WO2007008580A1

WO2007008580A1 - Increasing plant drought and cold resistance: aba + triazole

Info

Publication number: WO2007008580A1
Application number: PCT/US2006/026320
Authority: WO
Inventors: James Zhang; Joshua Armstrong; Neal Gutterson
Original assignee: Mendel Biotechnology, Inc.
Priority date: 2005-07-08
Filing date: 2006-07-06
Publication date: 2007-01-18

Abstract

The present application provides methods of imparting increased stress resistance on a plant by application of exogenous ABA and one or more triazole compounds, diniconazole, uniconazole and uniconazole-P.

Description

INCREASING PLANT DROUGHT AND COLD RESISTANCE:

ABA + TRIAZOLE

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/698,285, filed July 8, 2005, and U.S. Provisional Patent Application No. 60/739,231, filed November 23, 2005, the disclosures of which are hereby incorporated herein by reference in their entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention provides methods of imparting increased stress resistance, reduced foliar growth, and enhanced root growth on a plant by application of exogenous ABA and a triazole, including diniconazole, uniconazole and uniconazole-P.

BACKGROUND OF THE INVENTION

[0003] It is known that exogenous application of ABA results in increased cold and drought tolerance in plants. However, it has not been possible to use ABA in agricultural and horticultural settings because of its expense and rapid turn-over inplanta (Cutler and Krochko, (1999) Trends Plant Sd 4:472; Nambara and Marion-Poll, (2005) Annu Rev Plant Biol 56:165-185). The recent identification of fungi that produce ABA in large quantities through fermentation has considerably reduced the cost of ABA. However, ABA instability inplanta remains an obstacle to commercialization. ABA is catabolized inplanta into inactive forms by conjugation or through the predominant oxidative pathway. The oxidation reaction, catalyzed by a P450 hydroxylase, is initiated by the hydroxylation of ABA at the C-8' position to produce 8'-hydroxy ABA which spontaneously isomerizes to phaseic acid (PA) (Cutler and Krochko, supra; Nambara and Marion-Poll, supra. Thus, prolonging the half-life of ABA inplanta can be achieved in at least two ways - using analogs of ABA that are more resistant to oxidation instead of the natural ABA, or co-application with natural ABA (or ABA analogs degradable by the P450 8 '-hydroxylase) of a second compound that inhibits the P450 8 '-hydroxylase. An ABA analog such as 8'-methlene ABA that is oxidized more slowly while retaining bioactivity is an example of the first strategy (Abrams et al, (1997) Plant Physiol 114:89-97). This invention relates to the second strategy. Some nonspecific inhibitors of the P450s, such as tetcyclacis, also inhibit the ABA 8 '-hydroxylase (Krochko et al, (1998) Plant Physiol 118:849-860; Kushiro et al, (2004) EMBO J 23:1641- 1656). However, there are mixed reports on its efficacy (Churchill et al., (1998) Plant Growth Reg 25:35-45). More recently, diniconazole, a triazole fungicide that inhibits the fungus C14-demethylase in sterol biosynthesis (also a P450 enzyme) has been shown to be an even better inhibitor of the ABA 8 '-hydroxylase (Kitahata et al., (2005) BioorgMed Chem 13:4491-4498). We reason that such inhibitors will prolong the half life of ABA, and therefore maintain the desired effects of ABA treatment, inplanta. Furthermore, novel compounds can be identified in novel chemical genetics screens, and used with ABA to achieve the aforementioned benefits.

[0004] Accordingly, there remains a need to improved methods of imparting stress tolerance to a plant. This invention fulfills this and other needs.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention provides a method of imparting increased stress tolerance on a plant. The invention further provides methods of improving plant performance, for example, by reducing foliar growth, enhancing root growth, enhancing foliar color and/or enhancing fruit ripening.

[0006] Generally, the methods comprise contacting the plant with exogenous abscisic acid (ABA) and one or more triazole compounds of Formula I:

wherein the dashed line indicates an optional double bond, n is from 0 to 5 and R¹ is selected from the group consisting of alkyl, heteroalkyl, aryl, heteroaryl, wherein said aryl and heteroaryl groups are optionally substituted with from 1-5 Y substituents, and each X and each Y are independently selected from the group consisting of alkyl, alkoxy, heteroalkyl, halogen and cyano.

[0007] Li one embodiment, the triazole compound is at least one of diniconazole, uniconazole, and uniconazole-P.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figures IA-D illustrate how 4-40 μM diniconazole (D) and exogenous ABA (0.040- 0.400 nM) synergistically up-regulate the Rd29a promoter. Seedlings were germinated in 96-well plates and grown for 5 days prior to the addition of ABA and diniconazole.

[0009] Figure 2 illustrates how diniconazole (D) and exogenous ABA reduce growth and darken foliar tissue in Arabidopsis.

[0010] Figure 3 illustrates how diniconazole (D) and exogenous ABA synergistically enhance drought tolerance.

[0011] Figure 4 illustrates how 20 μM diniconazole (D) or tetcyclacis (T) display synergy with exogenously added ABA (1.25 μM) to up-regulate the Rd29a promoter. Nine day old seedlings (22⁰C, 24h 95μMol/m2/s) were transplanted to 24-well plates with media containing the indicated concentration of compounds. After an additional 3 days of growth, the plate was analyzed for GFP and chlorophyll fluorescence. Scan positions with a chlorophyll (CHL) level above the threshold were used to generate an average (GFP/CHL) value for each well. The average, per- well GFP/CHL metrics were used to calculate fold induction values relative to untreated seedling wells.

[0012] Figure 5 illustrates how 5μM and 20μM diniconazole (D) displays synergy with exogenously added ABA (0.4 μM) to up-regulate the Rd29a promoter. Nine day old seedlings (22°C, 24h 95μMol/m2/s) were transplanted to 24-well plates with media containing the indicated concentration of compounds. After an additional 3 days of growth, the plate was analyzed for GFP and chlorophyll fluorescence. Scan positions with a chlorophyll (CHL) level above the threshold were used to generate an average (GFP/CHL) value for each well. The average, per-well GFP/CHL metrics were used to calculate fold induction values relative to untreated seedling wells. [0013] Figures 6A and 6B illustrate how lOμM diniconazole (D) and exogenously added ABA (10 μM) retard plant growth, darken foliar tissue, increase stem thickness and enhance root growth in turf grass.

[0014] Figure 7 illustrates that the combination of uniconazole and exogenous ABA act synergistically to inhibit turf canopy elongation.

[0015] Figure 8 illustrates that the combination of uniconazole and exogenous ABA act synergistically to enhance root elongation and branching.

[0016] Figure 9 illustrates that the combination of diniconazole and exogenous ABA act synergistically to enhance root elongation and branching.

[0017] Figure 10 illustrates the results of a drought assay showing synergy between exogenous ABA and diniconazole, uniconazole or uniconazole-P.

DETAILED DESCRIPTION

Abbreviations and Definitions

[0018] "Contacting," "applying," "application" interchangeably refer to any mode of delivering a combination of exogenous ABA and one or more triazole compounds to a plant, including spraying, wiping, soaking, immersing. A combination of exogenous ABA and one or more triazole compounds can be applied to a whole plant, one or more plant parts (i.e., roots, stems, leaves, etc.), or to the soil surrounding the plant.

[0019] As used herein, "stress" or "stressor" refers to any noxious stimuli on a plant and the subsequent protective response of the plant. Oftentimes, such noxious stimuli will initiate cellular signaling pathways that result in transcription of stress response genes, including Rd29a. Exemplified stressors or noxious stimuli include, heat, dehydration, wind, freezing, temperatures above or below the viability range of a particular plant, soil salinity levels above or below the viability range of a particular plant, exposure to substances toxic to a particular plant, etc.

[0020] As used herein, "tolerance" or "resistance" to stress refers to the ability of a plant to withstand exposure to a noxious stimuli. Increased tolerance or resistance can be measured by, for example, increased viability of a population of plants treated according to the present methods in comparison to untreated plants; increased viability of one or more plants over an extended period of time; increased viability of one or more plants to higher levels of one or more noxious stimuli.

[0021] As used herein, "viability" of a plant refers to both whether the plant is living and the health of a living plant.

[0022] The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e. C_1-8 means one to eight carbons). If the number of carbon atoms is not indicated, the alkyl group will have from 1-8 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "cycloalkyl" refers to hydrocarbon rings having the indicated number of ring atoms (e.g., C_3-6cycloalkyl) and being fully saturated or having no more than one double bond between ring vertices. "Cycloalkyl" is also meant to refer to bicyclic and polycyclic hydrocarbon rings such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc.

[0023] The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by — CH₂CH₂CH₂CH₂ — . Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having four or fewer carbon atoms.

[0024] The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. Additionally, for dialkylamino groups, the alkyl portions can be the same or different and can also be combined to form a 3-7 membered ring with the nitrogen atom to which each is attached. Accordingly, a group represented as — NR^aR^b is meant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like. [0025] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C_1-4 haloalkyl" is mean to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0026] The term "aryl" means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon group which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) that contain from one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl, while non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5- oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2- furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, benzopyrazolyl, 5-indolyl, 1-isoquinolyl, 5- isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

[0027] For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like).

[0028] The above terms (e.g., "alkyl," "aryl" and "heteroaryl"), in some embodiments, will include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below. For brevity, the terms aryl and heteroaryl will refer to substituted or unsubstituted versions as provided below, while the term "alkyl" and related aliphatic radicals is meant to refer to unsubstituted version, unless indicated to be substituted.

[0029] Substituents for the alkyl radicals (including those groups often referred to as alkylene, alkenyl, alkynyl and cycloalkyl) can be a variety of groups selected from: -halogen, —OR', -NR¹R", -SR¹, — SiR'R"R"', -OC(O)R', -C(O)R', -CO₂R', — CONR'R", -OC(O)NR¹R", -NR¹¹C(O)R', — NR'- C(O)NR¹¹R"¹, — NR¹¹C(O)₂R', — NH- C(NH₂)=NH, — NR'C(NH₂)=NH, — NH- C(NH₂)=NR', -S(O)R¹, -S(O)₂R', -S(O)₂NR¹R", -NR¹S(O)₂R", — CN and — NO₂ in a number ranging from zero to (Im'+l), where m' is the total number of carbon atoms in such radical. R¹, R" and R¹¹' each independently refer to hydrogen, unsubstituted C_1-8 alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C_1-8 alkyl, C_1-8 alkoxy or C_1-8 thioalkoxy groups, or unsubstituted aryl-C^ alkyl groups. When R¹ and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, — NR¹R" is meant to include 1-pyrrolidinyl and 4-morpholinyl.

[0030] Similarly, substituents for the aryl and heteroaryl groups are varied and are generally selected from: -halogen, —OR', -OC(O)R¹, -NR¹R", -SR¹, — R¹, -CN, -NO₂, -CO₂R¹, -CONR¹R", -C(O)R¹, -OC(O)NR¹R", -NR¹¹C(O)R¹, -NR¹¹C(O)₂R', — NR¹- C(O)NR¹¹R"¹, — NH- C(NH₂)=NH, — NR'C(NH₂)=NH, — NH- C(NH₂)=NR', -S(O)R¹, -S(O)₂R¹, -S(O)₂NR¹R", -NR¹S(O)₂R", -N₃, ρerfluoro(Ci-C₄)alkoxy, and perfmorotd- C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R¹, R" and R"¹ are independently selected from hydrogen, C_1-8 alkyl, C_3-6 cycloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-C_1-4 alkyl, and unsubstituted aryloxy-Q.₄ alkyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene tether of from 1-4 carbon atoms.

[0031] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O) — (CH₂)_q — U — , wherein T and U are independently — NH — , — O — , — CH₂ — or a single bond, and q is an integer of from O to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH₂)_r — B — , wherein A and B are independently — CH₂-, —0—, -NH-, -S-, — S(O)-, -S(O)₂-, -S(O)₂NR¹ — or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula — (CH₂)_S — X — (CH₂)_t , where s and t are independently integers of from O to 3, and X is —0—, — NR¹- , -S-, -S(O)-, -S(O)₂-, or -S(O)₂NR'-. The substituent R' in — NR'- and -S(O)₂NR'- is selected from hydrogen or unsubstituted C_1-6 alkyl.

[0032] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

General

[0033] The present invention provides methods for the combined application of externally added ABA (or ABA analogs) and ABA degradation inhibitors, particularly triazoles, including diniconazole, uniconazole (i.e., racemic uniconazole), and uniconazole-P (i.e., pure active enantiomeric uniconazole), to plants or plant parts in order to achieve 1) freezing tolerance, 2) drought tolerance and 3) slowed growth of those plants. Surprisingly, the combined application of exogenously added ABA and one or more triazole compounds, including diniconazole, uniconazole, and uniconazole-P, synergize to impart increased stress resistance to a plant. One embodiment of the methods involves the combined application of ABA and diniconazole to turf so as to reduce frequency of mowing, reduce water use, and enhance tolerance to cold temperatures and general stresses. The chemicals can be applied in a liquid formulation, e.g., through irrigation water, or in a solid formulation, e.g., a slow- release formulation activated for release by irrigation. Any known or yet to be discovered compounds that inhibit ABA 8 '-hydroxylase activity can be used in conjunction with ABA to achieve the claimed benefits. For example, the known ABA 8 '-hydroxylase inhibiting compounds tetcyclacis (a norbornane compound) and diniconazole (a triazole compound) have very different chemical structures. This invention is also a method by which novel compounds acting synergistically with externally applied ABA can be discovered.

Description of the Embodiments

[0034] In a first aspect, the present invention provides a method of imparting increased stress tolerance on a plant. The invention further provides methods of improving plant performance, for example, by reducing foliar growth, enhancing root growth, and/or enhancing foliar color. The methods generally enhance the naturally occurring actions of ABA on a plant, including for example, enhancing storage reserve accumulation (i.e., seed storage protein, lipid, and long chain fatty acid accumulation); enhancing abscission of leaves, buds, petals, flowers and fruits as well as dehiscence of fruits; inducing or prolonging bud and/or seed dormancy; enhancing elongation growth, including tuberization and flowering; enhancing stomatal closure; enhancing root growth (i.e., elongation, lateral root development, geotropism, water uptake, ion transport); and enhancing fruit ripening.

[0035] Generally, the methods comprise contacting the plant with exogenous abscisic acid (ABA) and one or more triazole compounds encompassed by Formula I:

wherein the dashed line indicates an optional double bond, the wavy line indicates either rectus (R) or sinister (S) stereochemistry, n is from 0 to 5 and R¹ is selected from the group consisting of alkyl, heteroalkyl, aryl, heteroaryl, wherein said aryl and heteroaryl groups are optionally substituted with from 1-5 Y substituents, and each X and each Y are independently selected from the group consisting of alkyl, alkoxy, heteroalkyl, halogen and cyano. In some embodiments, the one or more halogen substituents are independently selected from the group consisting of Br, Cl, F, and I. In some embodiments, the alkoxy has from 1-5 carbon atoms. For example, the alkoxy can be a methoxy or an ethoxy.

[0036] Generally, the methods impart tolerance to stressors on a plant, including freezing temperatures, chilling temperatures, drought, and soil salinity that is abnormally high or abnormally low, particularly high soil salinity. [0037] In one embodiment, the triazole compound is at least one of diniconazole ((E)-I - (2,4-Dichlorophenyl)-4,4-dimethyl-2-(l,2,4-triazol-l-yl)-l-penten-3-ol), uniconazole (IH- 1 ,2,4-Triazole- 1 -ethanol, β-((4-chloroρhenyl)methylene)-α-(l ,1 -dimethylethyl)-, (E)-(+-)-), and uniconazole-P. In some embodiments, the methods are carried out by applying an exogenous ABA and diniconazole. In one embodiment, the methods are carried out with the proviso that the triazole compound is other than uniconazole. In a related embodiment, the methods include applying to a plant exogenous ABA and one or more triazole compounds selected from the group consisting of azaconazole (l-[[2-(2,4-dichlorophenyl)-l,3-dioxolan- 2-yl]methyl]-l H-1 ,2,4-triazole), cyproconazole (α-(4-chloroρhenyl)-α-(l -cyclopropylethyl)- lH-l,2,4-triazole-l-ethanol), difenoconazole (l-[2-[2-chloro-4-(4-chlorophenoxy)phenyl]-4- methyl-l,3-dioxolan-2-ylmethyl]-lH-l,2,4-triazole), diniconazole, epoxiconazole (rel-1- [[(2R,3S)-3-(2-chloroρhenyl)-2-(4-fluorophenyl)oxiranyl]methyl]-lH-l,2,4-triazole), fenbuconazole (α-[2-(4-chlorophenyl)ethyl]-α-phenyl-lH-l,2,4-triazole-l-propanenitrile), imazalil (l-[2-(2,4-dichlorophenyl)-2-(2-propenyloxy)ethyl]-lH-imidazole), ipconazole (2- [(4-chlorophenyl)methyl]-5-(l-methylethyl)-l-(lH-l,2,4-triazol-l-ylmethyl)cyclopentanol), myclobutanil (α-butyl-α-(4-chlorophenyl)- IH- 1 ,2,4-triazole- 1 -propanenitrile), tetraconazole (l-[2-(2,4-dichlorophenyl)-3-(l,l,2,2-tetrafluoroethoxy)propyl]-lH-l,2,4-triazole), trifiumizole (1-[(1E)-I -[ [4-chloro-2-(trifluoromethyl)phenyl]imino]-2-proρoxyethyl] - 1 H- imidazole), triticonazole ((5E)-5 -[(4-chlorophenyl)methylene] -2,2-dimethyl- 1-(1H- 1,2,4- triazol-l-ylmethyl)cyclopentanol), uniconazole, and uniconazole-P.

[0038] In some embodiments, the methods include applying to a plant exogenous ABA and a combination of diniconazole and uniconazole or uniconazole-P. This combination is advantageous to additionally impart enhanced drought tolerance and inhibit foliar growth. The tri-compound application provides a means to inhibit foliar growth via interference with two separate plant hormone pathways. Uniconazole acts to inhibit both the 8 '-hydroxylase and related cytochrome P450 enzymes that direct gibberellic acid (GA) biosynthesis. The plant hormone GA regulates shoot elongation and a reduced endogenous level slows foliar development. The simultaneous inhibition of GA biosynthesis and ABA degradation (with a concomitant application of exogenous ABA) will increase general stress tolerance while simultaneously retarding growth, a desirable combination for highly maintained crop species such as turf grass.

[0039] Typically, the diniconazole is applied to the plant at a concentration of about 0.04 μM to about 2000 μM or higher, for example, about 0.04, 0.05, 0.06, 0.07, 0.08, 0.10, 1.0, 2, 4, 8, 10, 15, 20, 40, 80, 10O₅ 200, 500, 1000, 2000, 3000 μM. Those of skill in the art can also prepare formulations for use in the present methods according to "parts per million" (ppm) units, usually from about 0.001 ppm to about 1000 ppm or higher, for example, about 0.001, 0.01, 0.1, 1.0, 2, 4, 8, 10, 15, 20, 40, 80, 100, 200, 500, 1000 ppm. Uniconazole or uniconazole-P can applied to the plant at a concentration of about 1.0 μM to about 2000 μM or higher, for example, about 1.0, 2, 4, 8, 10, 15, 20, 40, 80, 100, 150, 170, 200, 500, 1000, 2000, 3000 μM. Those of skill in the art can also prepare triazole formulations for use in the present methods according to "parts per million" (ppm) units, usually from about 0.001 ppm to about 1000 ppm or higher, for example, about 0.001, 0.01, 0.1, 1.0, 2, 4, 8, 10, 15, 20, 40, 80, 100, 200, 500, 1000 ppm. The exogenous ABA is usually applied to the plant at a concentration of about 0.04 μM to about 2000 μM or higher, for example, about 0.04, 0.05, 0.06, 0.07, 0.08, 0.10, 1.0, 2, 4, 8, 10, 15, 20, 40, 80, 100, 200, 500, 1000, 2000, 3000 μM. ABA can also be prepared in concentrations of "parts per million" (ppm) units, usually from about 0.001 ppm to about 1000 ppm or higher, for example, about 0.001, 0.01, 0.1, 1.0, 2, 4, 8, 10, 15, 20, 40, 80, 100, 200, 500, 1000 ppm.

[0040] In carrying out the methods, the combination of exogenous ABA and the one or more triazole compounds can be applied to the whole plant, a plant part (i.e., leaves, stems, roots), or the soil surrounding the plant, in either a liquid formulation or solid formulation. Usually, the combination of ABA and the triazole compound is applied to the whole plant, a plant part, or the soil surrounding the plant in a liquid formulation, either an aqueous solution or an organic solution, as appropriate. In some embodiments, the exogenous ABA and triazole compound are applied to the whole plant, a plant part, or the soil surrounding the plant in a solid formulation, for example, as solid pellets. In one embodiment, the solid pellets deliver the combination of exogenous ABA and triazole compound in a solid timed- release or sustained-release formulation. Timed-release or sustained-release formulations of potential use in the present methods for delivery of ABA and triazole compounds to a plant over an extended period of time are described, for example, in U.S. Patent Nos. 6,903,053; 6,900,162; 6,890,888; 6,858,634 and 6,746,684, each of which is hereby incorporated herein by reference.

[0041] In some embodiments, the exogenous ABA and the one or more triazole compounds are applied to the plant simultaneously. In other embodiments, the exogenous ABA and the one or more triazole compounds are applied to the plant sequentially. For example, the ABA can be applied first and the one or more triazole compounds can be applied subsequently. Alternatively, the one or more triazole compounds can be applied first, and the ABA can be applied subsequently.

[0042] The ABA and the one or more triazole compounds can be applied to the plant before or after the onset of the stress. Increased tolerance to stress can be imparted by application of ABA and one or more triazole compounds 1 day, 2 days, 3 days, 4 days, or even 5 days after the onset of the stress. The combination of ABA and one or more triazole compounds can be applied to a plant continuously, intermittently, or on an "as needed" basis to counteract one or more stressors. For example, the combination of ABA and one or more triazole compounds can be applied to a plant continuously or intermittently over a period of 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or longer, as needed. The combination of ABA and one or more triazole compounds can be applied to a plant every 24, 48, 72, or 96 hours, once weekly or less often, as needed. The concentrations and ratios of ABA and the one or more triazole compounds can be the same or varied (increased or decreased), as appropriate, over the course of treatment using the present methods.

[0043] The methods find use in imparting increased tolerance to stress in any kind of plant subject to one or more stressors, e.g., plants that reside indoors or outdoors, plants grown in inhospitable environments subject to chilling or freezing temperatures, inadequate water, and/or soil with abnormally high salinity. The methods find particular use in treating ornamental and amenity plants (both annuals and perennials), including but not limited to, azaleas, carnations, chrysanthemum, geraniums, gerberas, hydrangea, impatiens, pelargonium, petunia, poinsettia, rhododendrons, roses, ornamental solanum or tabacum varieties, turf grasses, as well as crop plants, for example, grain grasses {e.g., wheat, rye), corn, soybeans, cotton, sunflowers, Brassica species, grape vines, strawberry, grasses and vegetables, including but not limited to, beans, cucumbers, lettuce, melons, onion, peas, pepper, spinach, squash, and tomato. In one embodiment, exogenous ABA and one or more triazole compounds are applied to a turf grass, for example, either a warm season grass or a cool season grass. Warm season grasses suitable for treatment by the present methods include, for example, Bahiagrass, Bermudagrass, Blue gamma grass, Buffalo grass, Carpetgrass, Centipedegrass, Kikuyugrass, Seashore paspalum, Sideoates grama grass, St. Augustinegrass and Zoysiagrass. Cool season grasses suitable for treatment by the present methods include, for example, Bluegrass (Annual, Canada, Kentucky, Rough), Bentgrass (Colonial, Creeping, Velvet), Ryegrass (Annual, Perennial), Fescue (Chewings, Hard, Red, Sheep, Tall), Wheatgrass (Crested, Fairway, Western), Orchardgrass, Redtop grass, Smooth bromegrass, Timothy grass, and Weeping alkaligrass.

[0044] Those of skill in the art will appreciate that other nitrogen heterocyclic compounds will find use in the present methods, including, for example, pyrimidine and imidazole compounds. Exemplified pyrimidine compounds of use in the present methods include ancymidol (α-cyclopropyl-α-(4-methoxyρhenyl)-5-pyrimidinemethanol), fenarimol (α-(2- chlorophenyl)-α-(4-chlorophenyl)-5-pyrimidinemethanol), flurprimidol (α-(l -methylethyl)-α- [4-(trifiuoromethoxy)phenyl]-5-pyrimidinemethanol) and triarimol (α-(2,4-dichlorophenyl)- α-phenyl-5-pyrimidinemethanol). Exemplified imidazole compounds, include, for example, oxpoconazole (2-[3-(4-chlorophenyl)propyl]-3-(lH-imidazol-l-ylcarbonyl)-2,4,4- trimethyloxazolidine), prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-lH- imidazole-1-carboxamide) and triflumizole (l-[(lE)-l-[[4-chloro-2- (trifluoromethyl)phenyl]imino]-2-propoxyethyl] - 1 H-imidazole) .

Application Methods

[0045] A combination of exogenous ABA and one or more triazole compounds can be used in accordance with the invention with additional plant growth regulators, for example, chemical thinning agents.

[0046] As noted above, a variety of application methods can be employed to apply a combination of exogenous ABA and one or more triazole compounds. For example, by spraying a solution or suspension of the combination to whole plants, or plant parts (leaves, stems, roots, etc.) or by application to seeds, or roots or bulbs, corms or rhizomes (i.e., the soil around the plant), together with a suitable carrier, for example, for slow release. The addition of conventional adjuvants such as wetting agents and dispersants may prove to be beneficial in some agronomic situations. Examples of the effects on turf grass are the following: slowed growth, increased greenness, darkened foliar tissue, enhanced root growth, and enhanced tolerance to drought and cold. The increased tolerance to stress can be evaluated by comparing one or more plants before and after treatment with the present methods, or by comparing one or more plants receiving treatment with one or more plants that are not receiving treatment. Evaluation can be made by visual inspection or by measuring, for example, the change in leaf or root length, stem or root thickness, foliar color, viability, growth rate, etc. An increased tolerance to stress exposure is realized when a change can be determined by visual inspection or quantified, for example, by determining a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 1-fold, 2-fold, 3-fold or greater change of any of the parameters being quantified.

[0047] The agents according to the invention are also suitable for the general control and inhibition of undesired vegetative growth, such as the formation of sideshoots, without destroying the plants.

[0048] The mixing ratios of ABA and the one or more triazole compounds can vary within wide limits, for example between 250:1 and 1 :250. The choice of the mixing ratio depends on the type of components in the mixture, on the stage of development of the plants and on the desired degree of growth-regulating activity. Usually, mixing ratios of from 10:1 to 1:10 are chosen. The application rate of the compounds of the mixtures of active ingredients is in general between 5 and 5000 g per acre.

[0049] The agents according to the invention can either be formulations consisting of mixtures of the components (wettable powders or emulsion concentrates), which are then brought to use after dilution with water in a conventional manner, or be prepared in the form of so-called tank mixtures by diluting the separately formulated components with water. Wettable powders are preparations which can be dispersed homogeneously in water and which, in addition to the active ingredients and a diluent or inert substance, contain wetting agents, for example polyoxyethylated alkylphenols, polyoxyethylated oleylamines or stearylamines or polyoxyethylated fatty alcohols, alkylsulfonates or alkylphenylsulfonates, and dispersants, for example, sodium ligninsulfonate, sodium dinaphthylmethanedisulfonate, sodium dibutylnaphthalenesulfonate or sodium oleylmethyltaurate

EXAMPLES

[0050] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1. Diniconazole and ABA synergistically activate the Rd29a promoter.

Growth conditions

[0051] Transgenic Arabidopsis seedlings harboring the transcriptional fusion RD29a::GFP were arrayed in 96-well, flat-bottom polystyrene plates at a density of 5-10 seedlings/well in growth media (50% MS/B5, 0.05% MES (pH 5.7), 0.5% sucrose, 0.1% agar). After 5 days of growth at 25°C under continuous light (95μMol/m2/s), various concentrations of both abscisic acid (AB A, DMSO stocks, 1 μL/well) and diniconazole (D, DMSO stocks, 1 μL/well) were added with gentle mixing. The plates were returned to the growth chamber and GFP fluorescence levels were monitored every 24 hours post-addition using the Beckman-Coulter DTX800 microplate spectrofluorometer (area scan method; 3X3 grid).

Data Analysis

[0052] The fluorescence levels plateaued by the third day post-addition (Fig. IA) and all analysis was performed using the final GFP fluorescence readings. The arithmetic mean was computed using the 9 fluorescence scans for each well of the plate. These values were used to generate a "fold induction" by computing the ratio:

FL(experimental well)/FL(control wells)

FL(experimental well) = arithmetic mean of 9 scans/well

FL(control wells) = arithmetic mean of all negative control well scans

[0053] The average per- well fluorescence readings were logio-transformed and plotted (along with fold-induction data, Fig. IB) as a function of treatment conditions. The transformed data was subsequently modeled as a function of [diniconazole], [ABA] and the interaction effect, [diniconazole]* [ABA].

Conclusions

[0054] The model explains the majority of data scatter (R3=0.93) and the revealed a significant interaction effect between diniconazole and ABA (Fig. 1C). The synergy is clearly observed by contrasting the dose dependent induction of GFP fluorescence with diniconazole in the presence of ABA with the lack of induction in the absence of ABA (Fig. IB). Induction of expression of RD29a reporter in Arabidopsis is indicative of induction of the function of one or more stress tolerance pathways that include the native RD29a gene, and which are known to impart tolerance to drought, cold, high salinity, and freezing temperatures, along with reduced shoot growth rate with good root growth. These results of RD29a induction using diniconazole and ABA indicate that the combination provides the various desirable traits of drought, cold, high salinity and freezing tolerance, and reduced shoot growth with retained root growth. Example 2. Diniconazole and ABA act synergistically to delay growth, darken foliar tissue and up-regulate drought tolerance in Arabidopsis.

Growth conditions

[0055] Transgenic Arabidopsis seedlings harboring the transcriptional fusion RD29a::GFP were grown on solid media (50% MS/B5, 0.05% MES (pH 5.7), 0.5% sucrose, 0.8% agar) in a growth chamber at 22°C with continuous light (95μMol/m2/s) for 9 days. The seedlings were then transplanted onto media containing various concentration combinations of diniconazole and ABA (or DMSO controls) and returned to identical growth conditions for 3 additional days. After photographing the plates, the seedlings were desiccated within a laminar flow hood in a two-stage process. The lids were first removed from the plates for 4h with a 180° rotation at 2h. The seedlings were subsequently removed from the plates and allowed to dehydrate on the plate lid for an additional 3h. They were then transferred onto fresh media without compound and returned to the growth chamber. The plates were photographed after 4 days of recovery.

Conclusions

[0056] The combination treatment of diniconazole and ABA dramatically reduces developmental growth and darkens the appearance of foliar tissue (Fig. 2). ABA induces only limited drought tolerance at the low concentrations of 0.625 and 1.25μM (Fig. 3). This tolerance is enhanced by co-treatment with 20μM diniconazole.

Example 3. Combination treatments of diniconazole and ABA retard growth, darken foliar tissue and enhance drought tolerance in turf.

Growth Conditions

[0057] Penncross Creeping Bentgrass is sown as a 10: 1 (v/v) sand:seed mixture in various container types containing a known weight of an inert vermiculite/perlite substrate pre- wetted with a diluted 20:20:20 fertilizer solution (0.5g/L). The seeds are germinated and allowed to grow with regular watering to various stages of development (1-3 weeks) prior to treatment. The growth substrate is allowed to desiccate until barely moist (24-96h depending on the container format) and the seedlings are trimmed to a consistent height of 1-2 inches preceding the initial treatment. Various treatment regimes are utilized that vary in the frequency and concentration ratio of diniconazole and ABA. A two-stage treatment is typical consisting of two applications of the chemicals (concentration ratios from 1:10,000 to 10,000:1) separated by 48h. Average leaf elongation is measured along with the color of existing and new leaf growth and compared to control plants. For drought experiments, the containers are raised to facilitate air flow and the growth substrate is allowed to dry completely. Tissue death is monitored daily until all treated plant units display ubiquitous yellowing. Combined treatments of diniconazole and ABA yield the greatest drought tolerance with concomitant slow growth and darkening of both new and developed leaves post-treatment.

[0058] Similar experimental regimes are carried out using commercially available sod (e.g. hybrid Bermuda grass). The rolls are cut into identically sized pieces that are layered on the surface of vermiculite (or similar inert substrate) in a variety of different plastic growth containers (depending on experiment scale). The containers are bottom fed with a dilute 20:20:20 fertilizer solution (0.5g/L) and allowed to grow for at least 1 week prior to chemical treatments. After trimming to a uniform height (2 inches), similar concentration ratios of diniconazole and ABA as above are applied either as root drenches or top sprayed with various treatment regimes. Growth, color and drought tolerance are measured in a similar manner with parallel observations regarding the effects of diniconazole/ AB A combination treatments.

Example 4. Combination treatments of diniconazole and ABA retard growth, darken foliar tissue, increase stem thickness and enhance root growth in turf.

Growth Conditions

[0059] Penncross Creeping Bentgrass seeds were surface sterilized using 70% EtOH and 30% bleach using standard methods. Seeds (5-15) were then germinated on 4mL media (50% MS/B5, 0.05% MES (pH 5.7), 0.5% sucrose, 0.8% agar) and grown at 25°C with continuous light (95 μMol/m2/s) for 9 days. A single treatment of lOOμL (various concentrations, 0.4% DMSO in media w/o agar) was floated on the surface of the agar and the seedlings were returned to the chamber. Growth rates and tissue color were monitored daily. After 17 days the seedlings were removed and growth patterns noted for each treatment combination. Effects on leaf elongation, color and thickness were observed with chemical treatments with the most dramatic effects associated with the combination of diniconazole and ABA (Fig. 6A). In addition, roots from co-treated seedlings were longer and thicker than the singly treated samples (Fig. 6B). Example 5. Combination treatments of uniconazole and ABA inhibit creeping bentgrass growth rate.

Growth Conditions

[0060] Penncross Creeping Bentgrass seeds were sown on a peat-based potting mixture in plastic seed flats (0.14m²) at 7.5g/m² and grown under continuous light with regular watering at 28°C for 2 weeks. The grass was trimmed to 2 inches and sprayed (60mL/m²) with the indicated compounds and concentrations as shown in Figure 7. The turf was trimmed to 2 inches after 7d and at 14d the canopy height was measured. Little growth inhibition was apparent with isolated treatments of ABA or uniconazole alone. However, the combined treatment dramatically reduced canopy extension rates.

Example 6. Uniconazole and ABA act to stimulate root elongation in creeping bentgrass.

Growth Conditions

[0061] Penncross Creeping Bentgrass was sown in cone-tainers (5cm diameter, 21cm depth) containing a peat-based potting mixture and grown under continuous light with regular watering at 28°C for 3 weeks, trimming the canopy to 2 inches each week. The turf was then treated via root drench (9mL total volume, 6 replicates/treatment) with the indicated compounds and concentrations (Fig. 8). After 2 additional weeks of growth, the turf was removed from the pot and the roots washed extensively. Representative plants were aligned for photo-documentation. The combined treatments of uniconazole and ABA consistently triggered deeper root penetration and more significant root branching than either treatment alone.

Example 7. Diniconazole and ABA act to stimulate root elongation in creeping bentgrass.

Growth Conditions

[0062] Penncross Creeping Bentgrass was sown in cone-tainers (5cm diameter, 21cm deep) containing a peat-based potting mixture and grown under continuous light with regular watering at 28°C for 3 weeks, trimming the canopy to 2 inches each week. The turf was then treated via root drench (9mL total volume, 6 replicates/treatment) with the indicated compounds and concentrations (Fig. 9). After 2 additional weeks of growth, the turf was removed from the pot and the roots washed extensively. Representative plants were aligned for photo-documentation. The combined treatments of diniconazole and ABA consistently triggered deeper root penetration and more significant root branching than either treatment alone.

[0063] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of imparting increased stress tolerance on a plant comprising contacting the plant with exogenous abscisic acid (ABA) and one or more triazole compounds of Formula I:

2. The method of claim 1 , wherein the one or more triazole compounds is diniconazole.

3 . The method of claim 1 , wherein the one or more triazole compounds are selected from the group consisting of diniconazole, uniconazole and uniconazole-P.

4. The method of claim 1 , wherein the stress on the plant is one or more selected from the group consisting of drought, chilling temperatures, freezing temperatures and high soil salinity.

5. The method of claim 1 , wherein the plant has reduced foliar growth.

6. The method of claim 1 , wherein the plant has increased root growth.

7. The method of claim 1 , wherein the ABA and the one or more triazole compounds are applied to one or more of the leaves, stems or roots of the plant.

8. The method of claim 1 , wherein the ABA and the one or more triazole compounds are applied in a liquid formulation.

9. The method of claim 1, wherein the ABA and the one or more triazole compounds are applied in a solid formulation.

10. The method of claim 1 , wherein the ABA and the one or more triazole compounds are applied to the plant simultaneously.

11. The method of claim 1 , wherein the ABA and the one or more triazole compounds are applied to the plant sequentially.

12. The method of claim 11 , wherein the ABA is applied first and the triazole compound are applied subsequently.

13. The method of claim 11 , wherein the one or more triazole compounds are applied first, and the ABA is applied subsequently.

14. The method of claim 1, wherein the ABA and the one or more triazole compounds are applied to the plant before the onset of the stress.

15. The method of claim 1 , wherein the ABA and the one or more triazole compounds are applied to the plant after the onset of the stress.

16. The method of claim 1 , wherein the plant is a grass.

17. The method of claim 16, wherein the grass is a turf grass.

18. The method of claim 1, wherein the ABA is applied at a concentration of about 0.040 μM to about 2000 μM.

19. The method of claim 2, wherein the diniconazole is applied at a concentration of about 0.40 μM to about 2000 μM.

20. The method of claim 3, wherein the diniconazole is applied at a concentration of about 0.40 μM to about 2000 μM and the uniconazole or uniconazole-P is applied at a concentration of about 1.0 μM to about 2000 μM. .