Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audiences for this research are commercial greenhouse growers, students,and scientists. Changes/Problems:Greenhouse and lab faciltiies were effectively closed in March 2020 due to COVID19, and we are currently working at reduced capacity with other restrictions for research in the lab and greenhouse. This has resulted in significant delays to research progress. All greenhouse experiments underway in spring 2020 were discarded and now they are being started over in fall 2020. With limited access to facilities due to social distancing, it is not possible to operate at previous research efficiencies. Graduate student progress has been delayed and at least one of the PhD students working on this project has had a one semester delay to graduation. Teleworking time has been effectively used during this pandemic for scientific writing and to translate scientific results into practical solutions and recommendations to growers via trade publications, blogs, newsletters,and webinars. What opportunities for training and professional development has the project provided?This project has provided training in greenhouse and laboratory research, scientifc writing,and extension and scientific presentation skills for two PhD students. How have the results been disseminated to communities of interest?Results have been disseminated to scientific audiences through presentations at scientific conferences and articles in peer-reviewed scientific journals. Results have been disseminated to commericial growers and extension educators through trade magazine publications, websites, webinars, and in person presentations at industry and extension conferences. What do you plan to do during the next reporting period to accomplish the goals?The bacteria identified in these trials will be evaluated in additional greenhouse crops, including herbs and vegetables. We will optimize application timing and rates, and determine how environmental conditions and cultural inputs impact efficacy. We will continue to screen this collection and the OSU Pseudomonas collection, to identify plant growth promoting bacteria with potential for formulation into greenhouse biostimulants. The analysis of genomic DNA sequence data is also ongoing. Many of the experiments originally planned for 2020 were cancelled due to COVID19 closures and restrictions.
Impacts
What was accomplished under these goals?
Solenostemon scutellarioides (coleus), Petunia × hybrida (petunia), Pelargonium × hortorum (geranium), Catharanthus roseus (vinca), and Zinnia elegans (zinnia) plants at the marketable stage were collected from 15 commercial greenhouses throughout Ohio. All plants were brought to the research greenhouses at the Ohio Agricultural Research and Development Center in Wooster, Ohio. After all the plants were watered to saturation, water was withheld until plant were severely wilted. Plants were then irrigated with clear water (no fertilizer) until they regained turgidity. This cycle of wilting and recovery was repeated a total of three times to subject all the plants to severe water stress. This stress treatment was imposed to increase the likelihood of selecting bacteria from the rhizosphere that could withstand water deficit stress. The rhizosphere is the narrow region of soil near the root surface that is influenced by root exudates and associated microorganisms (including rhizobacteria). After the water stress treatment, growing media was removed from the roots and a 5 g root sample was collected from each plant. Roots were vortexed and sonicated in sterile water and serial dilutions (10-2, 10-3, and 10-4) were plated on King's Media B (KMB) agar plates. KMB plates were incubated at 28° C for 48 h for growth, followed by 48 h at 4° C to allow for color development in the different bacteria colonies. Colonies with unique morphologies were collected to create a library of potential plant growth promoting bacteria for evaluation in a series of lab- and greenhouse-based trials. This library of bacteria is called the greenhouse rhizosphere collection. Individual bacteria were cultured in yeast extract mannitol (YEM) broth amended with 30% PEG (polyethylene glycol) to identify bacteria that could grow under osmotic stress. Bacteria were grown on media containing 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole nitrogen source to identify bacteria that produced the enzyme ACC deaminase. ACC deaminase degrades ACC, the precursor to ethylene, a stress signal molecule in plants. Plant growth is inhibited by ethylene that is produced by plants under stress, and ACC deaminase producing bacteria can reduce this growth inhibition by degrading ACC and reducing the production of ethylene by plants. ACC deaminase activity was quantified in any bacteria that grew on ACC. Eighty bacteria were selected by these two in-lab assays. Initial greenhouse experiments were conducted to evaluate the effect of the 80 individual bacterial isolates on drought tolerance of petunia plants. Plants were arranged in the greenhouse in a randomized complete block design. The individual bacteria cultures were drenched on the growing media weekly, starting at transplant. After all plants had been treated for 3 weeks, plants were subjected to water stress by discontinuing irrigation. Once plants were wilted, irrigation resumed, and plant recovery was determined by evaluating shoot dry weight and flower numbers. Ten top bacterial strains were selected that improved plant recovery when compared to untreated control plants. The genomes of these bacteria were sequenced for taxonomic identification of the strains and to identify genes potentially involved in conferring drought stress tolerance. The top ten selected bacteria were evaluated in a greenhouse experiment that measured other parameters of plant growth and health before, during, and after recovery from water deficit stress. These greenhouse experiments used petunia and geranium plants that were treated weekly with a media drench of the individual bacteria. Control plants were drenched with sterile LB. Irrigation was withheld starting at 5 weeks after transplant. Once plants were wilted, irrigation was resumed and plants were grown for 3 weeks to assess plant recovery and to determine if there were any differences in plant growth and health in plants treated with bacteria compared to control, untreated plants. Electron transport rate, quantum yield, and quantum efficiency of photosystem II were measured to evaluate plant health. These measurements were taken the day before the water stress treatment began (T0), at severe water stress (T2), and 3 days after rewatering (T3; during recovery). Electrolyte leakage from the leaves was measured at T1 and T3. Flower numbers were counted and shoot dry biomass were determined as indicators of plant growth. Statistical analyses were conducted in R using ANOVA. Factors with a significant p-value (p < 0.05) were compared to the negative control using Dunnett's test. The application of beneficial bacteria improved plant recovery after severe water deficit stress. This was observed as improved growth and flowering and increased photosynthesis. All ten of the selected bacteria resulted in increased shoot growth in water-stressed petunia plants compared to untreated control plants. Treatment with seven of the 10 bacteria resulted in increased flower numbers. Treatment with Arthrobacter sp. strain C9C5 resulted in the greatest increase in both biomass and flower numbers. Eight of the 10 bacterial strains resulted in increased shoot biomass in geranium, while the number of inflorescences was not significantly different. Treating petunia plants with Pseudomonas corrugata strain C7D2 resulted in the highest values for electron transport rate, quantum yield, and quantum efficiency of photosystem II throughout the stress (T2) and during recovery (T3). This indicates that the bacterial treatment resulted in less damage to the photosynthetic apparatus and caused quicker recovery to prestress values for photosynthesis. Pseudomonas brassicacearum strain C2F7 and Pseudarthrobacter sp. strain C4D7 had the greatest impact on maintaining the photosynthetic health of geranium plants after severe water deficit. Overall, the difference in electrolyte leakage before stress and during recovery was much less in plants treated with beneficial bacteria, indicating that bacterial treatment protected membranes and the result was less leaking of electrolytes from the cells. The long-term recovery of the plant after stress is dependent on maintaining the integrity of the cell and cell membrane structures. This study has identified a large collection of bacteria from the rhizosphere of greenhouse-grown ornamentals. We have demonstrated that plant growth promoting bacteria that improve recovery from abiotic stresses can be identified from this collection. We have also established a pipeline for isolation, screening and validating the growth promoting effects of bacteria that can be used to identify other strains of interest. Greenhouse ornamentals may be exposed to various stresses during shipping and retailing that reduce plant quality. PGPR applications may be used to improve the retail quality and shelf life of ornamental plants. We plan to continue to evaluate this collection using both laboratory and greenhouse-based screens to identify consortia of bacteria that can be used to promote plant growth with low fertilizer inputs and to improve tolerance to pathogens and environmental stresses.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Nordstedt N., and M.L. Jones (2020) Isolation of rhizosphere bacteria that improve quality and water stress tolerance in greenhouse ornamentals. Frontiers in Plant Science. 11:826. doi: 10.3389/fpls.2020.00826
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Nordstedt N.P., Chapin L.J., Taylor C.G. and M.L. Jones (2020) Pseudomonas spp. increase floriculture crop quality during abiotic stress. Frontiers in Plant Science. 10:1754. https://doi.org/10.3389/fpls.2019.01754.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
South K.A., Hand F.P., and M.L. Jones (2020) Beneficial bacteria identified for the control of Botrytis cinerea in petunia greenhouse production. Plant Disease. 104(6): 1801-1810. https://doi.org/10.1094/PDIS-10-19-2276-RE.
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Nordstedt N. and M.L. Jones (2020) Prime your plants for success with biostimulants. When applied early, biostimulants can increase post-harvest quality of greenhouse crops. Greenhouse Grower, Focus on Biostimulants 38(8) 26-28.
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Chapin L. and M.L. Jones (2020) Cut flower vaselife is extended when plants are produced with microbial biostimulants. The Cut Flower Quarterly, 32(3): 22-23.
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Konjoian P. and M. Jones (2020) Biostimulants in crop production: Performance optimization. Greenhouse Product News, April: 13-15.
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
South K., Chapin, L., and M.L. Jones (2020) Biocontrol of Botrytis in cut flowers. The Cut Flower Quarterly, 32(2): 28-30.
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Jones M. and K. South (2020). How to rid your greenhouse crops of Botrytis blight. Greenhouse Grower, Biocontrol Report, 38(2): 10-11.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Research scientists, extension professionals,commerical greenhouse growers and allied industry members. Changes/Problems:No changes were made to the approach. What opportunities for training and professional development has the project provided?This project has provided training in applied greenhouse research, physiology and microbiology to a PhD student. The student has had the opportunity to present this research at International scientific conferences and industry trade conferences. How have the results been disseminated to communities of interest?Results have been presented at scientific conferences and industry trade conferences. The research has also been submitted for publication in peer-reviewed scientific journals and has been published in trade magazines for industry audiences. What do you plan to do during the next reporting period to accomplish the goals?The bacteria identified in these trials will be evaluated in additional greenhouse crops, including herbs and vegetables. We will optimize application timing and rates, and determine how environmental conditions and cultural inputs impact efficacy.
Impacts
What was accomplished under these goals?
Major activities completed / experiments conducted: A high-throughput greenhouse trial identified two pseudomonads, P. poae 29G9 and P. fluorescens 90F12-2, that increased petunia biomass and flower number under both drought and low-nutrient conditions. A large-scale greenhouse trial was then conducted to validate the observed growth promotion and stress responses in multiple plant species. The greenhouse validation trial utilized three economically important greenhouse ornamentals: Petunia x hybrida 'Picobella Blue' (petunia), Viola x wittrockiana 'Delta Pure Red' (pansy), and Impatiens walleriana 'Super Elfin Ruby' (impatiens). This trial included a drought experiment and a low-nutrient experiment. Seedlings were transplanted into 11.4 cm pots containing peat-based growing substrate (Pro-Mix PGX, Premier Tech Horticulture). The experiments utilized a randomized complete block design with at least 13 blocks. Diluted bacterial cultures were drenched on the growing substrate weekly. There were three treatments in each of the experiments: control (no bacteria), bacterial strain 29G9, and bacterial strain 90F12-2. For the drought experiment, irrigation was withheld starting at three weeks after transplant until plants were visibly wilted. Irrigation was then resumed, and plant recovery was evaluated. For the low-nutrient experiment, plants were grown with 25 mg L-1 N from 15N-2.2P-12.5K-2.9Ca-1.2Mg water soluble fertilizer (JR Peters Inc.) at every irrigation to induce low-nutrient stress. Data collected: After the drought stress treatment, irrigation and bacterial treatments were resumed and plants were grown to flowering to assess recovery. Plants were harvested when all plants had at least two open flowers. Flower numbers were counted and shoot dry weights were measured after drying tissue at 50° C for 3 days. For the low-nutrient experiment, plants were also grown to flowering. Shoot dry weights were measured and flower numbers were recorded at harvest. Tissue nutrient content was determined by the STAR lab (Wooster, OH). Summary statistics and discussion of results: Multi-species greenhouse validation trials confirmed that the two Pseudomonas strains (29G9 and 90F12-2) improved plant recovery and growth after drought stress in a greenhouse system. After irrigation was resumed, bacteria treated plants stayed dark green in color, while leaf yellowing was observed in the control (untreated) plants. Flower numbers were greater in the bacteria treated impatiens and petunias, with an average of 10 more flowers per plant. The biomass of the shoots was consistently higher after drought stress in all three plants species treated with 29G9 and 90F12-2 compared to control plants. Bacterial treatments also improved plant growth and nutrient uptake in plants grown under low-nutrient conditions. Shoot biomass was greater in all three plant species grown under low nutrient conditions when treated with bacteria, and flower numbers were higher in impatiens treated with 29G9. Pansy and impatiens control plants (no bacteria) had severe leaf chlorosis (yellowing), while bacteria treated plants were consistently dark green. Bacteria treated plants grown under low-nutrient conditions had higher leaf nutrient content compared to the untreated control plants. The nitrogen (N) content of leaf tissue showed the greatest increase in all three plant species. Tissue N content was increased by at least 41% in petunias and by 78% in both impatiens and pansies. In pansies, both bacterial strains also increased tissue phosphorus, potassium, calcium, magnesium, and sulfur content. Key outcomes or other accomplishments realized: These experiments provide a promising utility for these unique Pseudomonas strains to improve floriculture crop quality during times of environmental (abiotic) stress. The greenhouse trials identified two bacterial strains that could enhance growth and improve recovery after drought stress. This can be used to increase shelf life and reduce losses that are the result of water stress. These bacteria also improved nutrient uptake and enhanced growth with lower fertilizer inputs. Reducing fertilizer inputs can reduce fertilizer run off and environmental contamination. This research will contribute to the formulation of biostimulant products marketed for the greenhouse industry to enhance growth and abiotic stress tolerance. Microbial-based biostimulants can be used to reduce chemical inputs and make existing greenhouse practices more economically and environmentally sustainable.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Jones, M.L. and Nordstedt N.P. (2019) Application of Pseudomonas spp. Increases Floriculture Crop Quality During Abiotic Stress. HORTSCIENCE Vol. 54(9S): S103.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Chapin, L.J, Nordstedt N.P, and Jones M,L. (2019) Beneficial microorganisms can enhance greenhouse floriculture plant performance under reduced fertility. PHYTOPHATHOLOGY Vol. 109 (10S): S2.45. https://doi.org/10.1094/PHYTO-109-10-S2.1
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
M.L. Jones (2019) Identifying beneficial bacteria that enhance abiotic stress tolerance in horticulture crops. Global Conference on Plant Science and Molecular Biology, London, England.
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
Jones ML and N. Nordstedt. Sustainable Products, Broad Solutions: Biostimulants and Greenhouse Ornamentals. Greenhouse Grower Vol. 37 (No. 6), Pages 6- 8 (Special Report Biostimulants). June 2019.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Nordstedt N.P, Chapin L.J., Taylor C.G., and M.L. Jones (2019) Pseudomonas spp. increase floriculture crop quality during abiotic stress. Frontiers in Plant Science. Submitted.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2109
Citation:
Nordstedt NP and ML Jones (2019) Pseudomonas spp. Enhance Plant Growth Under Multiple Abiotic Stresses. American Society of Plant Biologists Midwest Regional Meeting.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Jones, M.L. and Nordstedt N.P. (2019) Pseudomonas spp. stimulate plant growth under multiple abiotic stresses. American Society of Plant Biologists Annual Meeting.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Jones M.L., Nordstedt N., and Mattson N. (2019) Biostimulants: Another Tool for the Growers Toolbox. AmericanHort Cultivate 19. Columbus, OH.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audiences for this project are plant science researchers interested in plant-microbe interactions, extension educators and greenhouse professionals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided research, extension and outreach training to two PhD students. How have the results been disseminated to communities of interest?Results have been disseminated through oral presentations at the American Society for Horticultural Sciences Annual Conference. What do you plan to do during the next reporting period to accomplish the goals?The strains identified from the high-throughput greenhouse trials and in-lab assays will be validated in a production-scale greenhouse trial that includes multiple economically-important floriculture crops. This trial will provide evidence for the utility of these beneficial bacteria in greenhouse production systems. The environmental conditions and cultural inputs that impact efficacy will be evaluated.
Impacts
What was accomplished under these goals?
Identifying beneficial bacteria: Major activities completed / experiments conducted: Two in-lab bioassays were optimized to screen a collection of 62 Pseudomonas strains and select for those with the ability to withstand osmotic stress or produce the enzyme ACC deaminase. In addition, a high-throughput greenhouse trial was developed to test these selected strains for their ability to improve floriculture crop quality when applied to plants growing under either low nutrient or drought conditions. A third in-lab assay was completed to screen the collection to identify those with the for the ability to reduce the growth of Botrytis cinerea, a fungal pathogen that causes large plant losses in the floriculture industry every year. Data collected: For the osmotic stress tolerance bioassay, data was collected on how well the bacteria were able to grow in liquid media with a low water potential. Strains that were able to grow over a predetermined threshold were selected for further evaluation. An enzyme activity assay was utilized to measure the amount of alpha-ketobutyrate produced (a byproduct of ACC deaminase activity), allowing for the quantification of ACC deaminase enzyme activity from each strain. For the high-throughput greenhouse trial, data was collected on plant size and flower number, two important factors of floriculture crop quality, to serve as an indicator of the bacteria's ability to promote plant growth. For the in-lab assay evaluating the control of Botrytis cinerea, each Pseudomonas strain was grown on a PDA plate with Botrytis. After incubating the plates, the horizontal and vertical growth of the Botrytis was measured. In addition, a zone of inhibition or the distance between the bacteria and Botrytis was measured. Summary statistics and discussion of results: The bioassays for osmotic stress tolerance and ACC deaminase activity resulted in the identification of fourteen and six strains, respectively. These strains were evaluated in the high-throughput greenhouse trial and three elite strains were identified to have the ability to increase flower number and plant biomass above the uninoculated control under both drought and low nutrient conditions. For the in-lab assay for Botrytis cinerea control, the growth of the Botrytis on plates containing bacteria were compared to those that did not contain bacteria (control). This comparison allowed the reduction of Botrytis growth to be determined for each bacteria strain evaluated. The top 20 performing strains were selected based on their ability to reduce the growth of Botrytis on PDA plates. Key outcomes or other accomplishments realized: These experiments provide a promising utility for these unique Pseudomonas strains to improve floriculture crop quality during times of abiotic and biotic stress. The in-lab bioassays and high-throughput greenhouse trials can be used to evaluate additional strains of interest in future work. Evaluating microbe-containing biostimulant products: Major activities completed / experiments conducted: A greenhouse trial was conducted with 11 commercially available microbe-containing products that are labeled for greenhouse plants and have been reported to have biostimulant effects. Petunia plants were treated with the products according to label recommendations starting at the time of transplant for a total of 6 weeks. Data collected: Plant performance was evaluated throughout the experiment by calculating plant growth index. At the termination of the experiment total flower number, shoot biomass, and chlorophyll content were measured. Summary statistics and discussion of results: Two products resulted in petunia plants with significantly larger growth indices, higher shoot biomass, and more flowers than the negative control (untreated) plants. The petunia plants treated with one of these products had higher chlorophyll content than negative control. Key outcomes or other accomplishments realized: Commercially available microbe-containing biostimulant products can enhance floriculture crop growth and quality, but the conditions needed to optimize their efficacy in the greenhouse must still be determined.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
South K.A., Taylor C., and M.L. Jones (2018) An in vitro study identifies possible bacteria candidates for Botrytis cinerea biocontrol. American Society for Horticultural Science Annual Conference, Washington, D.C. HortScience Supplement.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Nordstedt N., Taylor C., and M.L. Jones (2018) Identification of Rhizobacteria with the ability to increase floriculture crop quality. American Society for Horticultural Science Annual Conference, Washington, D.C. HortScience Supplement.
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