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1. (WO1991016820) PHOTODYNAMIC PLANT DEFOLIANTS AND CHLOROPHYLL BIOSYNTHESIS MODULATORS
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PHOTODYNAMIC PLANT DEFOLIANTS AND
CHLOROPHYLL BIOSYNTHESIS MODULATORS
This application is a continuation-in-part of copending application Serial No. 521,119, filed May 3, 1990, which in turn is a continuation-in-part of application Serial No. 895,529, filed August 11, 1986, which in turn is a continuation of abandoned application Serial No. 754,092, filed July 15, 1985, which in turn is a continuation-in-part of abandoned application Serial No. 634,932, filed July 27, 1984, and this application is a continuation-in-part of copending application Serial No. 895,529, filed August 11, 1986, which in turn is a Tntinuation of abandoned application Serial No. 754,092, .iled July 15, 1985, which in turn is a continuation-in-part of abandoned application Serial No. 634,932, filed July 27, 1984.
The invention described herein was made in the course of work supported by grants from the U.S. Department of Agriculture, the National Science Foundation, the University of Illinois, the University of Illinois Agriculture Experiment Station and the John P. Trebellas Photobiotechnology Research Endowment.
FIELD OF THE It.TVENTION
This invention pertains to plant desiccating compositions and methods, and more particularly to plant desiccating compositions and methods for the induction of the accumulation of photodynamic tetrapyrroles in the foliage of plants.
This invention also pertains to herbicidal and insecticidal compositions and methods, and more particularly to herbicidal and insecticidal compositions and methods for the induction of the accumulation of photodynamic tetrapyrroles in plants and for elevating endogenous tetrapyrrole levels in insects.
BRIEF DESCRIPTION OF THE FIGURES
The following terms, as used hereinbelow, have the following meaning unless expressly stated to the contrary: Alk ■= (C2-C10) alkyl group; ALA = δ-aminolevulinic acid; Chi *- chlorophyll; Chlide a. = chlorophyllide a.; coprogen = coproporphyrinogen; cv = cultivar; dicot = dicotyledenous plant; DP = dipyridyl; DV =• divinyl; E = ester; F.A1 = fatty alcohol; LMP = longer wavelength metalloporphyrins (the putative intermediates of ring E formation) ; M = methylation; ME = methyl ester; Me = methyl; Me.P = methylpropionate; monocot - monocotyledenous plant; MPE - Mg-protoporphyrin monoester; MP(E) = mixture of MPE and Mg-protoporphyrin IX; MV «= monovinyl; P - esterification with geranyl geraniol, followed by stepwise conversion of the latter to phytol; PBG = porphobilinogen; Pchl = protochlorophyll; Pchlide = protochlorophyllide; Phy = phytol; Proto = protoporphyrin IX; Protogen = protoporphyrinogen IX; Urogen = uroporphyrinogen, var = variety.
The invention will be understood more clearly and fully with reference to the accompanying figures, in which:
FIG. 1 depicts the six-branched Chi a biosynthetic pathway;
FIG. 2 depicts representative structures of some of the metallotetrapyrroles ("tetrapyrroles") depicted in FIG. 1;
FIG. 3 depicts the percent defoliation of apple seedlings as related to the accumulation of protoporphyrin IX (*coefficient of determination (r2) is significant at the 5% level) ;
FIG. 4 depicts the percent defoliation of apple seedlings as related to the accumulation of divinyl Mg protoporphyrin monoester (*coefficient of determination (r2) is significant at the 5% level) ; and
FIG. 5 depicts the percent defoliation of apple seedlings as related to the accumulation of monovinyl protochlorophyllide (*coefficient of determination (r2) is significant at the 5% level) .
BACKGROUND OF THE INVENTION
The elimination of undesirable plants by herbicides is critical to modern agricultural practice, and a great deal of time and money is currently dedicated to the discovery of efficient, environmentally safe herbicides. Usually this discovery begins with the screening of a spectrum of biochemicalε for herbicidal activity. Those chemicals which exhibit promising herbicidal activity are then subjected to further testing, aimed at defining their efficacy, selectivity, environmental impact, and toxic effects on fish, insects and animals. In this scheme, the understanding of the mode of action is irrelevant and is assigned a low priority. As a consequence the detailed mode of action for some of the widely used herbicides is still not completely understood. See, e.g., Herbicide Handbook. Beste, C.E., ed. (Weed Science Soc. of America, Champaign, IL 1983), pp. 1-469. There is neither a consistent scientific basis for the selection and/or design of safe, effective herbicides, nor a scientific rationale for the systematic elimination of compounds likely to have a deleterious effect on the environment or on non-target plants and animals.
The timing and manipulation of plant development have some important implications in deciduous tree and crop production. Two important aspects in the manipulation of plant development are defoliation and fruit drop. First, controlled defoliation of nursery stock is essential for the effective management of rootstocks and grafted trees of both fruit and woody ornamental crops. For example, in areas with long growing seasons, nurserymen need to hasten defoliation in order to facilitate autumn digging. Autumn digging is a process whereby trees are dug out of the ground or "undercut" and then placed in cold storage. In order for a tree to be undercut and placed in cold storage, it must be dormant, i.e., it is not producing new shoots. Defoliation triggers dormancy in the tree which is followed by hardening of the tree. Hardening protects the tree from cold injury. Young apple trees are usually undercut and removed from the nursery in the fall and placed in cold storage. However, very often, trees in the nursery become juvenile by continuing to produce new shoots and retaining foliage longer through the season which retards hardening of the trees, making them more susceptible to cold injury. Therefore, it is desirable to hasten defoliation of the young trees.
Prior to the advent of chemical sprays or dust treatments, defoliation was done by hand, sometimes causing damage to shoots, bark, and buds. However, hand defoliation is time consuming and adds to the cost of a nursery operation. Furthermore, nutrients that would normally be translocated into the shoots during leaf senescence are lost.
Second, control of fruit drop is important in harvesting of fruit from trees. For example, aerial and subterranean fruits and vegetables are presently harvested either manually or mechanically. Manual harvesting is labor-intensive and expensive. Aerial mechanical harvesting of fruits uses heavy equipment that shakes the fruit off the tree. This in turn causes both soil compaction and frequent limb and trunk injury resulting in shorter tree life. Such mechanical harvesting also results in fruit damage in the form of bruises, cuts and punctures.
Various naturally occurring and non-naturally occurring chemical substances have been used for the purposes of defoliation and enhancement of fruit drop. In the case of fruit drop, chemicals are used to reduce fruit-pedicel attachment strength, thus allowing tree shakers to drop fruit more easily. However, chemical fruit harvesting has not been completely successful and has been used only in order to facilitate mechanical harvesting.
It would be a significant and useful advance in the art to have a chemical composition capable of causing defoliation and/or defoliation and fruit drop in plants, particularly deciduous fruit trees, via a mechanism involving one or more naturally occurring intermediates of the chlorophyll biosynthetic pathway and which alleviates in turn the disadvantages associated with present methods for defoliation and fruit drop.
Chlorophyll biosynthesis is a major biological phenomenon in the biosphere and is mandatory for the biosynthesis of photosynthetic membranes during greening and for the repair and maintenance of the Chi in mature green plants. The chlorophylls are a group of Mg-tetrapyrroles which in green plants catalyze the conversion of solar energy into chemical energy via the process of photosynthesis. There are two basic classes of chlorophyll, designated chlorophyll a. (Chi a.) and chlorophyll b. (Chi b.) ; Chi a. is involved in the collection of solar energy and its conversion to chemical energy whereas Chi b_ is believed to be involved only in the collection of solar energy.
As shown in FIG. 1, ten species of Chi a are all synthesized via a multiple-branched pathway from one common precursor, δ-aminolevulinic acid (ALA) , via a series of porphyrin, Mg-porphyrin, and protochlorophyll intermediates, collectively referred to as tetrapyrroles or tetrapyrrole intermediates (see FIG. 2) .
As can be seen in FIG. 1, three of the branches of the synthetic pathway have been designated as divinyl (DV) pathways; the two monocarboxylic acid pathways predominate in dicots and in monocots in the presence of light. The remaining three branches have been designated the monovinyl (MV) pathways; the two monocarboxylic acid pathways predominate in monocots in the dark. Plants may be classified as "monovinyl" or "divinyl" plants, depending on which pathways predominate. A monovinyl plant is a plant species which in darkness accumulates MV Pchlide via the MV monocarboxylic acid biosynthetic routes and upon exposure to light initially forms Chi mainly via the MV monocarboxylic acid routes. Divinyl plants are plant species which accumulate mainly DV Pchlide in darkness and upon exposure to light initially form Chi preferably via the DV monocarboxylic biosynthetic routes. After several hours in daylight both MV and DV plants appear to form Chi via the DV monocarboxylic routes. This in turn has led to the classification of plants into four different greening groups (Rebeiz, CA. , Montazer-Zouhoor, A., Mayasich, J.M., Tripathy, B.C., Wu, S., and Rebeiz, C.C. CRC Crit . Rev. in Plant Sci . , 6:385-435 (1988)):
(a) Dark divinyl/liσht divinyl fDDV/LDV) . In this greening group, chlorophyll formation proceeds via the

DV-enriched protochlorophyllide pools at daybreak and in daylight.
(b) Dark onovinyl/light divinyl fDMV/LDV) . In this greening group, chlorophyll formation proceeds via the MV-enriched protochlorophyllide pools at daybreak and via the DV-enriched protochlorophyllide pools in daylight.

(c) Dark monovinyl/light monovinyl (DMV/LMV) . In this greening group, chlorophyll formation proceeds via the MV-enriched protochlorophyllide pools in darkness and via the MV-enriched protochlorophyllide pools at daybreak and in daylight.
(d) Dark divinyl/light monovinyl fDDV/LMV^ . In this pathological greening group, chlorophyll formation proceeds via the DV-enriched protochlorophyllide pools at daybreak and via the MV-enriched protochlorophyllide pools in daylight.

As can be seen from Fig. 2, δ-aminolevulinic acid (ALA) is a 5-carbon amino acid. ALA is found in most living animal and plant cells and is the primary tetrapyrrole precursor. It is available from a variety of specialty chemical sources, e.g., Sigma Chemical Co., St. Louis, MO and Biosynth International, Skokie, IL. It is known that excised plant tissues treated in the laboratory with small amounts of ALA will synthesize and accumulate Pchlide, which is the immediate precursor of Chlide a. and of Chi a., and that ALA will induce the accumulation of earlier tetrapyrrole intermediates of the Chi biosynthetic pathway, such as coproporphyrin, Proto, and MP(E). Once the ALA has stimulated the synthesis of the tetrapyrrole intermediates, they are normally converted in the presence of sunlight into the various forms of Chi a, as described in FIG. l. However, this rate-limiting conversion does not occur to any great extent in darkness; without sunlight, the tetrapyrrole intermediates accumulate in small amounts in their respective metabolic pools. Upon exposure to light, the conversion to Chi a. resumes and the pools are depleted. In 1974, Castelfranco, P.A. , Rich, P.M., and Beal, S.I., Plant Physiol . 53:615-618 noticed while studying the lag phase during greening of etiolated (dark grown) tissue that excised cucumber cotyledons soaked in ALA for 16 hours in the dark underwent visible tissue damage upon subsequent exposure to light, which was attributed to tetrapyrroles formed from exogenous ALA. This phenomenon was regarded as a nuisance to be avoided by illumination with red light of very low intensity or by illumination with intermittent light. It was believed that the accumulation of tetrapyrroles due to exogenous ALA was a phenomenon attributable to the peculiar circumstances of etiolation. Indeed, once the greening of etiolated tissue is initiated, the biosynthesis of chlorophyll proceeds at an abnormally high rate not found in normal green tissue.
Copending application Serial No. 07/294,132, the disclosure of which application is expressly incorporated herein by reference, describes insecticidal compositions comprising one or more compounds selected from the group consisting of δ-aminolevulinic acid, inducers of δ-aminolevulinic acid synthesis in insects and enhancers of δ-aminolevulinic acid conversion to tetrapyrroles in insects.
Copending application Serial No. 06/895,529, the disclosure of which application is expressly incorporated herein by reference, describes herbicidal compositions comprising one or more compounds selected from the group consisting of δ-aminolevulinic acid, inducers of δ-aminolevulinic acid synthesis in plants, enhancers of δ-aminolevulinic acid conversion to photodynamic tetrapyrroles in plants, and inhibitors of conversion of divinyl tetrapyrroles to monovinyl tetrapyrroles in plants; methods for inducing the accumulation of photodynamic tetrapyrroles in living plants using said compositions, and methods of controlling plants using said compositions. These compositions were discovered to have a herbicidal effect on plants as the result of the accumulation of tetrapyrroles in amounts greater than those normally found in the plants. This was surprising because mature green plants synthesize chlorophyll only at a rate sufficient to keep up with leaf expansion and repair, and it had not been previously believed that this rate would be sufficient to allow accumulation of amounts of tetrapyrroles large enough to result in photodynamic injury.
The accumulated tetrapyrroles photosensitize the formation of singlet oxygen, which is a very strong oxidant. The singlet oxygen rapidly oxidizes the lipoprotein components of the plant cellular membranes, thus setting in motion a highly destructive free-radical chain reaction, which can be summarized as follows (hv = photon of light; αTet = tetrapyrrole in the singlet ground state; 3Tet* = tetrapyrrole in the triplet excited state; -02 — oxygen in the triplet ground state; 102* = oxygen in the singlet excited state; UMLP = unsaturated membrane lipoproteins) :
(1) 2Tet + hv ► 3Tet*
(2) 3Tet* + 3022Tet + ~0
(3) 102* *+ (UMLP) ' hydroperoxides
(4) hydroperoxides ► free radicals
(5) free radicals + UMLP ► more hydroperoxides

(6) repetition of steps (4) and (5) until most of the UMLP are oxidized.
While photosensitization by injected tetrapyrroles had been described in animals and human tissues (see, e.g., Ellefson, R.D., Mayo Clinic Proc. 57:454-458 (1982); Christensen, T. , Sandquist, T. , Feren, K. , Waksvik, H. , and Moan, J. , Br. J. Cancer 48:35-43 (1983); Hopf, F.R., and Whitten, D.G., in The Porphyrinε , Vol. 2, Dolphin, D., ed. (Academic Press, New York, 1978), pp. 161-195; Sandberg, S., Romslo, I., Hovding, G. , and Bjorndal, T. , Acta Dermatovener (Stockholm) Suppl. 100:75-80 (1982); Latham, P.S., and Bloomer, J.R. , Photochem. Photobiol . 37:553-557(1983); Bickers, D.R. , Dixit, R. , and Mukhtar, H. , Biochem . Biophys . Res . Com . 108:1032-1039 (1982)), this phenomenon had not been demonstrated in whole green plants nor adapted to control undesirable susceptible plant species prior to the invention of Serial No. 895,529.
OBJECTS OF THE INVENTION
It is therefore an object of this invention to provide a model for the systematic design and formulation of herbicides and insecticides.

It is a second object of this invention to provide a class of herbicides and insecticides which will kill undesirable plants and insects via a predetermined and novel mode of action, based on sound biochemical principles.
It is an additional object of this invention to provide herbicides and insecticides which are environmentally safe, selective, and efficient at low concentrations.
It is also an object of the present invention to provide a composition which is capable of defoliating a plant without killing the plant by causing the foliage to accumulate levels of tetrapyrroles which are higher than those normally found in the foliage.
It is a further object of the invention to provide a composition for causing fruit drop in a plant, particularly in a deciduous fruit tree, without the need for mechanical harvesting.
It is yet another purpose of the invention to provide a composition capable of causing defoliation and fruit drop in a plant.
It is another object of the invention to provide compositions which are capable of defoliating and/or defoliating and causing fruit drop in a plant which are environmentally safe and efficient at low concentrations.
SUMMARY OF THE INVENTION
It has now been discovered that compositions comprising δ-aminolevulinic acid and/or inducers of δ- aminolevulinic acid and/or enhancers of δ-aminolevulinic acid conversion to photodynamic tetrapyrroles and/or inhibitors of conversion of divinyl tetrapyrroles to monovinyl tetrapyrroles can be safe, efficient, selective herbicides, when applied to plants which are subsequently exposed to light. The herbicidal compositions of the present invention result in death and destruction of the plant tissue by a process believed to involve the induced accumulation of photodynamic tetrapyrroles.
It has also been discovered that insects can be killed by administration of a composition comprising one or more compounds selected from the group consisting of

ALA, inducers of ALA in insects, and enhancers of ALA conversion to tetrapyrroles in insects.
The invention is broadly directed to compositions for causing defoliation and/or fruit drop in whole, living plants and methods for defoliating and/or causing fruit drop in whole, living plants. Thus, in one embodiment, the invention is a plant defoliating composition comprising a plant defoliating effective amount of δ-aminolevulinic acid or δ-aminolevulinic acid in combination with one or more chlorophyll biosynthesis modulators and a suitable carrier.
In another embodiment, the invention is a plant defoliating composition comprising a plant defoliating effective amount of δ-aminolevulinic acid in combination with one or more chlorophyll biosynthesis modulators which are selected from the group consisting of inducers of δ-aminolevulinic acid synthesis, enhancers of δ-aminolevulinic acid conversion to tetrapyrroles and inhibitors of conversion of divinyl tetrapyrroles to monovinyl tetrapyrroles.
Another embodiment of the invention is a method for defoliating a plant comprising the steps of contacting the plant with a defoliating effective amount of δ-aminolevulinic acid, one or more chlorophyll biosynthesis modulators, or δ-aminolevulinic acid in combination with one or more chlorophyll biosynthesis modulators and allowing the contacted plant to be exposed to light.
In another embodiment, the invention is a method for defoliating a plant comprising the steps of contacting the plant with a defoliating effective amount of δ-aminolevulinic acid in combination with one or more chlorophyll biosynthesis modulators and allowing the contacted plant to be exposed to light.
In still another embodiment, the invention is a method for defoliating a plant comprising the steps of contacting the plant with a defoliating effective amount of δ-aminolevulinic acid, one or more chlorophyll biosynthesis modulators, or δ-aminolevulinic acid in combination with one or more chlorophyll biosynthesis modulators, exposing the contacted plant to a substantial absence of light at wavelengths of 300 to 700 mM and then exposing the contacted plant to light.
A further embodiment of the invention is a composition for causing defoliation and fruit drop in a deciduous fruit tree comprising an amount effective to cause defoliation and fruit drop in a deciduous fruit tree of δ-aminolevulinic acid or δ-aminolevulinic acid in combination with one or more chlorophyll biosynthesis modulators and a suitable carrier.
Another embodiment of the invention is a composition for causing defoliation and fruit drop in a deciduous fruit tree comprising an amount effective to cause defoliation and fruit drop in a deciduous fruit tree of δ-aminolevulinic acid in combination with one or more chlorophyll biosynthesis modulators capable of causing the foliage of the tree to accumulate levels of tetrapyrroles which are selected from the group consisting of inducers of δ-aminolevulinic acid synthesis, enhancers of δ-aminolevulinic acid conversion to tetrapyrroles and inhibitors of the conversion of divinyl tetrapyrroles to monovinyl tetrapyrroles.
In a specific embodiment, the invention is a composition for causing defoliation and fruit drop in a deciduous fruit tree comprising an amount effective to cause defoliation and fruit drop in a deciduous fruit tree of δ-aminolevulinic acid in combination with ethyl nicotinate.
Still another embodiment of the invention is a method for causing defoliation and fruit drop in a deciduous fruit tree comprising the steps of contacting the tree with an amount effective to cause defoliation and fruit drop in the tree of δ-aminolevulinic acid, one or more chlorophyll biosynthesis modulators, or δ-aminolevulinic acid in combination with one or more chlorophyll biosynthesis modulators and allowing the contacted tree to be exposed to light.
A further embodiment of the invention is directed to a method for causing defoliation and fruit drop in a deciduous fruit tree comprising the steps of contacting tree with an amount effective to cause defoliation and fruit drop in the tree of
δ-aminolevulinic acid, one or more chlorophyll biosynthesis modulators, or δ-aminolevulinic acid in combination with one or more chlorophyll biosynthesis modulators, exposing the contacted tree to a substantial absence of light at wavelengths of 300 to 700 mM and then exposing the contacted tree to light.
DETAILED DESCRIPTION OF THE INVENTION
The invention will be understood more clearly and fully from the following detailed description.
It has now been discovered that foliage of whole, living plants can be induced by exposure to exogenous ALA to accumulate artificially high amounts of photodynamic tetrapyrrole intermediates in excess of levels normally found in foliage of living plants, and that such induced artificially high levels are sufficiently photodynamic so that subsequent exposure of the foliage to light causes desiccation and death of the foliage without killing the rest of the plant.
As a consequence of desiccation of the foliage of the plant, there is formation of an abscission layer between the branch and the leaf petioles (stems) which ultimately causes the leaves to fall from the branch. Similarly, desiccation of the foliage of fruit bearing plants, e.g., deciduous fruit trees, causes formation of an abscission layer between the branch and the fruit pedicel (stem) or the pedicel and the fruit which ultimately causes the fruit to drop from the branch.
The foliage of a plant can be induced to accumulate tetrapyrroles because foliage is capable of synthesizing tetrapyrroles via the chlorophyll biosynthetic pathway. In contrast, "woody" parts of a plant, e.g., bark or stalk, do not actively synthesize tetrapyrroles and have sufficient carbohydrate reserves to recover from desiccation so that, notwithstanding desiccation of the foliage, the plant does not die.
As used herein, the term "plant" means a tree, shrub, seedling, or herb, which is a living organism and which typically does not exhibit voluntary motion or possess sensory or nervous organs.
The term "woody" as used herein refers to plant tissue which does not actively synthesize tetrapyrroles and includes ligneous tissues, i.e., tissue containing wood, wood fibers or wood-like fibers.
The term "herbaceous plant" refers to a plant having little or no woody tissue.
The term "deciduous tree" broadly refers to the opposite of evergreen tree and includes trees whose leaves fall off seasonally or at a certain stage of development in the life cycle.
As used herein, the term "young leaf" refers to a leaf which is still expanding in size.
As used herein, the term "mature leaf" refers to a leaf which has stopped expanding in size.
As used herein, the term "old leaf" refers to a mature leaf which is approaching senescence.

As used herein, the term "desiccate" means broadly to dry and includes loss of cellular fluids, followed by degradation of chlorophyll (Chi) and other biomolecules such as proteins, lipoproteins, and nucleic acids, and disintegration of subcellular organelles such as vacuoles, nuclei, mitochondria, plastids, microsomeε and microbodies.
As used herein, the term "defoliate" meanε broadly to remove green plant tissue, in particular, foliage (leaves) and includes separation of leaves at their junction to petioles, or separation of leaves and petioles at their junction to branches, before or after complete leaf desiccation.
As used herein, the term "fruit drop" means broadly to remove fruit from branches of plants, in particular deciduous fruit trees, and includes separation of fruit at the junction to pedicels or separation of fruit and pedicels at their junction to branches before or after leaf desiccation.
As used herein, the term "fruit" means the mature ovary of a flower which includes either parts of the flower or inflorescence which are intimately associated with the mature ovary. Fruit includes but is not limited to apples, oranges, pears, peaches, cherries, tomatoes and the like.
As used herein, the term "chlorophyll biosynthesis modulator" refers to a compound other than exogenous ALA (ALA from sources outside the plant) which causes the green tissue of a plant, e.g., foliage, to accumulate levels of tetrapyrroles which are higher than levels of tetrapyrroles normally found in untreated green tissue. Such modulators are selected from the group consisting of inducers of ALA synthesis, enhancers of ALA conversion to tetrapyrroles and inhibitors of conversion of divinyl tetrapyrroles to monovinyl tetrapyrroles. In accordance with the invention, one or more modulators, or one or more modulators in combination with ALA can be used to effect defoliation or defoliation and fruit drop in plants.
Modulators of the invention include, e.g., o-phenanthroline, 1,7-phenanthroline, 4,7-phenanthroline, and phenanthridine, available from, e.g.. Alpha Products, Danvers, MA; 2 ,2 • -dipyridyl (2,2'-DP), 2 ,3 •-dipyridyl (2, 3' -DP), 2 , 4 ' -dipyridyl (2 ,4'-DP), 1,7 '-dipyridyl (1,7'-DP), 4 , '-dipyridyl (4,4'-DP), pyridine 2-aldehyde, pyridine 2-aldoxime, 2,2 '-dipyridylamine, 2 , 2 •-dipyridyl disulfide, 8-hydroxyquinoline, picolinic acid, nicotinic acid, 6-amino nicotinamide, ethyl nicotinate, 2-hydroxy-nicotinic acid, ethyl 2-methyl nicotinate, N-methyl nicotinamide, N-benzyl-N-nicotinoyl nicotinamide, 2-hydroxy-6-methylpyridine-3-carboxylic acid, 4-hydroxy-7-trifluoromethyl-3-quinoline carboxylic acid, 4-hydroxy-7-methyl-l,8-naphthyridine-3-carboxylic acid, diethyl 3,4-pyridine dicarboxylate, and niflumic acid, available from, e.g., Aldrich Chemical Co., Milwaukee, WI; and analogs thereof. Other modulators are listed in Table XVI below. It should be noted that nicotinic acid, an enzyme cofactor, occurs in all living cells and appreciable amounts are found in liver, yeast, milk, adrenal glands, white meat, alfalfa, legumes, whole cereals and corn. In addition, ethyl nicotinate is a vitamin derivative.
By "inducer of ALA synthesis" is meant a compound which, when applied to plants, stimulates the green tissue of the plant to produce a higher than normal amount of endogenous ALA ("native ALA,1* i.e., ALA normally found in a plant) which in turn causeε accumulation of tetrapyrroleε at levels sufficiently photodynamic so that upon subsequent exposure of the tissue to light, the tissue desiccates. Thus, an inducer results in a significant accumulation of a particular MV or DV tetrapyrrole when applied to a plant in the absence of exogenous ALA. Significant accumulation of a particular tetrapyrrole is defined as an amount of that accumulated tetrapyrrole which approaches or exceeds the net dark-conversion rate into that tetrapyrrole, brought about by a 5 mM exogenous ALA treatment. Furthermore, the inducer, in combination with ALA, results in the accumulation of higher levels of the particular MV or DV tetrapyrrole than when ALA or the inducer are applied to the plant separately. Thus, the compositions of the invention can comprise one or more inducers of ALA, or one or more inducers of ALA in combination with ALA.
By "enhancer of ALA conversion to tetrapyrroles" or "enhancer" is meant a compound which when applied to plants enhances the capability of the green tissues of the treated plants to convert exogenous or endogenouε ALA to photodynamic tetrapyrroleε. An enhancer does not result in a significant accumulation of a particular MV or DV tetrapyrrole when applied to a plant in the absence of exogenous ALA but, when used jointly with exogenous ALA, significantly enhances the dark conversion of exogenous ALA into the particular MV or DV tetrapyrroles over and beyond that caused by exogenous ALA alone, i.e., by ALA used as a control. A significant accumulation of a particular tetrapyrrole in this context is defined as the amount of that accumulated tetrapyrrole which approaches or exceeds the net dark-conversion rate into that tetrapyrrole brought about by a 5 mM exogenous ALA treatment. Enhancers of ALA conversion to tetrapyrroles fall into two groups: (1) enhancers of ALA conversion to MV Pchlide and (2) enhancers of ALA conversion to DV Pchlide and enhancers of ALA conversion to proto and MV- and DV-MPE. Thus, the compositions of the present invention can also comprise one or more enhancers of ALA, or one or more enhancers of ALA in combination with ALA or inducers of ALA.

By "inhibitor of the conversion of divinyl tetrapyrroles to monovinyl tetrapyrroles" is meant a compound which, when applied alone to plants, resultε in the inhibition of a particular MV tetrapyrrole in comparison to untreated controls and/or when applied to a plant in combination with ALA results in the inhibition of a particular MV tetrapyrrole in comparison to ALA-treated controls.
A modulator which functions as one of type of modulator (i.e., inducer, enhancer or inhibitor) in a specific plant or at a given concentration may function as a different type of modulator at a different concentration or in another plant, although a compound which is a modulator in one type of plant will be a modulator in most other types of plants.
For example, 2,2' -dipyridyl can be an enhancer in cucumber at concentrations less than 20 mM but can also be an inducer in cucumber at concentrations of 20 mM or greater. Further, in cucumber, a DDV/LDV plant species, 2 , 2 '-dipyridyl and o-phenanthroline are inducers; pyridine 2-aldoxime, pyridine 2-aldehyde, picolinic acid, 2 ,2 '-dipyridyl disulfide, 2,2'-dipyridylamine, ,4 '-dipyridyl, phenanthridine, nicotinic acid, 2-hydroxynicotinic acid, 2-hydroxy-6-methylpyridine-3-carboxylic acid, ethyl nicotinate, ethyl-2-methyl nicotinate and 4-hydroxy-7-trifluoro-8-quinoline carboxylic acid are enhancers; and 2,3'- dipyridyl, 2-4'-dipyridyl, 1,7-phenanthroline, 4,7-phenanthroline, diethyl 3,4-pyridine dicarboxylate and niflumic acid are inhibitors; in soybean, a DMV/LDV plant species, 2 , 4-dipyridyl, 2 , 2 '-dypiridylamine, phenanthridine, picolinic acid, pyridine 2-aldoxime, 2,3- dipyridyl, ,4 '-dipyridyl, 1,7-dipyridyl, pyridine 2- aldehyde, 2,2 -dipyridyl disulfide and 8-hydroxyquinoline are enhancers; and 4,7-phenanthroline and 1,7- phenanthroline are inhibitors; and in Johnsongrasε, a DMV/LMV plant species, 2,2 '-dipyridylamine, pyridine 2- aldoxime, pyridine 2-aldehyde, picolinic acid, 2,2'- dipyridyl, 2, 4-dipyridyl, 1,7-phenanthroline, 2,2*- dipyridylamine, 2,2 '-dipyridyldisulfide, 2,3-dipyridyl and 4,7-phenanthroline are enhancers; and 2,4-dipyridyl and 2,3-dipyridyl are inhibitors. One skilled in the art will be able to determine, without undue experimentation, whether a compound is a modulator and, if desired, will be able to determine the type of modulator based on the methods disclosed herein.
Advantageously, thiaflavin T can be used as a modulator in a photodynamic composition for defoliating a plant.
Various ALA and modulator combinations exhibit a significant degree of photodynamic herbicidal selectivity. This selectivity appears to be rooted (a) in the different tetrapyrrole accumulating capabilities of various plant tissues, (b) in the differential susceptibility of various greening groups of plants to the accumulation of various tetrapyrroles, and (c) in the differential response of various greening groups to photodynamic herbicide modulators.
Tetrapyrrole metabolism is not equally affected in various plant tissues by tetrapyrrole-dependent photodynamic herbicides (TDPH) treatment. For example, in green soybean seedlings, the stems, leaves and cotyledons exhibited different susceptibilities toward ALA + 2 , 2 '-dipyridyl treatments. The leaves which accumulated tetrapyrroles were quite susceptible to photodynamic damage, while the stems and cotyledons which were very poor tetrapyrrole accumulators exhibited resistance to treatment.
The dependence of TDPH susceptibility upon the greening group affiliation of treated plants as well as upon the nature of accumulated tetrapyrroles provides a basis to chemically modulate the activity of TDPH. This may be achieved with the use of chemicals that modulate the Chi biosynthetic pathway by forcing ALA-treated plants belonging to certain greening groups to accumulate the "wrong" type of MV or DV tetrapyrrole, while inducing other plant species belonging to other greening groups to accumulate the "right" type of MV or DV tetrapyrrole. A number of chemicals which acted in concert with ALA and which exhibited a definite modulating propensity toward the Chi a. biosynthetic pathway have been identified. These chemicals were therefore designated as TDPH modulators. They were classified into four groupε depending on their effects on the Chi a. biosynthetic pathway.
In order to determine whether a compound acts as a tetrapyrrole-dependent photodynamic herbicide modulator, the chemical is usually sprayed on a plant with and without ALA, and the treated plant is kept in darkness for several hours during which tetrapyrrole accumulation takes place. After dark incubation and prior to light exposure, the plant tissues are analyzed for tetrapyrrole content. Upon exposure to light, tissues that had accumulated tetrapyrroles in darkness, exhibit rapid photodynamic damage within the first hour of illumination. The classification of a modulator as an enhancer, inducer or inhibitor of tetrapyrrole accumulation is then determined from the pattern of tetrapyrrole accumulation in the presence and absence of

ALA and modulators.
Based on their mechanism of action TDPH modulators have been classified into four distinct groups: (a) enhancers of ALA conversion to DV Pchlide, which enhance the conversion of exogenous ALA to DV Pchlide, (b) enhancers of ALA conversion to MV Pchlide, which enhance the conversion of exogenous ALA to MV Pchlide, (c) inducers of tetrapyrrole accumulation, which induce the plant tissues to form large amounts of tetrapyrroles in the absence of exogenously added ALA, and (d) inhibitors of MV Pchlide accumulation, which appear to block the detoxification of DV tetrapyrroles by inhibiting their conversion to MV tetrapyrroles. Of all the aforementioned modulators, only inducers of tetrapyrrole accumulation are capable of cauεing tetrapyrrole accumulation in the absence of added ALA. The three other classes of modulators do not lead to significant levels of tetrapyrrole accumulation in the absence of added ALA. In all cases, the use of ALA together with a modulator resultε in enhanced tetrapyrrole accumulation and photodynamic damage over and beyond the levels caused by ALA alone.
It appears that (a) a modulator that acts in a certain way on the Chi biosynthetic pathway of one greening group of plants does not necessarily act the same way on plant species belonging to a different group, (b) different plant species belonging to the same greening group tend to exhibit similar Chi biosynthetic reactivities toward a given modulator and (c) modulators that belong to the same chemical category tend to exhibit the same Chi biosynthetic modulating activity toward a particular plant species. Thus, it may be possible to make certain predictions about the mode of action of a modulator toward a particular plant species belonging to a particular greening group, once the mode of action of the chemical category to which the modulator belongs has been determined for that particular group.
Thus, the compositionε of the preεent invention can also comprise combinations of ALA and one or more chlorophyll biosynthesis modulators selected from the group consisting of inducers, enhancers, and inhibitors, e.g., ALA + one or more inducers, ALA + one or more enhancers, ALA + one or more inhibitors, ALA + one or more inducers + one or more enhancers, ALA + one or more inducers + one or more inhibitors, ALA + one or more enhancers + one or more inhibitors, ALA + one or more inducers + one or more enhancers + one or more inhibitors, etc.
A consideration of one or more of the following factors will enable one skilled in the art to effect the desired defoliation and/or fruit drop for a given plant species: the species of the plant (monocot, dicot, annual, perennial, woody, non-woody) ; the age of the plant; the various tissues types present on the plant (cotyledons; stems, leaves, leaf petioles, growing points, fruit pedicels, bark, etc.); and the point of time in the growing season. For example, (a) spraying a plant with woody branches will result in the desiccation of the green leaves but not the woody branches because the woody branches are protected by suberized bark which does not respond to treatment by accumulating tetrapyrroles; (b) spraying a young plant with tender, succulent stems containing chlorophyll will desiccate both the leaves and the stems, while treatment of plants with branches protected by suberized bark will result in desiccation of the leaveε only; (c) spraying stems containing green leaves and unprotected growing points (e.g., leaf and flower buds) will desiccate both the leaves and the growing points, while spraying stems with leaves and growing points protected by suberized scales will only desiccate the leaves leaving the protected growing points unaffected; (d) spraying plants with young and old leaves may result in the desiccation of a larger proportion of the old or young leaves, depending on the nature of the modulator (i.e., inducer, enhancer or inhibitor) used with ALA; (e) spraying an annual plant with few carbohydrate reserves will result in desiccation followed by a slower rebound than a perennial plant with more carbohydrate reserves; and (f) spraying a woody plant, with carbohydrate reserves stored in the woody stems and roots, at the end of the growing season will result in desiccation of the leaves without resprouting of new leaves, while spraying the same plant early in the growing season will result in desiccation of the treated leaveε, but with regeneration of new leaveε from the carbohydrate reserves stored in the stems and roots, given proper temperature and daylength conditions.
The compositions of the present invention can contain one or more of the following: suitable carrier (s) (e.g., colloidal magnesium aluminum silicate, pumice, talc, or combinations thereof); solvent(s) (e.g., water, 0.45 acetone: 0.45 ethanol: 0.1 Tween 80:9 water (v/v/v/v) , 0.45 acetone: 0.45 methanol: 0.1 Tween 80:9 water (v/v/v/v), 0.1-1% Tween 80 in water (v/v) , 0.9 polyethylene glycol (PEG) : 0.1. Tween 80:9 water (v/v/v) , 0.1-0.7 PEG: 0.2-0.8 methanol: 0.1 Tween 80:9 water (v/v/v/v), 0.9 methanol: 0.1 Tween 80:9 water (v/v/v), 0.45 acetone: 0.45 ethanol: 0.2 Tween 80:0.9 ethylene glycol: 18 water (v/v/v/v/v) , or one or more of the following: benzene, toluene, xylene, kerosene, 2-methoxyethanol, propylene glycol, diethylene glycol, diethylene glycol diethyl ether, formamide, methylformamide, cyclohexanone, isophorone) ; buffer (s) (e.g., citric acid); wetting agent(s) (e.g., sodium N-methyl-N-oleoyltaurate, an alkylphenoxy polyoxyethylene ethanol, sodium olefin sulfonate, sodium isopropylnaphthalene sulfonate, polyoxyethylated vegetable oil); dispersing agent (ε) (e.g., sodium lignin sulfonate, the sodium salt of a naphthalene sulfonic acid-formaldehyde condensate, hydroxyethyl cellulose) ; defoaming agent(s) (e.g., silicone); emetic(s) (e.g., sodium tripolyphosphate, tetra potassium pyrophosphate, arecotine, apomorphine, copper sulfate); εtench(es) (e.g., pyridine); penetrant(s) ; surfactant (s) ; emulsifier(s) ; and adjuvant(ε) (e.g., phytoblend oils). Of course, any such additional component must be compatible with the active ingredients of the present invention and with the other ingredients in the mixture.
The compositions can be formulated in any manner conventionally used for plant preparations, e.g., as a solution, suspension, emulsion, flowable concentrate, emulsifiable concentrate, gel, paεte, foam, cream, aerosol, wettable powder, dust, dispersible granules, and the like, according to procedures known to those skilled in the art. Advantageously, the composition is a solution, suspension, emulsion, aerosol, flowable or emulsifiable concentrate, or wettable powder. Of course, the formulation must be such that the active ingredient (s) penetrate (s) the plant tissue and translocates to the sites of tetrapyrrole synthesiε. When the compositions are made in solution they can conveniently comprise concentrations of from about 1 to about 40 mM ALA, advantageously, 15 mM to 40 mM, and from about 5 to about 30 mM inducer, enhancer, or inhibitor, advantageously 15 to 30 mM.
The compositions of the present invention can be applied topically, e.g. , as a dust, soak, dip, spray, mist, or fog, in an amount sufficient to induce the accumulation of photodynamic tetrapyrroles. Alternatively, the compositions can be applied to the soil for uptake by plant roots and translocation to the vegetative part of the plant. The amount of composition to be applied will vary, depending on the particular active ingredient (s) selected, but in general will be an amount sufficient to supply from about 10 g to about 15 kg ALA per acre and/or from about 10 g to about 10 kg of an inducer, enhancer, or inhibitor per acre. Means of determining optimum application rates are within the purview of those skilled in the art.
Once the tissues of the plant have been induced to begin accumulating artificially high amounts of tetrapyrroles by exposure to the compositions of the present invention, the plant may be shielded from exposure to light to allow maximum tetrapyrrole accumulation. Such dark incubation is not required for activity but tends to optimize efficiency of the compositions. The plants can be shielded in any convenient manner, as by wrapping them in dark paper, cloth, or foil, or by placing them in a dark room or container. Under field conditions, the ideal method to provide a period of dark incubation is to apply the composition at dusk or during the night, at a time chosen to allow the plants to rest in the dark for at least one hour. It is to be understood that in order to facilitate tetrapyrrole accumulation, the dark need not be total absence of light, but rather substantial absence of light at wavelengths of from 300 to 700 nm. Advantageously, the plants are allowed to reεt in the dark for from about 1 to about 20 hours. One to 8 hours is particularly advantageous.
Thereafter the plants are expoεed to about 200 ft. candleε or more of light at wavelengths of about 300 to about 700 nm. The light can be supplied by any convenient source, e.g., an incandescent lamp, metal halide lamp, sunlamp, or a cool white or skylight fluorescent bulb. In the field, of course, the preferred source of light is sunlight. The plants are exposed to light for a period of time sufficient to oxidize most of the unsaturated membrane lipoproteins; a period of from about 1 to about 14 days is preferred.
Herbicidal activity is indicated by bleaching of the leaves, stems, and/or nodes, followed by wilting and death. If all the leaf buds are not treated, the plant may recover and require repeated treatment.
Insecticidal activity is indicated by alteration of the color of the skin, followed by desiccation and death.

A further understanding of this invention can be had from the following illustrative examples. Desiccating activity is indicated by tissue necrosiε and leaf abscission and/or fruit drop. As used hereinabove and below unless expressly stated to the contrary, all temperatures and temperature ranges refer to the centigrade system and the terms ambient and room temperature refer to about 20-25°C. The term percent or (%) refers to weight percent and the termε mole and moleε refer to gram moles. "Level of significance" refers to the probability that for a population for which the correlation coefficient (r) is equal to zero, a sample of size n can be taken, for which the correlation coefficient equals or exceeds the calculated value of r reported for the given sample. The abbreviation "n.s." stands for "not significant".

SECTION I PROTOCOL FOR DETERMINING PHOTODYNAMIC HERBICIDAL,
INSECTICIDAL AND DESICCATING COMPOSITIONS

The following examples describe model systems whereby personε skilled in the art can readily determine photodynamic compounds and compositions useful in the present invention.

Example 1
Photodynamic Effects of ALA

Cucumber (Cucumis sativus L. cv Beit Alpha MR) seedlings were germinated in the greenhouse in vermiculite in glass containers, 9 cm deep and 9 cm in diameter. The seedlings were watered periodically with Hoagland solution. The photoperiod was maintained at 14 hours of light per day with 50 ft. candles of incandescent light.

Six-day old green εeedlingε were thinned to 10 plantε per container and ALA (Sigma Chemical Co., St. Louis, MO) was applied as a fine spray. The ALA was dissolved at concentrations ranging from 0 to 20 mM in a solvent mixture made up of 0.45 acetone: 0.45 ethanol: 0.1 Tween 80:9 water (v/v/v/v), adjusted to pH 3.5 with dilute HC1. Each 9 cm-diameter glaεε container (approximately 63.6 cm2 leaf surface area) was sprayed with 0.25 ml of ALA (treated) or 0.25 ml of solvent (control) , which is equivalent to a spray rate of about 40 gallons/acre and a field application rate of ALA of about 0 to 524 g/acre. The εolutionε were delivered aε a very fine and uniform spray with a modified Pierce "Quixspray" aeroεol spray kit (Pierce Chemical Co. , Rockford, IL) , as follows: 0.25 ml of solution was placed in a sawed-off 10 ml conical centrifuge tube, which was placed inεide the Quixεpray εpray jar. The delivery of a very fine mist was achieved by pumping the solution through a fine bore polypropylene tubing (0.3 mm inside diameter, or 0.5 mm inside diameter for more viscouε solutions) . One end of the fine-bore tubing was inserted into the Quixspray intake hose, while the other end waε dipped into the solution in the conical centrifuge tube. In this manner it took 10-20 sec to deliver 0.25 ml spray, and this in turn provided ample time for thoroughly spraying the seedlings to leaf saturation. Each treatment was performed in duplicate. Average droplet size diameter was approximately 25 μm for the 0.3 mm tubing and about 50 μm for the 0.5 mm tubing.
After spraying, the plants were wrapped in aluminum foil and were placed inside a cardboard box which waε wrapped in two layerε of black plastic. The dark-boxes were then incubated overnight (17 hours) at 28 βC, in order to allow the biosynthesis and accumulation of tetrapyrroles to take place.

The next morning, the treated plants were sampled for their tetrapyrrole content. The plants were taken in the black boxes to a dark room equipped with a green safelight which permits the manipulation of the treated tissueε without affecting in any way their tetrapyrrole content. One of each two cotyledonε of every two replicates was excised. Two- to three-gram batches were then homogenized in a Sorval Omnimixer (DuPont Instruments, Newtown, CT) in acetone: 0.1 N NHOH (9:1 v/v) at a rate of 18 ml of solvent per 3 g of tissue. The resulting 80% acetone extract containing various tetrapyrroles was cleared from lipoproteins and cell debris by centrifugation at 39,000 x g for 10 min at 0 βC. Chlorophyll, a fully esterified tetrapyrrole, was removed from the aqueous acetone solution by extraction with hexane according to the method of Rebeiz, CA. , Mattheis, J.R. , Smith, B.B., Rebeiz, C.C, and Dayton, D.F. Arch . Biochem. Biophys . 166:446-465 (1975). The more polar mono- and dicarboxylic tetrapyrroleε εuch aε Proto, MP(E) , and Pchlide remained in the hexane-extracted aqueous acetone fraction. The chemical structure of these tetrapyrroles haε been discusεed at length in Rebeiz, CA. and Lascelles, J. , in Photosynthesis: Energy Conversion by Plants and Bacteria , Vol. 1, Govindjee, ed. (Academic Press, New York, 1982), pp. 699-780; and Rebeiz, CA. , Wu, S.M., Kuhadja, M. , Daniell, H. , and Perkins, E.J. Mol . Cellular Biochem . 57:97-125 (1983). The amount of Proto, MP(E), and Pchlide was determined spectrofluorometrically on aliquots of the hexane-extracted acetone fraction according to the method of Rebeiz, CA. , Mattheis, J.R. , Smith, B.B., Rebeiz, C.C, and Dayton, D.F., Arch. Biochem . Biophys . 171:549-567 (1975). A small aliquot of the hexane extract containing the Chi a. and _ was dried under N2 gas and the residue was redissolved in 80% acetone. The amount of Chi a. and b_ in this acetone solution was then determined spectrofluorometrically according to the method of Bazzaz, M.B., and Rebeiz, C.A., Photochem . Photobiol . 30:709-721 (1979).
Fluorescence εpectra were recorded on a fully corrected photon counting spectrofluorometer Model SLM 8000 DS (SLM-Aminco, Urbana, IL) equipped with two red- sensitive, extended S20 photomultipliers (EMI 9658) , and interfaced with a microcomputer system Model 9825 S (Hewlett-Packard, Sunnyvale, CA) . Tetrapyrrole solutions were monitored at room temperature on 0.3 ml samples, in cylindrical microcells, 3 mm in diameter. Conversion of the digital spectral data into concentrations was performed automatically by the microcomputer, following the recording of the pertinent spectra, according to the method of Rebeiz, CA. , Daniell, H., and Mattheis, J.R. , Biotech . Bioeng. Symp. No. 12:413-439 (1982). The emiεεion and excitation spectra were recorded at excitation and emission bandwidths of 2 nm.
Monovinyl tetrapyrroles were distinguished from divinyl tetrapyrroles by their well-established spectrofluorometric properties in ether at 77 °K (see Rebeiz and Lascelles, supra; Rebeiz, Wu, Kuhadja, Daniell and Perkins, supra ; Belanger, F.C, and Rebeiz, C . , J. Biol. Chem . 257:1360-1371 (1982); and Belanger, F.C, Duggan, J.X., and Rebeiz, C.A., J. Biol . Chem . 257:4849-4858 (1982)). The low temperature fluorescence emission and excitation spectra were recorded in cylindrical sample tubes as described in Cohen, C.E., and Rebeiz, CA. , Plant Physiol . 61:824-829 (1978).
Absorption spectra were recorded with an Aminco dual wavelength εpectrophotometer model DW-2 (SLM-Aminco, Urbana, IL) operated in the split-beam mode, at a slit width of 2 nm.
The acetone-insoluble residue which was left behind after centrifugation of the tissue homogenate waε suspended in distilled water with an all glass tissue grinder. Total proteins were determined on a small aliquot of the εuspenεion, after delipidation, according to the method of Rebeiz, CA. , Caεtelfranco, P.A. , and Engelbrecht, A.H. , Plant Physiol . 40:281-286 (1965).
After 17 hours of dark-incubation the treated plants accumulated 382.82 and 2.36 nmoles of Pchlide and MP(E) , respectively, per 100 mg protein, above and beyond the controls. The seedlings with half of their cotyledons still intact were then used for asseεεing photodynamic damage by light. The seedlings were exposed to daylight in the greenhouse (400 to 5000 ft. candles at noon, depending on cloud cover) and their growth was evaluated over a period of 10 days. In order to secure a permanent record of the growth behavior of the treated plants, the latter were photographed daily (Kodacolor, 400 ASA, Eastman Kodak Co., Rochester, NY) with a Pentax Super Program camera (Helix, Champaign, IL) equipped with an SMC Pentax-A 1:1.4 50 mm lenε and a digital back that imprinted on each photograph the date or time of day at which the photograph was taken. Per cent photodynamic damage was asseεsed as the percent death of the sprayed tissue, in response to exposure to sunlight. For example, if 10 out of 10 sprayed leaves or cotyledons died as a consequence of exposure to daylight, the photodynamic damage was considered to be 100%. If only five out of the ten sprayed leaves or cotyledons had died, the photodynamic damage was considered to be only 50%, etc.
The extent of photodynamic damage was related to the amount of accumulated tetrapyrroles by conventional correlation analysis. The amounts of tetrapyrrole that accumulated were expressed in nmoles per 100 mg of tissue protein.
The results of these experiments are shown in Table I.

TABLE I - Various Concentrations of ALA



1 In experiment A, the light intensity at noon during the first day of exposure to daylight was about about 5000 ft. candles; 2 The change in tetrapyrrole concentration is the difference between the tetr plants and that of the control plants which were sprayed with the solvent only, i.e. without added AL prior to exposing the plants to daylight. The control plants contained the following amounts of tetra prior to exposure to daylight: A: 99.6, 7.66, 17.33 and B: 22.69, 6.96, and 12.41 nmole Pchlide, MP(E protein; and 3 Pchlide + MP(E) + Proto

The symptoms of photodynamic damage asεumed two forms: bleaching of the green leafy tissue, which spread gradually; and severe bleaching of the hypocotyl. In both cases, this was accompanied by a severe losε of turgidity of the affected tiεsues. The photodynamic damage waε effected on the cell membraneε which became leaky and this in turn resulted in a rapid and severe dehydration of the tissues. For example, at ALA concentrations of 10-20 mM (262-524 g/acre) a large number of seedlings had undergone irreversible damage after four to five hours of exposure to daylight. The cause of death was usually due to severe dehydration, bleaching, and collapεe of the leafy and/or hypocotyl tissues. On the other hand, treated samples kept for the same period of time in darkness were unaffected.

Example 2
Photodynamic Response of Various Plant
Species to ALA + 2,2 '-DP Treatment
The procedure of Example 1 was performed on the following representative monocots and dicotε:

Cucumber (Cucumis sativus L. cv Beit Alpha MR)
Lambsquarter (Chenopodium album)
Muεtard (Braεεica kaber/iuncea)
Red root pigweed (A aranthuε retroflexuε)
Common purslane fPortulaca oleracea)
Tomato (Lycopersicon esculentum cv Jet Star)
Cotton (Gossvpium herbaciu cv Coker-315)
Red kidney bean (Phaseolus vulgaris L. cv.
California Dark Red)
Soybean (Glycine max cv Williams)
Perennial bluegrass (Poa pratensis cv Aspen)
Barley (Hordeum vulgare. var. Beacon Spring)
Sweet corn (Zea mays L. cv Gold Cup) Crabgrass (Digitaria sanguinalis L. and Digitaria ischaemuπO
Giant foxtail (Setaria faberii
Oat (Avena sativa cv Centennial)
Wheat (Triticum sativum cv Auburn)
The greenhouse-grown seedlings were treated with 0.25 ml of 5 mM (131 g/acre) ALA + 15 mM (402 g/acre) 2,2'-DP, pH 3.5. Controls were treated with solvent only. All plants were then incubated in the dark for 17 hourε. The next morning the seedlings were sampled in the dark for tetrapyrrole content using the procedure of Example I for dicots and the following procedure for monocots: the seedlings of one of the two replicates were excised into an upper half and a lower half. The two batches of excised tissue were then homogenized separately in a Sorval Omnimixer in acetone: 0. IN NH4OH (9:1 v/v) at a rate of 18 ml of solvent per 3 g of tissue. The other replicate was used to assess the photodynamic effect of light on the seedlings. For some dicots, the stems as well as the leaves were analyzed for tetrapyrroles. The results are given in Table II:


. .

An examination of reεultε of thiε εurvey revealed that plantε reacted in three different wayε to the ALA + 2,2 '-DP spray. One group of dicots, which is exemplified by cucumber, exhibited what is referred to as Type I herbicidal responεe in Table II. Thiε group of plants reacted to the ALA + 2, 2' -DP spray exactly as did cucumber. Leafy tissues, stems and growing pointε accumulated significant amounts of tetrapyrroles and were subject to severe photodynamic damage (Table II) . Usually, the seedlings died very rapidly, and the rapidity of the response depended on the light intenεity in the greenhouεe. For example, at the low εpray concentrations used in this work (131 g/acre ALA + 402 g/acre 2, 2* -DP), only 4 to 5 hours of exposure to daylight was sufficient to cause the death of the plantε on clear, bright dayε (4000 to 6000 ft. candleε at noon) . On the other hand, 2 to 3 dayε of inεolation were required on very cloudy days (400 ft. candles at noon) in order to achieve the same results. Some of the plant species that exhibited this type of photodynamic herbicidal response such as lambεquarter, muεtard, red-root pigweed and common purslane are considered to be serious weeds. While 13-day old tomato plantε, with fully expanded cotyledonε and with small developing primary leaves exhibited a Type I response (Table II) , younger 8- to 10-day old tomato seedlings were much less affected by the spray (approximately 40% photodynamic damage) .
Other dicots such as cotton, kidney bean and soybean exhibited a different responεe to the ALA + 2,2-DP treatment. This response is referred to as Type II in Table II. Plants belonging to this group accumulate significant amounts of tetrapyrroles in the leafy tissues, but not in the stems as in cotton and soybean. Other species such as kidney bean also accumulated some tetrapyrroles in the stems. Leaves that accumulate tetrapyrroles exhibit very severe photodynamic damage and die within a few hours. However, the cotyledonε, stems, and growing points remain unaffected. Such plants uεually recovered from the original photodynamic damage by producing new leaveε and may require a εecond application. In this group the Type II responεe alεo depended on the age of the seedlings. For example, 6-day old soybean in which the primary leaves were εtill enclosed within the cotyledons were completely unaffected by the ALA + 2,2' -DP treatment. On the other hand, 9-day old soybean plants, with expanded primary leaves, exhibited a typical Type II photodynamic herbicidal response. The only monitored monocot that exhibited this type of response was perennial blue graεε in which about 30-40% of the sprayed leaves died; the plants subsequently recovered and developed new leaves.
The third type of photodynamic herbicidal response elicited by the ALA + 2, 2' -DP treatment is referred to as a type III response. Based on available data, onocots exhibited this type of responεe. Although the ALA + 2,2* -DP treatment induced the accumulation of significant amounts of tetrapyrrole by the plantε, the photodynamic damage waε either imperceptible aε in wheat, oat, and corn, or when noticeable aε in barley, waε confined to the upper half of a small proportion of the sprayed plants. In that caεe the photodynamic damage conεisted of small necrotic regions. The seedlings continued to grow vigorously and developed into healthy plants.
The photodynamic formulations described in this example exhibited an excellent measure of species, age and organ-dependent selectivity. While dicotyledenous weedε such as lambsquarter, mustard, red root pigweed and common purslane were highly susceptible to tetrapyrrole-induced photodynamic damage, monocots such as corn, wheat, oats, and barley were not adversely affected by the spray. Other dicots were either unaffected by the spray at an early stage of development aε in soybean, or recovered fully from a rapid destruction of the primary leaveε by producing new and healthy leaves, as waε obεerved for kidney bean, εoybean and cotton. Furthermore some tissues which accumulated tetrapyrroles such as bean stems did not exhibit any photodynamic damage. The biochemical basiε of thiε organ, age and species-dependent photodynamic herbicidal selectivity appears to be dependent among other things on the rates of tetrapyrrole turnover and on a differential enhancement of the MV and DV tetrapyrrole biosynthetic pathways in any given plant species.

SECTION II EXAMPLES OF DEFOLIATION AND FRUIT DROP

Example 3
Defoliation Of "Red Delicious", "Golden Delicious",
"Winesap" and "Prima" Apple Cultivars
Under Greenhouse Conditions

Scion wood of "Red Delicious", "Golden Delicious", "Winesap", and "Prima" apple cultivars waε collected from the University of Illinois Pomology Research Farm. Theεe were "whip and tongue" grafted onto seedling rootstock (purchaεed from Pacific Coaεt Nursery, Portland, OR) . Following grafting, seedlings were placed in plastic bags containing moist vermiculite, and kept in a cold room for two weeks at 1°C to induce healing. The seedlings were then held in a cold chamber at 10°C until needed.
Seedlings were later planted in 15 cm plastic pots containing a 1:1:1:1 (v/v/v/v) mixture of soil, vermiculite, peat, and sand media. These were grown in a greenhouse at 28° C under a 14h light - lOh dark photoperiod. Light intensity was supplied by three, 1000W metal halide lamps. Light intensity was 24 W/m2.
Following five dayε of growth at 23°C, the apple seedlings were treated as follows: (a) solvent only (control); (b) 20 mM ALA (Biosynth Int'l, Skokie, IL) ; (c) 30 mM ethyl nicotinate (EN) (Aldrich Chem. Co., Milwaukee, WI) ; and (d) 20 mM of ALA plus 30 mM of EN. The solvent consisted of polyethylene glycol (Sigma Chem. Co., St. Louis, MO) :methyl alcohol:Tween 80:water at 7:2:1:90 (v/v/v/v) (Rebeiz, CA. , Montazer-Zouhoor, A., Mayasich, J.M. , Tripathy, B.C., Wu, S., and Rebeiz, C.C, CRC Crit . Rev. in Plant Sci . , 6:385-435 (1988)). pH was adjusted to 3.5 to facilitate the penetration of ALA into the leaves. Solutions were delivered as a fine and uniform spray with a spray gun nozzle. The solutionε were placed in a Binks Wren air brush, and the delivery of a fine mist (average diameter droplet size of 125 microns) was achieved by pumping the solution through metal tubing using compressed C02. One end of the tube was inserted into the gun intake hose, while the other end was dipped into the solution. Leaves were sprayed to a drip.
Treatment of the four apple cultivars was replicated three times in a randomized split plot design (four seedlings per replicate of each cultivar) . After spraying, seedlings were transferred to a dark-growth chamber at 28°C and kept from 6:00 p.m. to 9:00 a.m. the following morning in order to allow for the dark conversion of ALA to tetrapyrroles.
After 15 hours in darkness, plants were moved to the greenhouse for evaluation of growth and photodynamic damage over a period of 30 days. Plantε were periodically photographed to record response to treatment. A Pentax Super program camera, equipped with an SMC Pentax-A 1:1.4, 50 mm lens, and a digital back recording the date and time of each shot on the prints obtained, was used. A 400 ASA Kodacolor film was used.

First photographic records were taken before treatment and then at 1, 3, 8, 10, 17 and 30 days after exposure to light. Photodynamic damage consisted of the sum of tissue necrosis and leaf abscission.
A gram leaf sample (collected from both the top and the bottom of each of the seedlings) was homogenized in a Polytron homogenizer for one minute in 7 ml of acetone and 0.1 N ammonium hydroxide (9 : 1, v/v) . Acetone serves to extract the tetrapyrroles while ammonium hydroxide maintains the medium basic, thereby preventing losε of the Mg-atom from metalated tetrapyrroles. During homogenization, samples were handled under a green safelight which does not affect the tetrapyrrole content of the tissues.
After homogenization, extracts were centrifuged at 18,000 rpm for 12 min. at 1°C to separate lipoproteins and cell debriε from the supernatant containing tetrapyrroles. Chlorophyll and other fully esterified tetrapyrroles were removed by first extracting the supernatant with hexane followed by an equal volume of hexane and then with a 1/3 volume of hexane (Rebeiz, C.A., Mattheis, J.R., Smith, B.B., Rebeiz, C.C, and Dayton, D.F. Arch . Biochem . Biophys . 171:549-567 (1975)). Protoporphyrin IX (proto), Mg-protoporphyrin monoester (MPE) , and protochlorophyllide (Pchlide) remained in the hexane-extracted acetone fraction (HEAF) . The amount of tetrapyrroleε in the HEAF waε determined by spectrofluorometry.
Fluorescence emission and excitation spectra of the HEAF were recorded at room temperature (23.5° C) and at 77βK (frozen in liquid nitrogen) using a fully corrected photon counting spectrofluorometer (model SLM 8000 DS) equipped with two red-εenεitive, extended S 20 photomultipliers (EMI 9658) , and interfaced with an IBM Model 30 PC Room temperature determinations were performed on 0.3 ml aliquots in a cylindrical micro-cell of 3 mm in diameter. Analytical techniqueε used for precise quantitative determination of the various tetrapyrroles are described by Rebeiz, CA. , Mattheiε, J.R. , Smith, B.B., Rebeiz, C.C, and Dayton, D.F., Arch . Biochem . Biophys . 171:549-567 (1975); Smith, B.B., and Rebeiz, CA. , Photochem . Photobiol . 26:527-532 (1977); Bazzaz, M.B., and Rebeiz, CA. , Photochem . Photobiol .

30:709 (1979); and Rebeiz, C . , Daniell, H. , and Mattheis, J.R. , in 4th Sy p. Biotechnol . Energy Prod . Conserv. (Scot, CD., Ed.) John Wiley & Sons, New York, 413-439 (1982) . Recording of spectra and conversion of digital spectral data into concentrations were automatically performed by the microcomputer (Rebeiz, CA. , Daniell, H. , and Mattheis, J.R. , supra) . For room temperature spectra, both the emisεion and the excitation band width were set at 4 nm. The photon count was integrated for 0.5 seconds at each 1 nm increment. Ratios of MV MPE to DV MPE and of MV pchlide to DV Pchlide were calculated from spectra recorded in ether at 77βK (Tripathy, B.C., and Rebeiz, CA. , Anal. Biochem . 149:43-61 (1985)). The MV and DV tetrapyrrole content in a sample was determined from MV and DV tetrapyrrole ratios obtained at 77°K and from total pigment content determined at room temperature.
Total protein was determined using the bicinchoninic acid (BCA) method Smith, P.K. , Krohn, R.I., Hermanson, G.T. , Mallia, A.K. , Gardner, F.H. , Provenzano, M.D. , Fujimoto, E.K. Goeke, N.M. , Olson, P.J. and Klenk, D.C, Anal. Biochem . 150:76-85 (1985), after resuspending the pellet obtained after centrifugation in distilled water. Absorbance waε monitored on a Sequoia-Turner Model 340 spectrophotometer. Statistical analysiε waε conducted on an IBM P.C. Model 80, uεing a software package available from SAS Industries, Inc. Data were analyzed as a randomized split block design using the linear model. Testing for significant differences among cultivars and/or treatments was evaluated uεing the protected LSD teεt.
In general, leaveε exhibited photodynamic injury in both ALA + EN and ALA treatments just a few hours after exposure to light. This was expressed in the form of wilting and necrosis of leaves. Near complete defoliation of seedlings was achieved 8 days following treatment (Table III) .

Table III
Percent Defoliation Of "Golden Delicious"
Seedlings In The Greenhouse Over Time



Solvent only (polyethylene g_ycol: ethyl alcohol.Tween 80:water at 7:2:1:90 (v/v/v/v)).
2 30 mM ethyl nicotinate
20 πiM 5-sminolevuϋϊiic acid.
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate.

The experimentε indicate that the detachment of the leaveε from the stem is caused by the formation of an abscission layer at the leaf petiole level. The photodynamic phenomenology expresεed by the variouε apple cultivarε under greenhouse conditions is briefly described below.
For "Golden Delicious" seedlings, more than 75% of the leaves abscised by the third day following treatment with ALA + EN and defoliation was complete after 30 dayε (Table III) . Except for some negligible browning, EN-treated and control plants exhibited no photodynamic damage (Table III) .
"Red Delicious" seedlings lost 77% of their leaves 17 days after treatment with ALA + EN (Table IV) . Therefore, the response of "Red Deliciouε" seedlings to ALA + EN treatment was slower than that observed for "Golden Delicious". Control and EN-treated "Red Delicious" seedlings did not exhibit any noticeable photodynamic damage (Table IV) .

Table IV
Percent Defoliation Of "Red Delicious"
Seedlings In The Greenhouse over Time
Treatment Dav: 1 3 § 1£ 17 3β
% Defoliation
Control1 0.00 1.65 1.90 1.90 1.90 1.90 EN2 0.30 0.79 1.37 1.37 1.37 1.37 ALA3 27.15 41.03 55.30 56.69 59.47 60.86 ALA+EN4 21.47 29.70 50.11 54.06 77.14 88.83

1 Solvent only (polyethylene glycol: ethyl alcohol: ween 80: ater at 7:2:1:90 (v/v/v/v))
2 30 mM ethyl nicotinate
3 20 sK 5-aminclevulinic acid
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate.

The photodynamic phenomenology of "Winesap" seedlings differed from the previous two cultivars.
Overall, ALA treatment resulted- in continuously higher percentage leaf drop over ALA + EN treatment (Table V) . Almost 50% defoliation was achieved with ALA alone after the first day of treatment (Table V) .

Table V
Percent Defoliation Of "Winesap" Seedlings
in The Greenhouse Over Time
Treatment Dav; l 3 β 10 17 30
* Defoliation
Control1 0.30 0.58 1.17 1.17 1.17 1.17 EN2 0.89 1.62 2.13 2.13 2.62 2.62

ALA3 49.84 62.74 73.44 76.71 76.71 80.97

ALA+EN4 35.03 45.86 65.27 65.27 70.15 73.71

1 Solvent only (polyethylene glycol:methyl alcohol:Tween 80:water at 7:2:1:90 (v/v/v/v))
2 30 mM ethyl nicotinate
3 20 mM 5-aminolevulinic acid
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate Again, control and EN treated seedlings exhibited very small amounts of leaf browning (less than 2.6%) (Table

V) . "Prima" seedlings lost nearly 75% of their leaves 17 days after treatments with ALA and ALA + EN (Table VI) .
Table VI
Percent Defoliation Of "Prima" Seedlings
in The Greenhouse Over Time
Treatment Dav: J. 3 8 10 17 30

Control-1
EN2
ALA3
ALA+EN4

1 Solvent only (polyethylene glycol: ethyl alcohol:Tween 80:water at 7:2:1:90 (v/v/v/v))
2 30 mM ethyl nicotinate
3 20 mM 5-aminolevulinic acid
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate

Again, control and EN treatments exhibited negligible damage (Table VI) .
In general, the four apple cultivars
exhibited a similar response to treatment with ALA and EN. Indeed, no significant differences among cultivars were observed in protoporphyrin IX (Proto) , MV Mg-protoporphyrin monoester (MV MPE) , DV Mg-protoporphyrin monoester (DV MPE) , MV Protochlorophyllide (MV Pchlide) , and DV Protochlorophyllide (DV Pchlide) accumulation in leaves in all treatments (Tables VII-XI) .

Table VII
Leaf Accumulation Of Protoporphyrin IZ
In Each Of The Four Cultivars Following Treatment



Mean separation for various treatments within a cultivar by LSD at the 5% level of significance. Means followed by the same letter within a cultivar are not significantly different
Solvent only (polyethylene glycol: ethyl alcohol:Tween

80: ater at 7:2:1:90 (v/v/v/v))
2 30 M ethyl nicotinate
3 20 mM 5-aminolevulinic acid
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate

44/2
Table viii
Leaf Accumulation Of Monovinyl Mg-protoporphyrin In Monoester Bach Of The Four Cultivars Folloving Treatment
MV MPE accumulation



Mean separation for various treatments within a cultivar by LSD at the 5% level of significance. Means followed by the same letter within a cultivar are not
significantly different
Solvent only (polyethylene glycol: ethyl alcohol:Tween

80:water at 7:2:1:90 (v/v/v/v))
2 30 mM ethyl nicotinate
3 20 mM 5-aminolevulinic acid
4 20 M 5-aminolevulinic acid + 30 mM ethyl nicotinate

45/1
Table ιz
Leaf Accumulation of Divinyl Mg-protoporphyrin
Monoester in Each of The Four Cultivars
Folloving Treatment
DV XPE accumulation



Mean separation for various treatments within a cultivar by LSD at the 5% level of significance. Means followed by the same letter within a cultivar are not
significantly different
Solvent only (polyethylene glycol: ethyl alcohol:Tween

80:water at 7:2:1:90 (v/v/v/v))
2 30 mM ethyl nicotinate
3 20 mM 5-aminolevulinic acid
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate

45/2
Table X
Leaf Accumulation Of Monovinyl Protochlorophyllide
In Each Of The Four Cultivars Folloving Treatment
MVProtochlorophyllideaccumulation
in nmoles/100 mg protein



Mean separation for various treatments within a cultivar by LSD at the 5% level of signi icance. Means followed by the same letter within a cultivar are not
significantly different
Solvent only (polyethylene glycol: ethyl alcohol:Tween

80:water at 7:2:190 (v/v/v/v))
2 30 mM ethyl nicotinate
3 20 mM 5-aminolevulinic acid
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate

46
Table XI
Leaf Accumulation Of Divinyl Protochlorophyllide
In Each Of The Four Cultivars Folloving Treatment



Y Mean separation for various treatments within a cultivar by LSD at the 5% level of significance. Means followed by the seme letter treatments within a cultivar are not significantly different
1 Solvent only (polyethylene glycol:methyl alcohol:Tween

80:water at 7:2:1:90 (v/v/v/v))
2 30 mM ethyl nicotinate
3 20 mM 5-aminolevulinic acid
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate

However, significant differences among treatments were,-observed for proto, IX, DV MPE, and MV Pchlide accumulations (Tables VII-XI) . A significant cultivar x treatment interaction was obtained only in MV Pchlide accumulation. As for percent leaf defoliation, no significant differences were observed among cultivars. Significant differences were, however, observed among treatments along with a significant cultivar x treatment interaction (Table XII) .

47
Table XII
Percent Leaf Defoliation Of Four Apple Cultivars



Mean separat on for var ous treatments w thin a cultivar by LSD at the 5% level of significance. Means followed by the same letter treatments within a cultivar are not significantly different
Solvent only (polyethylene glycol: ethyl alcohol:Tween

80:water at 7:2:1:90 (v/v/v/v))
2 30 mM ethyl nicotinate
3 20 mM 5-aminolevulinic acid
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate

Overall, accumulation of proto and extent of leaf defoliation for the cultivars were not significantly different between ALA + EN and ALA; however, they were both significantly higher than EN and control treatments (Table

XIII) .

48
Table XIII
Mean Accumulation Of Tetrapyrroles in nmoles/10Omg
Protein In "Red Delicious", "Winesap", "Golden
Delicious" And "Prima" Cultivars And The Percent
Defoliation For The Four Treatments

Treatment Proto1 XV MPE2 DV MPE3 XV Pide4 DV Pide5 %Defol6
nmolea/100 mg protein
Control7 4.94 Bγ2.68 A 3.28 C17.70 C 0.82 A 2.95 B

EN8 7.52 B 3.50 A 3.77 C 9.01 C 0.55 A 2.41 B

ALA9 18.82 A 3.90 A48.19 E89.76 B 14.65 A 74.41 A ALA+EN 23.71 A 2.62 A69.58 £83.40 A 5.28 A 84.93 A

Signif.2 * NS * * NS *

Z Non-significant (NS) , significant at the 5% level (*) Y Mean separation within columns by LSD at the 5% level of significance. Means followed by the same letter for various treatments within a tetrapyrrole accumulation are not significantly different
1 Protoporphyrin IX
2 Monovinyl Mg-protoporphyrin monoester
3 Divinyl Mg-protoporphyrin monoester
4 Monovinyl protochlorophyllide
5 Divinyl protochlorophyllide
6 Percent defoliation
7 Solvent only (polyethylene glycol: ethyl alcohol: ween 80:water at 7:2:1:90 (v/v/v/v))
8 Ethyl nicotinate
9 5-aminolevulinic acid

The accumulation of DV MPE and MV Pchlide was significantly higher in ALA + EN treated seedlings than in the other three treatments (Table XIII) . However, ALA treated seedlings also exhibited significantly higher DV MPE and MV Pchlide accumulation in leaves than EN-treated and control plants. The EN treated seedlings and the controls were not significantly different (Table XIII) .

49
Since the control and the EN treated seedlings did not accumulate significant amounts of proto, MV MPE, DV MPE, MV Pchlide, and DV Pchlide, and since almost no defoliation waε obεerved in all four cultivarε, it can be stated that the exogenous ALA and ALA + EN applications were definitely responsible for the accumulation of tetrapyrroles and for the succesεful defoliation of apple seedlings. When applied with ALA, EN significantly increased the dark conversion of exogenous ALA to both DV MPE and MV Pchlide, beyond the level obtained by ALA alone (Table XIII) . Therefore, EN can be classified as an enhancer of ALA conversion to MV Protochlorophyllide and to DV MPE.
ALA+EN treatment induced a significantly higher proto accumulation in "Golden Delicious" than in the other treatments. However, ALA+EN treated "Golden Delicious" seedlings did not accumulate higher levels of proto than ALA treated seedlings in other cultivars. Furthermore, in "Winesap", ALA treatment resulted in a significantly higher level of Proto accumulation than that of the Control or EN treatments (Table VII) .
ALA-treated seedlings of "Prima" exhibited a higher MV MPE accumulation than ALA+EN treated plants. However, no significant differences in MV MPE accumulation were exhibited among any of the treatments of "Golden Delicious", "Red Delicious", and "Wineεap" cultivars (Table VIII) .
"Golden Delicious", "Prima", and "Winesap" cultivars did not exhibit any significant differences in DV MPE accumulation between the ALA and ALA+EN treated plants. However, ALA+EN treated "Red Delicious" seedlings exhibited a significantly higher DV MPE accumulation than ALA treated plants (Table IX) .
"Red Delicious" and "Winesap" ALA + EN-treated seedlings exhibited a significantly higher MV Pchlide accumulation than ALA-treated seedlings. The other two cultivars did not exhibit a significant difference between ALA and ALA + EN treatments (Table X) .

50
Only ALA-treated "Winesap" seedlingε exhibited a significant difference in their DV Pchlide accumulation when compared to the other three treatments (Table XI) .
For all four cultivars, there was no significant difference in defoliation between ALA and ALA + EN treated plants (Table XIII) .
Proto, DV MPE, and MV Pchlide accumulation in leaves of the various cultivars were positively correlated to leaf defoliation (FIGS. 3, 4 and 5). In other words, the higher accumulation of proto, DV MPE, and MV Pchlide, resulted in an increased rate of defoliation. It was observed, however, that beyond a certain tetrapyrrole concentration level, accumulation of proto, DV MPE and MV Pchlide inhibited defoliation (FIGS. 3, 4 and 5).
Because treated seedlings were not held in cold storage, and because of the favorable environmental conditions in the greenhouse, defoliated seedlings exhibited healthy, green regrowth. This in turn indicates that shoots and buds were not damaged aε a result of these treatments. Interestingly, more regrowth was observed with plants sprayed with ALA + EN than with those sprayed with ALA alone. In other words, in addition to enhanced tetrapyrrole accumulation, the addition of EN to ALA appeared to exert growth promoting effects on the defoliated shoots.

Comparison of ALA + EN
With Known Chemical Defoliants

Knight's (Knight, J.N., J. Hort. Sci . 58(4) :471-476 (1983)) results from use of FeEDTA and CuEDTA on "Cos's Orange Pippin" apple trees were comparable to results obtained with the composition according to the invention. Three percent FeEDTA and 2.1% CuEDTA resulted in 81 and 89% defoliation, respectively, of "Cox's Orange Pippin" apple trees 40 5 _.1_

days following treatment (Knight, J.N. , supra) , whereas the composition of ALA + EN according to the invention resulted in nearly 99% and 89% defoliation of "Golden Delicious" and "Red Delicious", reεpectively, 30 days following treatment (Table XII) . ALA+EN-treated "Prima" seedlings were 78.5% defoliated compared to 74% defoliation of "Winesap" seedlings (Table XII) .
Larsen's (Larsen, F.E., Proc. Jnternt . Plant Prop. Soc. 17:157-172 (1967)) use of 1% S,S,S,-tributylphosphoromtrithionate (DEF) resulted in a complete defoliation of "Winesap" four weeks following treatment and in 80% defoliation of "Rome Beauty" three weeks following treatment. However, DEF did not bring about defoliation of "Red King" apple seedlings (Larsen, F.E., supra) . Some excessive damage was reported on the seedlings as compared to non-noticeable damage on seedlings sprayed in accordance with the invention. Healthy regrowth followed spraying when seedlings were kept under favorable environmental conditions.
Defoliation of "Golden Delicious" with Ethrel® at varying concentrations waε significantly inferior to defoliation with the composition according to the invention because only 26% leaf drop was obtained (Jones, D.L., Nichols, D.G., Thompson, W.K. , and Jager, L.A. , Australian Journal of Experimental Agriculture and Animal Husbandry 13:460-464 (1973)). In another study by Larsen, 8,000 ppm Ethrel resulted in 95% defoliation of "Red Delicious" nursery seedlings 3-4 weeks following application (Larsen, F.E., J. Amer. Soc. Hort . Sci . 95:662-663 (1970)).

52
Example 4
Defoliation And Fruit Drop
of "Red Delicious" and "Golden Delicious" Apple
Cultivars Under Field Conditions

A total of sixteen apple trees growing at the Pomology Research Farm in Urbana and representing two cultivars, namely "Red Delicious" and "Golden Delicious", were subjected to four different treatments. Each treatment was applied to two individual trees. Three branches, 36 inches in length, were selected from each tree for treatment. Therefore, each of the four treatments was sprayed on a total of six branches. Fruits were counted on each branch before spraying.
The four treatments consisted of the following:
(a) control which consisted of solvent only (polyethylene glycol:Methyl alcohol:Tween 80:water at 7:2:1:90 (v/v/v/v));
(b) 40 mM ALA; and
(c) 30 mM EN; (d) 40 mM ALA + 30 mM EN.
Handling of solutions and sprays was similar to that described in Example 3. Trees of "Red Deliciouε" were sprayed on 9/15/88 starting at 2 p.m. and terminating at 6 p.m. It is important to' note that a heavy rainfall occurred in the following morning. Trees of "Golden Delicious" were sprayed on September 16, 1988 starting at 2 p.m. and ending at 6 p.m. No rain showers followed this set of treatments.
To evaluate the degree of photodynamic damage and extent of fruit drop, treated branches were photographed before spraying and on the 10th day after spraying for "Golden Delicious" and on the 11th day after spraying for the "Red Delicious". Photographic recording was conducted as described earlier, except that a 28-135 mm zoom lens was used. Number of fruit drop was recorded every day or every other day and percent fruit drop was calculated. Data were analyzed in a randomized complete block design.
Treatment of "Red Deliciouε" brancheε reεulted in neither significant defoliation nor in fruit absciεεion (Table XIV) , and treated fruitε did not exhibit skin injury. Because "Golden Delicious" and "Red Delicious" responded more or less in the same manner to the application of ALA and ALA + EN under greenhouse conditionε aε shown in Example 3 , and because both belong to the same DMV/LDV greening group, the observed difference in the field between the two cultivarε iε probably not due to differenceε in their susceptibility to ALA and ALA + EN treatments, but rather due to the heavy rain that fell the morning after having sprayed the "Red Delicious" trees.
"Golden Deliciouε" treeε treated with ALA or with ALA + EN underwent 100% defoliation by the 13th day after spraying. By that time, fruit drop was also 100% (Table XIV) . Visual examination of abscised fruits revealed the formation of a pronounced absciεεion layer at the base of the pedicel and some fruits exhibited pronounced skin browning. Branches sprayed with EN and solvent only (i.e., control) remained healthy and did not exhibit a significant level of defoliation or fruit abscission beyond normal levels. No injury was observed on sprayed fruit.
Significantly higher leaf defoliation and fruit absciεεion (Table XIV) were observed in ALA and ALA + EN treatments when compared to the control and EN treated branches. This was not so in the case of the "Red Delicious" cultivar where no significant difference between treatments was obεerved (Table XIV) .

54
Table XIV
Percent Fruit Drop Of "Red Delicious"
and "Golden Delicious" Cultivars
Folloving spray Treatments



x Mean separation within cultivars by protected LSD at the 1% level of significance
y Mean separation within treatments by protected LSD at the 1% level of significance
1 Solvent only (polyethylene glycol:methyl alcohol:Tween 80:water at 7:2:1:90 (v/v/v/v))
2 30 mM ethyl nicotinate
3 20 mM 5-aminolevulinic acid
4 20 mM 5-aminolevulinic acid + 30 mM ethyl nicotinate

Comparison With Other
Known Chemical Fruit Harvesters

In other studies (Hartmann, H.T., Fadl, F. , and Whisher, J. Calif. Agric. 21(7) :5-7 (1967); Unrath, CR. J. Amer. Soc. Hort. Sci . 94:387-391 (1967); Wilson, W.C., and Hendershott, CH. Proc. Amer. Soc. Hort . Sci . 90:123-129 (1967)) , chemicals were used to reduce the force separating the pedicel to facilitate mechanical harvesting. Therefore, the results from those studies are not comparable to those obtained in accordance with the invention where 100% defoliation and fruit drop were achieved following treatment of "Golden Delicious" trees (Table XXIX) without mechanical harvesting.

Example 5
Defoliation of Tomato under Greenhouse Conditions
A. MATERIALS AND METHODS

Seedlingε of tomato, a DMV/LDV plant species, were grown in a greenhouse under a fourteen hour light-ten hour dark photoperiod in vermiculite for twenty five days before spraying. (Tomato is accepted in the art as a model for potato) . Two days before spraying, the aging cotyledons were removed and the seedlings with old and young developing leaves were sprayed in the afternoon in the greenhouse with 20 mM ALA (about 500 g/acre) + 15 mM modulator (about 375 g/acre) at a rate of 40 gpa and an average droplet size of 75 μn Eleven different modulators, the identities of which are εhown in Table XXX, were tested. Studies on the mode of action of these modulators on cucumber revealed that the modulators acted as enhancers of the conversion of ALA to protochlorophyllide. Depending on the solubility of the particular modulator, ALA and the modulator were dissolved either in a solvent made up of polyethylene glycol:methanol:Tween 80:water (7:2:1:90 v/v/v/v) at a pH of 3.5 or in a solvent made up of polyethylene glycol: Tween 80:ethanol:methanol:water (7:1:45:45:2 v/v/v/v/v) at a pH of 3.5.
The plants were evaluated visually for growth and photodynamic damage over a period of ten days. The plants were periodically photographed to record responεe to treatment as described in the previous examples. The resultε of the treatment are shown in Table XV.

56
Table XV
Defoliation/Desiccation Of Tomato Seedlings By ALA
and Micotinic Acid or Nicotinamide Modulators
^Defoliation/desiccation
Older Leaves Younger Leaves
pa p ttor Treatment



2-hydroxy-6-methy1
pyridine-3-carboxylic
acid 95 95
+ ethyl 2-methyl nicotinate 95 95
+ N-methylnicotinamide 90 90

+ N-benzyl-N-nicotinoyl
nicotinamide 90 90 67 65
4-hydroxy-7-trifluoro
methyl-3-guinoline
carboxylicacid 90 90 85 85



An examination of Table XV reveals that tomato leaves were susceptible to combinations of ALA and nicotinic acid or nicotinamide modulators. Young developing leaves were less susceptible to treatment than the older, more mature leaves. Overall, ten modulators exhibited 88-90% or better defoliation of old leaves. Four of these modulators also exhibited 90% or better defoliation of young developing leaves.

57/1
Example 6
Defoliation Of Cotton Under Greenhouse Conditions

Twenty four day-old vermiculite-grown cotton seedlings were used. Two days before spraying, aging cotyledons were excised and we11-developed primary leaves

(old leaves) as well as developing secondary leaves (young leaves) were treated by spraying with a combination of ALA and a given modulator. The identities of the modulators tested are set forth in Table XVI. ALA and modulator concentrations were 20 and 15 mM, respectively. Spraying and evaluation of photodynamic defoliation was exactly as described in Example 5.
The results of the tests are reported in Table XVI.

Table XVI
Defoliation/Desiccation of Cotton
By ALA And Various Modulators
%DEATH
M0PULMOR YI,2T3P24 T 0L5D2 YL T D74 T OL D7
benzyl viologen dichloride monohydrate 87 75 IIID 111
di 2-pyridyl ketoneoxi e 69 81 111 111
2 , 2 ' -dithiobis (pyridine n-oxide) 100 100 111 111
6,6'-dithiodinicotinic acid 25 69 111 111
methyl5-(benzloxycarbonyl)-2,4-dimethyl-3- . . . . 50 57 111 111
1-methy1-2-pyrrole carboxaldehyde 25 37 111 111
l-methyl-2-pyrrole carboxylic acid 31 62 111 111 h
pyrrole-2-carboxaldehyde 25 87 111 111
pyrrole [l,2-a]guinoxaline 56 100 111 111
phenyl 2-pyridyl ketoxime 79 81 111 111
5-chloro-l,10-phenanthroline 100 100 100 100
dimidiu bromide 75 94 100 100
2 , 2 • -dipyridyl 100 100 100 100
ethidiu bromide 87 75 100 100
methyl viologen dichloride iodide 100 100 100 100
1,10-phenanthroline 100 100 100 100
4'-methyl-l,10-phenanthroline 100 100 100 100
l-(carboxymethyl)pyridinium chloride 94 100 94 100

*PEAT»
MODULATOR1 YL T3D24 T OL5D2 YL T D74 T OL D7

l,l'-diethyl-4,4'-cyanine iodide 87 100 94 100

4 , • -dimethyl-2 , 2 • -dipyridyl 94 100 94 100

1-dodecylpyridinium chloride monohydrate 81 100 94 100 l,l'-diethyl-2,2'-cyanine iodide 87 100 87 100 l,l'-diethyl-2,4,-cyanine iodide 87 100 87 100

SPEATH
MQPϋlATQR3 γL2τ3D24 T OL5D2 YL T D74 T OL D7

4,7-dimethyl-l,10-phenanthroline 87 100 87 100
5,6-dimethyl-l,10-phenanthroline 87 100 87 100
5-methyl-l,10-phenanthroline 87 100 87 100
2 , 2 • : 6 • , 6 ' • -terpyridine 87 100 87 100
3,4,7,8-tetramethyl-l,10-phenanthroline 87 100 87 100
6-amino nicotinamide 85 100 85 100 _ berberine hydrochloride hydrate 62 100 75 100
5-phenyl-2-(4-pyridyl)oxazole 75 100 75 100 o tert-butyl 4-acetyl3,5-dimethyll-2-pyrrolecarcoxylate 71 100 71 100
2-[4-(dimethylamino)styryl]-l-methyl pyridinium iodide71 100 71 100
sanguinarine chloride 71 100 71 100
1-furfurylpyrrole 69 100 69 100
2,4,6-collidine p-toluene sulfonate 62 100 62 100
3-ethyl-2-methyl-4,5,6,7-tetrahydroindol-4-one . . 62 100 62 100
3-amino-2,6-di-methoxypyridine onohydrochloride . 50 100 50 100
5-amino-2-methoxy pyridine 50 100 50 100
l-ethyl-3-OH-pyridinium bromide 44 100 44 100
6-amino nicotinamide 37 100 37 100
ethyl 2-methyl nicotinate 37 100 37 100
ethyl nicotinate 37 100 37 100

%PEATH
MODULATOR3 YL2T3D24 T OL5D2 YL T D74 T QL P7

2-hydroxy-6-methyl pyridine-3-carboxyiic acid . . . 37 100 37 100
2-hydroxynicotinic acid 25 100 25 100 niflumic acid 25 100 25 100 dibucaine hydrochloride . . . . 14 100 14 100 diethyl 3,4-pyridine dicarboxylate 12 100 12 100 ethyl nicotinate 91 95 91 95 P 2-hydroxy nicotinic acid 90 95 90 95
2-hydroxy-6-methyl pyridine-3-carboxylic acid . . . 74 95 74 95 ethyl 2-methylnicotinate 71 95 71 95
5-nitro-l,10-phenanthroline . . 100 94 100 94 phenanthridine 81 94 81 94 ethyl 3,5-dimethyl-2-pyrrolecarboxylate 75 94 75 94
2-[4-(dimethylamino)styryl]-l-ethyl pyridinium iodide 62 87 75 94 isocarbostyril 75 94 75 94
2,3-dihydroxypyridine 62 94 62 94
2-chloro-6-methoxypyridine 50 94 50 94
4-hydroxy-7-trifluoro methyl-3-quinoline carboxylic 50 94 50 94 l-(dimethylamino) pyrrole 44 94 44 94
4-hydroxy-7-methyl-l,8-naphthridine-3-carboxylic acid 43 93 43 93
N-methylnicotinamide 93 90 93 90

%DEATH
MODULATOR3 YL2T3D24 T OLSD2 YL T D74 T Qh P7

4-hydroxy-7-trifluoro methyl-3-quinoline carboxylic 85
N-benzyl-N-nicotinoylnicotinatearaide 65
4-hydroxy-7-methyl-l,8-naphthridine-3-carboxylic acid 95
citrazinic acid 81
propidium iodide hydrate 75
2-hydroxy-4-methyl quinoline 71
3-cyano-4,6-dimethyl-2-hydroσhloride 62


%DEATH
MODULATOR3 YL2T3D24 T OL5D2 YL T D74 T OL D7

N-benzyl-N-nicotinoyl nicotinatea ide 56
diethyl-2,4-dimethyl pyrrole-3 ,5-dicarboxylate . . 56
4-(dimethylamino)pyridinium bromide perbromide . . 56
l-(2-cyanoethyl)pyrrole 37
N-methylnicotinamide 25
diethyl3,4-pyridine dicarboxylate 72
niflumic acid 73
4,7-diphenyl-l,10-phenanthroline 75
8-hydroxy-5-nitroquinoline 75
5,7-dichloro-8-hydroxyquinoline 81
2,6-dimethoxypyridine 50
5-chloro-8-hydroxy-7-iodoquinoline 44
5,7-dibromo-8-hydroxyquinoline 44
bis-N-methyl acridinium nitrate 31
2,9-dimethyl-4,7-diphenyl-l,10-phenanthroline . . . 25
3-hydroxypicolinic acid 12
1-isoquinolinecarboxylic acid 12
picolinic acid 12

ALA and modulator concentrations were 20 and 15 mM respec
YL = young leaves
treated

D2, D7 = damage 2 and 7 days after spraying

%DEATH
MODULATOR* YL 2'πT,3JD2« T 0L3D2 YL T D7« T rOvLr. nDi7

6 The number 111 in each column refers to the development of nutrition deficiency symptoms
due to an error in the Hoagland solution. The plants died from nutritional toxicity
before the ten day photodynamic injury was evaluated.




60
Overall, the older mature leaves were more susceptible than the younger developing leaves, as was observed with tomato. Fifty seven of the tested modulators exhibited 90-100% defoliation of older leaves while twelve modulators exhibited similar performance on younger leaves. Seven of the modulators exhibited 100% defoliation of both young and old leaves.

SECTION III CHLOROPHYLL BIOSYNTHESIS MODULATORS

Example 7
TDPH Susceptibility of Corn, Soybean, and Ten Weed
Species toward ALA + Modulator Treatments
The molecular basis of the TDPH performance of ALA and four Chi biosynthesis modulators (picolinic acid, phenanthridine, 1,10-phenanthroline, and 4,7-phenanthroline) on corn (Zea roavs. , soybean (Glycine max) and 10 common weed species was investigated under greenhouse conditions. The plants were sprayed in the late afternoon and were placed in darkness for 12 h to allow tetrapyrrole accumulation to take place, before exposure to light. In the growth room, the photoperiod was set to 10 h dark and 14 h light. At the end of the dark incubation period, tetrapyrrole accumulation and the ensuing phytotoxicity which was observed upon exposure to light were subjected to statistical analysis.
Two solutions were freshly prepared on the day of spraying: (a) a solvent (-) , which consisted of polyethylene glycol 600, methanol, Tween 80 and water

(7:2:1:90 v/v/v/v/), and (b) a herbicidal solution (+) which consisted of the aforementioned solvent which in addition contained 40 mM ALA and 30 mM of one of the following modulators: 2-pyridine carboxylic acid (i.e. picolinic acid), 1,10-phenanthroline, phenanthridine, or 4,7-phenanthroline. The pH of the solvent and the herbicidal solutions were adjusted to 3.5 at room temperature with 1.2 N HC1 and/or 1.0 N KOH. The solutions were sprayed at a rate of 0.38 ml per container (9 cm in height and 9 cm in diameter) with a modified 61
aerosol kit which produced an average droplet diameter of 75 μm. Tetrapyrrole accumulation in darkness and photodynamic injury in the light were evaluated following a post-spray incubation period of 12h at 21βC.
The accumulation of tetrapyrroles is shown in

Table XVII. The accumulation was significantly dependent on the treatment, on the plant species, and on the interaction of treatment and plant species. The relatively low photodynamic injury sustained by johnsongrass and barnyardgrass (79-86%) , two DMV/LMV weeds, was attributed to their accumulating predominantly MV Pchlide. This form of Pchlide, which belongs to the prevailing MV monocarboxylic Chi a. biosynthetic routes in these two weeds, may be readily metabolized, and consequently does not remain long enough to generate damaging singlet oxygen upon exposure to light. Likewise the relatively low photodynamic injury sustained by velvetleaf (76%) and prickly sida (89%) , two DDV/LDV weeds, was attributed to their predominant accumulation of DV Pchlide, which may be rapidly metabolized by these two weeds in the presence of light. The relatively high levels of photodynamic injury (100-99%) sustained by all other weed species except tall morningglory, soybean, and corn was explained by their predominant accumulation of MV Pchlide. This form of Pchlide may not be readily metabolized by these DMV/LDV species at daybreak, because they primarily utilize the DV biosynthetic greening pattern in the light.
The overall low photodynamic susceptibility of soybean was attributed to rebounding caused by nearly intact cotyledons. Although the treated leaves of soybean, a DMV/LDV tissue, experienced 100% photodynamic damage in the light, due to the predominant accumulation of MV Pchlide in darkness, the cotyledons, being very poor accumulators of tetrapyrroles, were relatively unaffected by the spray. As a consequence the soybean cotyledons supported the development of new leaves which in turn lowered the overall photodynamic damage rating of the seedlings.

62
Corn, a DMV/LDV plant species which accumulated large quantities of tetrapyrroles, was the least affected among the 12 plant species. The reason for the relative resistance of corn to photodynamic injury is unknown. Morningglory, a DDV/LDV plant species, accumulated predominantly DV Pchlide and much less MV Pchlide, yet it sustained a mean photodynamic injury of 98%. Its extreme photodynamic susceptibility may be ascribed to very low tolerance to tetrapyrrole accumulation, irrespective of its greening group affiliation.

10

15

20



25 Means followed by the same letter within a column are not significantly different at the 5% level of significance based on overlap of upper and lower limits o the means.
Greening group affiliation: DDV « dark divinyl, LDV *= light divinyl, DMV » dark monovinyl, LMV = light monovinyl.

Example 8
Chlorophyll Biosynthesis Modulators For TDPH Activity

' Preliminary screening of putative modulators was performed under controlled experimental conditions using greenhouse-grown cucumber seedlings (cotyledon stage) , a DDV/LDV plant tissue. The seedlings were sprayed in the late afternoon with 5 mM ALA + 20 mM modulator at a rate of 40 gallons per acre and an average droplet size of 75 μ. Sprayed plants were wrapped in aluminum foil to maximize penetration of the active ingredients and were placed in darkness for 17 h at 28°C to induce tetrapyrrole accumulation. The next day the plants were unwrapped and were exposed to light in the greenhouse. Photodynamic damage was evaluated visually and photographically over a period of 10 days. Modulators that exhibited photodynamic damage of 88-90% or better were retained for further experimentation.
Sixty-six commercially available compounds exhibited 88-100% photodynamic kill on cucumber seedlings. These TDPH modulators belonged to 12 different templates. The 12 TDPH templates and the 66 corresponding analogs that exhibited TDPH activity are described in Table XVIII. In particular, nine modulators that belonged to the nicotinic acid and nicotinamide templates exhibited 88-100% photodynamic kill on cucumber when used in concert with ALA. These modulators, which are simple vitamin derivatives, allow the design of totally biodegradable and safe TDPH formulations made up of ALA and a vitamin derivative.
An additional eighty-nine commercially available 5-membered N-heterocyclic modulators exhibited minimal phytotoxicity on corn and 88% or better kill on cucumber, pigweed or johnsongrass. These TDPH modulators belonged to 11 different templates. The 11 TDPH templates and the 87 corresponding 5-membered N-heterocyclic modulators are described in Table XIX.

Tabla ZVIII
Primary Screening of Modulators Balonging to
13 Different Templates on Cucumber
Percent death was monitored 10 days after spraying;
ALA = ALA alone; Mod = modulator alone; A+M = ALA + modulator;
D10 = 10 days after spraying

TEMPLATE MODULATOR

1, 10-phenanthroline 2, 9-Dimethyl-4 ,7-diphenyl-l, 10-phen
1, 10-phenanthroline 3,4,7, 8-Tetramethyl-l, 10-phen
1, 10-phenanthroline 5-Chloro-l, 10-phen
1, 10-phenanthroline 5, 6-Dimethyl-l, 10-phen
1, 10-phenanthroline 5-Methyl-l, 10-phen
1, 10-phenanthroline 5-Nitro-l, 10-phen
1, 10-phenanthroline 4 , 7-Dimethyl-l, 10-phen
1, 10-phenanthroline 4 , 7-Diphenyl-l, 10-phen
1, 10-phenanthroline 1, 10-Phenanthroline
1, 10-phenanthroline 4-Methyl-l, 10-phen
2,2' -Bipyridine 4,4'-Dimethyl-2 • , 2 '-Dipyridyl
2,2'-Bipyridine 2 , 2 ' : 6 * , 2"-Terpyridine
2 , 2 '-Dipyridyl disulfide 2,2 •-Dithiobis(pyridine N-oxide)
2 , 2 ' -Dipyridyl disulfide 6,6-Dithiodinicotinic acid
2-Oxypyridine 5-amino-2-methoxypyridine
2-Oxypyridine 2, 3-Dihydroxypyridine
2-Oxypyridine 2-Hydroxy-4-methylpyridine
2-Oxypyridine Isocarbostyryl
2-Oxypyridine 3-Amino-2,6-dimethoxy pyridine, HC1

Tabla XVIII (continued)

TEMPLATE MODULATOR %DEATH %DEATH VDEATH
ALA MOD A+M
D10 D10 D10

2-Oxypy idine 2-Chloro-6-methoxypyridine 69 0 97 2rOxypyridine 3-Cyano-4 , 6-dimethyl-2-hydroxypyridine 50 0 97 2-Oxypyridine Dibucaine hydrochloride 50 38 97 2-Oxypyridine 2-Hydroxy-3rnitropyridine 50 0 94 2-Oxypyridine 2 , 6-Dimethoxypyridine 50 0 93 2-Oxypyridine Citrazinic acid 69 0 88

2-Pyridine aldoxi e Di-2-pyridyl ketone oxi e 50 25 100 2-Pyridine aldoxime Phenyl 2-Pyridyl Ketoxime 38 25 100

8-Hydroxyquino1ine 8-Hydroxy-5-nitroquinoline 56 100 100 8-Hydroxyquinoline 5-Chloro-8-hydroxy-7-iodoquinoline 25 75 100 8-Hydroxyquinoline 5, 7-Dichloro-8-hydroxyquinoline 25 83 100 8-Hydroxyquinoline 5,7, Dibromo-8-hydroxyquinoline 25 44 91

Nicotinamide N-Benzyl-N-nicotoyl nicotinamide 44 0 94 Nicotinamide N-Methylnicotinamide 44 0 93

Nicotinic acid Ethyl 2-methylnicotinate 50 0 100 Nicotinic acid Niflu ic acid 50 93 96 Nicotinic acid 2-Hydroxynicotinic acid 50 0 95 Nicotinic acid Diethyl 3,4-pyridine dicarboxylate 38 0 93 Nicotinic acid Ethyl nicotinate 30 0 93 Nicotinic acid 2-Hydroxy-6-methylpyridine-3-carboxylic acid 38 0 91 Nicotinic acid 4-Hydroxy-7-trifluoromethyl-3-quinolinecarboxy 44 65 88

Phenanthridine Dimidiu bromide monohydrate 44 97 100

Tabla XVIII (continued)

TEMPLATE MODULATOR

Phenanthridine Ethidiu bromide 44 94 100 Phenanthridine 6 mM Propidium iodide hydrate 44 94 100 Phenanthridine Phenanthridine 44 0 88 Phenanthridine 2 mM Sanguiηarine chloride 44 0 88

Picolinic acid 3-Hydropicolinic acid 40 88 100 Picolinic acid Picolinic acid 40 0 100 Picolinic acid 1-Isoquinoline carboxylic acid 50 0 95

Pyridinium 2-[4-(Dimethylamino) styryl]-l-ethylpyridinium 56 75 100 Pyridinium 2-[4-(Dimethylamino) styryl]-l-methylpyridinium 56 56 100
Pyridinium Berberine hydrochloride hydrate 38 81 100 Pyridinium Bis-N-methyl acridinium nitrate 38 100 100 ± Pyridinium l-(carboxymethyl)pyridinium chloride 38 0 100 Pyridinium 5-Phenyl-2-(4-pyridyl)oxazole 38 31 100 Pyridinium l,l-Diethyl-2,2-cyanine iodide 25 94 100 Pyridinium l,l-Diethyl-2,4-cyanine ibdide 25 63 100 Pyridinium l,l-Diethyl-4,4-cyanine iodide 25 97 100 Pyridinium 1-Dodecylpyridinium chloride onohydrate 25 100 100 Pyridinium 2,4,6-Collidine p-toluene sulfonate 25 0 97 Pyridinium l-Ethyl-3-OH-pyridinium bromide 56 0 94 Pyridinium 4- (Dimethylamino) bromide perbro ide 56 0 88

Quinoline 6-Nitroquinoline 30 30 100 Quinoline 8-Nitroquinoline 30 100 100 Quinoline 5-Nitroquinoline 25 95 100 Quinoline 4,7 Phenanthroline 35 40 95 Quinoline 1,7 Phenanthroline 25 35 95

Tabla XIX
Primary Scraaning of Modulators Belonging to Various
5-memberad N-hatarooyclic Templettea
Only modulators that exhibited a negligible effect on corn or a rate of kill of 88% or better the other test plants are reported. Percent death was monitored 10 days after spraying. DT

ALA = death due to 5 mM ALA treatment; DTH MOD = death due to 20 mM modulator treatment; DTH A

= death due to 5 mM ALA + 20 mM modulator treatment. Modulators that are effective by
themselves without ALA are probable inducers.
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEA ALA MOD +
D10 D10 D1

Thiazoline Methyl 3-Chlorocarbonyl-L-thiazolidine-4-carboxylate
Thiazoline Methyl 3-Chlorocarbonyl-L-thiazolidine-4-carboxylate
Thiazoline Methyl 3-Chlorocarbonyl-L-thiazolidine-4-carboxylate
Thiazoline Methyl 3-Chlorocarbonyl-L-thiazolidine-4-carboxylate
Thiazoline (-)-2-Oxo-4-thiazolidine carboxylic acid
Thiazoline (-)-2-Oxo-4-thiazolidine carboxylic acid
Thiazoline (-)-2-Oxo-4-thiazolidine carboxylic acid
Thiazoline (-)-2-Oxo-4-thiazolidine carboxylic acid
Thiazoline 5- (4-Diethylaminobenzylidene) -rhoda ine
Thiazoline 5- (4-Diethylaminobenzylidene) -rhodamine
Thiazole 5-Chloro-2-mercaptobenzothiazole
Thiazole 5-Chloro-2-mercaptobenzothiazole
Thiazole 5-Chloro-2-mercaptobenzothiazole
Thiazole 5-Chloro-2-mercaptobenzothiazole
Thiazole 5-(4-Dimethylamino benzylidine) rhodinine
Thiazole 5-(4-Dimethylamino benzylidine) rhodinine

Table XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEA ALA MOD A+
D10 D10 D1

Thiazole 5-(4-Dimethylamino benzylidine)rhodinine
Thiazole 5-(4-Dimethylamino benzylidine) rhodinine
Thiazole 4-(4-Biphenyllyi)2-methyl thiazole
Thiazole 4-(4-Biphenyllyi)2-methyl thiazole
Thiazole 4-(4-Biphenyllyi)2-methyl thiazole
_ Thiazole 4-(4-Biphenyllyi)2-methyl thiazole
Thiazole 3-(4-Chlorophenyl) -2-ethyl-2 ,3,5, 6-tetrahydro- imidazo[2, 1-b]thiazol-3-ola
Thiazole 3-(4-Chlorophenyl) -2-ethyl-2 ,3,5, 6-tetrahydro- i idazo[2 , 1-b]thiazol-3-ola
Thiazole 3- (4-Chlorophenyl) -2-ethyl-2, 3 , 5, 6-tetrahydro- imidazo[2, 1-b]thiazol-3-ola
Thiazole 3- (4-Chlorophenyl) -2-ethyl-2,3,5, 6-tetrahydro- imidazo[2, 1-b]thiazol-3-ola
Thiazole 3,3-Diethylthiocarbocyanine iodide
Thiazole 3,3-Diethylthiocarbocyanine iodide
Thiazole 3,3-Diethylthiocarbocyanine iodide
Thiazole 3,3-Diethylthiocarbocyanine iodide
Thiazole 2-Amino-6-fluorobenzothiazole
Thiazole 2-Amino-6-fluorobenzothiazole
Thiazole 2-Amino-6-fluorobenzothiazole
Thiazole 2-Amino-6-fluorobenzothiazole

Table XIX (continued)
% % %
TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+M
D10 D10 D10

Thiazole 2-Amino-5, 6-dimethylbenzothiazole Johnsongrass 40
Thiazole 2-Amino-5, 6-dimethylbenzothiazole Cucumber 50
Thiazole 2-Amino-5,6-dimethylbeηzothiazole Pigweed 70
Thiazole 2-Amino-5, 6-dimethylbenzothiazole Corn 0



Tabla XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEA ALA MOD A+
D10 D10 D1

Thiazole 2-(4-Aminophenyl) -6-methylbenzothiazole
Thiazole 2-(4-Aminophenyl) -6-methylbenzothiazole
Thiazole 2-(4-Aminophenyl) -6-methylbenzothiazole
Thiazole 2-(4-Aminophenyl) -6-methylbenzothiazole
Thiazole 2-Bromothiazole
. Thiazole 2-Bromothiazole
Thiazole 2-Bromothiazole
Thiazole 2-Bromothiazole
Thiazole (+)6-Aminopenicillanic Acid
Thiazole (+)6-Aminopenicillanic Acid
Thiazole (+)6-Aminopenicillanic Acid
1 Thiazole (+) 6-Aminopenieillanie Acid
Thiazole 2-Amino-6-nitrobenzothiazole
Thiazole 2-Amino-6-nitrobenzothiazole
Thiazole 2-Amino-6-nitrobenzothiazole
Thiazole 2-Amino-6-nitrobenzothiazole
Thiazole 2-Acetylthiazole
Thiazole 2-Acetylthiazole
Thiazole 2-Acetylthiazole
Thiazole 2-Acetylthiazole
Thiazole Basic blue 66
Thiazole Basic blue 66

Tabla XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+M
D10 D10 D10

Thiazole Basic blue 66
Thiazole Basic blue 66
Thiazole 3 , 6-Dimethylbenzothiazole
Thiazole 3 , 6-Dimethylbenzothiazole
Thiazole 3 , 6-Dimethylbenzothiazole
Thiazole 3 , 6-Dimethylbenzothiazole
Thiazole 4 , 5-Dimethylthiazole
Thiazole 4 , 5-Dimethy1thiazole
Thiazole 4, 5-Dimethylthiazole
Thiazole 4 , 5-Dimethylthiazole
Thiazole 2-[4-(Dimethylamino) styryl]-3-ethylbenzothiazolium
iodide
Thiazole 2-[4- (Dimethylamino) styryl]-3-ethylbenzothiazolium
iodide
yhiazole 2-[4-(Dimethylamino) styryl]-3-ethylbenzothiazolium
iodide
Thiazole 2-[4- (Dimethylamino) styryl]-3-ethylbenzothiazolium
iodide
Thiazole 2-Bromo-5-nitrothiazole
Thiazole 2-Bromo-5-nitrothiazole
Thiazole 2-Bromo-5-nitrothiazole
Thiazole 2-Bromo-5-nitrothiazole

Table XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEA ALA MOD A+
D10 DIO D1

Thiazole 2-Cyano-6-methoxybenzothiazole Pigweed 65
Thiazole 2-Cyano-6-methoxybenzothiazole Cucumber 45
Thiazole 2-Cyano-6-methoxybenzothiazole Johnsongrass 65
Thiazole 2-Cyano-6-methoxybenzothiazole Corn 0
Thiazole Ethyl 2-amino-4-thiazole acetate Pigweed 35
Thiazole Ethyl 2-amino-4-thiazole acetate Cucumber 30
Thiazole Ethyl 2-amino-4-thiazole acetate Johnsongrass 40
Thiazole Ethyl 2-amino-4-thiazole acetate Corn 0
Thiazole 3-Methylbenzothiazole-2-thione Corn 0
o Thiazole 3-Methylbenzothiazole-2-thione Johnsongrass 40

Tabla XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+M
D10 D10 D10

Thiazole 3-Methylbenzothiazole-2-thione Cucumber 30 0 10 Thiazole 3-Methylbenzothiazole-2-thione Pigweed 35 60 10

Thiazole 2-4-Thiazolidinedione Pigweed 35 50 9

Thiazole 2-4-Thiazolidinedione Cucumber 30 0 6

Thiazole 2-4-Thiazolidinedione Johnsongrass 40 0 3

Thiazole 2-4-Thiazolidinedione Corn 0 0

Thiazole 2- (4-Aminophenyl) -6-methylbenzothiazole Cucumber 40 0 10

Thiazole 2-(4-Aminophenyl) -6-methylbenzothiazole Pigweed 25 0 8

Thiazole 2-(4-Aminophenyl) -6-methylbenzothiazole Johnsongrass 20 0 8

Thiazole 2- (4-Aminophenyl) -6-methylbenzothiazole Corn 0 0

Thiazole 2-Amino-alpha-(methoxyimino) -4-thiazole acetic
acid hydrochloride Cucumber 55 0 7 Thiazole 2-Amino-alpha-(methoxyimino)-4-thiazole acetic
acid hydrochloride Pigweed 65 60 9 Thiazole 2-Amino-alpha-(methoxyimino)-4-thiazole acetic
acid hydrochloride Johnsongrass 30 0 8 Thiazole 2-Amino-alpha-(methoxyimino)-4-thiazole acetic
acid hydrochloride Corn 0 0

Thiazole 2-Aminobenzothiazole Corn 0 0

Thiazole 2-Aminobenzothiazole Cucumber 40 0

Thiazole 2-Aminobenzothiazole Pigweed 50 0

Thiazole 2-Aminobenzothiazole Johnsongrass 5 0

Tabla XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+M
D10 D10 D10

Thiazole 2-Amino-2-thiazoline Corn 0
Thiazole 2-Amino-2-thiazoline Cucumber 40
Thiazole 2-Amino-2-thiazoline Pigweed 50
Thiazole 2-Amino-2-thiazoline Johnsongrass 5
Thiazole 2-(4-Thiazolyl)benzimidazole Cucumber 60
Thiazole 2- (4-Thiazolyl)benzimidazole Pigweed 70
Thiazole 2- (4-Thiazolyl) benzimidazole Johnsongrass 50
Thiazole 2-(4-Thiazolyl)benzimidazole Corn 0
Thiazole Ethyl 2-(formylamino)-4-thiazolegloxylate Cucumber 60
Thiazole Ethyl 2-(formylamino)-4-thiazolegloxylate Pigweed 70
Thiazole Ethyl 2-(formylamino)-4-thiazolegloxylate Johnsongrass 50
Thiazole Ethyl 2-(formylamino)-4-thiazolegloxylate Corn 0
Thiazole Thiaflavin T Cucumber 100
Thiazole Thiaflavin T Pigweed 70
Thiazole Thiaflavin T Johnsongrass 20
Thiazole Thiaflavin T Corn 0
Thiazole Ethyl 2-amino-alpha-(methoxyimino)-4-thiazole acetate Pigweed 70
Thiazole Ethyl 2-amino-alpha-(methoxyimino)-4-thiazole acetate Johnsongrass 20
Thiazole Ethyl 2-amino-alpha-(methoxyimino)-4-thiazole acetate Corn 0
Thiazole Ethyl 2-amino-alpha-(methoxyimino)-4-thiazole acetate Cucumber 100
Thiazole 2-(Tritylamino) -alpha-( ethoxyimino) -4-thiazole
acetic acid hydrochloride Cucumber 70

Tabla XIX (continued)
% % %
TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+M
D10 D10 D10

2-(Tritylamino) -alpha-(methoxyimino) -4-thiazole
acetic acid hydrochloride
2- (Tritylamino) -alpha-(methoxyimino) -4-thiazole
acetic acid hydrochloride
2- (Tritylamino) -alpha-(methoxyimino) -4-thiazole
acetic acid hydrochloride

l-Phenyl-3-(2-thiazolyil-2-thiourea)





Tabla XIX (continued)
% % %
TEMPLATE MODULATOR PLANT

l-Phenyl-3-(2-thiazolyil-2-thiourea) Pigweed 70
l-Phenyl-3-(2-thiazolyil-2-thiourea) Johnsongrass 80
l-Phenyl-3-(2-thiazoly^l-2-thiourea) Corn 0



Blue tetrazolium Cucumber 20
Blue tetrazolium Pigweed 0
Blue tetrazolium Johnsongrass 20
Blue tetrazolium Corn 0
2,3,5-Triphenyl-2H-tetrazolium chloride Cucumber 50
2,3, 5-Triphenyl-2H-tetrazolium chloride Pigweed 50
2,3, 5-Triphenyl-2H-tetrazσlium chloride Johnsongrass 30

2,3, 5-Triphenyl-2H-tetrazolium chloride Corn 0

Tabla XIX (continuad)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+N
D10 D10 D10

Pyrroline N-(4-Dimethylamino-3 , 5-dinitrophenyl) -maleimide
Pyrroline N-(4-Dimethylamino-3,5-dinitrophenyl) -maleimide
Pyrroline N-(4-Dimethylamino-3, 5-dinitrophenyl) -maleimide
Pyrroline N-(4-Dimethylamino-3,5-dinitrophenyl) -maleimide
Pyrroline Bilirubin
Pyrroline Bilirubin
Pyrroline Bilirubin
Pyrroline Bilirubin
- Pyrrolidine Trans-4-hydroxy-L-proline
j Pyrrolidine Trans-4-hydroxy-L-pro1ine
^ Pyrrolidine Trans-4-hydroxy-L-proline
2 Pyrrolidine Trans-4-hydroxy-L-proline
B Pyrrolidine Alpha-methyl-alpha-propyl-succinimide
Pyrrolidine Alpha-methyl-alpha-propyl-succinimide
Pyrrolidine Alpha-methyl-alpha-propyl-succinimide
Pyrrolidine Alpha-methyl-alpha-propyl-succinimide
Pyrrolidine N-Hydroxysuccinimidyl acetoacetate
Pyrrolidine N-Hydroxysuccinimidyl acetoacetate
Pyrrolidine N-Hydroxysuccinimidyl acetoacetate
Pyrrolidine N-Hydroxysuccinimidyl acetoacetate
Pyrrolidine N-(9-Fluorenylmethoxycarbonyloxy) succinimide
Pyrrolidine N-(9-Fluorenylmethoxycarbonyloxy) succinimide

Tabla XIX (continued)
% % %
TEMPLATE MODULATOR PLANT DEATH DEATH DEA ALA MOD A+
D10 D10 D1

Pyrrolidine N-(9-Fluorenylmethoxycarbonyloxy) succinimide Johnsongrass 15
Pyrrolidine N-(9-Fluorenylmethoxycarbonyloxy) succinimide Corn 0
Pyrrolidine 4-Pyrrolidinopyrridine Cucumber 60
Pyrrolidine 4-Pyrrolidinopyrridine Pigweed 0
Pyrrolidine 4-Pyrrolidinopyrridine Johnsongrass 10
Pyrrolidine 4-Pyrrolidinopyrridine Corn 0
o



O


Tabla XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+
D10 D10 D10

Pyrrolidine 1-[2-(4-Bromophenoxy) ethy1]pyrro1idine
Pyrrolidine 1-[2-(4-Bromophenoxy)ethyl]pyrrolidine
Pyrrolidine l-[2-(4-Bromophenoxy)e hyl]pyrrolidine
Pyrrolidine 1-[2-(4-Bromophenoxy) ethyl] yrrolidine
Pyrrolidine S) -(+) -Ethyl-2-pyrrolidine-5-carboxylate
Pyrrolidine s) "(+) -Ethyl-2-pyrrolidine-5-carboxylate
Pyrrolidine S) -(+) -Ethyl-2-pyrrolidine-5-carboxylate
o. Pyrrolidine S) - (+) -Ethyl-2-pyrrolidine-5-carboxylate
Pyrrolidine -)-Cotinine
Pyrrolidine -)-Cotinine
Pyrrolidine -)-Cotinine
Pyrrolidine -)-Cotinine
Pyrrole Tert-butyl 4-acetyl-3 , 5-dimethyl-2-pyrrolecarboxylate
Pyrrole Pyrrolo (1,2-a) quinoxaline
Pyrrole Pyrrole-2-carboxaldehyde
Pyrrole Ethyl 3 , 5-dimethyl-2-pyrrolecarboxylate
Pyrrole 3-Ethyl-2-methyl-4 ,5,6, 7-tetrahydroindol
Pyrrole l-Methyl-2-pyrrolecarboxylic acid

Tabla XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEA ALA MOD A+
D10 D10 D1

Pyrrole l-Methyl-2-pyrrolecarboxaldehyde
Pyrrole 1-Furfurylpyrrole
Pyrrole 1-(Dimethylamino) pyrrole
Pyrrole 1-(2-Cyanomethy1)pyrrole
o Pyrrole Diethyl 2,4-dimethylpyrrole-3 , 5-dicarboxylate
Pyrrole Methyl 5-(benzoxycarbonyl) -2, 4-dimethyl-3-pyrr
o Pyrazolene 4-Methyl-2-pyrazolin-5-one
Pyrazolene 4-Methyl-2-pyrazolin-5-one
Pyrazolene 4-Methyl-2-pyrazolin-5-one
Pyrazolene 4-Methyl-2-pyrazolin-5-one
Pyrazolene 3 ,4-Dimethyl-l-phenyl-3-pyrazolin-5-one
Pyrazolene 3 , 4-Dimethyl-l-phenyl-3-pyrazolin-5-one
Pyrazolene 3 ,4-Dimethyl-l-phenyl-3-pyrazolin-5-one
Pyrazolene 3 , 4-Dimethyl-l-phenyl-3-pyrazolin-5-one
Pyrazole Pseudothiohydrantoin
Pyrazole Pseudothiohydrantoin
Pyrazole Pseudothiohydrantoin
Pyrazole Pseudothiohydrantoin

Tabla XIX (continued)
% % %
TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+
D10 D10 D10

Oxazole 3,3' -Dipropyloxacarbocyanine iodide Cucumber 50
Oxazole 3,3' -Dipropyloxacarbocyanine iodide Pigweed 20
Oxazole 3,3' -Dipropyloxacarbocyanine iodide Johnsongrass 50
Oxazole 3,3*-Dipropyloxacarbocyanine iodide Corn 0
Oxazole 3,3'-Di ethyloxacarbocyanine iodide Cucumber 50
Oxazole 3,3*-Dimethyloxacarbocyanine iodide Pigweed 20
Oxazole 3,3' -Dimethyloxacarbocyanine iodide Johnsongrass 50


Tabla XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEA ALA MOD A+
D10 D10 D1

Oxazole 3,3'-Dimethyloxacarbocyanine iodide Corn 10
Oxazole 2, 5-Diphenyloxazole Cucumber 50
Oxazole 2, 5-Diphenyloxazole Pigweed 20
Oxazole 2,5-Diphenyloxazole Johnsongrass 50
g Oxazole 2, 5-Diphenyloxazole Corn 0

o Oxazole 2-Mercaptobenzoxazole Cucumber 50
Oxazole 2-Mercaptobenzoxazole Pigweed 20
Oxazole 2-Mercaptobenzoxazole Johnsongrass 50
Oxazole 2-Mercaptobenzoxazole Corn 0
4 Oxazole 3-Methyl-2-oxazolidinone Cucumber 50
Oxazole 3-Methyl-2-oxazolidinone Pigweed 15
5 Oxazole 3-Methyl-2-oxazolidinone Johnsongrass 60
Oxazole 3-Methyl-2-oxazolidinone Corn 0
Oxazole 2-Chlorobenzoxazole Cucumber 90
Oxazole 2-Chlorobenzoxazole Pigweed 100
Oxazole 2-Chlorobenzoxazole Johnsongrass 50
Oxazole 2-Chlorobenzoxazole Corn 0
Oxazole 2-(4-Biphenylyl) -5-phenyl-oxazole Cucumber 90
Oxazole 2-(4-Biphenylyl) -5-phenyl-oxazole Pigweed 100
Oxazole 2-(4-Biphenylyl) -5-phenyl-oxazole Johnsongrass 50
Oxazole 2-(4-Biphenylyl) -5-phenyl-oxazole Corn 0

Tabla XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+M
D10 D10 D10

2-Benzoxazolinone
2-Benzoxazolinone
2-Benzoxazolinone
2-Benzoxazolinone
2,5-Bis(4-biphenylyl)oxazole
2, 5-Bis(4-biphenylyl)oxazole
2, 5-Bis(4-biphenyly1)oxazole
2,5-Bis(4-biphenylyl)oxazole
3,3'-Dihexyloxacarbocyanine iodide
3,3'-Dihexyloxacarbocyanine iodide
3,3'-Dihexyloxacarbocyanine iodide
3,3'-Dihexyloxacarbocyanine iodide
3,3'-Diethyloxacarbocyanine iodide
3,3*-Diethyloxacarbocyanine iodide
3,3'-Diethyloxacarbocyanine iodide
3,3'-Diethyloxacarbocyanine iodide
2, 5-Dimethyl-benzoxazole
2,5-Dimethyl-benzoxazole
2,5-Dimethyl-benzoxazole
2, 5-Dimethyl-benzoxazole
2-Mercaptoimidazole

2-Mercaptoimidazole

Tabla XIX (continued)
% % %
TEMPLATE MODULATOR PLANT DEATH DEATH DEA ALA MOD A+
D10 D10 D1

Imidazole 2-Mercaptoimidazole Johnsongrass 10
Imidazole 2-Mercaptoimidazole Corn 0





Table XIX (continued)

TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+M
D10 D10 D10

Imidazole 6-Thioxanthine
Imidazole 6-Thioxanthine
Imidazole 6-Thioxanthine
Imidazole 6-Thioxanthine
O Imidazole 2,4, 5-Triphenylimidazole
Imidazole 2,4,5-Triphenylimidazole
O Imidazole 2,4, 5-Triphenylimidazole
Imidazole 2,4, 5-Triphenylimidazole
Imidazole 4 ,5-Diphenylimidazole
Imidazole 4, 5-Diphenylimidazole
O Imidazole 4,5-Diphenylimidazole
Imidazole 4 , 5-Diphenylimidazole
Imidazole Guanosine hydrate
Imidazole Guanosine hydrate
Imidazole Guanosine hydrate
Imidazole Guanosine hydrate
Imidazole 2-Ethyl-4-methyl-imidazole
Imidazole 2-Ethyl-4-methyl-imidazole
Imidazole 2-Ethyl-4-methyl-imidazole
Imidazole 2-Ethyl-4-methyl-imidazole
Imidazole 4 , 5-Dicyanoimidazole
Imidazole 4 , 5-Dicyanoimidazole

Tabla XIX (continued)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEA ALA MOD A+
D10 D10 D1

4 , 5-Dicyanoimidazole
4 , 5-Dicyanoimidazole
1-(Mesitylenesulfonyl) -imidazole
1-(Mesitylenesulfonyl) -imidazole
1- (Mesitylenesulfonyl) -imidazole
1-(Mesitylenesulfonyl) -imidazole
2,2'-Dithiobis(4-tert-butyl-l-isopropylimidazole)
2,2'-Dithiobis(4-tert-butyl-l-isopropylimidazole)
2,2'-Dithiobis(4-tert-butyl-l-isopropylimidazole)
2,2'-Dithiobis (4-tert-butyl-l-isopropylimidazole)
Inosine-5'-triphosphate, disodiu salt dihydrate
Inosine-5'-triphosphate, disodium salt dihydrate
Inosine-5'-triphosphate, disodium salt dihydrate
Inosine-5'-triphosphate, disodium salt dihydrate
1- (2, 4 , 6-Triisopropylbenzenesulfonyl) imidazole
1- (2, 4 , 6-Triisopropylbenzenesulfonyl) imidazole
1-(2 , 4, 6-Triisopropylbenzenesulfonyl) imidazole
1- (2, 4 , 6-Triisopropylbenzenesulfonyl) imidazole
Nitrofurantoin
Nitrofurantoin

Nitrofurantoin

Tabla XIX (continuad)
% % %

TEMPLATE MODULATOR PLANT DEATH DEATH DEAT ALA MOD A+M
D10 D10 D10

Furfural Kinetin
Furfural Kinetin
Furfural Kinetin

74
Exam le 9
Substituted 1,10-Phenanthrolines as Potent
Photodynamic Herbicide Modulators
1, 10-Phenanthroline, also known as orthophenanthroline (OPH) , is a potent photodynamic herbicide modulator. It induces the accumulation of large amounts of tetrapyrroles in dark treated cucumber, in the absence and presence of exogenous ALA. In the presence of ALA it exhibits very potent photodynamic herbicide modulating activities against a large number of weed species. Whether the mode of action of OPH as a Chi biosynthesis modulator is affected by the introduction of peripheral substituents was investigated.
Compounds 1-7 and 11 (Table XX) were dissolved in acetone:methanol: ween 80:water (4.5:4.5:1:90, v/v/v/v) . Compounds 8-10 and 12 (Table XX) were dissolved in ethanol:methanol:Tween 80:Polyethylene glycol 600 (45:45:1:9, v/v/v/v). All solutions were freshly prepared on the day of spraying. Treatments consisted of solvent treated controls, 5 mM ALA, and 2, 4, and 6 mM modulator with or without 5 mM ALA. The pH of all solutions was adjusted to 3.5 at room temperature with 1.2 N HC1 or 1.0 N KOH.
Cucumber seeds were planted in moist vermiculite. Germination was carried out in a growth room under a 14/10 h light/dark photoperiod. The temperature ranged from 27°C in the light to 21°C in darkness. Seedlings were watered with Hoagland1 s nutrient solution. Prior to spraying, seedlings were thinned to 6 per container.
The plant foliage was sprayed at a rate of 0.35 ml per container with a modified aerosol kit. The sprayed plants were wrapped in aluminum foil and were incubated in darkness for 14 to 16 h at 21βC prior to unwrapping the plants, determining the tetrapyrrole content and exposing to light.
Following postspray dark incubation, one cotyledon from each plant was excised under a dim green safelight and used for the determination of tetrapyrrole content. The plants with one remaining cotyledon per 75
seedling were then exposed to light in the growth chamber for the induction of photodynamic injury. The latter was monitored visually and photographically over a period of 10 days. At day 10, the top growth was excised at soil level, dried, and the dry weight was recorded.
Substitutions at the periphery of the OPH macrocycle resulted in a pronounced modulation of the photodynamic herbicidal activity as ALA and various OPH analogs contributed singly and in combinations to photodynamic injury. The results are described in Table XX.

76
Table XX
Photodynamic Damage in cucumber Seedlings caused by

Various PHTN and OPH analogs Applied Alone or in
Combination with 5 mM ALA.



Mod ■ Modulator 77
SECTION IV EFFECT OF TDPH ON TISSUES LACKING CHLOROPHYLL

Example 10
Insecticidal Compositions
The insecticidal system described in co-pending application Serial No. 07/294,132 describes certain modulators of the porphyrin-he e biosynthetic pathway, which when used singly or in combination with ALA, induced the massive accumulation of Proto in treated insects. The uncontrolled Proto biosynthesis and accumulation caused death of the treated insects in darkness (dark death) via an unknown mechanism, and in the light (light death) probably via singlet oxygen formation. Proto is a transient metabolite which does not accumulate to any large extent in normal tissues. It is an immediate precursor of protoheme which in turn is the prosthetic group of cytochromes in mitochondria and chloroplasts. It is also the prosthetic group of catalases and peroxidases. Twenty-three modulators that belonged to nine TDPH templates exhibited 88-100% photodynamic death on four insect species when used in concert with ALA. The TDPH templates and the corresponding modulators are described in Table XXI.

Tabla XXI
Modulator Kill (88-100%) on Four Insect Species
Modulators were incorporated in the diet at the following rates: 4 mM ALA + 3 mM modulator fo
T. ni and H. zea, 6 mM ALA + 8 mM modulator for A. grandis, and 24 mM ALA + 18 mM modulator f
B. germanica. The insects were placed on the diet in darkness for 17 h. Photodynamic death w
evaluated after 6 days of exposure to 14h light-lOh dark in the growth chamber.

TEMPLATE MODULATOR INSECT 6DL

Orthophenanthroline Bathophenanthroline disulfonic acid Trichoplusia ni 8
4,4'-Dipyridyl Benzyl viologen dichloride onohydrate Anthonomus grandis 10
4,4'-Dipyridyl Benzyl viologen dichloride monohydrate Heliothus zea 9
Orthophenanthroline 5-Chloro-l,10-Phenanthroline Trichoplusia ni 9
Pyridinium 1, l-Diethyl-4,4-carbocyanine iodide Heliothis zea 9
Pyridinium 1, l-Diethyl-4,4-carbocyanine iodide Anthonomus grandis 10 o
Pyridinium 1, l-Diethyl-2 , 4-cyanine iodide Blattella germanica 9 fl Pyrrole 3-Ethyl-2-methyl-4 , 5, 6, 7-tetrahydroindol-4-on Trichoplusia n
4,4'-Dipyridyl l,l'-Diheptyl-4,4'-Bipyridinium dibrσmide Heliothus zea 10
Pyridinium 2-(4-(Dimethylamino) styryl) -1-ethylpyridinium Anthonomus grandi
Orthophenanthroline 4, 7-Dimethyl-l, 10-Phenanthroline Heliothus zea 9
Orthophenanthroline 4, 7-Dimethyl-l, 10-Phenanthroline Blattella germanica 9
Orthophenanthro1ine 4 , 7-Dimethyl-l, 10-Phenanthroline Trichoplusia ni 10
Orthophenanthroline 5, 6-Dimethyl-l, 10-Phenanthroline Blattella germanica 9
Orthophenanthroline 5, 6-Dimethyl-l, 10-Phenanthroline Trichoplusia ni 9

Tabla XXI (continuad)

TEMPLATE MODULATOR INSECT 6DL

Orthophenanthroline 4,7-Diphenyl-l, 10-Phenanthroline Trichoplusia ni 96
2,2'-Dipyridyl Blattella germanica 100
2,2' -Dipyridyl Heliothus zea 100
2,2'-Dipyridyl Trichoplusia ni 100
2,2' -Dipyridyl Anthonomus grandis 10
2-Methoxy-5-nitropyridine Blattella germanica 10
5-Methyl-l, 10-Phenanthroline Trichoplusia ni 9
Methyl viologen dichloride hydrate Blattella germanica 10
5-Nitro-l, 10-Phenanthroline Trichoplusia ni 9
1,10 Phenanthroline Heliothis zea 10
1,10 Phenanthroline Anthonomus grandis 9
1,10 Phenanthroline Blattella germanica 10
1,10 Phenanthroline Trichoplusia ni 10
Poly(4-Vinylpyridinium) dichromate Trichoplusia ni 8
2 , 2 ' : 6 ' , 2 '-Terpyridine Heliothus zea 10


4-Pyrrolidinopyrridine Blattella germanica 8

Tabla XXI (continuad)

TEMPLATE MODULATOR INSECT 6DL

Imidazole 4,5-Dicyanaimidazole Blattella germanica 10

Thiazole Thioflavin T Blattella germanica 10

Orthophenanthroline 4-Methyl-l, 10-Phenanthroline Heliothis zea 9 Orthophenanthroline 4-Methyl-l, 10-Phenanthroline Trichoplusia ni 9


80
Example 11
Effect of TDPH on Roots
Since non-chlorophyllous plant tissues such as roots consist of cells containing an abundance of mitochondria which in turn contain cytochromes and presumably an active porphyrin-heme biosynthetic pathway, it was investigated whether some TDPH formulations would be effective against plant roots, in the same manner they were effective against insects. In particular it was interesting to determine whether plant roots would be susceptible to TDPH-dependent dark death since in their natural environment in the soil, roots are usually shielded from light. The effects of ALA and four modulators belonging to four different templates on excised and intact cucumber roots were investigated.
Excised cucumber roots were incubated overnight in darkness with ALA and one of several modulators, namely; DPY, 1,10-phenanthroline, 4,7-dimethyl-l,10-phenanthroline, 4,7-phenanthroline, phenanthridine, picolinic acid, and ethyl nicotinate. At the end of dark incubation the tissue was analyzed for tetrapyrrole accumulation and was exposed to light for evaluation of photodynamic damage. The latter was evaluated visually, and polarographically via the decrease in oxygen consumption of treated roots as compared to controls. Excised roots incubated with ALA and TDPH modulators accumulated massive amounts of tetrapyrroles in darkness. Although Proto was the main tetrapyrrole that accumulated, significant amounts of MPE and Pchlide were also formed. This in turn suggested that some extramitochondrial, plastidic tetrapyrrole biosynthesis may also be taking place. In the light, the excised roots that accumulated tetrapyrroles exhibited significant phytotoxicity.
To determine the effects of TDPH treatments on the roots of intact seedlings, cucumber seedlings were watered once with a solution consisting of 4 mM ALA + 3 mM modulator. The treated seedlings were kept in darkness for various periods of time prior to tetrapyrrole analysis and exposure to light. As was 81
observed with excised roots, the roots of intact seedlings watered with a solution of ALA + 1,10-phenanthroline accumulated massive amounts of tetrapyrroles. In this case too the major tetrapyrrole pool that accumulated in darkness consisted of Proto. However, this Proto accumulation was not toxic to the root cells in darkness. Even after two days in darkness no apparent damage to the root system was observed. Altogether these results indicated that although plant roots do react to treatment with ALA and TDPH modulators by accumulating tetrapyrroles, they do not exhibit the phenomenon of dark tetrapyrrole-dependent death which was observed in insects.
* * *
These examples serve to demonstrate the novel concept of the present invention. The photodynamic mode of action is different from other known modes of action in two main respects: (a) it is dependent on the biosynthesis and accumulation of tetrapyrroles in the foliage of living plants; and (b) the accumulated tetrapyrroles render the foliage of the plants light- sensitive so that upon subsequent exposure to light, a very damaging photodynamic effect is produced which results in death of susceptible undesirable plants or desiccation of the foliage without death of desirable plants.
In the insecticidal system, accumulation of Proto is induced in treated insects. The uncontrolled Proto biosynthesis and accumulation caused death of the treated insects in darkness and in light.
5-Aminolevulinic acid is a natural metabolite present in all living cells; it is a natural component of the biosphere and is readily biodegradable. The same is true for the products of ALA dark-metabolism, i.e., the tetrapyrrole intermediates of the Chi biosynthetic pathway, which have been demonstrated to disappear very rapidly upon exposure of the plant to light. Similarly, modulators which are naturally occurring vitamins or derivatives thereof, e.g., ethyl nicotinate, are expected to be readily biodegradable and to have no adverse impact 82
on the environment. It therefore appears that the photodynamic desiccating compositions and methods of the present invention employing ALA and/or vitamins or derivatives thereof are likely to have no adverse impact on the environment.
Further examples of compositions and applications within the spirit and scope of this invention are described in copending applications Serial Nos. 06/895,529 and 07/294,132 and in Rebeiz, CA. et al., CJ?C Critical Reviews in Plant Sciences , 6(4) :385-436 (1988) .