Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Plant Science
Non Technical Summary
Foliar plant diseases that cause necrotic lesions on foliage diminish the plant's photosynthetic ability and can lead to reduced grain yield. Fungal leaf blights of small grains are usually promoted by cool and wet or humid weather. The primary disease inoculum source is contaminated seed or infested crop residue. Another aerial portion of small grains that can be attacked by pathogens is the wheat head. For example, the fungus Fusarium graminearum causes Fusarium head blight (FHB) of wheat, which can cause serious reductions in grain yield. Moreover, FHB can produce mycotoxins in infected grain that are harmful to both livestock and humans. Grain with more than 2 ppm mycotoxin can be docked or rejected at the grain elevators. Spores of the FHB fungus are carried by wind to infect wheat heads at flowering (anthesis). The fungus usually infects the anthers first, then uses the anthers and pollen as a food base for further growth by which ovaries and developing kernels can be infected.Recent FHB epidemics in the United States have caused dramatic reductions in yield and quality of wheat and barley. Head blight development is affected by many factors including host genetics, favorable environmental conditions, and prevalence, survival, and spread of the pathogen. Currently, readily available and affordable measures for controlling FHB, including resistant varieties, cultural practices, and foliar fungicides are only partially effective in disease control. Cultivars grown commercially often have only moderate levels of FHB resistance, and the most commonly grown cultivars are often the most susceptible. Also, because of the pathogen's wide host range, especially on grasses, benefits of crop rotation can sometimes be minimal. Host plant resistance may ultimately be an effective management solution, but it has been difficult to obtain resistance in adapted cultivars to date.Until more resistant wheat varieties are obtained, growers need methods or products to prevent FHB from causing severe economic loss. Of the billions of dollars spent annually in the United States for fungicides, approximately $4.16 billion are spent to control foliar plant-pathogenic fungi. Although chemical fungicides are currently a dominant means of controlling foliar plant pathogens, government regulations are placing increasing restrictions on the use of synthetic chemical pesticides. Moreover, fungicide resistance in the FHB pathogen has been reported in a few states. Therefore, other means of controlling plant diseases are needed. Biological control agents (BCAs) are one alternative, in which selected microbial species are used to antagonize plant pathogens. BCAs have the potential to enhance the effectiveness of integrated pest-management systems, in which cultural and biological control measures are used in concert with synthetic chemicals. Development of biological-control measures will also help reduce concerns about risks to human health associated with residues of certain pesticides in food, and prevent the development of pathogen resistance to synthetic pesticides.Currently about 40 biocontrol products are commercially available for controlling plant diseases, however these products have shown inconsistent results in field situations. This may be due to the variable environmental conditions in field situations, and to the polycyclic nature of many of these diseases and their ability to cause infection throughout the host's growth cycle. Thus there is a need to continue isolating, characterizing, and formulating other BCAs with improved ability to antagonize plant pathogens.Previous work in my laboratory has isolated several microbial strains from South Dakota soils, wheat residue, and foliage that can antagonize both the tan-spot and head-scab fungi using in vitro plate assays. Most of the research has focused on four Bacillus strains isolated from wheat foliage in South Dakota. These bacilli have demonstrated stable, reproducible antagonism of both the tan-spot and head-scab fungi in plate assays. The small subunt rRNA sequences indicate the strains are Bacillus amyloliquefaciens. Two strains of B. amyloliquefaciens, 1BA and 1D3, have shown the most dramatic effects in reducing FHB when they are applied in combination with a fungicide. When applied in tandem with fungicide these BCAs have, at times, resulted in significantly lower amounts of DON toxin in harvested grain and higher grain yields. The BCAs have only been applied at or near anthesis, where the pathogen is most likely to infect the host plant. The BCA literature has shown that some BCAs can elicit induced resistance in crops to help combat disease. To observe such an effect, the BCA should be applied many weeks before infection by the pathogen. Therefore one goal of the work proposed herein will be to assess whether these previously isolated BCAs are capable of eliciting induced resistance. Another goal will be to isolate and screen for new BCAs that exhibit in vitro antagonism against small grain pathogens. Finally, we will conduct greenhouse and field plot trials to assess performance of BCAs under real world conditions. This will entail developing optimal strategies for formulating and applying the BCAs.Economical and easy-to-use commercial formulations of the BCAs will hopefully result from this project, to provide growers with an alternative or supplement to the use of synthetic chemical fungicides for the control of foliar and head diseases of wheat. This work should also reduce levels of fungal toxins such as DON in wheat and barley, which would aid the marketability and value of grain.
Animal Health Component
75%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Goals / Objectives
Evaluate the Bacillus amyloliquefaciens strains we have previously studied as biological control agents (BCAs) that are useful in biological control of plant diseases of small grains, including Fusarium Head Blight (FHB) and Tan Spot, in the presence of fulvic acid.Screen for new BCAs that exhibit in vitro antagonism against small grain pathogens including Fusarium graminearum and Pyrenophora tritici-repentis.Conduct greenhouse and/or field trials with BCAs in different combinations, formulations, and application methods/timing, including treatments with and without fulvic acid.
Project Methods
Objective 1. We have previously shown that several B. amyloliquefaciens strains can antagonize certain fungal plant pathogens. We will continue this assessment using in vitro plate assays where BCA antagonist and target pathogen are inoculated on the same Potato Dextrose agar (PDA) plate. Fungal inoculum will be obtained by culturing fungi on water agar (WA) for 7-14 days at 25o C, then cutting circular agar plugs for inoculum to be placed onto PDA. Bacterial inoculum cultured for 48-72 hours on PDA at 25o C will be applied as linear streak lines a few mm distant from the fungus. Growth inhibition of fungi will be assayed over 7-14 days incubation in the same conditions as above. This work is needed to assure that the bacterial strains produce similar fungal antagonism as they were initially found to, ensuring there has not been decline or loss of antagonism by mutation. Fulvic acid has potential for use in biological control of plant pathogens. Unlike other humic substances, fulvic acid is small enough to cross cell membranes, and directly affect cell processes. Depending on its origin and preparation, fulvic acid may either stimulate or suppress microbial growth; and may increase plant growth in some cases. Fulvic acid will be prepared by extracting soil with aqueous dilute base, followed by centrifuging to remove soil. The humic acid fraction will be precipitated by acidification; then filtering it out, with fulvic acid still in solution. Since fulvic acids from different soils/management systems can have different biological effects/properties, soils from various field plot studies suggested by Dr. Byamukama will be used. Fulvic acid samples will be filter sterilized; then incorporated in PDA or other media at different concentrations, to assay if it affects microbial growth. Cultures grown on plates without fulvic acid will be controls. Fungal cultures grown on WA as described above will be used as inoculum. Bacteria from 48-72 hour-old agar medium cultures will be inoculated onto fulvic acid-containing agar by spot inoculation in a circular area of 3-4 mm. Radial growth of microbes will be assayed over 7-14 days with incubation as above. Growth of BCAs and fungi will be assessed in relation to exposure to fulvic acid. We will then explore potential synergistic antagonism of fungal pathogens by B. amyloliquefaciens/fulvic acid combinations by plate assay methods.Objective 2. New bacterial cultures will be isolated from selected soils/locations as suggested by Dr. Byamukama during the first two years of the project. Culture isolations will be done by standard spread plating, then evaluating number of colony morphologies present, then streaking for isolation until colony purity is assured. Isolates will be screened for antagonism against selected small grain pathogens, including F. graminearum and P. tritici-repentis, using in vitro plate assays. Isolates with promise as BCAs based on plate assays will be sent to MidiLabs for partial sequencing of 16S rDNA allowing identification. The compatibility of new isolates with the two B. amyloliquefaciens strains we have used as BCAs in the past will be via plate assays. Sensitivity of each BCA to fulvic acid, alone or in combination, will also be done using plate assays as above.Objective 3. i. Greenhouse Trials: Greenhouse trials will be conducted as follows: Wheat seeds of 'Select" hard red spring wheat cultivar will be planted in "cone-tainers" (Stuewe & Sons, Inc. Tangent, OR) of a diameter of 3.8 cm and depth of 20 cm filled with a soil mix (Pro-mix® BX mycorrhizae, Greenhouse Megastore, Danville, IL). Five seeds per cone will be planted and thinned to 4 seedlings after emergence. A plot (treatment) will consist of 2 cones. Plants will be maintained in a greenhouse kept at 25-28 C and day length of 14 hours with supplemental lighting. We will employ a randomized complete block design, with 4 replications of each treatment. Treatments will include: A: (without fulvic acid): not inoculated with pathogen; inoculated with pathogen; inoculated with pathogen and treated with chemical fungicide; and inoculated with pathogen and treated with individual pure cultures of BCAs. B; (with fulvic acid): not inoculated with pathogen; inoculated with pathogen; inoculated with pathogen and treated with chemical fungicide; and inoculated with pathogen and treated with individual pure cultures of BCAs.Pathogen: Tan spot pathogen will be inoculated by spraying at tillering growth state, while FHB pathogen will be inoculated by spraying onto grain heads at flowering.Experiments with BCAs and/or fulvic acid applied twice: In experiments to check for ability of BCAs and/or fulvic acid to induce resistance in the wheat, application of the BCAs and/or fulvic acid will be done twice; initial application for Tan spot studies will be 10-17 days before tillering, and initial application for FHB studies will be 10-17 days before flowering. Second applications of BCAs or fulvic acid will be administered as for the single application studies; i.e. 10 days prior to pathogen inoculation. Fulvic acid concentrations will be as concentrated as possible based on volumes/amounts obtained from the preparation method above, with minimal dilution. Pathogen inoculation will be as above.Experiments with single applications of BCAs and/or fulvic acid: For studies of efficacy of single applications of BCAs and/or fulvic acid, Tan spot pathogen and FHB pathogen will be applied as described above. Tryptic soy broth cultures of BCAs grown for 7-10 days at 25o C with shaking at 150 rpm, reaching cell densities of ~ 107 CFU/ml will be applied by spraying 10 days prior to pathogen inoculation. Fulvic acid concentrations will be as concentrated as possible as described above. Disease ratings will be done as per standard methods described below for field plots. Data will be analyzed by ANOVA. Some testing of individual BCAs or combinations and their effect on wheat cultivar seedlings will also be done using Magenta boxes.ii. Field Trials: Field trials will be established at the Volga Research Farm. The hard red spring wheat cultivar 'Select' will be planted in plots of 1.2 m x 6 m at approximately 1.2 m seeds per acre, with 2 replications. Standard agronomical practices for weed and soil fertility management will be employed. Weather information (temperature, rainfall, relative humidity) will be obtained from nearby Mesonet weather station. In field plots, a set of foliar treatments will be administered, compared to an untreated check. These treatments will be applied at various crop stages, including before anthesis and at anthesis foliar application (in the case of FHB). Foliar agents will be applied using a CO2 pressurized backpack sprayer at 40 psi with three TJ 8002VS nozzles spaced at 0.38 m apart. Plots will be inoculated with FHB pathogen and misted to enhance FHB disease development after BCAs are applied. BCAs for foliar diseases will be applied at green-up growth stage. Treatments using BCAs with and without fulvic acid will be the same as described for the greenhouse experiment above. Microbial inoculum preparation, rates of pathogen and BCA application, and concentration of fulvic acid applied will be the same as for greenhouse experiments above. Disease ratings will be taken at the soft dough stage of kernel development (Feekes 11.2), and will include FHB incidence, FHB head severity, and flag leaf disease severity. To assess foliar disease severity, 10 random flag leaves per plot will be individually assessed for tan spot severity as percentage leaf area covered with tan spot lesions on each leaf. A mean of the 10 flag leaves will be used as a score. Harvesting will be done using a Wintersteiger small-plot combine. Plot weights will be adjusted at 13.5% moisture content. Yield and quality (DON, test weight and protein content) will be determined Data will be analyzed by ANOVA.