Progress 10/01/15 to 09/30/19
Outputs
Target Audience:The target audience will include stakeholders in academia interested in enhancing the efficacy of biological control strategies, people in agriculture and aquaculture who want to promote the growth of animals and plants, and people in industry who might want to commercialize the technologies to get this into the hands of farmers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Deepak Shantharaj was the primary researcher conducting these studies. Dr. Shantharaj was involved in all aspects of this work including design, execution and analysis of the results, and was provided with the opportunity to write and serve as a co-PI for a USDA Crop Protection and Management proposal. How have the results been disseminated to communities of interest?We have now published one peer-reviewed article and a second article should be published by next year. In addition, we hope to advance the discovery of a Xanthomonas biocontrol agent to the stage of field tests if we are successful in acquiring extramural support for this project. That would be the next needed step to advance the outcome of this project for agricultural application. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Using the strategy of cultivating rhizobacteria using a root extract, we identified a total of 117 unique rhizobacteria isolates and completed a phylogenetic analysis of these isolates (Objective 1). The phylogenetic analysis revealed that these isolates were highly diverse, with over 30 bacterial genera and many phyla represented, with some potentially novel species of bacteria. Some of these isolates were previously recognized to have roles in promoting plant growth (e.g. Rhizobium spp.) whereas other isolates have not been previously described to prmote plant growth or prevent plant disease. We screened in greenhouse assays for the ability of the isolates (that were not potential plant pathogens) to promote soybean growth when applied as a seed treatment. Among our isolates, the Bacillus spp. showed the most potential as plant growth-promoting rhizobacteria (Objective 2) and these are being further evaluated for their synergy with pectin-rich amendments in plant growth promotion. In assessing rhizobacteria for their ability to inhibit plant pathogens, we observed very significant inhibition of both bacterial and fungal pathogens by many rhizobacteria (Objective 3); in particular, we observed that the pathogen Xanthomonas perforans was inhibited by several isolates, in particular the Bacillus and Burkholderia spp. isolates. Due to the need for alternative treatment for bacterial spot of tomato due to X. perforans, we evaluated multiple rhizobacteria isolates for X. perforans biocontrol in tomato. Our best-performing biocontrol agent, Burkholderia gladioli C101, was able to reduce disease severity by about 90% when compared to water-treated plants when a cell-free supernatant was applied as a foliar application. This has now been replicated, and shows consistent reduction in tomato bacterial spot due to copperresistant isolates of X. perforans. We have furthermore shown that the secondary metabolites produced by B. gladioli C101 are heat-resistant. This is important since some Burkholderia species (like B. cepacia) can be opportunistic human pathogens particularly among immunocompromised individuals, it would be improtant to heat-treat a culture thereby removing any viable cells, in a cost-effective way to provide this biocontrol agent to farmers.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Hassan, M.K., McInroy, J.A., Jones, J., Shantharaj, D., Liles, M.R. and Kloepper, J.W. (2019) Pectin-rich amendment enhances soybean growth and nodulation mediated by Bacillus velezensis strains. Plants, 8(5), doi: 10.3390/plants8050120.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Shantharaj, D., Williams, M.A., Potnis, N.S. and M.R. Liles. Burkholderia gladioli C101 metabolites protect tomato plants against Xanthomonas perforans infection
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience will include stakeholders in academia interested in enhancing the efficacy of biological control strategies, people in agriculture and aquaculture who want to promote the growth of animals and plants, and people in industry who might want to commercialize the technologies to get this into the hands of farmers. Changes/Problems:One significant issue brought up by reviers of the USDA Crop Protection and Management Proposal was the difficulty in using a potential human opportunistic pathogen like Burkholderia species for crop protection. Our approach to overcome this difficulty is two-fold: 1) To explore the ways in which we could treat the Burkholderia spp. cultures to elminate any viable cells while retaining the metabiolites active against X. perforans; fortunately, we have found that for all 3 of these Burkholderia spp. cultures that produce metabolites active against X. perforans, that the metabolites have significant heat-stability so that we can heat treat a culture and remove ALL viable Burkholderia cells while retaining X. perforans inhibitory activity. We want to further refine the conditions that can be used to lyse Burkholderia cells while providing a low-cost and safe biocontrol treatment to prevent X. perforans in tomato. 2) An alternative approach would be to identify the novel biosynthetic gene clusters encoded in Burkholderia isolates that could be cloned and expressed in an alternative heterologous host, that would not be a potential human pathogen. We are exploring this concept through the use of a newly developed collaboration and this could provide another source of funding to develop this concept. What opportunities for training and professional development has the project provided?Dr. Deepak Shantharaj was the primary researcher conducting these studies. Dr. Shantharaj was involved in all aspects of this work including design, execution and analysis of the results, and was provided with the opportunity to write and serve as a co-PI for a USDA Crop Protection and Management proposal. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will continue our research in understanding the nature of the secondary metabolites produced by our best-performing rhizobacteria biocontrol agents, particularly among the Burkholderia species. This will include analysis of draft genome sequences to identify predicted biosynthetic gene clusters and compare these with known biosynthetic pathways, as well as some preliminary mass spectrometry analyses to identify potentially novel secondary metabolites that have the ability to inhibit the growth of plant pathogens. We will also complete Objective 4 in identifying additional anti-fungal active metabolites expressed from biosynthetic gene clusters obtained from a soil metagenomic library.
Impacts
What was accomplished under these goals?
Using the strategy of cultivating rhizobacteria using a root extract, we identified a total of 117 unique rhizobacteria isolates and completed a phylogenetic analysis of these isolates (Objective 1). The phylogenetic analysis revealed that these isolates were highly diverse, with over 30 bacterial genera and many phyla represented, with some potentially novel species of bacteria. Some of these isolates were previously recognized to have roles in promoting plant growth (e.g. Rhizobium spp.) whereas other isolates have not been previously described to prmote plant growth or prevent plant disease. We screened in greenhouse assays for the ability of the isolates (that were not potential plant pathogens) to promote soybean growth when applied as a seed treatment. Among our isolates, the Bacillus spp. showed the most potential as plant growth-promoting rhizobacteria (Objective 2) and these are being further evaluated for their synergy with pectin-rich amendments in plant growth promotion. In assessing rhizobacteria for their ability to inhibit plant pathogens, we observed very significant inhibition of both bacterial and fungal pathogens by many rhizobacteria (Objective 3); in particular, we observed that the pathogenXanthomonas perforans was inhibited by several isolates, in particular the Bacillus and Burkholderia spp. isolates. Due to the need for alternative treatment for bacterial spot of tomato due to X. perforans, we evaluated multiple rhizobacteria isolates for X. perforans biocontrol in tomato. Our best-performing biocontrol agent, Burkholderia gladioli C101, was able to reduce disease severity by about 90% when compared to water-treated plants when a cell-free supernatant was applied as a foliar application. This has now been replicated, and shows consistent reduction in tomato bacterial spot due to copper-resistant isolates of X. perforans. We have furthermore shown that the secondary metabolites produced by B. gladioli C101 are heat-resistant. This is important since some Burkholderia species (like B. cepacia) can be opportunistic human pathogens particularly among immunocompromised individuals, it would be improtant to heat-treat a culture thereby removing any viable cells, in a cost-effective way to provide this biocontrol agent to farmers.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2019
Citation:
Cultivation of rhizobacteria using a root extract medium and identification of Burkholderia spp. isolates that produce metabolites that protect plants from Xanthomonas perforans infection
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience for this research are scientists in the field of plant pathology and in the agricultural sciences, as well as extension specialists in plant sciences. We have completed a field trial at an agricultural field station during the past year, engaging the extension scientists at the agricultural research station, as well as given scientific presentations at an agricultural workers conference, an Auburn University College of Agriculture symposium and a student research symposium. Changes/Problems:A principal issue is the cost/benefit for using a pectin amendment to stimulate PGPR efficacy. Clearly it will not be economically feasible for farmers to use purified pectin purchased from Sigma Chemical Company to apply to their soybean or other crop. To solve this problem we will be doing experiments to evaluate low-cost, pectin-rich agricultural waste products (e.e. beet pulp) to see if this can enhance PGPR-mediate plant growth promotion and/or disease biocontrol. What opportunities for training and professional development has the project provided?A graduate student, Mohammad Hassan, has led these research efforts in collaboration with a Postdoctoral scientist Dr. Deepak Shantharaj (not supported from this source of funding). The PhD student has given presentations in multiple venues and is now actively writing a manuscript to publish these results. How have the results been disseminated to communities of interest?Our efforts to disseminate this information are in their beginning stages, once we have evaluated a low-cost source of pectin (see below) that we think can be adopted for sustainable agricultural purposes then we will actively communicate this to stake holders. What do you plan to do during the next reporting period to accomplish the goals?A key aspect of this work has been the synergy between beneficial microorganisms and the use of complex carbohydrates such as pectin in stimulating their bioactivities - resulting in enhanced plant growth and disease control. In the next reporting period we plan to evaluate pectin-rich amendments that would be low cost and could be adopted by farmers. By using a pectin-rich "waste stream" such as beet pulp instead of a purified pectin purchased from a scientific supply source, this will importantly develop this research along the applied direction needed for it to be adopted to promote agricultural sustainability. The studies will involve greenhouse experiments at first, and if successful we hope to conduct a field study subsequently. In addition, we will continue our research concerning rhizobacteria that can prevent plant disease and promote plant growth. We plan to have more data concerning their bioactivie metabolites and identify a small subset of strains that have demonstrated efficacy in plant growth promotion and disease control that will be studied in more detail and with a greater number of replications.
Impacts
What was accomplished under these goals?
This year we have completed a soybean field trial at the Gulf Coast Research and Extension Center. The experimental design included soybean seeds planted with and without pectin amendment, and with and without the addition of a plant growth promoting rhizobacteria (PGPR) strain. All posssible combinations were evaluated with 2 PGPR strains (i.e. no PGPR strain, + PGPR, + pectin or + PGPR and + pectin) in evaluating soybean growth, yield and effects on soybean nodulation. While moderate increases in soybean growth and yield were observed when comparing PGPR + pectin vs. PGPR alone, the most significant finding was an increase in soybean nodulation as a result of pectin amendment during planting. This has been a consistent finding in greenhouse and now in field studies, and in this case we also included a Bradyrhizobium inoculum for all treatments as opposed to stimulation of the indigenous Bradyrhizobium populations in soil. These experiments supported the benefit of pectin in stimulating PGPR-mediated increases in plant growth and through as yet unknown mechanisms stimulating soybean nodulation. In addition, we have been able to characterize a collection of new rhizobacteria isolates that were isolated using complex carbohydrate (root extract) amended growth medium, and have identified many isolates that have the ability to inhibit the growth of plant pathogenic bacteria and fungi. These rhizobacteria have also shown some benefit in preliminary greenhouse experiments at controlling disease due to Xanthamonas under greenhous conditions. Experiments have also been initiated to characterize the metabolites produced by these isolates that have bioactivity.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2018
Citation:
M. K. Hassan, J. A. McInroy, J. Jones, D. Shantharaj, M. R. Liles, and J. Kloepper. Pectin amendments enhance soybean yield and nodulation mediated by plant growth promoting Bacillus velezensis strains. Manuscript in preparation for submission to Applied and Environmental Microbiology.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
3. M. K. Hassan, J. A. McInroy, J. Jones, D. Shantharaj, M. R. Liles, and J. Kloepper. Effect of pectin amendments on root colonization and plant growth-promotion by PGPR. 75th Professional Agricultural Workers Conference (PAWC), Tuskegee University, Tuskegee, AL, December 3-5, 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
2. M. K. Hassan, J. A. McInroy, J. Jones, D. Shantharaj, M. R. Liles, and J. Kloepper. Efficiency of pectin supplement in root colonization and plant growth-promotion of soybean plants by Bacillus velezensis. College of Agriculture poster showcase, Auburn University, Auburn, AL, October 26, 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
1. M. K. Hassan, J. A. McInroy, D. Shantharaj, M. R. Liles, and J. Kloepper. Effect of pectin amendments on root colonization and plant growth-promotion by PGPR. Student symposium, Auburn University, Auburn, AL, April 13, 2017.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students, one in the lab of each PI (Liles and Kloepper) have worked together on this study. The grad student in the Kloepper lab completed his Master's thesis on this project, and is now working on this project for his PhD dissertation. A manuscript based on his Master's thesis is in preparation. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In addition to plant growth promotion, another key aspect of this project will be to evaluate the combination of bacterial and pectin inocula on the prevention of plant disease due to bacterial or fungal plant pathogens. Other studies will evaluate the dose-response of the pectin-and bacterial mediated enhancement of plant growth and the production of bacterial derived bioactive compounds (plant hormones) in the different treatment groups.
Impacts
What was accomplished under these goals?
We have now tested the hypothesis that the complex carbohydrate pectin will enhance the bacterial (Bacillus amyloliquefaciens subsp. plantarum, or "Bap")-mediated plant growth promotion, in two highly replicated trials in soybean. The experimental design in both trials included soybean seeds planted in soil that contained Bap strains with or without 0.1% pectin supplement, along with a water control, and after 28 days the plants were harvested and analyzed for statistical significance. The data indicate that Bap strains with pectin supplement enhanced soybean shoot and root length in greenhouse conditions at a statitistically significant higher levels compared to the water, pectin only, or Bap only treatment groups. An unexpected and exciting result, observed in both trials, is that the combination of Bap strains with a pectin supplement in soil significantly increased soybean root nodulation. We observed an over 10-fold increase in nodulation, with the size of the nodules produced in plants that had a Bap inoculum and pectin supplementation greatly increased compared to a Bap strain alone. Whereas we had predicted the increased rate of root and shoot growth with the combination of bacterial and pectin inocula, the finding that nodulation was enhanced was a serendipitous discovery. We hypothesize that increased pectinolytic activity from the Bap plus pectin combination may result in enhanced Bradyrhizobium infection of soybean roots, since it has been shown before in the model legume Lotus japonicus that a mutant lacking pectate lyase activity was also deficient in nodulation. Based on the increased nodule frequency and size, we predict that the rhizobia metabolic activity within the nodules is increased relative to the no pectin controls, and that this results in greater nitrogen fixation rates. We further anticipate that the use of a pectin-rich soil amendment together with bacterial inoculants will be a cost-effective and sustainable method to promote plant growth and reduce the variability inherent in the use of beneficial bacterial strains in field soils. However, rhizobacterial populations recovered from the rhizosphere of soybean plant roots were not significantly different.
Publications
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