EP1253826A1 - Diphenyl ether induction of systemic resistance in plants - Google Patents

Abstract

The invention relates to a method for inducing systemic resistance in plants, thereby protecting plants against a broad range of plant pathogens and disease. The method of the invention comprises the application of a biologically active formulation, comprising a diphenyl ether, to a plant. In accordance with the invention, it has been observed that use of this formulation results in induced systemic resistance in a target plant. Also in accordance with the method of the invention, the formulation has been shown to trigger long-lasting, non-specific systemic resistance in the plant to a variety of pathogens and disease. Furthermore, the method of the invention results in an increase in the levels of plant isoflavones.

Description

DIPHENYL ETHER INDUCTION OF SYSTEMIC RESISTANCE IN PLANTS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit and priority from the following three applications: U.S. Provisional Application No. 60/181,933, filed February 11, 2000; U.S. Provisional Application No. 60/181,707, filed February 11, 2000; and U.S. Provisional Application No. 60/181,686, filed February 1 1, 2000.

FIELD OF THE INVENTION

The present invention relates to the field of inducing disease resistance in plants. More specifically, this invention relates to the induction of natural plant disease resistance, through the use of a formulation comprising a diphenyl ether. In certain embodiments, the present invention relates to a method for combating plant pathogens by inducing the productions of isoflavones in a plant.

BACKGROUND OF THE INVENTION Sclerotina sclerotiorum (white mold) damage in soybeans accounts for an estimated average annual loss of roughly 26 million dollars in the United States alone. Losses resulting from other crop diseases, such as sudden death syndrome (Fusarium species), brown stem rot, Phytophthora species, etc., add significantly to the 26 million dollar loss estimate resulting from white mold each year. Attempts to control white mold and other diseases of soybeans have included the use of chemicals and biological control methods applied to the surface of the plant. These methods strive to block the growth and development of the disease-causing organism before it can enter the plant. While these methods can be effective, their duration is typically short term and their efficacy can depend on environmental conditions. A second method of plant disease control is the use of disease-resistant cultivars.

Typically, these plants are genetically engineered to produce compounds toxic to disease- causing organisms. However, the toxic compounds generally do not occur naturally in these plants. While this method of disease control can be very effective, and can be an improvement over the use of chemicals sprayed onto crops in both terms of time and safety. there has been resistance by the gen ral public to the use of genetically engineered crops, both in the U S and abroad

Recently, researchers have fccused on a new method of plant disease control, through the augmentation of natural plant defenses Plants innately resist pathogenic attacks in two general manners, through pieformed barriers and induced mechanisms The former include physical barriers and continuously-expressed defense proteins These seive to stop initial pathogen entry and provide the means of minimizing deleterious effects if a barrier is breached The latter are activated only upon challenge or breach of the preformed baiπeis For example, localized infection by a pathogen results in the induction of physical changes at the site of infection (including cell wall lignification and papilla formation) (reviewed in Kessmann, H , Staub, T , Hofmann, C , Maetzke, T , Herzog, T , Ward, E , Uknes, S , and I Ryals 1994 Induction of systemic acquired lesistance in plants by chemicals Annual tew oj Phytopathology 32 439-459, Schneider, M , Schweizer, P , Meuwly, P , and I P Metraux 1996 Systemic acquned resistance in plants International Journal of C\ tolog\ 168 303- 340, Sticher, L , Mauch-Mam, B , and J P Metraux 1997 Systemic acquired lesistance

Annual Review of Plant Pathology 35 235-270) Additionally, signal transduction pathways aie activated that lead to systemic resistance in uninfected parts of the plant (reviewed in Mauch-Mam, B , and J P Metraux 1998 Salicylic acid and systemic acquired resistance to pathogen attack Annals Botanx 82 535-540) Thus, the first infection conditions the plant to resist futuie insults, similar to the vaccination of humans and other animals Importantly, though, this systemic resistance is broad spectrum, against widely different pathogens such as fungi, bactena or viruses, and not meiely the pathogen that caused the initial infection While the conditioned state associated with systemic lesistance is often transient, under some circumstances it can be sustained Conditioning has also been termed "priming," "activation," "potentiation," and "competency "

At least two different signal transduction pathways appeal to be involved in systemic resistance, although both similarly condition the plant to resist further pathogenic attacks Systemic acquired resistance (SAR) is chaiactenzed by an accumulation of salicylic acid (SA) in plant tissues, and an increase in a class of proteins termed pathogenesis-i elated (PR) proteins (reviewed in Kessmann, H , Staub, T , Hofmann, C , Maetzke, T , Heizog, I , Ward, E , Uknes, S , and 1 Ryals 1994 Induction of systemic acquired resistance in plants by chemicals Annual Review of Phytopathology 32 439-459, Hunt, M D , and I A Ryals 1996 Systemic acquπ ed resistance signal transduction Cr itical Review in Plant Science 15 583- 606, Ryals, J , Neuenschwander, U , Wil ts, M , Molina, A , Sterner, H Y , and M Hunt 1996 Systemic acquired resistance Plant Cell 8 1809-1819, Schneidei, M , Schweizer, P , Meuw ly, P , and J P Metraux 1996 Systemic acquired lesistance m plants International Journal of Cy tology 168 303-340, Yang, Y O , Shah, J , and D F Klessig 1997 Signal perception and transduction in defence responses Genes and Development 1 1 1621 -1639) SA has been pioposed to act by increasing cellular hydrogen peroxide concentrations (Chen, Z , Silva. H , and D F Klessig 1993 Active oxygen species in the induction of plant systemic acquiied resistance by salicylic acid Science 262 1883-1885), triggering hpid peroxidation (l eviewed by Goodman, R N , and A J Novacky 1994 The hypeisensitive reaction plants to pathogens St Paul APS Press), inducing alternativ e oxidase and theimogenesis (Raskin, 1 . and B J D Meeuse 1987 Salicylic acid A natui al inducer of heat pioduction in Arum lilies Science 231 1601 , Rhoads, D M , and L Macintosh 1 992 Salicylic acid legulation of respiration in higher plants alternative oxidase expression Plant Cell 4 1 121 - 1 139), and enhancing the subsequent response to ehcitoi treatment (reviewed in Mauch-Mani, B , and J P Metraux 1998 Salicylic acid and systemic acquired resistance to pathogen attack Annals Botany 82 535-540) Thiough these mechanisms, and moi e directly , SA induces the expiession of a numbei of defense related genes and proteins (Hunt, M D , and J A Ryals 1996 Systemic acquired resistance signal tiansduction C ritical Re\ in Plant Scienc e 1 5 583-606, Schneidei , M , Schweizer, P , Meuwly, P , and I P Metraux 1996 Systemic acquii ed lesistance in plants International Journal of Cytology 168 303- 340, Stichei , L , Mauch-Mani, B , and J P Meti aux 1997 Systemic acquired lesistance Annual oj Plant Pathology 35 235-270, Van Loon, L C 1997 Induced l esistance in plants and the l ole of pathogenesis-related pioteins European Journal of Plant Pathology 103 753-765, Yang, Y O . Shah, J , and D F Klessig 1997 Signal peiception and transduction defence responses Genes and Development \ \ 1621 - 1639) While most reports agi ee that SA is an initial element of these signal transduction pathw ays, it is not cleai if it is the pnmai y systemic signal, or secondarily activated (Mauch-Mani, B , and 1 P Metraux 1998 Salicylic acid and systemic acquired resistance to pathogen attack Annals Botany 82 535-540)

A second signal transduction pathway, termed induced systemic lesistance (ISR), operates independently of the SAR pathway An illustrative example is a study that demonstiated the plant growth-promoting rhizobacteπa (PGPR) induced a systemic resistance-like phenomena without accumulation of SA accumulation or PR gene expiession (Pieterse, C M J , Van Wees, S C M , Hoffland, E , Van Pelt, J A , and L C Van Loon 1996 Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-i elated gene expiession Plant Cell 8 1225-1237) Additional studies have also shown that neither SA or PR protein levels increase upon induction of an ISR response Thionins and the small, cysteine-πch plant defensins (PDFs) are found to accumulate upon induction of an ISR response, and aie believed to be effectors of the response (Epple, P , Apel, K , and H Bohlmann 1995 An Aiabtdopsis thaltana thiomn gene is inducible via a signal transduction pathway diffeient from that for pathogenesis-related protein Plant Physiology 109 813-820, Penmnckx, I A M A , Eggermont, K , Terras, F R G , Thomma, B P H I , DeSamblanx, G W , Buchala, A , Metraux, I P , Manners, J M , and W F Broekaeit 1996 Pathogen-induced systemic activation of a plant defensm gene in Arabidopsis follows salicylic acid-independent pathway Plant Cell 8 2309-2323) These studies also suggested that methyl jasmonate may be a mediator of ISR It was shown that the addition of methyl jasmonate to arabidopsis resulted in the induction of the thiomn 2 1 gene, but that SA did not have the same effect (Epple, P , Apel, K , and H Bohlmann 1995 An Arabidopsis thaltana thio n gene is inducible via a signal tiansduction pathway different from that foi pathogenesis-ielated protein Plant Physiology 109 813-820) Similarly, while methyl jasmonate, ethylene, paraquat and rose bengal were found to induce the accumulation of the antifungal plant defensm PDF! 2 in arabidopsis leaves, none of these chemicals had any effect on levels of PR-1 mRNA (Pen nckx, I A M A , Eggermont, K , Terras, F R G , Thomma, B P H I , DeSamblanx, G W , Buchala, A , Metraux, J P , Manneis, J M , and W F Broekaeit 1 W( Pathogen-induced systemic activation of a plant defensm gene in Arabidopsis follows salicylic acid-independent pathway Plant Cell 8 2309-2323) In contiast, SA induced the accumulation of PR-1 mRNA but not the defensm or its mRNA (Pen nckx. I A M A , Eggermont, K , Terras, F R G , Thomma, B P H J , DeSamblanx, G W , Buchala, A Metraux, I P , Manners, J M , and W F Broekaert 1996 Pathogen-induced systemic activation of a plant defensm gene in Arabidopsis follows salicy lic acid independent pathway Plant Cell 8 2309-2323) In aiabidopsis plants o\ eι-expιessιng the nahG gene (charactei lzed by very low levels of SA) (Delaney, T P , Uknes, S , Vernooij B , Fπediich L , Weymann, K , Negrotto, D , Gaffney, T , Gutrella, M , Kessmann, H Waid, E and I Ryals 1994 A central role of salicylic acid m plant disease resistance Science 266 1247- 1250), and in the npr 1 mutant (no detectable PR.-1 protein expression) (Cao, H , Bowling, S , Gordon, A , and X Dong 1994 Characterization of Arabidopsis mutant that is non- responsive to inducers of systemic cquired resistance Plant Cell 6 1583-1592), induction with an avirulent fungus led to accum ilation of defensm, demonstrating that plants with defective SAR pathways maintain functional ISR pathways Moreover, two arabidopsis mutants demonstrated that an inability to respond to ISR inducers results in decreased expression of ISR-associated proteins, but not SAR-associated proteins While coif which does not respond to methyl jasmonate (Feys, B J F , Benedetti, C E , Penfold, C N , and I G Turner 1994 Arabidopsis mutants selected for resistance to the phytotoxin coronatme are male sterile, insensitive to methyl jasmonate, and resistant to a bacteiial pathogen Plant Cell 6 751 -759), and eι/ι2, which does not respond to ethylene (Guzman, P , and J Eckei 1990 Exploiting the triple response of Arabidopsis to identify ethylene-related mutants Plant Cell 2 513-523) both produce PR-1 , both also show a highly reduced ability to accumulate the PDF1 2 plant defensm after fungal induction treatment These studies, and others, demonstrate that the ISR and SAR responses are unique and different

Not all plants possess both of these signal transduction pathways For example, soybeans ai e believed to lack the elements required for an SAR response While treatment ol soybean cotyledon tissues with either methy l jasmonate oi 1 -amιnocyclopropanecaιboxylιc acid gives use to protection of cells distal from the point of application (Paik, D -S 1998 Proximal cell competency and distal cell potentiation soybean resistance Ph D Thesis The Ohio State University), SA does not induce any detectable changes in sovbean defense pathways

In addition to this ISR pathway, it has been suggested that soybeans may have a response that may "substitute" for the SA response seen in most plants This substitute response is chaiacteπzed by a high accumulation isoflavones including daidzein and conjugates of the lsoflavone gemstein (present in the apoplast of soybean seedling tissues as a malonyl glucosyl conjugate (MGC), likely released by a highly lsoflavone specific apoplastic β-glucosidae (Hsieh, M -C 1997 Purification and characteiization of an lsoflavone specific β-glucosidase from soybean Ph D Thesis The Ohio State Univ ersity)) Gemstein is then thought to act m a manner somewhat similar to SA activating the defense potential of soybean cells (T L Graham and M Y Graham 2000 Defense Potentiation and Ehcitation Competency Redox Conditioning Effects of Salicylic Acid and Gemstein, pp 181 -219, Plant- Microbe Intei actions, G Stacey and N Keen, eds)

Isoflavones aie phytoestiogens that are naturally produced in plants including those belonging to the family Legummosae, particulai ly in plants belonging to the subfamily the Papihonoidease which includes soybeans Recent studies have shown that plants which do not belong to the family Legummosae can be genetically engineei ed to pi oduce isoflavones For example, At abtdopsis thaltana has been tiansformed with a single enzyme which allo s it to produce gemstein (Yu, Ohvei , Jung, Woosuk, Shi, lune, Ci oes, Robert A , Fadei , Gai y M , McGomgle, Brian, Odell, Joan T 2000 Production of the isoflavones gemstein and daidze in non-legume dicot and monocot tissues Plant Physiology 124 781 -793)

Isoflavones exist an inactive foim in plants, attached to a sugai molecule such as glucose 7 he fiee lsoflavone form, which is know n as an "aglycone" is i clcased upon wounding oi infection by a pathogen Once l eleased, the aglycones play multiple l olcs in the establishment of the capacity of the cell to mount an eff ectiv e defense i csponse Foi example, the lsoflav one daidzein is a piecursor of the plant antibiotic "phytoalexm" glyceollin, and the lsoflavone gemstein aids in the priming of the soybean's capacity (competency) to l ecogmze pathogen-derived "ehcitoi s" that tnggei glyceollin pi oduction Fuithermoi e, gemstein itself has some antibiotic activity Thus, the simple l eleasc of these two aglycones enhances thi ee cπtical and complementai y aspects of plant defense Application of methyl jasmonate greatly potentiates this i espouse (Gi aham, 7 L , and M Graham 1996 Signaling in soybean phenylpiopanoid response dissection of pnmai y, secondaiy and conditioning effects of light, wounding and ehcitoi ti eatments Plant Physiology 1 10 1 123-1 133) The accumulation of lsoflavone conjugates thus "loads" the capacity of the soybean to lespond to a pathogen The formation of glyceollin f i om l eleased daidzem "taps" into this pool of isoflavones

Unfortunately, levels of the isoflavones in soybean ai e not alw ays adequate to effectively launch these resistance l esponses Some tissues (foi instance matin c leav es) hav e relatively low lev els of isoflav ones and the lsoflavone content of tissues aie deci eased undei certain envn onmental conditions such as low light (cloudv eathei ) As a l csult of this lack of adequate lsoflav one lev els, the plants become less resistant to attack by phytopathogens

Methods of ' priming" a plant to l esist attack by phytopathogens, both thi ough the triggering of ISR and inducing an inci ease in the lev el of plant isoflavones, would sei v e to increase the choices available to plant growers, from farmers to backyard gardeners, for combating plant pathogens. The present invention presents an environmentally safe, effective and convenient formulation and method for triggering ISR and increasing the levels of plant isoflavones

SUMMARY OF THE INVENTION

The present invention relates to a method of triggering induced systemic resistance in a plant comprising applying an effective amount of a biologically active formulation compπsing a diphenyl ether to the surface of at least a part of the plant, triggering activation of induced systemic resistance in the plant, thereby inducing systemic resistance to at least one pathogen or disease

In another embodiment, the present invention relates to a method of increasing plant yield comprising, applying an effective amount of a biologically active formulation comprising a diphenyl ether to the surface of at least a part of the plant, triggering activation of induced systemic resistance in the plant, and maintaining or increasing the general health of the plant, thereby increasing crop yield

In yet another embodiment of the invention, the present invention provides a method for increasing the levels of isoflavones in plants comprising, applying an effective amount of a biologically active formulation comprising a diphenyl ether to the surface of at least a part of the plant, triggering release or production of isoflavones, thereby increasing the levels of isoflavones in plants Advantageously, the present method also enhances the glyceollin ehcitation competency of the treated plant

The active diphenyl ether of the present invention preferably has the structure

(I) wherein R_t is a hydrogen, fluorine, or chlorine atom, or a trifluoromethyl group, R₂, R and R-, are independently a hydrogen, fluorine, or chlorine atom, R₄ is a hydrogen atom, NR₍₎, NR₆R₆, OR₆, COOR₆, COOCHR₆CO₂R₆, CONHSO₂R₍„ or a cyclic ether, wherein R is a hydrogen atom, a branched alkyl group of 1 to 4 carbon atoms or a linear alkyl group of 1 to 4 carbon atoms

The active diphenyl ether of the present invention also preferably has the structure

wherein R₇ is an oxygen or nitrogen atom, and R_x is a hydrogen atom, CTh, an aliphatic chain comprising 2 to 5 carbon atoms, or HSO₂CH-,

In each of the above embodiments, the diphenyl ether is more preferably acifluorfen, aclonifen, bifenox, chlomethoxyfen, chlornitrofen, fluorodifen, fluoroglycofen, fliioronitrofen, fomesafen, furyloxyfen, halosafen, lactofen, nitrofen, nitro fluorfen or oxyfluorfen Most preferably, the diphenyl ether is lactofen.

In other preferred embodiments, the biologically active formulation furthei comprises one or moie adjuvants selected from crop oil concentrates, surfactants, fertilizers, emulsifleis, dispersing agents, foaming activators, foam suppressants, and correctives In a more preferred embodiment, the one or more adjuvants m the biologically active formulation ai e a crop oil concentrate, a surfactant and a fertilizer

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a working model for the establishment of ehcitation competency in soybeans via release of lsoflavone conjugates Details of this model can be found in T L Graham and M Y. Graham. 1999. Role of hypersensitive cell death in conditioning ehcitation competency and defense potentiation Physiol Mol Plant Pat ol 55 13-20

DETAILED DESCRIPTION OF THE INVENTION

The biologically active formulation of the present invention has unexpectedly been found to tπggei ISR and increase the levels of isoflavones in plants In one embodiment ol the invention, treatment of plants with the biologically active formulation leads to deci eased incidence of pathogen or disease-caused plant damage, exhibiting the beneficial effects of ISR In another embodiment of the invention, plants treated with the biologically active formulation are more robust and pi oduce a higher yield upon harvest, suggesting ISR is broad-based and non-specific, allo ving a plant ΪO grow unimpeded throughout the growing season. In a third embodiment of Ihe invention, plants treated with the biologically active formulation had a higher level of isoflavones than found in non-treated plants. This increase was found in all plant parts tested, including seeds, cotyledons, leaves and stems.

In each of these embodiments, the biologically active compound is a diphenyl ether, which is preferably encompassed by formula (I) and/or formula (II).

In the present invention, the term "induced systemic resistance" or "ISR" refers to an inducible, plant-wide resistance to the growth and pathogenic effects of pathogenic organisms and disease. Such resistance may be total or somewhat less than total. Furthermore, such resistance may be induced in a therapeutic or prophylactic manner. ISR is also used interchangeably with the terms "immunity," "resistance," "disease resistance," and "induced disease resistance."

As used herein the term "plant" encompasses all forms and organs of a monocotyledonous or dicotyledonous plant, including but not limited to the seed, the seedling, and mature plant.

1. Production of biologically active formulations comprising a diphenyl ether

In the present invention, the biologically active formulation is comprised of at least one diphenyl ether compound (i.e., a compound having the core structure of

©~®

with desired substitutions on one or both of the phenyl rings) as the active ingredient. As would be understood by one of ordinary skill in the art, the term "diphenyl ether" as used herein encompasses any active form of the compound, including acid and salt forms, metabolites, racemic mixtures of stereo- or optical isomers, purified isomers, etc. Preferably, for triggering induced systemic resistance in a plant and/or increasing plant yield, the diphenyl ether has the structure shown as (I) above. Non-limiting examples of diphenyl ethers suitable for use in the present invention include acifluorfen, aclonifen, bifenox, chlome hoxyfen, chlornitrofen, fluorodifen, fluoroglycofen, fluoronitrofen, fomesafen, furyloxyfen, halosafen, lactofen, nitrofen, nitrofluorfen and oxyfluorfen.

In another embodiment, the method comprises administering to the plant an agricultural chemical composition comprising a phytologically acceptable carrier or diluent and an effective amount of a diphenyl ether, preferably having the structure shown as (II) above. The structures of several preferred diphenyl ethers which are useful in this method and which have been tested are shown below:

Lactofen

Acifluorfen

Fomesafen

In yet another embodiment, the diphenyl ether has a structure represented by form it 1

(III):

wherein RQ is H, Cl, I, Br or CF ; and Rio is a branched aliphatic chain comprising 1 -5 carbon atoms. Compounds of formulas (II) and (III) are particularly useful in the method of increasing the levels of isoflavones in plants. The biologically active formulation of the present invention is produced by mixing the active ingredient into water One or a mixture of diphenyl ether compounds can be used as the active ingredient Although one of ordinary skill in the art will understand that various volumes of the biologically active formulation may be prepared, depending on the size of the area to be treated, 15 gallons is a useful volume As such, the biologically active formulation of the invention can be produced in preferred embodiments by mixing between about 0 0050 and 0 50 pounds of the active ingredient into 15 gallons of water, more preferably, between about 0 050 and 0 125 pounds, most preferably, about 0 1 pounds Of course, these limits aie not absolute, and the outer boundaries could be readily determined by one of ordinary skill in the art

A preferred diphenyl ether used as the active ingredient in the pi esent invention is lactofen (C|<,H,,ClF₃NO₇) (2-ethoxy-l -ethyl-2-oxoethyl 5-[2-chloro-4-

(tπfluoromethyl)phenoxy] -2-nιtrobenzoate) A biologically active foimulation compnsed of lactofen as the active ingredient is typically produced by mixing lactofen into water Preferably, between about 0 0050 and 0 50 pounds of lactofen is mixed into 15 gallons of water, moie preferably, between about 0 050 and 0 125 pounds, most preferably, about 0 1 pounds

An exemplary form of commercially-available lactofen is the heibicide Cobra®, produced by Valent U S A Corporation Cobia® has been approved foi use as a selective, broad spectrum herbicide for pre-emergence and post-emergence contiol of susceptible bioadleaf weeds (EPA Reg No 59639-34) Cobi a® is commercially available in a concentrated form comprised of 23 2% lactofen by weight and 76 8% other giedients, and is sold as a liquid containing 2 pounds of lactofen per gallon A biologically active formulation comprised of Cobra®, containing lactofen as the active ingiedient, is pioduced by mixing Cobia® into water As used in the Examples below, and as typically used in the field, prefeiably between about 0 25 and 50 fluid ounces of Cobra® is mixed into 15 gallons of watei, more pieferably, between about 2 5 and 10 fluid ounces, most preferably, about 6 fluid ounces

Other useful forms of lactofen include the herbicide Stellar®, also produced by Valent U S A Corporation (EPA Reg No 59639-92) Stellar is comprised of 26 6% lactofen by weight, 7 6% flumiclorac pentyl ester, and 65 8% other ingredients Flumiclorac pentyl estei is the active ingredient in Resource® herbicide While the biologically active formulation of the present invention may be comprised of a diphenyl ether alone, it is preferred that the formulation also includes one or more adjuvants. Useful adjuvants include, without limitation, crop oil concentrates, surfactants, fertilizers, emulsifiers, dispersing agents, foaming activators, foam suppressants, and correctives Adjuvants generally facilitate the entry of the diphenyl ether active ingredient through plant cell walls. A phytologically acceptable carrier is a physiologically acceptable diluent or adjuvant The term "phytologically acceptable" means a non-toxic material that does not interfere with the effectiveness of the diphenyl ether The usefulness of a particulai adjuvant or carrier depends on, among other factors, the species of the plant being treated with the formulation of the invention, the plant's growth stage and the related environmental conditions, the route of administration and the particular compound oi combination of compounds the composition In a more preferred embodiment, the one or more adjuvants in the biologically active formulation are a crop oil concentrate, a surfactant and a fertihzei Preparation of such formulations is withm the level of skill in the art A biologically active formulation comprised of a diphenyl ether, a crop oil concentrate, a surfactant and a fertilizer, is produced by mixing each of the compounds into water in the following order: fertilizer, diphenyl ether, crop oil concentrate, surfactant Although one of ordinary skill in the art will understand that various volumes of the biologically active formulation may be prepared, depending on the size of the area to be treated, 15 gallons is a useful volume As such, this embodiment of the biologically activ e formulation can be produced by mixing between about 0 1 and 10 pounds of ammonium sulfate into 15 gallons of water, more preferably, between about 1 and 4 pounds, most preferably, about 2 pounds. Exemplary fertilizers found to be useful in formulations of this embodiment of the invention include ammonium sulfate A second exemplary fertihzei found to be useful in formulations of this invention is urea ammonium nitiate In an embodiment utilizing urea ammonium nitrate, preferably, between about 1 and 200 fluid ounces of uiea ammonium nitrate are mixed into 15 gallons of water, more preferably, between about 25 and 100 fluid ounces, most preferably about 50 fluid ounces Preferably , between about 0 0050 and 0.50 pounds of the diphenyl ether active ingredient are next mixed into the formulation, more preferably, between about 0 050 and 0 125 pounds, most preferably, about 0 1 pounds As noted above, one or a mixture of diphenyl ethci compounds can be used as the active ingredient Then, preferably between about 1 and 100 fluid ounces of a crop oil concentrate are next mixed into the formulation, more preferably, between about 5 and 25 fluid ounces, most prefer, bly, about ' 0 fluid ounces Crop oil concentrates are generally comprised of from 65-9t % by weight of a hydrocarbon oil or solvent with the balance being a surfactant The hydrocarbons may be petroleum or vegetable based Exemplary crop oil concentrates fouiiα to be useful m the formulations of this invention include methylated seed oil, Dyne-Amic® (Helena Chemical Co ) and Herbimax® (Loveland Industries Inc ) A preferred amount of a non-ionic surfactant, generally in the range of about 0 1 to 25 fluid ounces, are finally mixed into the formulation, more preferably between about 2 and 10 fluid ounces, most preferably about 5 fluid ounces Surfactants aie also available from a variety of commercial souices Useful forms include anionic, cationic, nonionic and ampholytic suifactants Exemplaiy surfactants found to be useful in the formulations of this invention include Kinetic® (Helena Chemical Co ) and Induce® (Helena Chemical Co )

In a further embodiment of the inv ention, the biologically active formulation may also contain one oi moie other active chemicals, such as herbicides, insecticides fungicides, bacteπocides, and plant growth legulators As used in the present invention, the term "othei active chemicals" refeis to those chemicals having activ ities other than the ability to triggei ISR in plants, such as insecticidal, herbicidal, fungicidal, bacteriocidal, etc In a preferred embodiment, the one or more other active chemicals in the biologically activ e formulation is a heibicide Non-limiting examples of acceptable herbicides include 2,4-DB, Assure®/Assure II, Basagran®, Classic®, Firstrate®, Fusilade® DX, Option E), Passport®, Pinnacle®, Puisuit®, Pursuit Plus®, Reliance™ STS®, Roundup Ultra®, Select^ 2 EC,

Scepter®, and Synchrony™ STS® A biologically active formulation containing a heibicide is produced by mixing the herbicide into w atei, followed by a fertihzei (if any ), the diphenyl ether active mgiedient, a crop oil concentrate (if any), and a surfactant (if any), in that oidei For a 15 gallon biologically active formulation, the mixture can be produced by mixing between about 0 005 and 10 pounds of the herbicide active ingredient into 15 gallons of water, more preferably between about 0 5 and 5 pounds, most pieferably about 1 pound The remaining ingredients aie then mixed into the formulation as directed abov e An exemplaiy herbicide found to be useful in the formulation of this invention is Roundup Ultra® (Monsanto Corp ), a post-emergence, non-selective systemic herbicide One of ordinary skill in the art will understand that other inert ingredients may be included m all embodiments of the biologically acti e formulation of the inv ention to pio ide a more satisfactoiy formulation, piovided the inert ingredients do not detract fiom the effect of the essential components of the invention. The composition may further contain other agents which either enhance the activity of the diphenyl ether or complement its activity. Such additional factors and/or agents may be included in the composition to produce a synergistic effect with the diphenyl ether, or to minimize side effects. The composition may further comprise fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

2. Application of biologically active formulations to plants

One of ordinary skill in the art will understand that the methods of the invention may be practiced by applying a formulation comprising a diphenyl ether alone, although it is preferred that at least one adjuvant is present in the formulation. The methods of the invention may be practiced by applying a formulation comprising a diphenyl ether, one or more adjuvants, with or without other active chemicals, and with or without other inert ingredients. Furthermore, it will be understood that the diphenyl ether, one or more adjuvants, other active chemicals, and other inert ingredients may be applied concurrently or sequentially (in any desired sequence) so long as each component will perform as intended in accordance with the invention. If applied sequentially, the individual components may be applied over a short or long time frame.

The biologically active formulation of this invention may be applied to seed, to roots, or to leaves and stems. The composition may be administered to seed by coating the seed with a powdered composition, which may include a "sticker", to the soil, or to seed and/or the root zone either as a liquid or in a granulated form. The composition may be applied to the surface of the plant in a single application until the leaves of the plant are partially wetted, fully wetted or until runoff. The treatment of the plant may also involve adding the composition to the water supply of the plants, or in the case of plants grown by tissue culture. to the culture media. The formulation may be applied at any time of day or night with good resistance resulting, but preferentially on actively growing plants and at least 30 minutes before a predicted rainfall. The application can be repeated as often as considered useful, with one or more "booster" applications applied to bolster resistance should the previously induced resistance begin to fade, as evidenced by the onset of disease symptoms. Thus, the formulation may be considered "prophylactic" as well as "therapeutic." In a preferred embodiment, the formulation is applied by spraying the formulation onto the plants. Non- limiting examples of means for spraying the formulation onto plants include a tractor boom sprayer, a hand held aerosol sprayer, airblast sprayer, and helicopter or fixed-wing aircraft boom sprayer Preferably, the sprayer is calibrated to deliver the formulation at between about 1 and 100 gallons per acre, more preferably between about 3 and 50 gallons per acre, most preferably about 15 gallons per acre It will be apparent to one of ordinary skill in the art that the "effective amount" of the diphenyl ether compound required to trigger ISR in a plant will be largely variable, depending on many factors, including the species of plant and its growth stage, row and plant spacing, environmental conditions, weather, etc In general however, it has been determined that a biologically active formulation comprised of a diphenyl ether, applied in amounts generally between about 0 001 and 10 pounds active ingredient per acre, adequately tnggers ISR in plants to which it is applied More preferably, between about 0 01 and 1 pounds active ingiedient pei acre is used to triggei ISR Most pieferably, about 0 05 to about 0.25 pounds acti e ingredient per acre is used to trigger ISR.

An effectiv e amount of diphenyl ether for the induction of increased levels of isoflavones is an amount sufficient to increase the levels of an lsoflavone, such as gemstein and daidze , in the treated plant above levels found in control untreated plants Such amounts can be determined by routine testing such as measurement by high performance liquid chromatography as noted below. The effective amount can be achieved by one application of the composition. Alternatively, the effective amount is achieved by multiple applications of the composition to the plant The amount of the diphenyl ether in the composition will depend upon the particular compound or mixture of compounds being employed, the plant tissue being treated, and the ability of the plant to take up the composition For instance, young plant leaves take up most compositions more leadily than older leaves It is contemplated that the various compositions used to practice the method ol the present invention should contain from about 200 micromolar to 2 millimolar per dose of the diphenyl ether

In a preferred embodiment, the biologically active formulation applied to plants is comprised of a fertilizer, a diphenyl ethei, a crop oil concentrate, and a surfactant Preferably, the fertilizer is added to the formulation in an amount so as to be applied at a rate of between about 0 1 and 10 pounds per acre, more preferably between about 1 and 4 pounds per acre, most preferably about 2 pounds per acre. Preferably, the diphenyl ether of this preferred formulation is applied within the range discussed above Preferably, the crop oil concentrate of the formulation is applied at a rate of between about 1 and 100 fluid ounces per acre, more preferably between about 5 and 25 fluid ounces per acre, most preferably about 10 fluid ounces per acre. Preferably, the surfactant of the formulation is applied at a rate of between about 0.1 and 25 fluid ounces per acre, more preferably between about 2 and 10 fluid ounces per acre, most preferably about 5 fluid ounces per acre. Again, it is anticipated that within these general guidelines, one of ordinary skill in the art would be readily able to select an appropriate formulation and application volume per acre, to achieve the objects and advantages of the present invention.

The ISR and/or increased levels of isoflavones triggered by the biologically active formulation of the invention results in plant resistance to pathogens and disease, and depending on the application method and conditions of application, the present methods will provide specific and/or broad spectrum disease control including prevention of fungal infections and also infection by bacterial, viral and nematode pathogens. Non-limiting examples of plant pathogens include insects (e.g., diptera, hymenoptera, coleoptera, lepidoptera, orthoptera, heimptera, and homoptera), bacteria (in soybeans, for example,

Pseudomonas syringae pv. glycinea and Xanthomonas campestris pv. phaseoli), viruses (in soybeans, for example, Bean Pod Mottle Virus, Cowpea Chlorotic Mottle Virus, Peanut Mottle Virus, Soybean Dwarf Virus, Soybean Mosaic Virus, Tobacco Ringspot Virus, Tobacco Streak Virus, Bean Yellow Mosaic Virus, Black Gram Mottle Virus, Cowpea Mild Mottle Virus, Cowpea Severe Mosaic Virus, Indonesian Soybean Dwarf Virus, Mung Bean Yellow Mosaic Virus, Peanut Stripe Virus, Soybean Chlorotic Mottle Virus, Soybean Crinkle Leaf Virus, Soybean Yellow Vein Virus, and Tobacco Mosaic Virus), fungi ( in soybeans, for example, Cercospora sojina, Chaetomiuin cupreum, Colletotrichum truncatum, Dtaponlie- Phomopsis Complex, Fusariuπi spp., Macrophoinina phaseolina, Peronospora manschurica), and nematodes (in soybeans, for example, Soybean Cyst Nematode, Lance Ncmatodes, Lesion Nematodes, Reniform Nematode, Root-Knot Nematodes, and Sting Nematodes). Non-limiting examples of plant diseases include 1 ) infectious diseases such as a) bacterial diseases (in soybeans, for example, Bacterial Blight, Bacterial Pustule, Bacterial Tan Spot. Wildfire, Bacterial Wilts, and Crown Gall), b) mycoplasmalike diseases (in soybeans, for example, Machismo, Bud Proliferation, Witches'-Broom and Phyllody), c) fungal diseases of foliage, upper stems, pods, and seeds (in soybeans, for example, Alternaπa Leaf Spot and Pod Necrosis, Anthracnose, Brown Spot, Cercospora Blight and Leaf Spot, Choanephora Leaf Blight, Downy Mildew, Frogeye Leaf Spot, Phyllosticta Leaf Spot, Powdery Mildew, Red Leaf Blotch Rhizoctoma Aerial Blight, Rust, Scab, and Target Spot), d) fungal diseases of roots and low x stems (in soybeans, for example, Brown Stem Rot, Charcoal Rot, Fusaπum Blight or Wilt, Poot Rot, and Pod and Collar Rot, Phytophthora Stem Rot, Pod and Stem Blight and Fhomopsis Seed Decay, Stem Cankei, Pythium Rot, Red Crown Rot, Rhizoctonia Diseases, Sclerotima Stem Rot, Sclerotium Blight, and Thielaviopsis Root Rot), e) viral disease (m soybeans, for example, bud blight, soybean mosaic, f) nematode diseases, g) seedborne bacteria and bacterial diseases of seeds (m soybeans, for example, Bacillus Seed Decay), h) seedborne fungi and fungal diseases of seeds (in soybeans, for example, Alternaπa Pod and Seed Decay, Purple Seed Stain, Yeast Spot (Nematospoia Spot), and Phomopsis Seed Decay), I) seedborne viruses, 2) diseases of unknown or uncertain cause (in soybeans, for example, Foliage Blight, Sudden Death Syndrome, and Yellow Leaf Spot), and 3) noninfectious or stress diseases (e g , crusting and compaction, frost, hail, heat cankei, lightning, sunburn, water stress, mineral deficiencies and toxicities, heibicide damage, insecticide damage, and air pollutants) Specific examples of administration would be for control of phytophthora root lot, sclerotima white mold, brown stem rot and the soybean cyst nematode

The ISR and/or increased levels of isoflavones triggered by the biologically active formulation of the invention has also been unexpectedly discovered to result in increased plant yields In the present invention, the term "yield" refers to the useable plant pioduct produced by the plant In the present invention, plant yield is expressed as a alue of diy weight in bushels per acre When properly applied, the biologically active formulation of the invention can increase plant yield a minimum of about 0 5%, moie preferably the increase is at least 5%, and most preferably, the increase is 30% or more, in comparison to the same plant grown under the same environmental conditions but without application of the active formulation of the invention Even an increase in yield of 0 5% is an economically significant inciease on a large, multi-acre farm The same general guidelines for piepanng biologically acti e formulations, and effective application rates to the plants, as set forth above, can be used to achieve this objective of the invention

Plants capable of producing isoflavones include those plants that naturally produce isoflavones, such as plants in the family Legummosae, subfamily Papihonoidease, as well as plants that have been genetically engineered to produce isoflavones In a further embodiment, the invention provides plants, especially crops, which have ISR. In a particularly useful aspect, the ISR is long lasting, often persisting until harvest time. If desired, a booster immunization can be applied at a later date after initial application of the formulation. The booster immunization may be applied if the initial resistance appears to be fading, that is, if the plants develop disease symptoms.

The method of the invention may be used to trigger ISR and/or increased levels of isoflavones in a great variety of plants, including vegetable and fruit crops, legumes, cereals, fruit trees, berries, forestry trees, ornamental plants, and other plants such as coffee and cotton. In a preferred embodiment of this invention, the method is used to trigger ISR and/or increased levels of isoflavones in legume plants such as soybeans, lima beans, pinto beans, green beans, peas, chickpeas, peanuts and mung beans.

The scope of this invention also applies to crops where ISR and/or increased levels of isoflavones are important. By applying a biologically active formulation comprising a diphenyl ether, the plants are rendered systemically competent to attacks from a wide range of pathogens and disease. This has various advantages over current methods of plant protection. These advantages include, but are not limited to: 1 ) broad spectrum control because ISR is less specific than most fungicides or bactericides. and 2) less frequent applications because ISR is more systemic and longer lasting than the protection most fungicides or bactericides provide. It has been observed that the triggering of ISR and/or increased levels of isoflavones in accordance with the invention results in a systemic resistance that lasts at least several weeks (for instance, 4 to 6 weeks) and may last throughout the growing season and/or throughout the life of the plant. Alternatively, treatment with the method of the invention may result in something less than total resistance to disease. Such reduced resistance may still provide the plant with resistance to pathogens and diseases. Reduced resistance may not be a total resistance, but will reduce the growth of pathogenic organisms adequately and will reduce the pathologic effects of those organisms.

It is an unexpected advantage of the invention that the resistance induced by the method of the invention is non-specific. Plants treated in accordance with the method of the invention have been found to be resistant to pathogen growth and disease from a broad range of pathogens, including bacteria, fungi, and viruses. This non-specificity is in contrast to the specificity of resistant cultivars and to other chemical methods of disease control. Because of this non-specificity, ISR can protect plants from pathogens against which no other treatments are yet known

3. Methods of Assessing the Effect of the Diphenvl Ethers on Production of Isoflavones and the Activation of Defense Elicitors in the Soybean System. The soybean cotyledon assay is the standard assay for assessing the activity of defense elicitors in the soybean system There are two adaptations of this assay which can be used to determine the effective concentration of the nuclear leceptoi hgands

Cut cotyledon assay

The cut cotyledon assay is used to investigate both the ability of a compound (effectoi ) to activ te basal ehcitation competency in plants, and to evaluate the ability of a sccondaiy compound (elicitor) to enhance glyceollin ehcitation competencv in plants in which the lsoflavone pools were "loaded" by the action of the effectoi

The level of isoflavones in the cotyledon tissues aie measured aftei the addition of different diphenyl etheis to determine the effectiveness of each in inducing the basal pioduction of isoflavones in cotyledon tissues (effector studies) In the elicitor studies, the addition of diphenyl ether first "primes" the cut cotyledon That is, the competency for the ehcitation of the phytoalexin glyceollin in response ^fo the glucan elicitor from Phy tophthoi a sojae is aheady partially activated by the pπoi addition of a diphenyl ether As a lesult, the diphenyl ether-induced, increased levels of daidzem, which is the pi ecuisoi for glyceollin, aie lapidly conv erted into glyceollin in the piesence of the glucan Thus, the ability of a compound to enhance glyceollin ehcitation competency by "loading" the lsoflavone pools can also be measured

Cotyledons from 7-8 day old soybean seedlings are removed from the plant and cut on the lo ei surface to expose subepidermal tissues In experiments designed to study only the actions of the effectoi cotyledons are treated with a 15 ul dose of a diphenvl ethei or watei (control) In the ehcitoi studies, the cotyledons are furthei treated with 15 ul of the glucan defense ehcitoi (30 ug/nil) from the fungal pathogen Phy tophthoi a sojae oi watei (control) immediately aftei the addition of the diphenyl ether Ten cotyledons are used per tieatment and arranged in a petn plate containing a wet filter paper to keep the cotyledons moist After incubation at room temperature under approximately 200 uEinste s of light foi 48 h cotyledon tissues aie harvested for analysis Tissues for analysis are harv ested by cutting a vertical column of cells from the cotyledon using a numbei 1 cork borer The column of cells is then subsampled by cutting slices of cells progressively away fio the original cut surface The fust section is approximately 4 cell layers thick and the second two are approximately 8 cell layers thick These allow the examination of proximal and distal effects of tieatments, respectively Tissues are analyzed by HPLC as noted below Full details of this assay can be found in the publication Graham, T L , and Graham, M Y 1991 Glyceollin Ehcitoi s Induce Major But Distinctly Different Shifts in Isoflavonoid Metabolism in Local and Distal Cell Populations Mol Plant Microbe Intei 4 60-68

Snapped cotyledon assay The snapped cotyledon assay is a minimal wound assay used to niv estigate the effects of test compounds m a non-primed background The assay is perfoimed by snapping cotyledons in two and placing the petiole side down in 0 5% water agai Ten snapped cotyledons are used pei treatment, and the subepidermal cells exposed by snapping are treated with glucan defense ehcitoi and/or the effector (I e , diphenyl ethei ) being examined The cotyledons aie incubated in the light for 48 h as in the cut cotyledon assay Both proximal (fust cell layer) and distal (second and thud cell layeis) are harvested foi analysis by HPLC (see below) Full details of this assay can be found in the publication Giaham, T L and Giaham, M Y 1996 Signaling in soybean phenylpiopanoid lesponses dissection of primary, secondaiy and conditioning effects of light, wounding and ehcitoi tieatments Plant Phvstol 1 10 1 123-1 133

The snapped cotyledon assay is "naive " That is, it is not pic-disposed oi primed foi competency foi the ehcitation of phytoalexm glyceollin in lesponse to the glucan ehcitoi Thus, treatment with the glucan elicitor induces the formation of the isoflav ones daidze and gemstein, but veiy little glyceollin This is an excellent assay to study the effects of a chemical tieatment on isoflavone metabolism by itself oi in combination with the glucan In the absence of the glucan, it gives an excellent picture of the effects of the compound alone on isoflavone metabolism In the piesence of the glucan, it tells us if the test compound can induce ehcitation competency foi the glyceollin response to the glucan

4. HPLC analysis of isoflavone lev els in cotyledons treated with diphenv l ethers High peiformance liquid chromatogiaphy (HPLC) is the method of choice foi determining the levels of isoflavone defense compounds in soybean With a single HPLC analysis, one gets a complete and q lantitative profile of up to 50 or more aromatic compounds, including all the isofla ones and their conjugates and the phytoalexms, including glyceollin. As little as 20 mg of pi; nt tissue is needed and the method can be readily applied to cotyledon, leaf or any soybean tissue This analytical method allows us to determine the nmoles/g of each metabolite, which can then readily be processed to compare the percent increase or decrease of a given metabolite in comparison to either water or glucan-treated control tissues Routinely, tissues are extracted in 80% ethanol and subjected to water/acetonitπle gradient elution from a Cl 8 reverse phase HPLC column Full details of this procedure can be found in the publication: Graham, T L 1991. A Rapid, High Resolution High Performance Liquid Chromatography Profiling Procedure for Plant and Microbial Aromatic Secondary Metabolites Plant Physiol 95 584-593

EXAMPLES

The following examples are merely illustrative of the preferred aspects of the invention and are not to be construed as limiting in any way

EXAMPLE 1 - FIELD STUDIES ON THE EFFECTS OF LACTOFEN ON SOYBEAN CROPS

A) This example demonstrates the effects of a formulation, comprising lactofen, a surfactant and ammonium sulfate, in triggering ISR in soybeans, as evidenced by soybeans protection against attack by the pathogen S sclerotiorwn. The form of lactofen used the example was Cobra® The form of the surfactant used was Induce® Roundup® Ultra was also included in some of the formulations for weed control in the test plots Five different formulations were prepared, each in 15 gallon batches The identity and concentration of the ingredients in each formulation is listed in Table 1, Column 1 Foui treatments were arranged in a randomized complete block design (RCBD) so that statistical analysis of variance (ANOVA) could be performed on the results Foui replications (plots) were established for each of the four treatments Each plot measuied 25 feet by 200 feet Each plot received an application of a different formulation on Day 40, when three of the trifoliate leaves had opened and the fourth was cupped, at the V3 giowth stage Each formulation was applied using a tractor boom sprayer, calibrated to deliver 15 gallons per acre Formulations 1 , 2, 3 and 4 were applied to each of the four plots in treatments 1 , 2, 3 and 4, respectively, on day 40. On day 47, the four plots in treatment 4 received an application of formulation 5.

On Day 104, individual soybean plants in five row sections (each section measured 5 feet) were randomly selected in each of the four treatments, and inspected for evidence of S. sclerotiorwn attack. Evidence of S. sclerotiorwn attack includes both an area of the stem that is brown with white mycellium and general plant wilting. After it was found that the area of the field in which one replication of each of the treatments was located had no incidence of disease, inspections were made only in the other three replications of each of the four treatments. The mean results of the inspections in each of the four treatment (three replications) are outlined in Table 1 , Column 4. On Day 1 10, individual soybean plants in each of the four treatments (three replications) were again inspected for evidence of S. sclerotiorwn attack. The mean results of the inspections of the three replications of each treatment are outlined in Table 1 , Column 5. Analysis of variance was conducted using the Student-Newman-Keuls test. Means followed by the same letter do not significantly differ. On Day 142, the soybean plants from each of the four treatments were harvested.

Yield means from the three replications of each treatment, adjusted to 13% moisture, are outlined in Table 1 , Column 6. The mean moisture content of plants from each plot was also determine on the same date, and the results are outlined in Table 1 , Column 7.

These results demonstrate that the formulations applied to treatments 2, 3 and 4 each significantly reduced the incidence of S. sclerotiorwn attack, compared to treatment 1 . While treatment 2, 3, and 4 each received a formulation containing the diphenyl ether lactofen, treatment 1 did not. Moreover, ISR protection was maintained for the length o f the growing season in treatments 2 and 3, 60 days after application of the formulations to the individual plots. The results also demonstrate that the plants harvested from treatments 2 and 3 produced numerically higher yields of soybeans that treatment 1 , and plants harvested from treatment 4 produced significantly higher yields than treatment 1 . This indicates that the induction of systemic resistance results in a plant that is more healthy and vigorous, leading to higher yields. As seen in Table 1 , the percent moisture in plants harvested from each of the four plots was not significantly different, indicating that the infestation and yield results were not influenced by the ability of the plants to absorb and maintain moisture.

?? TABLE 1

Formulations in italics arc the controls Means followed by the same lettci do not significantly differ (P = (h Student Newman kculs)

B) Table 2 summarizes the results from a number of experimental field studies performed on vaiious farms in Ohio, Illinois and Pennsylvania While a numbei of factors varied from farm to farm (e g form of lactofen used, composition of the formulations, composition of the controls, weather conditions, soil conditions, planting conditions, etc ), as can be seen from Table 2, soybean crops treated with lactofen-containing formulations had significantly I educed incidences of S sclerotiorwn damage compared to soybeans tieated with control foπnulations lacking lactofen Moieover, crop yields from lacto fen-treated plots were generally higher than that of control plots Moistuie content did not significantly vaiy between those plants receiving lactofen and the controls

The form of lactofen used in these field trials was eithei Cobra® or Stellar® The adjuvants used in one or more formulations of this example were crop oil concentrates, non ionic suifactants, ammonium sulfate, and urea ammonium nitrate Other active chemicals used in one or moie formulations of this example were the herbicides Roundup® Ultra, Python®, Select®, Firstrate®, and Pinnacle® TABLE 2

Woodstock, 2 2 IL

Formulations in italics are the contiols

C) Additional field studies demonstrated that ISR triggered by lactofen protected soybean crops against attack by Phytophthora sojae. Soybean seeds (Pioneer 93B01 RR) were planted m plots measuring 25 by 300 feet Treatments were replicated three times in a RCBD Two separate trials were established in the same field Lactofen was applied at the Rl growth stage (Day 1) On Day 64, ten samples were taken from each plot, each sample compnsed of the plants in a five foot long section of a row Each plant in each sample was examined for evidence of P sojae Table 3 summarizes the results of this study As can be seen from the lesults, lactofen significantly reduced P sojae giovvth and increase overall crop yield

TABLE 3

Formulations in italics are the contiols Means followed bv the same lettei do not diftci smnificantK (P= 05 Student

Newman-Keuls

D) Further field studies demonstrated that ISR triggered by lactofen piotected soybean crops against sudden death syndrome, caused by F solani f sp gly cines Soybean seeds (BSR 101 , Asgrow A 3701 (RR) variety, or Pioneer P9344 (RR)) ere planted in soil infested with F. solani f.sp. glycines. Treatments were at 1 X rate and applied at the vegetative state. As can be seen from the results below, lactofen significantly reduced damage cause by F. solani f.sp. glycines.

BSR 101 A3701 (RR) P9344 (RR) V arietv

E) Greenhouse studies further demonstrated that ISR triggered by lactofen protected soybean crops against sudden death syndrome caused by F. solani f.sp. glycines. Soybean seeds (BSR 101 , Asgrow A 3701 (RR) variety, or Pioneer P9344 (RR)) were planted in soil infested with F. solani f.sp. glycines. Treatments were at 1 X rate and applied at the vegetative state.

Periodic isolation of the pathogen from roots of these plants showed significant reduction of root infection, measured by root rot severity. The reduction in soybean root colonization of /⁷, solani f.sp. glycines after foliar application of lactofen indicates that the induced resistance to F. solani f.sp. glycines is systemic.

BSR 101 43701 (RR) 1^»9344 (RR) Varict

EXAMPLE 2 - VARIATION IN TIME OF LACTOFEN APPLICATION TO SOYBEANS A field study was conducted on a farm near Muncy, Pennsylvania to determine whether varying the time at which lactofen application took place had any effect on ISR, as evidenced by suppression of S. sclerotiorwn growth. Soybean seeds (Pioneer 9352) were planted in rows 14" apart. Four treatments were arranged in a RCBD with four replications per treatment. Treatment 1 was the untreated control. Treatment 2 received only the crop oil concentrate Treatment 3 received Cobra® and crop oil concentrate when soybean plants were at the V4 growth stage (Day 1) Treatment 4 received Cobra® and crop oil concentrate when soybean plants were at the Rl growth stage (Day 12) As can be seen from the results, summarized in Table 4, the timing of lactofen application had no significant effect on the ISR-inducing ability of lactofen

TABLE 4

Foi mulations in italics are the controls Means followed bv the same letter do not differ significantly ( P 05 Student Newman euls)

EXAMPLE 3 - FIELD STUDIES ON THE EFFECTS OF LACTOFEN ON SOYBEAN CROP YIELDS

Table 5 summarizes the results of crop yield measurements, comparing lacto fen- treated soybeans with control plants The soybeans weie hai vested from farms in Ohio While a number of factors varied from farm to farm (e g form of lactofen used, composition of the fomuilations, composition of the contiols, weather conditions, soil conditions, etc ), as can be seen fiom Table 5, soybean crops treated with lactofen-contaimng fomuilations geneially had inci eased yields compared to control plots Moistuie content did not significantly vary between those plants receiving lactofen and the controls The form of lactofen used in these field trials as either Cobra® oi Stellai &> The adjuvants used in one or moie formulations of this example wei e crop oil concentrates and ammonium sulfate Roundup® Ultra was also used in a number of the formulations TABLE 5

Formuations in italics aie the controls EXAMPLE 4 - INDUCTION OF IMOFLAVONE LEVELS AS SHOWN BY TREATMENT OF SNAPPED COTYLEDONS

Plant material: cotyledons were isolated from 7 day old Williams variety soybean; 10 cotyledons were assayed per treatment. Formulation composition: diphenyl ethers tested include lactofen, fomesafen, and acifluorfen. The diphenyl ether was dissolved either in water or first in isopropanol to give a saturated solution followed by rapid dilution in water. Final concentration of isopropanol did not exceed 0.5%. The diphenyl ethers were tested over a concentration range of about 10 uM to 1 mM, using serial 3 fold dilutions from 1 mM. Diphenyl ethers were tested both alone and in the presence of 30 ug/ml of the glucan elicitor from the fungal pathogen Phytophthora sojae. Concentrations of the diphenyl ether and glucan noted here were final concentrations on the treated cotyledon.

Treatment: the exposed surface of each snapped cotyledon was treated with 7 uL of the diphenyl ether being tested, followed by 7 uL of the glucan or water. Cotyledons were incubated in constant light (200 uEinsteins) for 48 h.

Analysis: at 48 h, a thin (translucent) section was harvested from the treated cotyledon surface. Sections from the 10 cotyledons for each treatment were pooled and extracted in 80% ethanol (400 uL for each 50 mg fresh weight). The extract was then subjected to HPLC as described above. The results, shown in Table 6, indicate that each of the diphenyl ethers were capable of both inducing basal levels of isoflavones, and "priming" glyceollin competency. Values are the average of two separate experiments. The standard error was less than 15% of the average for all values.

TABLE 6

Compound Total Isoflavone Induction Glyceollin Induction

Fomesafen +85% +233%

Lactofen +64% + 181%

Acifluorfen +56% + 122%

Additional diphenyl ethers were tested in the cut cotyledon assay (Williams cotyledons) over a range of concentrations from 50-500 uM. The activity of thyroxine, expressed as the range of increase of isoflavones over the concentration range was 20-40%. EXAMPLE 5 - INDUCTION OF ISOFLAVONE LEVELS IN OTHER PLANTS AND PLANT ORGANS

A) Legumes including lima bean, mung bean, green bean, peanuts, and chickpeas were treated with compositions comprising lactofen in the cut cotyledon assay as described above in Example 4. The composition was applied to cotyledons of plants of appropriate age for the assay, that is, tissues which were fully expanded, green and non-senescent.

Lactofen was applied at a concentration of 100-200 uM. When 1 00 uM lactofen was applied to the cut cotyledons, increases ranging from 20-500% in aromatic metabolites in lima bean, chickpea, green bean and peanuts were observed. These aromatic metabolites were not identified specifically. However, their ultraviolet spectra suggest that many are isoflavones.

B) True leaves of soybean plants were also treated as described above in Example 4. For tests on true leaves of soybean or other plants, two methods were used. In the laboratory, a vacuum was used to infiltrate a small spot on the leaves using a filter disc platform hooked to a water aspirator. The leaf was placed on the filter disc platform and mild suction applied. A drop of the glucan elicitor and/or chemical treatment was then placed on the exposed surface of the leaf and allowed to infiltrate the tissue through the leaf stomatal pores. For the greenhouse or field, the chemical was applied in a formulation with a surfactant to emulsify and disperse the chemical. In each assay, the tissues w ere again analyzed by HPLC as described above.

Applied by infiltration into soybean leaf tissue, 100 uM lactofen caused a 6-fold increase in conjugates of the isoflavone genistein, and at least a 1 0-fold increase in the isoflavone daidzein, which is at nearly undetectable levels in mature soybean leaves.

C) Field and greenhgouse studies were also performed to determine whether direct application of lactofen to soybean leaves would induce increased levels of the aglycones of daidzein and genistein, and conjugates of genistein. Table 7 below shows the response of leaf tissues to lactofen as a percent increase over the appropriate control treatment. In the field (Ohio or Pennsylvania). Cobra was applied at 6 oz/acre and leaves were analyzed 8 days after treatment. In the greenhouse, lactofen was applied at 50 micromolar and the leaves were analyzed 48 hours after treatment.

As is evident from the results, soybean leaves respond to lactofen by producing very large increases in the aglycones of daidzein and genistein, and conjugates of genistein. TABLE 7

EXAMPLE 6 - INDUCTION OF ISOFLAVONE LEVELS IN SOYBEAN SEEDS

Analyses were made to determine daidzein, conjugated daidzein, genistein, and conjugated genistein levels separately at two node locations per plot (3 soybean plants weie sampled per plot) Results aie given as a total amount of the four named isoflavones The values determined for the lower node samples were added to the values of the uppei node samples, and the lesultmg value was divided by 2 to give a mean isoflavone level for each plot A total of 3 field replications (randomized complete block design) were made of each treatment times 2 samples per plot = 6 samples per treatment

A) Chemgro 2289 was planted in a field at Port Farms, Waterford, PA Applications were made June 24, 1999, at stage V4/V5 Soybeans were harvested on October 6, 1999, 104 days after treatment Leaf samples were taken on 4 DAT and 45 DAT for analysis No ratable disease occurred in these plots Yields were measured on October 7, 1999 No yield differences between treatments was found For total isoflavone calculations LSD (P = 0.05) = 1951.141 , SD = 555 345, CV = 2 24, treatment probability (F) = 0 0125

TABLE 8

Treatment Total isoflavone (nM)

Fu st, ate 84 WDG 0 3 oz/aci e 22770 67 b

Select 2EC 6 fl oz/aci e

D\ n-amiL 0 4 % V/V

Fustiate 84 WDG 0 3 oz/acie 26795 67 a

Cobra 2EC 6 fl oz/acre

Select 2EC 6 fl oz/acie e the controls Means followed by the same letter do not differ significantly (P= 05 Student-New man- euls) B) Garst 261 RR was planted m a field at Port Farms, Waterfoid, PA Applications were made June 24, 2000, at early Rl Soybeans ere harvested on October 6, 1999 No ratable disease occurred in these plots Yields were measured on Octobei 7, 1999 No yield diffeiences between treatments was found For total isoflavone calculations LSD (P = 0 05) = 3025 34, SD = 1334 75, CV = 7 4, treatment probability (F) = 0 6493

TABLE 9

Foi mulations in italics ai e the conti ols Means followed by the same lettei do not diftci smnificantlv ( P O Student-New man culs)

C) Group 1 9 RR soybeans were planted in a field at Port Farms, Watei loid, PA

Applications weie made June 24, 1999, at early Rl Soybeans weie harv ested on October 6, 1999 No latable disease occurred in these plots Yields were measured on Octobei 7, 1999 No yield diffeiences between treatments was found Foi total isoflavone calculations LSD (P = 0 05) = 2536 3, SD = 721 9, CV = 3 57, tieatment piobabihty (V) = 0 6225 TABLE 10

I reatment I otal isoflav one (nM)

Roundup Ulti a l e/t/acu 20402 67 a AMS 2 0 lbs/acι e

Roundup Ultia 1 qt/aci e 20062 83 a Cobra 2EC 6 fl oz'acie AMS 2 0 lbs/acie

Formulations in italics are the conti ols Means f ollow ed bv the same lettei do not differ siunificantK ( P- 0ι Student New man Kculs )

D) Pioneer 93B01 RR was planted in a field at Spπngei Faun, Mt Vei non, OH Applications weie made June 30, 1999 Soybeans were harvested on Octobei 26, 1999 Phytophthora in was present and was rated on Septembei 2, 1999 Cobi a tieatments provided significant I eduction in Phy tophthoi a incidence and resulted in a significant yield mciease Foi total isoflavone calculations LSD (P = 0 05) = 3835 87, SD = 1 19 86, CV = 9 36, treatment piobabihty (F) = 0 153 TABLE 11

E) Pioneei 93B01 RR was planted in a field at Springer Farm, Mt Vernon, OH Applications weie made June 30, 1999. Soybeans were harvested on October 26, 1999 Phytophthora in was present and was rated on September 2, 1999. Cobra treatments provided significant reduction in Phytophthora incidence and resulted in a significant yield increase For total isoflavone calculations LSD (P = 005) = 418986, SD = 209706, CV = 1047, treatment piobabihty (F) = 0.2878

TABLE 12

t e same etter o not i ei smni icantv (P= 0 tu ent- e man- eus) F) Ohio FG1 was planted in a field at Profit Farm, Van Wert, OH. Applications were made July 20, 2000. Soybeans were harvested at the end of the normal growing season. For total isoflavone calculations: LSD (P = 0.05) = 524.66; SD = 233.18.

TABLE 13

Treatment Total isoflavone (nM)

No treatment 4479 5 a

Cobi a 2EC 2 fl oz/acre 4761 3 a Fertilize! 3 0 gal/acι e

Formulations in italics ai e the conti ols Means followed by the same letter do not diffei significantly (P= ()ϊ Student-New man-Keuls)

G) Ohio FG1 was planted in a field at Profit Farm, Van Wert, OH Applications were made July 13, 2000. Soybeans were harvested at the end of the normal growing season. For total isoflavone calculations: LSD (P = 0.05) = 407.05; SD = 180.91.

TABLE 14

the same lettci do not diffei sι«nιf ιcantl\ ( P 05 Student-New man-Keuls)

H) Ohio FG1 was planted m a field at Profit Farm, Van Wert, OH. Applications were made July 10, 2000. Soybeans were harvested at the end of the normal growing season For total isoflavone calculations: LSD (P = 0.05) - 1 137.79; SD = 505.68.

TABLE 15

1 reatment Total isoflav one (nM)

No ti eatment 4816 8 a

Cobia 2EC 4 fl oz/acie 5046 a Feitihzei 3 0 gal/acie

Formulations in italics are the conti ols Means follow ed b\ the same letter do not differ significantly ( P= 05 Student-New man-Keuls) I) Ohio FG1 was planted in a field at Profit Farm, Van Wert, OH Applications were made July 10, 2000. Soybeans were harvested at the end of the normal growing season For total isoflavone calculations: LSD (P = 0.05) =1355.94; SD = 602.64

TABLE 16

Treatment Total isoflavone (nM)

No ti eatment 5164 3 a

Cobia 2EC 2 fl oz/acre 5135 5 a Feiti zer 3 0 gal/acre

Formulations in italics are the controls Means followed by the sa e letter do not differ significantly (P= 05, Student-Newman-Keuls)

J) Pioneer 9305 was planted in a field at St Charles Seminary, Coldwater, OH Applications were made July 1 , 2000 Soybeans were harvested at the end of the normal growing season For total isoflavone calculations LSD (P = 0 05) = 296 53, SD = 131 79

TABLE 17

Treatment Total isoflavone (nM)

Select 2 EC 6 fl oz/aci e 3648 a

Dynamic 2 c/uai ts/100

Cobra 2EC 6 fl oz/acre 4009 8 b

Select 2EC 6 fl oz/acie

Firstiate 0 3 oz/acre

Dynamic 2 quai ts/100

Foi mulations m italics are the contiols Means followed by the same letter do not differ significantly (P= 05, Student-New man Keuls)

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one of skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof

Claims

CLAIMSWhat is claimed is:

1. A method of triggering induced systemic resistance in a plant comprising, applying an effective amount of a biologically active formulation comprising a diphenyl ether to the surface of at least a part of the plant, triggering activation of induced systemic resistance in the plant, thereby inducing systemic resistance to at least one pathogen or disease.

2. The method of claim 1 , wherein said diphenyl ether has a structure represented by one of the following formulas:

(I)

wherein R_\ is a hydrogen, fluorine, or chlorine atom, or a trifluoromethyl group; R₂, R₃ and R₅ are independently a hydrogen, fluorine, or chlorine atom; R₄ is a hydrogen atom, NR₆, NR₆R₆, OR₍₎, COOR₆, COOCHR₆CO₂R₆, CONHSO₂R₆, or a cyclic ether, wherein R„ is a hydrogen atom, a branched alkyl group of 1 to 4 carbon atoms or a linear alkyl group of 1 to 4 carbon atoms; wherein R₇ is an oxygen or nitrogen atom; and R₈ is a hydrogen atom, CH₃, an aliphatic chain comprising 2 to 5 carbon atoms, or HSO₂CH₃; and wherein R₉ is H, CI, I, Br or CF₃; and Rio is a branched aliphatic chain comprising 1-5 carbon atoms.

3. The method of claim 2, wherein said diphenyl ether has a structure represented by formula (I).

4. The method of claim 3, wherein the diphenyl ether is acifluorfen, aclonifen, bifenox, chlomethoxyfen, chlomitrofen, fluorodifen, fluoroglycofen, fluoronitrofen, fomesafen, furyloxyfen, halosafen, lactofen, nitrofen, nitrofluorfen or oxyfluorfen.

5. The method of claim 4, wherein the diphenyl ether is lactofen.

6. The method of claim 1 , wherein the formulation further comprises one or more adjuvants selected from phytologically acceptable carriers, crop oil concentrates, surfactants, fertilizers, emulsifiers, dispersing agents, foaming activators, foam suppressants, and correctives.

7. The method of claim 6, wherein the adjuvant is a surfactant.

8. The method of claim 7, wherein the adjuvant is a non-ionic surfactant.

9. The method of claim 6, wherein the adjuvant is a crop oil concentrate.

10. The method of claim 6, wherein the adjuvant is ammonium sulfate or urea ammonium nitrate.

1 1. The method of claim 1 , wherein the formulation further comprises one or more other active chemicals.

12. The method of claim 1 1 , wherein the one or more other active chemicals is a herbicide.

13. The method of claim 1, wherein the plant is a legume selected from lima bean, pinto bean or soybean.

14. The method of claim 13, wherein the legume is soybean.

15. The method of claim 1, wherein induced systemic resistance is triggered before the onset of disease due to said pathogen.

16. The method of claim 1 , wherein induced systemic resistance lasts until the plant is harvested.

17. The method of claim 1 , which further comprises applying to the surface of the plant a booster application of said fomiulation subsequent to the initial application, thereby inducing continued resistance to the pathogen.

18. A method of increasing plant yield comprising, applying an effective amount of a biologically active formulation comprising a diphenyl ether to the surface of at least a part of the plant, triggering activation of induced systemic resistance in the plant, and maintaining or increasing the general health of the plant, thereby increasing crop yield.

19. The method of claim 18, wherein said diphenyl ether has a structure represented by one of the following formulas:

(I)

wherein R| is a hydrogen, fluorine, or chlorine atom, or a trifluoromethyl group; R₂. R₃ and R₅ are independently a hydrogen, fluorine, or chlorine atom; R4 is a hydrogen atom, NR₆, NR₆R₍₎, OR₆, COORo, COOCHR₆CO₂R₆, CONHSO₂R₍„ or a cyclic ether, wherein R₍₎ is a hydrogen atom, a branched alkyl group of 1 to 4 carbon atoms or a linear alkyl group of 1 to 4 carbon atoms; wherein R₇ is an oxygen or nitrogen atom; and R₈ is a hydrogen atom, CH₃, an aliphatic chain comprising 2 to 5 carbon atoms, or HSO₂CH₃; and wherein R₉ is H, CI, I, Br or CF₃; and Rio is a branched aliphatic chain comprising 1-5 carbon atoms.

20. The method of claim 19, where said diphenyl ether has a structure represented by formula (I).

21. The method of claim 20, wherein the diphenyl ether is acifluorfen, aclonifen, bifenox, chlomethoxyfen, chlornitrofen, fluorodifen, fluoroglycofen, fluoronitrofen, fomesafen, furyloxyfen, halosafen, lactofen, nitro fen, nitrofluorfen or oxyfluorfen.

22. The method of claim 21 , wherein the diphenyl ether is lactofen.

23. The method of claim 18, wherein the fomiulation further comprises one or more adjuvant selected from phytologically acceptable carriers, crop oil concentrates, surfactants, fertilizers, emulsifiers, dispersing agents, foaming activators, foam suppressants, and correctives.

24. The method of claim 23, wherein the adjuvant is a surfactant.

25. The method of claim 24, wherein the adjuvant is a non-ionic surfactant.

26. The method of claim 23, wherein the adjuvant is a crop oil concentrate.

27. The method of claim 23, wherein the adjuvant is ammonium sulfate or urea ammonium nitrate.

28. The method of claim 18, wherein the formulation further comprises one or more other active chemicals.

29. The method of claim 28, wherein the one or more other active chemicals is a herbicide.

30. The method of claim 18, wherein the plant is a legume selected from lima bean, pinto bean or soybean.

31. The method of claim 30, wherein the legume is soybean.

32. The method of claim 18, wherein induced systemic resistance is formed before the onset of disease due to said pathogen.

33 The method of claim 18, wherein induced systemic resistance lasts until the plant is harvested

34 The method of claim 18, which further comprises applying to the surface of the plant a booster application of said fomiulation subsequent to the initial application, thereby inducing continued resistance to the pathogen

35 A method for increasing levels of an isoflavone in a plant comprising, applying an effective amount of a biologically active fomiulation comprising a diphenyl ethei to the surface of at least a part of the plant, inducing release or production of an isoflavone in the plant, thereby increasing levels of an isoflavone in the plant

36 The method of claim 35, wherein said diphenyl ether has a sti uctuie repiesented by one of the following formulas

(I)

wheiem R, is a hydiogen, fluoπne, or chlorine atom, or a trifluoromethyl gioup, R₂, R₃ and R₅ aie independently a hydrogen, fluonne, or chlorine atom, R4 is a hydiogen atom, NR₆, NR₆R₆, OR_ϋ, COOR₆, COOCHR₆CO₂R₆, CONHSO₂Rr„ 01 a cyclic ether, wheiem R„ is a hydrogen atom, a branched alkyl group of 1 to 4 carbon atoms or a lineai alkyl group of 1 to 4 carbon atoms, wherein R₇ is an oxygen or nitrogen atom; and R₈ is a hydrogen atom, CH₃, an aliphatic chain comprising 2 to 5 c;ιrbon atoms, or HSO₂CH₃; and wherein R₉ is H, CI, I, Br or CF₃; and Rio is a branched aliphatic chain comprising 1-5 carbon atoms.

37. The method of claim 36, wherein said diphenyl ether has a structure represented by formula (I).

38. The method of claim 37, wherein the diphenyl ether is acifluorfen, fomesafen or lactofen.

39. The method of claim 38, wherein the diphenyl ether is lactofen.

40. The method of claim 35, wherein the formulation further comprises one or more adjuvants selected from phytologically acceptable carriers, crop oil concentrates, surfactants, fertilizers, emulsifiers, dispersing agents, foaming activators, foam suppressants, and correctives.

41. The method of claim 40, wherein the adjuvant is a surfactant.

42. The method of claim 41 , wherein the adjuvant is a non-ionic surfactant.

43. The method of claim 40, wherein the adjuvant is a crop oil concentrate.

44. The method of claim 40, wherein the adjuvant is ammonium sulfate or urea ammonium nitrate.

45. The method of claim 35, wherein the fomiulation further comprises one or more other active chemicals.

46. The method of claim 45, wherein the one or more other active chemicals is a herbicide.

47. The method of claim 35, wherein the plant is a legume selected from lima bean, pinto bean or soybean.

48. The method of claim 47, wherein the legume is soybean.

49. A method for increasing the levels of isoflavones in plants, comprising applying to the plant a composition comprising a phytologically acceptable carrier and an effective amount of a diphenyl ether of formula (II): wherein R₇ is an oxygen or nitrogen atom; and R₈ is a hydrogen atom, CH₃, an aliphatic chain comprising 2 to 5 carbon atoms, or HSO₂CH₃.