Progress 10/01/02 to 09/30/07
Outputs
OUTPUTS: Significant activities included conducting and analyzing experiments in the laboratory and field plots with the bacteria used as biological control agents (BCAs) of Fusarium Head Blight (FHB); and teaching and mentoring four undergraduate students and three Master's students who worked on the project. Significant events included attending and presenting research results at five National Fusarium Head Blight Forums, two General Meetings of the American Society for Microbiology, and two workshops organized and conducted by the U.S. Wheat and Barley Scab Initiative. Products included new fundamental and applied knowledge about the BCAs; collaborations with Marty Draper and Kay Ruden of the SDSU Plant Science Department, Scott Halley at North Dakota State field station in Langdon, North Dakota, and with Gary Yuen of the University of Nebraska-Lincoln; new methods for growing and applying the BCAs to grain heads; and the completion of the Master's degree by two graduate students
who worked on the project. Information was disseminated by presenting posters, publishing abstracts and reports in the FHB Forum Proceedings, and talking with attendees at the scientific meetings.
PARTICIPANTS: Individuals who worked on the project included the PI/PD (Bruce Bleakley). Four undergraduate students (Laura Fuhrman, Jennifer Morgan, Jennifer Vogel, and Adam Burthus) worked on the project, aiding the PI and the graduate students in microbiological procedures in the laboratory and in field plot work. Three Master's students (Nichole Baye, Amanda Gill, and Jennifer Morgan) worked on the project, conducting both laboratory and field studies with the BCAs. Partner organizations included the University of Nebraska at Lincoln; the University of Missouri at Columbia; North Dakota State University field station at Langdon; and the USDA-ARS National Center for Agricultural Utilization Research (NCAUR) at Peoria, Illinois. Collaborators and contacts included Martin Draper and Kay Ruden in the Plant Science Department; and Neil Reese in the Biology/Microbiology Department at South Dakota State University; Gary Yuen and Christy Jochum in the Plant Pathology Department at the
University of Nebraska at Lincoln; Laura Sweets of the Division of Plant Sciences at the University of Missouri at Columbia; Scott Halley of the North Dakota State University Langdon Research Extension Center; and Chris Dunlap of the USDA-ARS NCAUR in Peoria, Illinois. Training and professional development of the undergraduate and graduate students mentioned above was part of the project.
TARGET AUDIENCES: Target audiences included other researchers investigating Fusarium Head Blight, especially in the area of chemical and biological control of the disease; and wheat and barley producers who are part of the U.S. Wheat and Barley Scab Initiative.
Impacts
Outcomes/impacts: Changes in knowledge resulting from the project included new fundamental knowledge verifying that the Bacillus strains used as BCAs in our studies produced lipopeptide antibiotics including iturin and surfactin, and that patterns and amounts of these antibiotics were different in different broth growth media. Finding that the BCAs were both thermotolerant and salt tolerant, elevated temperature and elevated salt concentration were used in plating media to select for populations of BCAs sprayed onto grain heads. These studies showed that the applied BCAs persisted and grew on grain heads after spray application, although BCA numbers fluctuated greatly over time. Field plot studies provided evidence that the BCAs can be used in combination with fungicide sprays to control FHB and reduce the amounts of the fungal toxin deoxynivalenol in grain. Based on the new fundamental knowledge, changes in actions included using new broth medium formulations to grow
BCAs for spray application, and using new plating formulations for recovering and counting BCAs applied to grain heads.
Publications
- Morgan, J., B.H. Bleakley, and C.A. Dunlap. 2007. Field and laboratory studies to monitor cell populations, lipopeptides and lipopeptide genes of Bacillus 1BA, a biocontrol agent active against Fusarium Head Blight. Abstract, pg. 106. In: S.M. Canty, A. Clark, D. Ellis, and D. Van Sanford (eds.), Proceedings of the 2007 National Fusarium Head Blight Forum, Dec. 2-4, 2007, Kansas City, Missouri. East Lansing; Michigan State University.
- Ruden, K.R., B.H. Bleakley, and B.E. Ruden. 2007. 2007 Uniform trials for the performance of biological control agents in the suppression of Fusarium Head Blight in South Dakota. Abstract, pg. 136. In: S.M. Canty, A. Clark, D. Ellis, and D. Van Sanford (eds.), Proceedings of the 2007 National Fusarium Head Blight Forum, Dec. 2-4, 2007, Kansas City, Missouri. East Lansing; Michigan State University.
- Yuen, G.Y., C.C. Jochum, S. Halley, G. Van Ee, V. Hoffman, and B.H. Bleakley. 2007. Effects of spray application methods on biocontrol agent viability. Pp. 149-152. In: S.M. Canty, A. Clark, D. Ellis, and D. Van Sanford (eds.), Proceedings of the 2007 National Fusarium Head Blight Forum, Dec. 2-4, 2007, Kansas City, Missouri. East Lansing; Michigan State University.
- Gill, A. 2006. Development of an isolation method that uses tolerance to salt and heat, to select for Bacillus strain 1BA, a biocontrol agent used to control Fusarium Head Blight. M.S. thesis, South Dakota State University, Brookings, SD.
- Baye, N.L. 2007. Bacillus strains used for biological control of Fusarium Head Blight: identification, growth studies, and lipopeptide production. M.S. thesis, South Dakota State University, Brookings, SD.
- Yuen, G.Y., C.C. Jochum, K.R. Ruden, J. Morgan, B.H. Bleakley, and L.E. Sweets. 2007. Results from the 2007 standardized evaluation of biological agents for the control of Fusarium Head Blight on wheat and barley. Pp. 153-157. In: S.M. Canty, A. Clark, D. Ellis, and D. Van Sanford (eds.), Proceedings of the 2007 National Fusarium Head Blight Forum, Dec. 2-4, 2007, Kansas City, Missouri. East Lansing; Michigan State University.
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Progress 01/01/06 to 12/31/06
Outputs
Selected strains of Bacillus can be used as biocontrol agents (BCAs), to antagonize Fusarium graminearum which causes Fusarium Head Blight (FHB) of wheat and barley. To assay numbers of the biocontrol agent Bacillus strain 1BA, after spraying its cells onto wheat heads in field plots, a selective and/or differential growth medium is needed to allow differentiation of Bacillus 1BA from native wheat microflora. The ability of Bacillus 1BA to tolerate temperature and salt stresses was exploited to allow most probable number (MPN) estimates of its numbers on inoculated wheat heads over time in field plots. Little or no growth of the native microflora occurred with these conditions, which led to continued studies involving the recovery of Bacillus 1BA from wheat heads after spray application in the field. To produce BCA inoculum for field plot application, Bacillus 1BA was grown in a variety of different broth media, including Field Defined Medium (FDM), FDM plus 8.5
percent NaCl, Tryptic Soy Broth and Yeast Extract (TSB/YE), TSB/YE plus 8.5 percent NaCl, half-strength TSB/YE, and half-strength TSB/YE plus 8.5 percent NaCl. Cell counts of the Bacillus 1BA in these media were estimated using a MPN procedure, on the day cells were sprayed onto wheat heads at flowering. For the MPNs, a selective medium of TSA plus 8.5 percent NaCl was used, with an incubation temperature of 47 C . Counts of the BCA on inoculated wheat heads were done at days 0, 3, 6, 9, 12, 15, and 20 after inoculation. Numbers of bacterial cells recovered from control plots over time were much lower than for plots sprayed with 1BA; but results from replicate plots (both control and treated) were highly variable. Data from treated plots were also variable, but trends suggested that growth of 1BA occurred on wheat heads after application. Statistical analysis of non-transformed MPN data using one way ANOVA (P less than 0.05) did not show significant differences between treatments, or
over time within a treatment; probably because of the large variability between replicates in most treatments. ANOVA of log transformed data (P less than 0.05) revealed some significant differences between treatments, and over time within some treatments. On Day 6, there was a significant difference between MPN values for the control (with low numbers), and half strength TSB/YE (with higher numbers); and a significant difference between half strength TSB/YE (with higher numbers) and FDM plus Agridex (with lower numbers). By Day 20, several differences between log-transformed MPN results of treatments were significant. FDM had significantly higher MPN values than the control, TSB/YE plus 8.5 percent NaCl, and FDM plus Latron CS7. Within half strength TSB/YE plus 8.5 percent NaCl, MPN numbers were significantly higher on Day 20 than on Day 15. Inocula produced in different broth media behaved differently over time on wheat heads, with some experiencing little or no population increase,
and others showing a dramatic increase in numbers several days after spray application. Evidence for the growth of the BCA Bacillus 1BA on wheat heads in field plots after spray application was obtained.
Impacts
Better understanding of the ability of these biological control agent bacteria (BCAs) to colonize, persist, and grow on inoculated wheat heads is needed to evaluate differences in these abilities among different BCAs, and to correlate presence and growth of BCAs on inoculated wheat heads to their ability to reduce occurrence or severity of FHB, or reduce the levels of DON (vomitoxin) in grain. Better understanding of how survival and growth of BCAs on inoculated wheat heads correlates with the type of growth medium the BCAs were grown in is also needed for optimization of the growth medium the BCAs are cultured in before spray application onto wheat heads. The effect of spray additives including surfactants on numbers of BCAs on inoculated wheat heads over time also needs to be evaluated, to see if these chemicals act to reduce or increase numbers of BCAs. Optimizing methods for foliar application of the bacterial strains used for biological control of FHB may, alone
or in combination with fungicide application, result in reduction of DON (vomitoxin) levels to that meeting guidelines for human consumption. This is very important for wheat producers, as well as for grain millers and bakers.
Publications
- Yuen, G.Y., C.C. Jochum, K.R. Ruden, L.E. Sweets, B.H. Bleakley, and M.A. Draper. 2006. 2006 Results from the standardized evaluation of biological agents for the control of Fusarium Head Blight on wheat and barley. Pp. 27-30. In: S.M. Canty, A. Clark, and D. Van Sanford (eds.), Proceedings of the 2006 National Fusarium Head Blight Forum, Dec. 10-12, 2006, Research Triangle Park, North Carolina. East Lansing; Michigan State University.
- Gill, A.L., and B.H. Bleakley. 2006. The use of chemical and physical stressors, 8.5 percent NaCl and 47 C, to assay populations of a Bacillus strain used to control Fusarium Head Blight on wheat heads in field plots. Abstract, pg. 12. In: Proceedings of the 66th Annual Meeting of the North Central Branch of the American Society for Microbiology. South Dakota State University, Brookings, SD.
- Gill, A.L., and B.H. Bleakley. 2006. The use of chemical and physical stressors, 8.5 percent NaCl and 47 C, to assay populations of a Bacillus strain used to control Fusarium Head Blight on wheat heads in field plots. Abstract, pg. 9. In: S.M. Canty, A. Clark, and D. Van Sanford (eds.), Proceedings of the 2006 National Fusarium Head Blight Forum, Dec. 10-12, 2006, Research Triangle Park, North Carolina. East Lansing; Michigan State University.
- Ruden, K.R., B. Bleakley, S.M. Thompson, K. Maxson-Stein, and M.A. Draper. 2006. 2006 Uniform trials for the performance of biological control agents in the suppression of Fusarium Head Blight in South Dakota. Abstract, pg. 20. In: S.M. Canty, A. Clark, and D. Van Sanford (eds.), Proceedings of the 2006 National Fusarium Head Blight Forum, Dec. 10-12, 2006, Research Triangle Park, North Carolina. East Lansing; Michigan State University.
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Progress 01/01/05 to 12/31/05
Outputs
Selected strains of Bacillus can be used as biocontrol agents (BCAs), to antagonize Fusarium graminearum which causes Fusarium Head Blight (FHB) of wheat and barley. The ability of the BCA strain 1BA to survive during storage at different temperatures was examined. Cells of strain 1BA grown for five days at 27 degrees C in tryptic soy broth were stored at either 4 degrees C, minus 20 degrees C, or room temperature (27 degrees C) for one month, then enumerated for viable cells. Best survival of cells was at 27 degrees C (a 2 log decrease), with a 4 log decrease in cell numbers at minus 20 degrees C and at 4 degrees C. This was unexpected, since bacterial numbers usually remain more stable at refrigerator temperature rather than at room temperature. This has implications for using these BCAs for control of FHB, since due to unpredictable weather conditions or maturation of wheat or barley in the field, storage of BCAs before their use in the field is often required. The
production of endospores by strain 1BA at different temperatures was examined, using nutrient agar amended with manganese sulfate to help encourage spore production. Large numbers of spores were produced at selected incubation temperatures, ranging from 27 degrees C to 50 degrees C. Endospores survived pasteurization at 80 degrees C for 10 minutes, indicating that endospores of this BCA could remain viable at elevated storage temperatures. Using plate assays, amylase but not chitinase activity was verified at all examined temperatures for 1BA. To assay numbers of 1BA after spraying its cells onto wheat or barley heads in the field, a selective and/or differential growth medium is needed to either discourage growth of native wheat or barley microflora while allowing growth of this BCA. Temperature and salt stresses on 1BA were examined to develop such a medium. Strain 1BA grew on tryptic soy agar (TSA) and nutrient agar (NA) at various temperatures, ranging from 27 degrees C to 50
degrees C. 1BA also grew on TSA and NA amended with various NaCl concentrations, ranging from 2.5 percent NaCl to 10 percent NaCl . Strain 1BA also grew at elevated temperature and salt concentrations in the defined broth medium used for producing inoculum for field application. There were distinct colonial morphology changes in 1BA depending on temperature or NaCl concentration. The elevated temperature and NaCl concentrations that 1BA could withstand were used in preliminary plate counting of the microflora of wheat heads. Little or no growth of the native microflora was noted with these conditions, suggesting that we will be able to apply and count 1BA inoculum that has been sprayed onto wheat heads using a plate count methodology. The ability of these BCAs to grow at elevated temperature and salt concentration indicates these stresses can be used to select for these BCAs in plate count assays, and possibly to enrich for them on plant surfaces by spraying salt solutions on aerial
plant parts.
Impacts
Better understanding of the ability of these biocontrol bacteria to withstand environmental stresses such as elevated temperature and salt concentrations, may allow media to be formulated to count the number of these bacteria on wheat heads after spray appliction, allowing estimation of whether the bacteria grow or not after being sprayed onto wheat. Better understanding of how best to store these bacteria will help optimize handling of bacterial inoculum prior to spray application in the field. Optimizing methods for foliar application of the bacterial strains used for biological control of FHB may, alone or in combination with fungicide application, result in reduction of DON (vomitoxin) levels to that meeting guidelines for human consumption. This is very important for wheat producers, as well as for grain millers and bakers.
Publications
- Bleakley, B.H., and N.L. Baye. 2005. Production in different broth media of iturin and surfactin by a Bacillus strain used in the biological control of Fusarium Head Blight. Abstract N-108. In Abstracts of the 105th General Meeting of the American Society for Microbiology. American Society for Microbiology, Washington, D.C.
- Dangel, A.L., and B.H. Bleakley. 2005. Characteristics, including tolerance to elevated heat and elevated salt concentration, of a Bacillus strain used as a biocontrol agent to control Fusarium Head Blight. Abstract, p. 187. In S.M. Canty, T. Boring, J. Wardwell, L. Siler, and R.W. Ward (eds.), Proceedings of the National Fusarium Head Blight Forum; 2005 December 11-13; Milwaukee, WI. Michigan State University, East Lansing, MI.
- Draper, M.A., B. Bleakley, K.R. Ruden, S.M. Thompson, and D.S. Wittmeier. 2005. 2005 Uniform trials for the performance of biological control agents in the suppression of Fusarium Head Blight in South Dakota. Abstract, p. 189. In S.M. Canty, T. Boring, J. Wardwell, L. Siler, and R.W. Ward (eds.), Proceedings of the National Fusarium Head Blight Forum; 2005 December 11-13; Milwaukee, WI. Michigan State University, East Lansing, MI.
- Yuen, G.Y., C.C. Jochum, B.H. Bleakley, M.A. Draper, K.R. Ruden, and L.E. Sweets. 2005. Standardized evaluation of biological agents for the control of Fusarium Head Blight: 2005 results. Pp. 237-239. In S.M. Canty, T. Boring, J. Wardwell, L. Siler, and R.W. Ward (eds.), Proceedings of the National Fusarium Head Blight Forum; 2005 December 11-13; Milwaukee, WI. Michigan State University, East Lansing, MI.
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Progress 01/01/04 to 12/31/04
Outputs
Selected strains of bacteria in the genus Bacillus can antagonize Fusarium graminearum in laboratory, greenhouse, and field-plot studies. In some field plot studies where Bacillus spp. have been sprayed on to wheat or barley, symptoms of Fusarium Head Blight (FHB) have been reduced, and/or deoxynivalenol (DON) levels in grain have declined. The mechanism of the antagonism is not understood, but may depend in part on bacterial antibiotics, such as cyclic lipopeptides in the iturin family. We have cultured Bacillus sp. strain 1BA in a variety of defined (synthetic) and semi-defined broth media that lack glucose (which can suppress iturin production). The three broth media that were studied were: (1) a basal defined medium (BDM) containing mannitol, glutamic acid and inorganic salts; (2) a defined medium (DM) similar to BDM but containing increased amounts of mannitol and glutamic acid; and (3) a defined medium with the same composition as (2) but with increased
concentrations of calcium and manganese, two elements which are known to be important in regulating different aspects of Bacillus metabolism. Broth cultures of Bacillus strain 1BA were grown for different time periods in these three media. At selected time intervals culture samples were aseptically removed for measurement of optical density at 600 nm, and for iturin analysis using high pressure liquid chromatography (HPLC), to see if different phases of bacterial growth resulted in differences in iturin production. Standard curves of iturin A (Sigma) were linear in a range from 50 ug/ml up to 250 ug/ml, with absorption maxima for iturin occurring at 214 nm and 275 nm for each iturin peak. In the BDM broth, maximum optical density at 600 nm near 2.0 was reached after about 7 days of growth, and maximum iturin production occurred at this time (about 720 ug/ml). After this time, iturin levels declined greatly. In the DM broth having increased levels of mannitol and glutamic acid, maximum
optical density at 600 nm of 2.5 was not reached until 14 days incubation. However, maximum iturin production was reached around 5 days of growth (optical density at 600 nm of 1.3; iturin production of 400 ug/ml). After 5 to 6 days of growth, iturin production sharply declined in this DM broth. In broth medium (3) containing the same components as (2) but with increased levels of Ca and Mn, maximum optical density at 600 nm of 3.6 was reached after 8 days of growth, giving the highest cell yield of any broth medium. Iturin production in this medium had a much different pattern than the other two, with greatest iturin levels found within the first 24-48 hours of growth (132 ug/ml), and then declining sharply. Production of iturin in medium (3) was greatest during exponential growth, not stationary phase, and increased levels of Ca and Mn allowed iturin to be produced sooner, during early log to mid log phase of growth. Different growth media will result in different amounts of iturin;
and the time of incubation will also affect iturin levels. This has implications for growing these bacteria for field application, in determining medium composition and incubation time.
Impacts
Optimizing methods for foliar application of the bacterial strains used for biological control of FHB may, alone or in combination with fungicide application, result in reduction of DON (vomitoxin) levels to that meeting guidelines for human consumption. This is very important for wheat producers, as well as for grain millers and bakers.
Publications
- Draper, M.A., Bleakley, B., Ruden, K.R., Glover, K.D., Schilling, S.M., Wittmeier, D.S., and Lammers, G. 2004. "2004 uniform trials for the performance of biological control agents in the suppression of Fusarium Head Blight in South Dakota." In:Canty, S.M., Boring, T., Wardwell, J. and Ward, R.W. (Eds.), Proceedings of the 2nd International Symposium on Fusarium Head Blight; incorporating the 8th European Fusarium Seminar; 2004, 11-15 December; Orlando, FL, USA. Michigan State University, East Lansing, MI. p. 297.
- Yuen, G.Y., Bleakley, B.H., Draper, M.A., Jochum, C.C., Milus, E.A., Ruden, K.R., and Sweets, L.E. 2004. "Results from the 2004 standardized evaluation of biological agents for the control of Fusarium Head Blight." In:Canty, S.M., Boring, T., Wardwell, J. and Ward, R.W. (Eds.), Proceedings of the 2nd International Symposium on Fusarium Head Blight; incorporating the 8th European Fusarium Seminar; 2004, 11-15 December; Orlando, FL, USA. Michigan State University, East Lansing, MI. pp. 380-382.
- Bleakley, B.H., and Baye, N.L. 2004. "Use of HPLC in examining culture supernatants of bacteria used in biological control of FHB for the presence of iturin." In:Canty, S.M., Boring, T., Wardwell, J. and Ward, R.W. (Eds.), Proceedings of the 2nd International Symposium on Fusarium Head Blight; incorporating the 8th European Fusarium Seminar; 2004, 11-15 December; Orlando, FL, USA. Michigan State University, East Lansing, MI. p. 292.
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Progress 01/01/03 to 12/31/03
Outputs
Progress was made in formulation of the defined growth medium for growing bacterial strains that can biologically control Fusarium Head Blight (FHB). A modified medium containing 2.3 times more mannitol as carbon source and 2.1 times more glutamic acid as nitrogen source compared to an initial broth formulation resulted in increased bacterial cell numbers compared to the original broth formulation. The increased bacterial numbers obtained with this modified defined medium (with up to one hundred million cells per ml) were similar to bacterial numbers that other research groups have obtained with their bacterial biocontrol agents. Increased cell numbers of the bacteria applied as a foliar spray to wheat plants should allow better biological control of FHB. In field plot trials in South Dakota, 'Oxen' hard red spring wheat was planted in a factorial randomized complete block design with four replications. Foliar application of broth cultures of bacterial strains 1BA and
1BC at initial anthesis of wheat was carried out. Although disease severity of the wheat was not as high as desired (being below 10 per cent), application of bacterial biocontrol agents resulted in significantly lower vomitoxin (also known as deoxynivalenol or DON) content as compared to the Folicur treatment (based on LSD at P of 0.05). In Folicur-treated wheat the DON level was 1.5 ppm, whereas the DON levels in wheat treated with biocontrol bacteria ranged from 1.1 to 0.9 ppm depending on the bacterial strain. Controlling or reducing DON levels in grain is very important, due to current guidelines that wheat flour for human consumption should contain less than one ppm DON.
Impacts
Optimizing methods for foliar application of the bacterial strains used for biological control of FHB may, alone or in combination with fungicide application, result in reduction of DON (vomitoxin) levels to that meeting guidelines for human consumption. This is very important for wheat producers, as well as for grain millers and bakers.
Publications
- Draper, M.A., Bleakley, B.H., Baye, N.L., Ruden, K.R., Calli, E., and Schilling, S.M. 2003. 2003 Uniform trials for the performance of biological control agents in the suppression of Fusarium Head Blight in South Dakota. pp. 67-68. In: Canty, S.M., Lewis, J., Siler, L., and Ward, R.W. (eds.), Proceedings of the National Fusarium Head Blight Forum; 2003 December 13-15, Bloomington, MN. MSU, East Lansing, MI.
- Yuen, G.Y., Jochum, C.C., Bleakley, B.H., Ruden, K.R., Draper, M.A., Schisler, D.A., Zhang, S., Boehm, M.J., Lipps, P.E., and Bergstrom, G.C. 2003. Cooperative multistate field tests of biological agents for control of Fusarium Head Blight in wheat and barley. pp. 113-115. In: Canty, S.M., Lewis, J., Siler, L., and Ward, R.W. (eds.), Proceedings of the National Fusarium Head Blight Forum; 2003 December 13-15, Bloomington, MN. MSU, East Lansing, MI.
- Baye, N.L., and Bleakley, B.H. 2003. Comparison of complex and defined growth media on antifungal activity of bacterial strains used as biological control agets of Fusarium Head Blight. Abstract N-212, p. 436. In: Abstracts of the 103rd General Meeting of the American Society for Microbiology. ASM, Washington, D.C.
- Baye, N.L., Bleakley, B.H., Draper, M.A., and Calli, E. 2003. Laboratory studies with purified iturin A, and with Bacillus spp. grown in complex and defined growth media, to ascertain the identity and ability of compounds that inhibit Fusarium graminearum. Abstract, p. 60. In: Canty, S.M., Lewis, J., Siler, L., and Ward, R.W. (eds.), Proceedings of the National Fusarium Head Blight Forum; 2003 December 13-15, Bloomington, MN. MSU, East Lansing, MI.
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