a randomized complete block pattern. The weeds were 2-6 inches (5-15 cm) in height at thetime of treatment. Atrazine was applied from aqueoils solution at rates equivalent to either 1 or 2 pounds of active ingredient--" per acre. BP01 concentrate was added to the atrazine in an amount equivalent to either 0.5 or 1 fluid ounce per acre (0.5-1 X 100 CPU per acre). For WO 98/23157PCUS/116- .PCT-IUS96/19116-- 1 0* comparison, crop oil concentrate (about 85 paraffinic oil and about 17 surfactant) was used as is conventional with triazine hembicides to increase their efficacy. Tables 4-8 report the degree of control for Florida Pusley (Table 4), Bull Grass (Table Bermuda gr ass (Table Dog Fennel (Table and Primrose (Table 8).

Table 4 Florida Pusley Control After Treatment Treatnitj 4 days j 9 days J16 days (23 days Cntr 0 0 0 0 Atrazine (1 lb.) -3 33 42 Atrazine (2 lb.) 17 57 72 83 Atrazine (1 Ib) crop oil conc. (1 6 68 68 Atrazine (1 Ib) 0.5 oz. BPO1 27 43 60 Atrazine (I Ib) 1 oz. BPO1 22 53 65 67 Table 5 gull Grass T Control-After Treatmen t Treatment 4 days ]9 days J16 days 23 days Control 0- 0 0 0 Atrazine (I lb.) -10 40 40 -Atrazine (2 lb.) 25 70 80 Atrazine (1 Ib) crop oil conc. (I gal.) 25 -40 65- Atrazine (1 Ib) 0.5 oz. BPO1 35 40 55 63 *Atrazine (1 lb) I oz BPO1 30 60 80 Table 6. Bermuda grass7 %-Control After Treatment j Treatment 4 days 9 days 16 days 23 days] Control 0 0 0 0 WO-98/23107 PCTIUS96/19116 1o Atrazine (1 lb.) 0 10 15 Atrazine (2 lb.) 8 15 22 27 Atrazine (1 lb) crop oil cone. (1 gal.) 25 27 30 43 Atrazine (1 lb) oz. BP01 7 20 37 68 Atrazine (1 1 oz. BP01 10 23 53 63 Table 7 Dog Fennel Control After Treatment STreatment -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. Atrazine (1 lb) crop oil conc. (1 gal.) 25 75 93 98 Atrazine (1 lb) 0.5 oz. BPO1 33 88 96 99 Atrazine (1 lb) 1 oz. BP01 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 Atrazine (2 lb.) 30 65 70 Atrazine (1 lb) crop oil conc. (1 gal.) 20 83 88 93 Atrazine (1 lb) 0.5 oz. BP01 20 60 -72 88 Atrazine (1 lb) 1 oz. BP01 23 70 65 From Tables 4-8, it can be seen that BPO1 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 WO 98/23157 PCT/US96/19116 37 atrazine with BP01 was the control rate reduced relative to the 2 lb/acre treatment with atrazine..

-Similarly, the BP01 also improved the control rate of atrazine relative to a mixture 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.

Claims (2)

1. 55675. 24 A method for enhancing the effectiveness of an agriculturally effective active ingredient by applying to a plant: an agriculturally effective active ingredient of a plant growth stunting agent, CD/01145003.0 41 ethephon, or a herbicide; and an enhancer of spores, cultures, or suspensions of a suitable Bacillus or soil bacteria. The method according to claim 24 wherein said agriculturally effective active ingredient is a triazine, glyphosate, or sulfosate. 26 The method of claim 24 wherein said active ingredient is mepiquat chloride or chlormequat chloride. 27 The method of any one of claims 24 to 26 wherein said enhancer contains a strain of bacteria from the genus Bacillus. 10 28 The method of any one of claims 24 to 26 wherein said enhancer contains a strain of soil bacteria. 29 The method of any one of claims 24 to 26 wherein said enhancer contains a B. I cereus. S* 30 The method of any one of claims 24 to 26 wherein said enhancer contains a B. S" 15 cereus having the characteristics of ATCC 55675. 31 The method of any one of claims 24 to 26 wherein said enhancer contains a B. subtilis. 32 The method of any one of claims 24 to 26 wherein said enhancer contains a B. subtilis having the characteristics of ATCC 55675. 33 The method of any one of claims 24 to 26 wherein said enhancer contains B. megaterium. 34 The method of any one of claims 24 to 26 wherein said enhancer contains ATCC R N 55675. CD/003840840v02.doc 42 A composition comprising a Bacillus in spore, culture or suspension form and a plant growth stunting agent, wherein the composition does not contain ATCC 55675. 36 The composition of claim 35 wherein said plant growth regulating agent comprises mepiquat chloride. 37 The composition of claim 35 wherein said plant growth regulating agent comprises chlormequat chloride. 38 The composition of any one of claims 35 to 37 wherein the Bacillus is a B. cereus. The composition of any one of claims 35 to 37 wherein the Bacillus is a B. subtilis. 40 The composition of any one of claims 35 to 37 wherein the Bacillus is a B. megaterium. 41 A composition for enhancing the activity of an agriculturally effective active ingredient according to claim 1, substantially as described herein with reference to the examples. 15 42 A nonliquid composition according to claim 13, substantially as described herein with reference to the examples. 43 A method for increasing the number of fruiting sites on a fruiting plant according to claim 16, substantially as described herein with reference to the examples. 44 A method for enhancing the effectiveness of an agriculturally effective active ingredient according to claim 24, substantially as described herein with reference to the examples. A composition according to claim 35, substantially as described herein with reference to the examples. Micro Flo Company By its Registered Patent Attorneys Freehills Carter Smith Beadle
2. 2 August 2001