S1001: Development of Plant Pathogens as Bioherbicides for Weed Control (S268)
(Multistate Research Project)
Status: Inactive/Terminating
S1001: Development of Plant Pathogens as Bioherbicides for Weed Control (S268)
Duration: 10/01/2001 to 09/30/2006
Administrative Advisor(s):
NIFA Reps:
Non-Technical Summary
Statement of Issues and Justification
Project's Primary website is at http://plantpath.ifas.ufl.edu/s1001/main.htm (direct link can be found under LINKS)
There is a clear need for the proposed research. Weed control is required to sustain and maximize agricultural productivity. Crop losses due to weeds in the United States are estimated to be nearly $6 billion per annum, which is up from an estimate of $ 4.1 billion about a decade ago (Bridges, 1992). Losses due to weeds in major crops in the United States, such as wheat, maize, rice, and soybean are estimated at 11% to 21% of the attainable yields (Oerke et al., 1994). In the Southern Region, losses due to weeds are said to exceed $1.7 billion. In the absence of weed control, it is estimated that crop losses in the United States would amount to $19.6 billion. These figures do not include the loss of productivity encountered in livestock production due to invasive weeds, the costs of weed control in natural areas (e.g., waterways and rangelands), recreational areas (e.g., golf courses and parkland), or urban and home landscapes.
Chemical herbicides and cultivation are used as the primary means of weed control in the United States. Based on pesticide sales figures, more herbicides are used than any other type of pesticide (Kearney, et al., 1988; USDA-ERS, 1988). Worldwide, chemical herbicides account for approximately 44% of the chemical pesticides used, compared with 29%, 21%, and 6% for insecticides, fungicides, and other pesticides (i.e., nematicides, plant growth regulators, etc.), respectively (Klassen, 1995). The pesticide production figures in the United States also generally support these figures since the proportion of the acreage treated with herbicides or insecticides in 1988 were, respectively, 96% and 40% for corn, 95% and 4% for soybean, and 94% and 63% for cotton (USDA-ERS, 1988). It is estimated that cessation of all pesticide use in the United States would reduce agricultural output by 30% (Fernandez-Cornejo et al., 1998). Furthermore, the economic benefit to the growers from the use of traditional chemical pesticides has steadily declined during the last four decades. For example, it is estimated that whereas in 1949 every $ 1.00 spent on pesticides returned $ 7.96 in benefits, the returns in 1991 amounted to only $ 4.16 per $ 1.00 spent (Teague and Brorsen, 1995).
Recently there has been a notable reduction in the amounts of chemical pesticides used, but the rate of decline for chemical herbicides use has been less than that for other chemical pesticides (Gianessi and Anderson, 1993). This decrease in pesticide use is likely to continue in the United States partly due to economic realities of global agricultural production and marketing as well as the national initiative for the reduction in the use of chemical pesticides. For instance, the passage of the Food Quality Protection Act (FQPA) in 1996 has mandated sweeping changes in the way pesticides are registered and re-registered by the EPA. When fully in force, the FQPA will initiate a progressive review of existing pesticides and in this process several older classes of herbicides (including several important herbicide chemistries) are likely to be phased out either due to environmental and human health considerations or voluntary withdrawal from the markets by companies. The FQPA also requires that incentives be developed for nonchemical alternatives, reduced-risk pesticides, and for greater implementation of integrated pest management (IPM) (EPA-OPP, 2001), thus providing a powerful incentive to develop bioherbicides, for example.
The use of herbicide-tolerant transgenic crops is also expected to drastically change the way weeds are managed, in spite of the fact that this new technology has limitations. Among the major unknowns of this technology are the general uncertainties about acceptance of transgenic crops by the public and the high cost of transgenic seeds that small farmers may not be able or willing to afford. There is also the possibility of escape of the herbicide tolerance trait to desirable relatives of the weeds (e.g., canola and wild rape, among Lolium spp., and others.) Thus, the use of herbicide-tolerant transgenic crops is not a panacea.
Weed control becomes even more complicated when considering management of invasive weeds in natural and urban areas, where economic, environmental, or human risk concerns may entirely preclude the use of chemical herbicides.
Although chemical pesticides are an integral part of modern food production, there has been a significant cost to the society and the environment associated with the widespread use of these chemicals (Pimentel and Greiner, 1997). Thousands of cases of accidental poisonings and numerous accidental deaths from chemical pesticides, including some herbicides, are reported each year (Ecobichon, 1998). Extensive use of herbicides and other pesticides has resulted in groundwater contamination (Abdalla and Libby, 1987), and the use of certain herbicides has been linked to some types of cancer (Hoar et al., 1986). Other preventable problems, mainly from misuse of chemical herbicides can result, such as nontarget damage from spray drift and carry-over problems in soil and produce.
Finally, despite the extensive use of herbicides, many weed species continue to cause losses in agriculture, and the current strategies for control of these weeds are inadequate. One reason for this is the emergence of herbicide-resistant weeds (Holt and LeBaron, 1990). Many present-day herbicides have single-site modes of action and therefore are prone to promote rapid development of resistance among weeds. It took approximately 25 years for the first atrazine resistant weeds to be identified, but weeds resistant to sulfonylurea and imidazolinone appeared in less than 10 years after these herbicide chemistries were introduced and widely used. The intensive cultivation of herbicide-tolerant crops is likely to further accelerate the emergence of herbicide-resistant weeds consisting of mutant weed biotypes as well as naturally resistant weeds, the latter due to weed-shifting.
A different problem exists in some situations, such as vegetable crops (i.e., minor crops) and range lands, where the economic realities of the marketplace preclude the development and/or use of conventional chemical herbicides. Organic farming is gaining a foothold in the mainstream American agriculture, but this industry suffers from the general lack of biologically based weed control options. Foods consumed by infants, children, and elderly and commodities that rely on a few classes of herbicide chemistries for weed control are particularly vulnerable in this regard. Problems related to the current restrictions in the use of certain chemical herbicides and the impending loss of methyl bromide as a broad-spectrum soil fumigant will further exacerbate weed problems in crops such as vegetables, strawberry, and many others. Development of herbicide-tolerant crops, along with the consolidation of major agrochemical companies and increasing globalization of agricultural production and marketing further limit the choices available to manage weeds on local and regional scales. Many of these developments disproportionately constrain small and mid-sized farmers, especially given the recent depressed prices for commodities.
Invasive weeds are emerging as another major threat to agricultural and natural areas and to the long-term health and biodiversity of our nations land and water resources. Weed problems become more complicated in natural and urban areas, where economic, environmental, or human-health risks may entirely preclude the use of chemical herbicides. Lastly, weeds such as pigweeds (Amaranthus spp.), nutsedges (yellow nutsedge [Cyperus esculentus] and purple nutsedge [C. rotundus]), purslanes (Portulaca spp.), spurges (Euphorbia spp.), kudzu (Pueraria lobata), and various grasses and invasive weeds are not controlled effectively by available methods. Therefore, development of newer weed-management agents and technologies, including biologically based approaches, is of greater importance now than ever before.
Importance of the proposed multistate research and the consequences of not undertaking this cooperative endeavor are as follows. As explained above, the need for developing diversified weed management tools and strategies is more acute than ever. One of the alternatives to chemical weed control is biological control by using plant pathogens (Charudattan and Walker, 1982; TeBeest, 1991; Rosskopf et al., 1999). "Biological control" is defined as "management of natural enemies (predators, parasites, and pathogens of pests) and selected beneficial organisms (antagonists, competitors, and allelopaths) and their products to reduce pest populations and their effects" (USDA-ARS, 1988). Plant pathogens used in an augmentative or inundative, biopesticide mode are referred to herein as bioherbicides.
In 1995, the S-268 Regional Research Project was initiated to evaluate and develop plant pathogens for biological control of weeds. S-268 succeeded two previous projects, S-136 and S-234, and collectively these cooperative projects served as the foundation for research and evaluation of nearly two dozens of pathogens as potential bioherbicides. These projects also helped to develop epidemiological and risk-analysis models to understand the performance and safety of two previously registered and commercialized bioherbicidal pathogens, Colletotrichum gloeosporioides f. sp. aeschynomene (Collego.), used for the control of northern jointvetch (Aeschynomene virginica), and Phytophthora palmivora (DeVine.) used for the control of stranglervine (Morrenia odorata) in Florida, understand some aspects of the genetics of these fungi, and develop systems to integrate their use in rice and citrus production (Bowers, 1986; Kenney, 1986; Ridings, 1986; Smith, 1986; TeBeest, 1982, 1988; TeBeest and Dickman, 1989; Cisar et al., 1996). It also provided a forum to develop scientific and technical concepts helpful in the registration but not commercialization of Puccinia canaliculata as Dr. BioSedge. (Phatak, 1992).
Several other bioherbicide candidates were developed through regional trials, notably Alternaria cassiae (Charudattan et al., 1986), although registration and commercialization did not materialize. While in some cases this was simply be due to a lack of consistency of the bioherbicide agent or technical feasibility, the availability of efficacious chemical alternatives, coupled with the lack of coordinated efforts by researchers and commercial enterprises were also contributory factors. Nonetheless, it has been amply demonstrated that bioherbicide products are practical and economically sustainable in the marketplace. Moreover, their use over the past 25 years has not led to any risks to human health or the environment. The basic and applied research done through these cooperative projects have helped to develop and validate the concept of inundative biological control of weeds by the bioherbicide strategy (TeBeest, 1991; Rosskopf et al., 1999; Charudattan, 2000).
The success of the S-268 project and its predecessors can be measured also in terms of the scientific and technical knowledge gained. The members of this group have written two books, hundreds of refereed papers, reviews, and popular articles on the subject of biological control of weeds with plant pathogens. About twenty-five graduate students have been trained since the inception of the first cooperative project on this topic, S-136. Three bioherbicides have been registered and a classical biocontrol introduction of a rust fungus has resulted during this period. Several prospective bioherbicides have been patented.
Just during the last five years, under S-268, three pathogens have been cooperatively developed: Colletotrichum truncatum (COLTRU) for control of hemp sesbania (Sesbania exaltata), Alternaria destruens (Smolder) for dodders (Cuscuta spp.), and Pseudomonas syringae pv. tagetis for control of several weeds in the Asteraceae. Smolder and Mallet (Colletotrichum gloeosporioides f.sp. malvae) are currently under EPA review for registration. In addition, these regional projects have also helped to stimulate research on the use of novel materials for formulation, or as surfactants and spray adjuvants to improve the efficacy (Boyette, 1994) and host range of bioherbicide agents (Zidack et al., 1992; Boyette and Abbas, 1994), improve production methods (Chandramohan and Charudattan, 1998; Connick et al., 1991; Daigle and Cotty, 1994; Jackson, 1994; Jackson et al., 1994; Jackson and Schisler, 1994; Shabana et al., 1997; Quimby et al., 1994; Wyss et al., 1999; Yandoc and Charudattan, 1998), and develop effective delivery systems (Rosskopf et al., 1996; DeValerio et al., 2000). During this period, 97 refereed papers, 19 book chapters, 33 papers in conference proceedings, 51 abstracts, and 9 patents, and several miscellaneous publications were produced by the members and other participants of S-268 project. These contributions would not have been possible without the facility to engage in cooperative regional research under the auspices of S-268. In addition, the project has enabled the discovery of numerous new pathogens or pathogen records on weeds and clarification of weed and pathogen taxonomies, understanding of the epidemiology of several diseases, development of methods for microbial fermentation, formulation, and delivery, risk assessment, and molecular biology of host-pathogen relationships. Moreover, the development Smolder and Mallet as possible commercial products, methods development for COLTRU, and cooperative testing of several other agents have been greatly facilitated by this cooperative research endeavor. Given this history, the consequences of not undertaking this proposed multistate research project include:
- Further development of potentially useful bioherbicides will be abandoned because no single SAES has all the resources necessary to develop a pathogen from its discovery to the status of a commercial product.
- The concept of regional research (i.e., multistate research project) will be key to the development and registration of bioherbicides in the next 5- to 10-year term.
- There are many common weed problems throughout the continental United States, especially the Southern Region. Our ability to develop and register bioherbicides will be greatly aided by testing candidate pathogens in different states and regions, in North America. Also, strains of candidate pathogens may be distributed in several locations in the United States that differ in virulence, fitness, and other traits. Hence, bioherbicides can be developed more quickly more efficiently if scientists from several states cooperate toward common goals.
- Although this new project is proposed for the Southern Region, participation by states and institutions outside this region will be beneficial for two reasons: A) Expertise available in the different institutions listed herein will be essential for the success of this project. B) Bioherbicide agents proposed to be developed under this project will need to be tested for efficacy against the target weeds in several regions within and outside the Southern Region, under different climatic and edaphic conditions.
- A multistate research project organization, such as the one proposed, also offers a forum to develop new information and ideas. Recent annual meetings of S-268 have fostered participation by scientists from SAES, ARS, small colleges, and industry. Scientists from other countries (e.g., Canada, South Africa, and U.K.) have also frequently participated in the annual meetings.
The research proposed under this multistate project relates to national priorities established by the Experiment Station Committee on Organization and Policy (ESCOP), the Southern Region Strategic Plan (SAAESD, 2000), and the USDA-ARS. The Southern Region Strategic Plan has stressed the need to develop and integrate biological control with current pest control practices. Biologically based pest management has been recognized in this plan as the second most critical need for agriculture in the Southern Region. A similar emphasis on biological control has been also made by the National Research Councils Board on Agriculture (NRC, 1996) and the U.S. Congress Office of Technology Assessment (OTA, 1995). Several SAES and the USDA-ARS are committed to biological control of weeds as a top research priority. In 1984, a Research Planning Conference on Biological Control attended by a broad group of stakeholders identified targets for research in biological control (USDA-ARS, 1984). Weed species identified for research in biological control included: velvetleaf (Abutilon theophrasti), cocklebur (Xanthium strumarium), the morningglories (Ipomoea spp.), the nutsedges (Cyperus spp.), sicklepod (Cassia obtusifolia), johnsongrass (Sorghum halepense), prickly sida (Sida spinosa), and spurred anoda (Anoda cristata). Areas of research priority for biological control for the USDA-ARS also have been determined (USDA-ARS, 1988). These include: mass propagation, harvesting, packaging, storage, and distribution of biological agent populations, systematics, genetic improvement through biotype selection, conventional crosses, and genetic engineering, and exploration for new, more effective biological agents (USDA-ARS, 1988). A recent review of the USDA-ARS Weed Science program has further highlighted the need to increase efforts on biological control of weeds (USDA-ARS, 2001).
Related, Current and Previous Work
The first weed-control pathogens were discovered and deployed between 1969 and 1973 (Daniel et al., 1973; Smith, et al., 1973 a, b; TeBeest and Templeton, 1985; Ridings, 1986; Templeton et al., 1986). By 1982, 49 plant species were reported as targets for control by bacteria, fungi, nematodes, and viruses (Templeton, 1982). Recent analyses indicate that nearly 200 fungal pathogens have been screened as potential bioherbicides (Charudattan 1991, 2001). Of these, eight pathogens have been registered worldwide and about 50 are rated as having good to excellent potential for commercialization. About 46 different weeds have been identified as potential targets for bioherbicide research in different countries. About a third of these weeds are also the primary targets in the United States.
Several ongoing regional research projects address various aspects of biological control, including some in the Southern Region. However, only one other project, S-303, deals with biological control of weeds and complements this proposed multistate project on the development of plant pathogens as bioherbicides. The two projects are different in that the S-303 project deals with biocontrol of introduced insect pests and weeds through classical importation of biocontrol agents, while the proposed project is aimed at the use of indigenous plant pathogens in augmentative and inundative biocontrol strategies.
A CRIS search was done to see whether similar projects exist in other regions. Only one other multistate project related to weed biocontrol is in existence. This project, S-303, deals with classical biological control of arthropod pests and weeds through importation of natural enemies from abroad. This project has four objectives, including survey and importation of natural enemies of invasive pests; integration of exotic and indigenous natural enemies with other pest management approaches; evaluation of the effects of exotic natural enemies on nontarget organisms; and characterization of the role of indigenous natural enemies in suppressing pest and beneficial species. These objectives and the general aim and scope of this project do not conflict or duplicate the objectives proposed herein. The bioherbicide project proposed here deals exclusively with indigenous microbial plant pathogens used in the inundative biocontrol strategy. Accordingly, the proposed multistate project can compliment the S-303 project.
Objectives
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To evaluate and develop bioherbicide agents to control nutsedges, pigweeds, grasses, purslanes, spurges, kudzu, weeds in Asteraceae, and others
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To develop and evaluate formulations to improve performance and standardization of selected bioherbicides
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To evaluate bioherbicides in multistate field trials in different crops and as alternatives to methyl bromide.
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To safely enhance the virulence of bioherbicides by selection of variants of the plant pathogen that overproduce a target amino acid.
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Methods
Objective 1: To evaluate and develop bioherbicide agents to control nutsedges, pigweeds, grasses, purslanes, spurges, kudzu, weeds in Asteraceae, and others.The first objective will address the development of bioherbicide agents and a bioherbicide mixture to control several highly important weeds including nutsedges, pigweeds, grasses, purslanes, spurges, kudzu, and others. The biocontrol agents proposed here have all been previously shown to be effective in weed control and are critically in need of further research and development to realize their registration and utilization.
1-A. Development of Dactylaria higginsii as a bioherbicide for nutsedges (Cyperus spp.).
1-B. Development of Microsphaeropsis amaranthi and Phomopsis amaranthicola as broad-spectrum bioherbicides to control pigweeds.
1-C. A multiple-pathogen approach to control several weedy grasses.
1-D. Myrothecium verrucaria as a broad-spectrum bioherbicide for purslanes, spurges, kudzu, and other weeds.
1-E. Development of Pseudomonas syringae pv. tagetis as a bioherbicide for weeds in the Asteraceae.
Objective 2: To develop and evaluate formulations to improve performance and standardization of selected bioherbicides.
The second objective will focus on the development and evaluation of suitable formulations for the delivery of the microbial weed-management agents selected for multistate trials. Successful application of microbial weed-management agents requires the development of suitable formulations. Considerable research has been done with various weed-pathogen systems to develop suitable formulations, adjuvants, and carriers. Research on this aspect is essential to move the microbial agent forward to the marketplace. Since each weed-pathogen system is influenced by specific biotic and epidemiological interactions, formulation research, by necessity, requires empirical approaches to the development of methods and materials. Few laboratories in the nation have the expertise to develop microbial formulations, but the USDA-ARS laboratory in New Orleans, LA is one of the premier institutions in this field, and we have enlisted two leading experts from this laboratory (Drs. Connick and Daigle) to participate in this project.
Proper formulations, adjuvants, and carriers should be developed to ensure effectiveness and feasibility of the bioherbicides selected for this project. Formulations enable spreading of very small amounts of active ingredients (e.g., microbial propagules) over a wide area, prolong the shelf-life, improve efficacy, and facilitate transportation, all without adversely affecting the effectiveness of the agent. In some cases, an adjuvant or a carrier may serve as the formulation. This is usually the case with dry pellet or powder formulations. In addition to the basic formulation, materials that act as surfactants may be added to the formulated product to further enhance the activity or usability of the product. Formulation of biological agents is complicated by the need for the organism to remain alive and virulent through processing. Therefore, usually the emphasis is on improving the agents longevity and efficacy. In addition, formulation of a microbial agent may enable combinations of bioherbicides as well as bioherbicide agents and chemical pesticides to be used simultaneously without affecting performance.
The most commonly used additive in bioherbicide formulations is a surfactant, such as Silwet L-77, an organosilicone compound that reduces the surface tension and enables small spores, cells, and mycelia to enter natural openings in the foliage (Zidack and Backman, 1996). Materials capable of forming aqueous gels when added to spore suspensions have also been explored. Such materials help to stick the spores to the leaf surface, while providing a hydrophilic matrix in which the spores may germinate more readily in the absence of an adequate dew period (Shabana et al., 1997). In the case of the bioherbicide Collego (Colletotrichum gloeosporioides f.sp. aeschynomene; Encore Technologies), a dried spore preparation is resuspended in water and a prepackaged amount of sugar, as an osmoticum and nutrient source, is added to allow for the gradual rehydration and germination of spores.
Formulations that encapsulate the microbial propagules in starch granules and other materials have been developed and tested. Encapsulation allows the biocontrol agent to be delivered in a conveniently applied form; however, adherence to the leaf surface requires that the surface be prewetted, which is not realistic for large-scale commercial use (McGuire and Shasha, 1992). Two additional formulations that have become increasingly popular for microbiological control agents of weeds and for diseases are alginate pellets and one termed Pesta.. These solid formulations can be easily adapted to a number of different fungi (Daigle and Cotty, 1992; DeLucca et al., 1990). Organisms that have been formulated in this manner have excellent shelf life compared to unformulated organisms, and the formulation itself can actually act as a spore-production medium.
Invert emulsions have shown considerable promise as carriers for bioherbicides (Womack et al., 1996; Yang and Schaad, 1996). The knowledge gained from invert-emulsion formulation has led to the extensive use of vegetable oils, with the oils apparently causing no alteration to the cuticular layer of the leaf surfaces (Egley and Boyette, 1995). Work with Alternaria spp. has indicated that invert emulsions can cancel the need for prolonged free moisture on plant foliage (Amsellam et al., 1990). Control of hemp sesbania was dramatically improved by the use of invert emulsion in the absence of dew, again indicating that high levels of weed control may be achieved by providing a prolonged period of water availability (Boyette, 1994). Recently, the use of sunscreens has been shown to be useful in protecting spores that are not melanized (Prasad, 1993). For instance, inoculum of Chondrostereum purpureum formulated with an ultra-violet protectant was able to withstand a wider range of environmental conditions than unprotected inoculum.
Thus, the aim of this objective is to discover and develop appropriate solid and liquid materials for the delivery of bioherbicide agents chosen for this project. Materials and methods will be evaluated cooperatively to develop appropriate formulations for pathogens undergoing testing under this multistate project. Suitable solid matrices and liquid formulations will be tested and developed for stabilizing bacterial and fungal weed pathogens during processing, storage, and application.
Roles and responsibilities: the following stations will participate in this objective: USDA-ARS-SWSRU, MS; Sylvan; FL SAES; USDA-ARS-SRRC, LA; IN SAES; USDA-ARS-EBCL, France; USDA-ARS-USHRL, FL; and Macdonald College, PQ. Individuals from these stations (See Appendix-Projected Participation) will follow the common protocols described below.
Objective 3: To evaluate bioherbicides in multistate field trials in different crops and as alternatives to methyl bromide:
The third objective will involve multistate trials to field test experimental and pre-commercial bioherbicide formulations in various crops (e.g., vegetables, strawberry, floriculture, turf, rangelands, etc.) and as methyl bromide alternatives.
Methyl bromide is a low molecular weight, volatile, organic compound that is highly toxic to most living organisms. One of the principal uses of this compound is in agriculture as a fumigant to disinfest soil prior to the planting of high value crops (EPA, 1997). Methyl bromide is a Class 1 ozone depleting substance (ozone depletion potential > 0.2). Recent legislative actions taken by the U.S. Congress in October 1998 have made specific changes to the Clean Air Act, essentially phasing out the pest-control use of methyl bromide, in accordance to the Montreal Protocol phase-out schedule for developed countries. The final methyl bromide phase-out date in the United States is now set as 2005. In the United States, producers of fresh-market vegetables and fruits, including tomato, pepper, and strawberry utilize production systems that are highly dependent upon methyl bromide for soil disinfestation. Soilborne pathogens and weeds are the primary pests controlled with the use of methyl bromide and several regions in the United States depend on methyl bromide for cost-effective production of several crops. Florida alone accounts for 25% of the total methyl bromide consumption in the United States (EPA, 1997). Without methyl bromide, production of these crops is projected to decline by 61.4% to 63.5%, resulting in an annual loss of $ 388 million to Florida growers alone (Spreen et al., 1995).
Generally, very few registered herbicides are available for use in minor crops. While some new chemistries are being introduced for minor-crop use, the sensitivity of certain crops to these chemicals is a constraint. Therefore, growers are urgently seeking effective replacements that will have no or minimal nontarget effects and can be used in an integrated pest-management approach.
Roles and responsibilities: the following stations will participate in this objective: Sylvan; FL SAES; CA SAES, Riverside; and USDA-ARS-USHRL, FL. Individuals from these stations (See Appendix-Projected Participation) will follow the common protocols described below.
The bioherbicide agents identified under Objective 1 and developed according to Objective 2 will be evaluated in different cooperating states as possible alternatives to methyl bromide. The choice of crops, cropping systems, weed targets, and bioherbicide candidates will be made according to the local needs of participating states and institutions.
Objective 4: To safely enhance the virulence of bioherbicides by selection of variants of the plant pathogen that overproduce a target amino acid.
Molecular and physiological mechanisms underlying host-pathogen interactions are complex and each pathosystem needs to be studied in detail to identify genetic and physiological mechanisms that could be used to improve pathogens virulence, environmental fitness, host specificity / host range, and other desired attributes. A few studies have been done to improve the efficacy of certain bioherbicide agents, including Sclerotinia sclerotiorum, Colletotrichum gloeosporioides f.sp. aeschynomene, and C. gloeosporioides f.sp. malvae (Sands et al., 1990; and reviewed in TeBeest et al., 1992; Goodwin, 2001). Some ideas, highly speculative at this time, have been proposed (Gressel, 2001) that aim to improve fungal bioherbicides by specific transgenic means. Goodwin and coworkers (reviewed in Goodwin, 2001) are examining host and pathogen genes expressed during infection of round-leaved mallow (Malva pusilla) by C. gloeosporioides f.sp. malvae. Pathogen genes coding for pectinase, arylsulfatase, and catalase (involved pathogenesis), and protein kinase (involved in the regulation of fungal metabolism), as well as plant genes encoding actin (involved in cellular processes such as cell division and cytoplasmic streaming) and glutathione S-transferase (induced in the host plant during the pathogens transition from the biotrophic to necrotrophic phases) are being studied (Goodwin, 2001). While these studies represent a hopeful start, methods based on conventional strain improvement techniques, which can be used without costly regulatory delays, should also be examined.
Researchers at Montana State University have developed a promising strain-improvement technology to greatly enhance the virulence and efficacy of bioherbicides by selecting variants of pathogens that overproduced and excreted amino acids that were inhibitory to the respective target weed (Tiourebaev, 1999; Sands et al., 1999). This approach was modeled after "Frenching disease," a naturally occurring disease of tobacco (Steinberg, 1946). Steinberg et al. (1950) discovered that saprophytic bacteria growing on the roots of symptomatic plants were overproducing a single amino acid, isoleucine. Isoleucine is synthesized in plants via the branched chain amino acid pathway. The end-products of the pathway (valine, leucine and isoleucine) allosterically regulate the activity of acetolactate synthase (ALS). In "Frenching disease," overproduction of isoleucine in the rhizosphere by the saprophytic bacteria inhibited the activity of ALS in tobacco, shutting down synthesis of valine and leucine which disrupted essential protein metabolism.
Interestingly enough, several modern chemical herbicides also inhibit single biosynthetic enzymes in plants, rendering treated plants incapable of producing a metabolite essential for plant growth. For example, acetolactate synthase (ALS), the key enzyme in the branched chain amino acid biosynthesis, is inhibited by the sulfonylurea herbicides. The herbicide glyphosate inhibits 5enolpyruvylshikimate 3-phosphate synthase (EPSP), the key enzyme in the shikimic acid pathway (Amrhein, 1986).
Given this knowledge, scientists exposed Fusarium oxysporum f. sp. cannabis, a host-specific pathogen of marijuana plants (Cannabis sativa) (Tiourebaev et al., in press) to the valine analogs, norvaline and penicillamine, and selected spontaneous mutants that overproduced the amino acid valine (Tiourebaev, 1999). Valine is a product of the branched chain amino acid pathway and by feedback regulates ALS. Host plants were inoculated with the valine-overproducing mutants in growth-chamber studies. The virulence and rate of mortality were greater than that caused by the wild type strain of the pathogen. The host range of the valine-producing strain was unaffected.
Amino acid overproduction also enhances the virulence of Fusarium oxysporum f. sp. papaveris for control of Papaver sp. and Pseudomonas syringae pv. tagetis for control of houndstongue (C. officinale). Thus, this method is a novel, yet simple method, for increasing virulence of plant pathogens for weed biocontrol. The method can be easily adapted to any host-specific weed pathogen. Since this technique does not require recombinant genetics and is based simply on overproduction of amino acids, biosafety considerations are significantly reduced. It simply needs to be applied to obtain efficacious bioherbicides.
Roles and responsibilities: the following stations will participate in this objective: FL SAES; IN SAES; MT SAES; and USDA-ARS-USHRL, FL. Individuals from these stations (See Appendix-Projected Participation) will follow the common protocols described below.
Under this objective, amino acids or combinations of amino acids that are most inhibitory to the growth and development of the target weeds listed under Objective 1 will be determined. Spontaneous variants of plant pathogenic fungi or bacteria that are resistant to amino acid analogs and screen for overproduction of the selected amino acid(s) will be identified and selected. The virulence and host range of the amino acid overproducing bioherbicides will be confirmed in growth-chamber studies. Finally, the amino acid overproducing bioherbicide agents will be tested in multistate field trials.
Feasibility of the Proposed Work:
The technical feasibility of the bioherbicide approach to weed control has been well established, as evidenced by the continued use of two registered bioherbicide products, Collego and DeVine, since the early 1980s, albeit intermittently and on a relatively small scale. A wealth of scientific and technical information is available (e.g., Charudattan and Walker, 1982; TeBeest, 1991; and dozens of review articles and book chapters) that can guide and enable the proposed project. Expertise in plant pathology, weed science, microbial fermentation, formulation, and delivery systems, which are needed for this project, will be provided by the participants named. Participation by commercial industries will be crucial to the success of the project, and the recent incentives for the development of reduced risk pesticides (under the FQPA) have prompted companies to reinvest the bioherbicide technology. Some small biotech companies and longstanding agrochemical companies have agreed to (or will be invited to) participate in this proposed multistate project.
The project is realistic and not ambitious. Since the bioherbicide prospects of the agents proposed herein for development have been already established, the project can focus fully on the technology development aspects.
[In depth discussion of sub-objectives under Objective 1 are found in the Attachment below.]
Measurement of Progress and Results
Outputs
- One to five new, registered bioherbicides are expected to result from this multistate project. Even if the output results in one new registration, it will be a good achievement and the project will have a notable impact on the available weed control options. The project will target several highly important and difficult-to-control weeds such as purple nutsedge, pigweeds, grasses, kudzu, composite weeds, and others. Development and registration of commercial bioherbicides for these weeds will fill an important market niche for weed control products. This work will also provide for new biologically based, reduced risk pesticides.
Outcomes or Projected Impacts
- Development and registration of bioherbicides for economically important weeds, such as nutsedges, pigweeds, grasses, purslanes, and others named in this project will benefit growers in the Southern Regions and beyond. The scientific output from this study will stimulate further research investment in nonchemical weed-control alternatives. Reduction in the use of chemicals and development of an alternative weed control technology are two additional direct benefits that are likely for this project. The outcomes from this project will have a direct bearing on some of the mandates of FQPA, such as development of reduced-risk pesticides and implementation of IPM.
Milestones
(2006): Most of the background information and materials necessary to begin this project are already in place. The project is expected to meet its stated objectives by the year 2006. Tentative milestones are as follows: Years 1-3: Development of common protocols and completion of multistate field trials of the agents selected. Years 1-3: Development of formulations and delivery systems to facilitate field trials and to establish prototype commercial formulations. Years 2-5: Development of efficacy and label data for possible registration of promising agents. Years 4-5: Registration of promising bioherbicide products. Given the need to develop methods and materials for each agent-weed system, these milestones are realistic and achievable.(0):0
Projected Participation
View Appendix E: ParticipationOutreach Plan
Information derived from this project will be made available to scientific audiences and to stakeholders through refereed journal articles, meeting abstracts, conference proceedings, extension bulletins, popular articles, and field demonstrations. A web page will be developed and maintained by the Chairman of the Technical Committee. Patentable materials and technologies developed through this project will be submitted for patenting through the appropriate institutions of the lead scientists/inventors.
Successful bioherbicide agents (i.e.: registered) will be made available through cooperating state and federal agencies and scientists. News releases, field demonstrations, and user-education workshops will be used to publicize and distribute these agents. Extension personnel at State Cooperative Extension Services will be trained in the use of registered agents through educational and demonstration material.
The success of this project will be determined in terms of the number of successful multistate field demonstrations, registered agents, and users trained in this technology.
Organization/Governance
The organization and conduct of this project will be in accordance with the rules and procedures set forth in the "Format for Multistate Research Proposals," September 26, 2000 (http://www.msstate.edu/org/saaesd). This project will use the standard form of governance. A technical committee composed of one voting member from each SAES, one voting member from each USDA-ARS site, and as many participating members as approved from each SAES, USDA-ARS, and other institutional units. The Technical Committee will be responsible for planning the research project, coordinating the research activities of the participants, developing appropriate research methods and procedures for cooperative field trials, reporting of results, and for reviewing of research progress through annual meetings. The Executive Committee will consist of a Project Chair, a Secretary, 3 Objective Chairs, an Annual Meeting Chair, and the Administrative Advisor. The Project Chair, the Secretary, and the Objective Chairs will serve five-year terms. The Annual Meeting Chair will serve a one-year term during which he/she will serve as the local host for the annual meeting of the Technical Committee.
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