Towards an ecofriendly herbicide: (-)-catechin, a natural flavonol secreted by spotted knapweed roots, is a potent inhibitor of plant growth

TOWARDS AN ECOFRIENDLY HERBICIDE: (-)-CATECHIN, A NATURAL FLAVONOL SECRETED BY SPOTTED KNAPWEED ROOTS, IS A POTENT INHIBITOR OF PLANT GROWTH

Sponsoring Institution

State Agricultural Experiment Station

Project Status

TERMINATED

Funding Source

STATE

Reporting Frequency

Annual

Accession No.

0191089

Grant No.

(N/A)

Project No.

COL00185

Proposal No.

(N/A)

Multistate No.

(N/A)

Program Code

(N/A)

Project Start Date

Dec 1, 2001

Project End Date

Jun 30, 2007

Grant Year

(N/A)

Project Director
Vivanco, J. M.

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
HORTICULTURE & LANDSCAPE ARCHITECTURE

Non Technical Summary
Invasive plants are among the greatest threats to croplands, rangelands, and wild lands in North America. Conventional weed control measures rely on frequent applications of chemical herbicides, which may contribute to soil/water pollution, and pose risks to human health. As an alternative, strategies that utilize endogenous plant allelopathy can reduce our reliance on synthetic herbicides, and improve the economics of food production. Our finding that (-)-catechin secreted by C. maculosa roots accounts for a broad-spectrum phytotoxic activity holds promise for such applications.

Animal Health Component

(N/A)

Research Effort Categories

Basic

60%

Applied

30%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
213	2420	1000	30%
213	2420	1040	10%
213	2420	1140	60%

Knowledge Area
213 - Weeds Affecting Plants;

Subject Of Investigation
2420 - Noncrop plant research;

Field Of Science
1000 - Biochemistry and biophysics; 1040 - Molecular biology; 1140 - Weed science;

Goals / Objectives
Determine the mechanism behind the herbicidal activity of (-)-catechin, and producing derivatives of (-)-catechin with improved herbididal activity and solubility. Additionally, we will purify and characterize novel allelochemicals from other invasive weeds to demonstrate the value and use of this approach. Background - We developed a system whereby knapweed roots, grown in vitro, can secrete and be induced to secrete allelochemicals into sterile media comparable to secretion into the rhizosphere. We have recently identified such a chemical from C. maculosa for the first time, and have shown that the flavanol (-)-catechin, is the compound responsible for the allelopathic activity. Centaurea maculosa Lam. (spotted knapweed) is one of the most invasive and destructive introduced weeds in North America, particularly in fields and pastures of the northwest U.S. Although it has long been proposed that C. maculosa achieves its dominance through secretion of a phytotoxic allelochemical, no such chemical had been previously discovered. We have determined that an exudate of C. maculosa roots is indeed phytotoxic, that it inhibits seed germination, and that production of the exudates is dramatically increased upon elicitation. The active fraction of the exudate was found to be composed of racemic catechin, a 50:50 mixture of (+)-catechin and (-)-catechin. Although (+)-catechin is widespread throughout the plant kingdom, both (-)-catechin and the racemic form have only rarely been isolated. Further, we found that the phytotoxicity and germination inhibiting action of the exudate was entirely due to (-)-catechin, while (+)-catechin was inactive. Conversely, (+)-catechin had antimicrobial properties, while (-)-catechin did not. Thus, C. maculosa produces an enantiomeric mixture, with one enantiomer having phytotoxic properties and the other being an antimicrobial. (-)-Catechin was toxic to a variety of model and crop plant species and was also toxic to C. diffusa (diffuse knapweed), a close relative of C. maculosa. (-)-catechin did not affect seedlings of C. maculosa itself. Catechins are common components of foods and are among the most widespread flavanols. Their antioxidant properties are believed to be responsible for positive bioactivity against a variety of diseases and human pathologies, but the discovery of phytotoxicity for a catechin is unprecedented. The results of this study could be valuable in the design and use of natural and ecologically benign herbicides. (+)-(2R,3S)-catechin (-)-(2S,3R)-catechin.

Project Methods
Aim 1: To determine the scope of flavanol and other root exudates as phytotoxins and seed germination inhibitors. A. Structure-Activity Relationships: catechin chemical derivatives. The relationship between stereochemistry and phytotoxicity will be studied. This will be done with the aid of two related natural flavanols (diastereomers of (+)-catechin and (-)-catechin), which are the pair of enantiomers (-)-epicatechin (3) and (+)-epicatechin (4). We will also determine whether or not small or large changes in polarity or functional group changes in 2 can affect activity. B. Purification and characterization of allelochemicals from other aggressive exotic weeds. Among the knapweeds (Centaurea species), C. maculosa, C. diffusa Lam. (diffuse knapweed) and C. repens L. (russian knapweed) are important species to study. An investigation of possible allelopathy in diffusa Lam. and C. repens will be an important extension of the studies on C. maculosa. A first goal will be to use the same methods employed in our study on spotted knapweed to illuminate the allelopathic activity of diffuse knapweed. Aim 2: To delineate the detailed biochemical mechanism(s) of flavanol phytotoxicity. We will attempt to answer the questions 1) why is (-)-catechin phytotoxic to most plant species? and 2) why is Centaurea maculosa itself not affected by (-)-catechin? A. Identification of membrane and/or cytoplasmic receptor target(s) that interact with (-)-catechin. The receptor site(s) or cytoplasmic proteins that interact with (-)-catechin (2) will be identified. Arabidopsis, one of the most sensitive plants to 2 activity, will be used as an experimental system due to the genomic and proteomic information available. Identification of the protein(s) that interact with 2 will make use of affinity chromatography. B. Determination of the fate of (-)-catechin in the cell. As a complementary approach to affinity chromatography, bioactive 2 derivatives will be labeled with a fluorescent dye to monitor the fate of this compound in Arabidopsis roots and cells using confocal microscopy. Additionally, we will explore other labeling procedures such as labeling with specific fluorescent groups supplied by Pierce (Amca-hydrazide). We will analyze whether (-)-catechin-fluorescent dye penetrated cells or if it was instead attached to the cell membrane. This information will allow us to begin dissecting the herbicidal mechanism of 2 at the biochemical level. C. Dissection of the pathway involved in the production of catechin and the ability of knapweed to detoxify this compound. The hypothesis that enantiomer 1 is formed in the usual way, but that a racemase enzyme is present in the roots which racemizes enantiomer 1 to produce enantiomer 2 (or vice versa 2 to produce 1), resulting in (+/-)-catechin. If racemization is successful, chromatography of C. maculosa root proteins combined with precursor incubation experiments will be performed to isolate the enzymes(s) involved in this catalysis. In another approach, we will attempt to isolate the two precursor flavones (5 and 6) from roots of C. maculosa and determine if they are racemic or chiral.

Progress 12/01/01 to 06/30/07

Outputs
We have continued our research on C. maculosa allelopathy and we have found that the secretion of the allelochemical, catechin, tends to be rather low throughout the year but can reach very high concentrations at some points of the year. These studies suggest that invasive plants might selectively deploy allelochemicals at particularly advantageous times; however, at present we don't know what the triggers for induction might be. We have also reported that C. maculosa not only wields allelopathy against other plants but that it literally eliminates soil microbes in its vicinity and that this effect extends to the rhizosphere of other plants, suggesting that antimicrobial root exudates are involved in this process. Genomic resources in the form of an EST library have been developed for this invasive weed to help us understand the genetic makeup of this plant. These molecular resources combined with our investigations into the biochemistry of this invasive weed should help us dissect the genetics of invasion. On a different front, we have reported that gases released by flowers could also have phytotoxic effects on top of their reported ability to attract pollinators. These studies should provide ideas for the study of allelopathy coming from flowers as well as from roots.

Impacts
Our studies have helped understand the real role of allelopathy in natural environments.

Publications

Perry, L.G., Thelen, G.C., Ridenour, W.M., Callaway, R.M., Paschke, M.W., Vivanco, J.M. (2007) Concentrations of the allelochemical catechin in Centaurea maculosa soils. Journal of Chemical Ecology 33:2337-2344
Broz, A.K., Manter, D.K., and Vivanco, J.M. (2007) Soil fungal abundance and biodiversity: another victim of the invasive plant Centaurea maculosa. The ISME Journal 1:763-765
Broz, A.K., Broeckling, C.D., He, J., Dai, X., Zhao, P.X. and Vivanco, J.M. (2007) A first step in understanding an invasive weed through its genes: an EST analysis of invasive Centaurea maculosa. BMC Plant Biology 7:25
Horiuchi, J., Badri, D., Kimball, B.A., Negre, F., Dudareva, N., Paschke, M., and Vivanco, J.M. (2007). The floral volatile, methyl benzoate, from snapdragon (Antirrhinum majus) triggers phytotoxic effects in Arabidopsis thaliana. Planta 226:1-10 This research was featured on the cover of the journal for six consecutive issues.
Prithiviraj, B., Paschke, M.W., and Vivanco, J.M. (2007). Root communication: the role of root exudates. Encyclopedia of Plant and Crop Science 1:1, 1-4 DOI: 10.1081/E-EPCS-120042072
Perry, L.G, Alford, E.R., Horiuchi, J., Paschke, M.W., Vivanco, J.M. (2007). Chemical signals in the rhizosphere: root-root and root-microbe communication. In The Rhizosphere (Second Edition). Pinton, R., Varanini, Z. and P. Nannipieri, eds. CRC Press. Boca Raton, FL. pp. 297-330.

Progress 01/01/06 to 12/31/06

Outputs
We have continued our research on the identification of native plants that are resistant to the allelochemical produced by Centaurea maculosa, and identified a potential mechanism of resistance. We found that the native plants Gaillardia and Lupinus respond to the allelochemical (catechin) by increasing the exudation of organic acids, which counteracts catechin's toxicity. Further, after determining that catechin concentrations can vary throughout the year, dropping to low or undetectable levels at certain times, we found that lower concentrations of catechin can induce growth and defense responses in some species, which is the first report that the exudation of any compound produced by the roots of a plant can have a positive effect on other plants. These results may explain the positive role of this compound in the native range as compared to the introduced ranges, and offer clues for mitigation responses among North American land managers.

Impacts
Our Planta paper (#7 below) was highlighted by the Editor of Science ("Editors choice" in Science Vol 311), and received press coverage in Chemical Engineering News.

Publications

Prithiviraj, B., Perry, L.G., Badri, D., Vivanco, J.M. (2007) Chemical facilitation and induced pathogen resistance mediated by a root-secreted phytotoxin. New Phytologist doi:10.1111/j.1469-8137.2006.01964.x
Inderjit, Callaway, R.M., and Vivanco, J.M. (2006) Can plant biochemistry contribute to understanding of invasion ecology? Trends in Plant Sciences 11:574-580
Qin, B., Perry, G.L., Broeckling, C.D., Du, J., Stermitz, F.R., Paschke, M.W., and Vivanco, J.M. (2006) Phytotoxic allelochemicals from roots and root exudates of Leafy Spurge (Euphorbia esula L.). Plant Signaling and Behavior 1: 323-327
Weir, T.L., Perry, L.G., and Vivanco, J.M. (2006) Phytoxins produced by invasive weeds and their applications in agriculture and the restoration of natural areas. In Natural Products for Pest Management. Rimando, A. M. and S.O. Duke, eds. Symposium Series No. 927. Washington, D.C. American Chemical Society. pp. 99-112.
Broz, A. K., Vivanco, J. M., Schultz, M. J., Perry, L. G., and Paschke, M. W. (2006) Secondary Metabolites and Allelopathy in Plant Invasions: A Case Study of Centaurea maculosa. In Plant Physiology, Fourth Edition, L. Taiz and E. Zeiger, eds. On line Essay 13.7. http://4e.plantphys.net/article.php?ch=e&id=377

Progress 01/01/05 to 12/31/05

Outputs
To date progress has been made on several fronts, including the potential identification of a new toxin produced by Leafy Spurge as well as definitive proof that Yellow Starthistle does not produce allelochemicals. Current research is focused on studying whether North American native plants that produce allelochemicals could be used to displace exotic allelopathic weeds. Other research has focused on understanding the chemical ecology of Spotted Knapweed and Russian Knapweed, two species that produce allelochemicals. It has been found that the production, accumulation and persistence of allelochemicals are related to biological, environmental and soil factors, and current studies are determining the kinetics of allelochemical production and accumulation throughout the year. Native plants have been identified that are resistant to the allelochemical produced by Spotted Knapweed and these plants are currently being tested in greenhouse and field competition experiments with Spotted Knapweed. Furthermore, molecular and biochemical studies are determining why some native species are resistant to the allelochemical produced by Spotted Knapweed.

Impacts
Some of the technologies developed in this project (i.e., allelochemical-resistant species) are being tested in military facilities for their effectiveness in preventing the spread of exotic weeds.

Publications

Weir, T.L., Bais, H.P., Stull, V.J., Callaway, R.M., Thelen, G.C., Ridenour, W.M., Bhamidi, S., Stermitz, F.R., and Vivanco, J.M. 2005. Oxalate contributes to the resistance of Gaillardia grandiflora and Lupinus sericeus to a phytotoxin produced by Centaurea maculosa. Planta DOI 10.1007/s00425-005-0192-x
Perry, L.G., Johnson, C., Alford, E.R., Vivanco, J.M., and Paschke, M.W. 2005. Screening of grassland plants for restoration after spotted knapweed invasion. Restoration Ecology 13:725-735
Perry, L.G., Thelen, G.C., Ridenour, W.M., Weir, T.L., Callaway, R.M., Paschke, M.W., and Vivanco, J.M. 2005. Dual role for an allelochemical: (+/-)-catechin from Centaurea maculosa root exudates regulates conspecific seedling establishment. Journal of Ecology 93:1126-1135
Callaway, R.M., Ridenour W.M., Laboski, T., Weir, T., and Vivanco, J.M. 2005. Natural selection for resistance to the allelopathic effects of invasive plants. Journal of Ecology 93:576-583

Progress 01/01/04 to 12/31/04

Outputs
Through this project we have continued our investigations into the chemical, biological and ecological modes of action of catechin, the phytotoxin produced by spotted knapweed (Centaurea maculosa) and secreted in the soil by the roots of this plant in order to gain competitive advantage. Using analytical and organic chemistry techniques, we have investigated sites in the catechin molecule that may account for phytotoxicity, and our results indicate that the antioxidant properties of catechin are not a determining factor for phytotoxicity. Through a long-standing collaboration with the ecologist Professor Ray Callaway (University of Montana), we have found that root insect herbivory attack stimulates the secretions of catechin into the soil, which subsequently may stimulate the competitive ability of spotted knapweed. These results should be taken into account when considering the use of biocontrol species as part of management programs to control certain weeds. Finally, we have begun in-depth study of another invasive weed, diffuse knapweed (Centaurea diffusa), isolating a novel allelochemical produced by its roots and integrating that knowledge with ecological data to increase our understanding of the role of this phytotoxin in diffuse knapweed invasive behavior.

Impacts
These studies have led to the award of a major research grant from the Department of Defense, and opened a new interdisciplinary field by linking biology, ecology and biochemistry to study plant invasions. Through better understanding of invasiveness, the knowledge gained from this research will also be of value to commercial horticulturists as more "new" plants are introduced through such programs as Plant Select.

Publications

Thelen, G.C, Vivanco, J.M., Newingham, B., Good, W., Bais, H.P., Landres, P., Caesar, A., and Callaway, R.M. (2005) Insect herbivory stimulates allelopathic exudation by an invasive plant and the suppression of natives. Ecology Letters 8:209-217
Vivanco, J.M., Bais, H.P., Stermitz, F.R, and Callaway, RM (2004) Root allelochemistry strongly contributes to Centaurea diffusa invasive behavior. Ecology Letters 7:285-292
Veluri, R., Weir, T.L., Bais, H.P., Stermitz, F.R., and Vivanco, J.M. (2004) Phytotoxic and antimicrobial activities of catechin derivatives. Journal of Agricultural and Food Chemistry 52:1077-1082

Progress 01/01/03 to 12/31/03

Outputs
A related group of invasive plants called knapweeds (Centaurea maculosa Lam., Centaurea diffusa Lam., and Acroptilon repens (L.) DC.) are some of the most destructive invasive plants in western North America. Since 1842, ecologists have known that knapweeds achieve their competitive advantage through the secretion of phytotoxic allelochemicals into the soil. However, no such chemical had been identified until 2001, when our laboratories determined that an exudate of C. maculosa roots is indeed phytotoxic. We have determined that (-)-catechin, the phytotoxic compound, inhibits seed germination and root growth by causing a rapid Ca2+ signaling cascade leading to progressive cell death and ultimately phytotoxicity. Using molecular genetics techniques combined with genomics tools available in the model plant Arabidopsis, we have found Arabidopsis mutants that are resistant to catechin. Currently, the genes that confer resistance to catechin are being studied with the goal of eventually transferring these traits to plants. If this technology is successful we predict developing "catechin-READY" plants that will be resistant to the effect of this chemical. CSURF is initiating conversations with agro-chemical companies about this technology. A subsequent series of experiments has demonstrated the allelopathic effects of C. maculosa in the soil by extracting (-)-catechin from different rhizosphere samples in Colorado and Montana, and testing the phytotoxic effect of this compound in soil amendments. Furthermore, we have found that root biocontrol enhances secretion of (-)-catechin into the soil. We have recently undertaken preliminary greenhouse studies, which show that (-)-catechin is phytotoxic when sprayed on foliage of susceptible plants. In these studies, (-)-catechin significantly reduced the height of some weed species and significantly reduced the height of broadleaf weeds. Furthermore, we have developed catechin derivatives and tested the effect of these compounds as herbicides and as antimicrobials. CSURF is currently exploring the patenting and licensing of these new compounds. Using similar strategies as used for C. maculosa, we have recently isolated the allelochemical compound secreted by C. diffusa (diffuse knapweed). This chemical was identified as 8-hydroxyquinoline, which is a chemical compound not related to (-)-catechin. 8-Hydroxyquinoline was found to be phytotoxic against a variety of plants including C. maculosa. However, C. diffusa was resistant to 8-hydroxyquinoline. Recently, we have also identified the allelochemical produced by Russian knapweed (A. repens): a-naphthoflavone. It seems that all the different Centaurea species produced different allelochemicals, and thus there is a great potential to develop these systems for the isolation of ecologically benign herbicides.

Impacts
Our studies have been highlighted in popular newspapers, magazines, and TV news shows, including The New York Times, Scientific American Magazine, National Geographic, CNN News, and the Discovery Channel. As a result of our studies a company has licensed one of the allelochemical compounds as an ecologically-benign herbicide. Two other companies are interested in licensing this technology as well. At the local level, the development of the goals proposed in this project will facilitate the progression of this research into practical applications for weed management in Colorado, and surrounding states. We have developed strong contacts with the office of the State of Colorado Weed Coordinator, and based on these interactions, weed managers in Colorado are starting to use our information in decision making for weed control. We are currently developing contacts with State Weed Coordinators in Utah, Wyoming and Montana, and park and city weed managers who are very interested in implementing the practical applications of our research.

Publications

Walker, T.S., Bais, H.P., Halligen, K.M., Stermitz, F.R., and Vivanco, J.M. (2003) Metabolic profiling of non-polar compounds in root exudates of Arabidopsis thaliana in vitro; study of dynamic interface for the comprehensive characterization of rhizospheric interactions. Journal of Agricultural and Food Chemistry 51:2548-2554
Bais, H.P., Walker, T.S., Kennan, A.J., Stermitz, F.R., Vivanco, J.M. (2003) Structure-dependent phytotoxicity of catechins and other flavonoids; flavonoid conversions by cell-free protein extracts of Centaurea maculosa (spotted knapweed) roots. Journal of Agricultural and Food Chemistry 51: 897-901
Weir, T.L., Bais, H.P., and Vivanco, J.M. (2003) Intraspecific and interspecific interactions mediated by a phytotoxin, (-)-catechin, secreted by the roots of Centaurea maculosa (spotted knapweed). Journal of Chemical Ecology 29:2397-2412
Bais, H.P., Vepachedu, R., Gilroy, S., Callaway, R.M., and Vivanco, J.M. (2003) Allelopathy and exotic plant invasion: from molecules and genes to species interactions. Science 301:1377-1380
Stermitz, F.R., Bais, H.P., Foderaro, T.A., and Vivanco, J.M. (2003) 7,8-Benzoflavone: a phytotoxin from root exudates of invasive Russian knapweed. Phytochemistry 64:493-497 (Memorial Issue for J. B. Harborne)
Walker, T.S., Bais, H.P., Grotewold, E., and Vivanco, J.M. (2003) Root exudation and rhizosphere biology. Plant Physiology 132:44-51

Progress 01/01/02 to 12/31/02

Outputs
A related group of invasive plants called knapweeds (Centaurea maculosa Lam., Centaurea diffusa Lam., and Acroptilon repens (L.) DC.) are some of the most destructive invasive plants in western North America. Since 1842, ecologists have known that knapweeds achieve their competitive advantage through the secretion of phytotoxic allelochemicals into the soil. However, no such chemical had been identified until last year (2001), when our laboratories determined that an exudate of C. maculosa roots is indeed phytotoxic. We have determined that (-)-catechin, the phytotoxic compound, inhibits seed germination and root growth by causing a rapid Ca2+ signaling cascade leading to progressive cell death. A subsequent series of experiments has demonstrated the allelopathic effects of C. maculosa in the soil by extracting (-)-catechin from different rhizosphere samples in Colorado and Montana, and testing the phytotoxic effect of this compound in soil amendments. Furthermore, we have found that root feeding by biocontrol insects enhances secretion of (-)-catechin into the soil. We have recently undertaken preliminary greenhouse studies, which show that (-)-catechin is phytotoxic when sprayed on foliage of susceptible plants. In these studies, (-)-catechin significantly reduced the height of some weed species and significantly reduced the height of broadleaf weeds. Using similar strategies as used for C. maculosa, we have recently isolated the allelochemical compound secreted by C. diffusa (diffuse knapweed). This chemical was identified as 8-hydroxyquinoline, which is a chemical compound not related to (-)-catechin. 8-Hydroxyquinoline was found to be phytotoxic against a variety of plants including C. maculosa. However, C. diffusa was resistant to 8-hydroxyquinoline. Interestingly, under natural conditions C. maculosa and C. diffusa do not share the same habitats and rarely displace each other. The secretion of different allelochemicals can presumably explain this observation. Recently, we have also identified the allelochemical produced by Russian knapweed (A. repens): a-naphthoflavone.

Impacts
This project has already produced significant impact for the State of Colorado and for the country. For instance, two pesticide companies have signed confidentiality agreements with CSU regarding the potential licensing of (-)-catechin as an environmentally friendly herbicide. CSU is currently negotiating with these companies to expedite licensing of this chemical. Similar strategies will be pursued for the other two chemicals. Furthermore, basic knowledge derived form our research is currently being implemented as IPM practices for weed control. For instance, we have recently found that root biocontrol (insect feeders) may increase secretion of (-)- catechin into the soil, and affect revegetation strategies. Weed managers are starting to use our information in decision making for weed control.

Publications

No publications reported this period