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S1001: Development of Plant Pathogens as Bioherbicides for Weed Control (S268)

Annual/Termination Reports (SAES-422): [04/14/2003] [03/31/2004] [09/07/2006]

Date of Annual Report: 04/14/2003

Report Information:
  • Annual Meeting Dates: 02/09/03 to 02/09/03
  • Period the Report Covers: 01/2002 to 12/2002

    • Participants:
    Brief Summary of Minutes of Annual Meeting:


    URL: Copy of minutes

    Accomplishments:
    Progress was made towards field testing and evaluation of efficacy of Dactylaria higginsii, Phomopsis amaranthicola, pathogens of weedy grasses and Pseudomonas syringae pv. tagetis. Advances were also made in mass production and formulation of bioherbicides. A strain selection method based on nutritional conditioning of fungi was developed.

    Impact Statements:
    1. Bioherbicides to manage purple nutsedge, pigweeds, weedy grasses and weeds in the Asteracae family are under development.
    Last Modified: unknown

    Date of Annual Report: 03/31/2004

    Report Information:
  • Annual Meeting Dates: 02/08/04 to 02/09/04
  • Period the Report Covers: 10/2002 to 09/2003

    • Participants:

    URL: Copy of participant list
    Brief Summary of Minutes of Annual Meeting:

    URL: Copy of minutes
    Accomplishments:
    Objective 1-A: Efficacy of Dactylaria higginsii (DH), a bioherbicide agent for purple nutsedge (Cyperus rotundus), was evaluated in field trials with bell pepper and onion as crops. Application of DH at different days after nutsedge emergence as well as single, double, and triple applications improved crop yield and quality. However, DH?s efficacy must be enhanced (i.e., increased fungal virulence, secondary infection, and conidia survival) and/or must be applied more than twice in order to suppress purple nutsedge interference to acceptable levels (<10% yield loss). Higher levels of suppression of purple nutsedge and higher yields are likely if DH were applied under favorable weather conditions.

    Objective 1-B: Phomopsis amaranthicola (PA) was evaluated as a post-emergence bioherbicide to control Amaranthus lividus in bell pepper (C. annuum) and A. dubius in Caribbean-bonnet pepper (C. frutescens) and eggplant (S. melongena). Pigweeds that survived infection by PA were allowed to interfere with the crops season-long. In eggplant and Caribbean-bonnet pepper, spraying PA 10 days after weed emergence (DAE) resulted in about 30% mortality in different population densities of A. dubius and yield-loss reductions of about 25% in pepper and 16% in eggplant, as compared to the untreated weedy crops. In the bell pepper experiment, the results were similar when using a Psyllium mucilloid or an agricultural oil (PCC-588) as a surfactant in the spraying mix. In bell pepper, two applications of PA (10 and 20 DAE) were more effective than one application (10, 20, 30, or 40 DAE) in suppressing A. lividus growth and interference with the crop. When PA was applied more than twice, improvements in pigweed control and pepper yield were negligible. Maximum weed mortality, growth suppression, and yield-loss reduction in these crops were obtained with 1 or 2 early applications of PA (10 DAE in eggplant and Caribbean-bonnet pepper and 10 and 20 DAE in bell pepper). Further enhancement in the efficacy of PA as a post-emergence bioherbicide is possible through the use of improved formulations.

    Waterhemp (Amaranthus tuberculatus) is a late-emerging species that is well suited to no-till cropping systems and remedial postemergence weed control strategies. Populations of waterhemp have developed resistance to chemical herbicides including the ALS-inhibitors, the s-triazines, the diphenylethers, and glyphosate. Resistance to the ALS-inhibitor herbicides has been found in Illinois, Indiana, Iowa, Kansas, Michigan, Missouri, Ohio, and Wisconsin. Approximately 70% of the waterhemp in Illinois is resistant to ALS-inhibitor herbicides, and it is advised not to use these herbicides where waterhemp is present. An isolate of Microsphaeropsis amaranthi (MA) has been shown to be pathogenic to a wide range of Amaranthus spp. The potential to exploit the interactions between MA and chemical herbicides for the control of waterhemp was studied.

    Disease expression by MA was greatest with a dew period of > 12 h and around 18°C. Younger plants were more susceptible to MA, and greater impact was achieved with the highest inoculum levels. When waterhemp plants at the 2-4 leaf stage were sprayed with different rates of glyphosate (0, 0.16, 0.32, and 0.63 kg ae ha^-1) and then with conidia of MA after 24 h, a strong interaction was seen between the herbicide and MA at the higher glyphosate rates. A similar level of control was achieved with either a manufacturer-recommended rate (X = 0.63 kg ae ha^-1) of glyphosate alone or 0.5X rate of glyphosate combined with MA.

    By screening putative glyphosate-resistant populations of waterhemp from different accessions in Indiana, Illinois, Missouri, and Iowa, the most susceptible and the most resistant individuals were selected and cloned by vegetative propagation. A response between glyphosate and MA existed for both the susceptible and resistant clones, but in each case the interaction was only apparent with rates of glyphosate that caused visible plant damage alone ? approx 0.32 kg ae ha^-1 for the susceptible clone and approx 1.25 kg ae ha^-1 for the resistant clone.

    Objective 1-C: Torpedograss (Panicum repens) is susceptible to three indigenous fungi, Drechslera gigantea, Exserohilum longirostratum, and E. rostratum. The feasibility of using these fungi as a bioherbicide mixture was tested under field conditions. An experiment was designed (randomized split-plot design; three replicates per main-plot and sub-plot treatments) and done twice at a site with dense, uniform growth of torpedograss. The main-plot treatments were: 1) chemical herbicide, imazapyr (Arsenal) at 64 oz per acre, 2) mowing, once, and 3) not mowed, no chemical herbicide applied. The sub-plot treatments were: 1) the bioherbicide mixture in an emulsion (30% Sunspray 6E in water, spores of each fungus at 1:1:1 v/v, final total spore concentration 1x10^6 per ml; BHE), 2) control emulsion only (E), and 3) untreated torpedograss (U). The chemical herbicide treatment was applied aerially (Trial 1), or manually (Trial 2), and the bioherbicide mixture with a CO2-propelled backpack sprayer set at 20 psi pressure. The bioherbicide was reapplied twice (Trial 1) or once (Trial 2) at three to five weeks after the initial application (WAI). At different time intervals, healthy plant species present every 10 cm along a diagonal transect of 3.5 m in each plot were counted. Chemical herbicide application killed all plant species present at the site by 19 weeks after chemical application (WAC) (Trial 1) or 8 WAC (Trial 2), compared to plots not treated with chemical. The BHE treatment provided maximum level of damage severity on both mowed and unmowed torpedograss (70-72% (Trial 1); 60-65% (Trial 2)), and was safe to the native plant species present. In both mowed and no treatment (unmowed) plots at 8 WAI (Trial 1) and 9 WAI (Trial 2), torpedograss population was significantly reduced, when treated with the bioherbicide mixture in emulsion. Simultaneously, numbers of total native plant species censused increased. The chemical control (nonselective) of torpedograss lasted for about 6 months, and bioherbicidal control (selective) for 7 to 9 months without any significant regrowth of torpedograss. Thus, the bioherbicide agents have potential to be used in integrated management of torpedograss.

    Objective 1-D: A strain of Myrothecium verrucaria (MV) isolated from sicklepod in Louisiana shows strong bioherbicidal activity against a wide range of weed species across a number of plant families. Grasses (Poaceae), sedges (Cyperaceae), and a range of tree species associated with kudzu in the southeast appear to be relatively resistant/immune to MV.

    Members of the genus Myrothecium are known producers of a variety of toxic secondary metabolites with biological activity against plants, fungi, nematodes, and mammals. The spectrum of biological activity of some secondary metabolites is known. To separate the biological activity of spores versus culture filtrate, cultures of MV were harvested, and the different treatments prepared as follows: 1) ?Washed Spores?: Spores were separated from culture filtrate by centrifuging four times at 4,000 g for 5 minutes and resuspending in water, 2) ?Cell-free Filtrate?: Crude filtrate centrifuged once at 4,000 g for 5 min and then filtered sequentially through 5.0 ?m and then 0.45 ?m millipore filters. Absence of fungal material was verified by plating onto PDA ? no growth observed, 3) ?Crude Filtrate?: Untreated harvest from PDA plates containing live fungus and its metabolites. The effect of MV was dominated by toxins with little effect of fungal infection per se, although infection was a significant factor in Chenopodium album (lambsquarters).

    A range of dilutions of washed spores (0, 10^4, 10^5, 10^6, 10^7, and 10^8) and a range of dilutions of cell-free filtrate (0, 0.01%, 0.1%, 1%, 10%, and 100% concentration of original filtrate) were prepared and applied to hemp sesbania (Sesbania axaltata) and lambsquarters in a factorial design. The impact of MV conidia upon hemp sesbania was very small at all concentrations tested. Conidia of MV caused significant biomass reductions in lambsquarters at concentrations higher than 10^6 conidia ml^-1. In contrast, large biomass reductions were caused by culture filtrates, and this effect increased directly with increasing concentration.

    Objective 1-E: Pseudomonas syringae pv. tagetis (PST), is being considered as a biological control agent of Canada thistle (Cirsium arvense). It produces tagetitoxin, an inhibitor of RNA polymerase that causes chlorosis of developing shoot tissues. While the mode-of-action of tagetitoxin is well known, little is known of the genes required for the production of the toxin. In an attempt to identify the genes required for tagetitoxin production, a strain, EB037, isolated from common ragweed was mutagenized using Tn5, and 17 nontoxigenic mutants were identified. DNA sequences of affected genes were obtained and BLAST searches conducted which demonstrated homologies to genes in other P. syringae pathovars.

    Gene sequences from two of the mutants, Tox-9 and Tox-10, were used to develop the PCR protocols that can distinguish PST from closely related Pseudomonads, including those that produce toxins that cause apical chlorosis symptoms. The Tox-9 mutant has a mutation in a gene with homology to exbB, which codes for an auxiliary protein in the TonB/ExbD/ExbB, a membrane-transport system involved in iron transport. The mutation does not affect the growth rate of the mutant, which is identical to the wild-type strain in iron-rich or iron-deficient media. What role iron plays in tagetitoxin production is unknown. The Tox-10 mutant has a mutation in a gene with homology to asnB, which codes for an asparagine synthetase. Growth of the Tox-10 mutant with or without asparagine is comparable to that of the wild-type strain, indicating that this strain is not an asparagine auxothroph. Although growth is stimulated by the addition of asparagine to the growth medium, toxin production in Tox-10 is not restored. Additional experiments are planned to examine the effect of other nitrogenous amino acids and amine donors on tagetitoxin production. Knowledge of the genes required for tagetitoxin production may enable development of toxin over-producing strains. The production gene cluster may also be useful in modifying other bacterial weed pathogens with the ability to produce tagetitoxin, which may increase the efficacy of the weed pathogens.

    Objective 3: Control of purple and yellow nutsedge (C. rotundus and C. esculentus) continues to be ranked as one of the greatest problems facing growers in the southern United States. It has been shown that the competitive ability of nutsedge can be significantly decreased with the application of the fungus, DH. A field experiment was designed to use the fungus as a component in an integrated approach to pest management as an alternative to methyl bromide fumigation. A tomato production system utilizing multiple treatment combinations was conducted using fallow season treatment as the main plot and production practice as the sub-plot treatment. Fallow season treatments of D. higginsii, glyphosate, and disk fallow were implemented in summer, followed by a fall tomato crop. Significant disease incidence was seen in the fungus-treated plots and no significant difference was found in tomato yield or nutsedge density in the following production season. Tomato yield from fumigant/fungus-treated plots was similar to yields in the fumigant/herbicide-treated plots.

    Objectives 4 and 5: No new reports.

    Impact Statements:
    1. Objectives 1-3: considerable progress was made towards the development of bioherbicides for purple nutsedge, pigweeds, waterhemp, weedy grasses, and weeds in the Asteracae family. This work will enable commercial development and introduction of bioherbicides to manage some of the difficult-to-control weeds affecting agricultural and natural areas.
    Last Modified: unknown

    Date of Annual Report: 09/07/2006

    Report Information:
  • Annual Meeting Dates: 02/12/06 to 02/12/06
  • Period the Report Covers: 06/1905 to 06/1905

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    The meeting opened at 7:00 pm with results presented by objective.

    Objective 1A , R. Charudattan, University of Florida presenting.

    Reviewed Dactylaria higginsii biology and disease process. From 1997-1999, several field trials conducted in Florida, Puerto Rico, and the Dominican Republic. D. higginsii is a highly stable fungus. However, requires an 8 h dew period, slow growing, requires spores for infection. May be useful as postemergence for purple nutsedge in greenhouse-grown tomatoes. Further development as a postemergent sprayable should be accomplishable. Need to develop a formulation that can address the moisture requirements and speed up the infection process.

    Objective 1B Microspaeropsis amaranthi and Phomopsis amaranthicola vs. Amaranthus spp. , Steve Hallett, Purdue University and Yasser Shabana, El-Mansoura University, presenting.

    Reviewed host range, growth and interactions with chemicals. There is synergy with glyphosate (technical material), but surfactants in formulations are antagonistic. Organism needs 12 h dew peroid at temperatures around 20C. Application as fine mist best. When application conditions are favorable, it can kill small plants. Unfortunately, the optimum temperature and moisture conditions are uncommon during the growing season. Target species did not show any sensitivity to amino acids. So modifying the pathogen to overproduce amino acids might not prove useful in this system. Reviewed disease development of M. amaranthi in water hemp and conidia development on different substrates. Corn leaves and husks proved to be good media for spore production. Spores produced in this manner had a high viability and infectivity.

    Objective 1C. Multiple pathogens vs. grasses. S. Chandramohan presenting.

    Reviewed the activity of Drechslera gigantea, Exserohilum longirostratum, and E. rostratum. All have similar activities. Reviewed scale-up of mycelial production. A 50 gallon run costs about $20.00 for media and labor. Preparation stored under oil (Sunspray oil). Reviewed efficacy, functions like a contact herbicide. Preparation can be autoclaved without loss of activity. Consequently, live material is not needed, the activity is due to phytotoxins produced by the fungus. With cogongrass, one to two applications per year give good control. No residual control, i.e. would need to be applied each growing season. Enhanced activity with 10% oil and 0.1% herbicide. Studies on glumegrass in progress.

    Objective 1D. Myrothecium verrucaria vs. multiple hosts. Mark Weaver, USDA/ARS/SWSL, Stoneville, MS presenting.

    Reviewed interest and rational for using M. verrucaria as a bioherbicide. Interest is in the use of the organism for the control of weedy vines (kudzu, red vine, and trumpet vine). Need to optimize formulation and application. Seedlings can be controlled but need to demonstrate utility on plants in the perennial stage. Some advantages of M. verrucaria are that it is indigenous and has a broad spectrum of activity. The big concern about the organism as a biological control agent is that I produces a number of mycotoxins. There have been concerns about genetic variability as well. Problems with sectoring were resolved by successive single-spore culturing.

    Objective 1E. Pseudomonas syringae pv. tagetis vs. Asteraceae. John Lydon presenting. No progress was made on this objective during 2005.

    Featured Speaker. Alan Watson, McGill University, Is Fusarium the Achilles heel of Striga?

    Reviewed the biology of Striga and its impact on food production and society in Africa. Presented a review on the development of Fusarium oxysporum M12-4A for the contorl of Striga. The fungus can kill seeds in the soil, even before they sprout. By coating seeds, the application rates can be as low as 80 g/ha. Busness Meeting

    The S-1001Multistate Project has reached its 5 yr limit. Following discussion, the decision was made to renew the project as a Coordinating Committee.

    The meeting was adjourned at 9:40 pm.

    Accomplishments:
    The S-1001 Regional Committee initiated in 2001 (following the S-136, S234 and S-268 committees initiated in the late 1970s) has involved scientists from twelve states, primarily from the Land Grant Universities and USDA-ARS, and from other countries, primarily Canada. Researchers have cooperated and collaborated on bioherbicides research projects with four broad objectives related to the development of specific bioherbicidal organisms or bioherbicide technologies. The committee has met, with a significant activities in bioherbicides research; more than 100 journal articles have been symposium and discussion session, yearly throughout its tenure, and has promoted published by the group in the last five years. Significant contributions continue to be made through the field of bioherbicides with a number of organisms in development towards market. Also significant, has been the contribution of this field to our understanding of a range of aspects of integrated weed management, plant pathogen interactions, plant disease epidemiology and soil microbiology. Summaries and highlights of the groups activities are listed below. More detailed information is available in individual reports by participants that have been submitted each year; all of which are available at the S-1001 website at www.btny.purdue.edu/S1001.

    Impact Statements:
    1. In 2002, a number of field experiments were performed with varying results, with the best effects found following high field humidty and moisture. With more than one application of D. higginsii, significant nutsedge control enabled yield increases in pepper fields in Florida, but did not increase onion yield in a similar experiment in Puerto Rico. Lab experiments demonstrated that some chemical herbicides were antagonistic to the germination and growth of the fungus. A range of formulations were found to enhance the efficacy of spray applications of D. higginsii in the field.
    2. Phomopsis amaranthicola gave good control of spleen amaranth (Amaranthus dubius) in the Dominican Republic resulting in significantly improved pepper yields compared to a weedy check. In a field experiment in Illinois, P. amaranthicola caused very severe disease in a range of weedy amaranths, including Palmer amaranth (A. palmeri), redroot pigweed (A. retroflexus) and common waterhemp (A. rudis).In field experiments, M. amaranthi caused severe necrosis of a number of weedy amaranths, including Palmer amaranth (A. palmeri), redroot pigweed (A. retroflexus) and common waterhemp (A. rudis), resulting in plant death when conditions were warm and moist.Potential remains for the integration of P. amaranthicola and/or M. amaranthi into cropping systems where they have potential to provide remedial control of weedy amaranths where herbicides are inadequate.
    3. Dreschlera gigantea, isolated from large crabgrass bipolaris sacchari isolated from cogon grass,Exserohilum rostratum isolated from johnsongrass,and E. longirostratum isolated from crowsfootgrass infect a range of weedy grasses, and can be applied in various combinations as a cocktail. The bioherbicide cocktails tested did not affect a number of important grass crops, including corn and a e range of native grasses. Potential may exist for the control of invasive grasses in non-crop land. In field experiments in Florida, selective control of torpedograss and cogongrass using mixtures of the bioherbicidal fungi was superior to control with chemical herbicides since reduced disturbance of competing flora was minimized.
    4. Spray applications of conidia of Myrothecium verrucaria caused severe necrosis and high levels of mortality to a wide range of weeds from agricultural and horticultural (e.g. waterhemp, velvetleaf) and natural (e.g. kudzu, leafy spurge) systems. In most cases, monocots were resistant or immune and dicots were susceptible. Spray applications were reliant upon the inclusion of a wetter in the formulation. When the fungus was grown in liquid culture and then removed by filtration, the culture filtrate retained strong herbicidal activity.
    5. Suspensions of conidia separated from the culture filtrate by washing showed little or no activity against most of the weeds tested. Thus, we conclude that the herbicidal activity of M. verrucaria is dominated by the activity of secondary metabolites rather than by infection by the fungus per se.
    6. The bacterial pathogen Pseudomonas syringae pv. tagetes (PST) causes chlorosis of several Asteraceae, and selected isolates have been shown to cause this disease when applied as foliar sprays. Spray applications in formulations containing Silwett caused 100% mortality in Canada thistle, and severe disease in common ragweed, common sunflower, horseweed, prickly lettuce and common cocklebur. PST produces tagetitoxin, an inhibitor of RNA polymerase.
    7. Collaboration at S-1001 meetings has enabled exchange of information on bioherbicide formulations under study in different labs. As a result, numerous labs have tested bioherbicides in oil emulsions, invert emulsions, wetters such as Silwett L-77 and granular formulations such as pesta.A number of groups have shared info. regarding the interaction on various bioherbicides w/chemical pesticides. In most cases, bioherbicides were effectively integrated with a range of herbicides but were generally antagonized by many other pesticides, particularly fungicides.
    8. The S-1001 project has enabled significant coordination of development activities for each of the bioherbicide systems studied and each of the pathogens has been studied in multiple labs, and has been field tested in multiple sites. Such sharing of pathogens and analysis in different regions has permitted the development of a much broader understanding of the limitations of the pathogens under different conditions, and the potential for their development in different cropping systems.
    9. Bioherbicide virulence can be enhanced by the selection of strains of a plant pathogen that overproduce a particular amino (e.g. valine) acid causing feedback suppression of an amino acid pathway (e.g. ALS pathway; valine, leucine, isoleucine; feedback by valine causes depletion of leucine and isoleucine resulting in plant stress). The S-1001 project has enabled sharing of this methodology as the Montana State University group (D. Sands, A. Pilgeram) have offered training in the selection and testing of amino acid overproducing plant pathogens studied by other groups associated with the project.
    Last Modified: 18-Jan-2008
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