Progress report for GNE19-207
Fluctuations in climate and sea-level rise have increased the frequency and harshness of drought and other stresses impairing agricultural production. Kale, Brassica oleracea, is a good source of health-beneficial substances. However, locally produced kale only fulfilled 20.9% of the amount consumed in Maryland in 2012. Ensuring adequate and reliable production of kale under drought and high salinity conditions are crucial for increasing net farm income and diversifying produce available to consumers. The purpose of this project is to support the agricultural production of safe and nutritious kale in Maryland in a way that sustains soil health, improves productivity, and enhances resilience to abiotic stresses such as drought and high soil salinity. This project proposes to investigate PGPR as a way to mitigate abiotic (drought and salinity) and biotic (human pathogen) stresses.
This study will assess the effects of PGPR on biomass accumulation and nutritional value of kale plants, as well as resilience to abiotic stresses and S. enterica association under regular watering, irrigation-withholding conditions, and salt stress. Plant growth-promoting effects of P. putida will be assessed by measuring plant biomass accumulation and leaf chlorophyll content. Levels of bioactive substances and antioxidant capacity will be measured between plants colonized with PGPR and control plants. Untargeted metabolome profiling and quantification of phenolics, flavonoids, and glucosinolates in leaf tissue and exudates will help understand the interaction between physiological changes induced by abiotic stresses, PGPR, and S. enterica colonization.
As for now, the results showed that shoot biomass accumulation of PGPR inoculated kale plants did not increase. This might due to the short growth time, thus we decide to extend the growth time for kale plants to 59 days post-germination when the kale plants are usually ready for harvest. The inoculation of PGPR has caused changes to the antioxidant capacity, total phenolics, and glucosinolates under both watering regimes. After 6 days of drought treatment, the surface survival of Salmonella Newport on the P. putida S4-treated regularly watering kale plants was significantly lower than negative control kale plants. However, there was no difference regarding the survival of S. Newport on drought-treated negative control and PGPR-treated kale plants. After 24 hours, the growth of Salmonella Newport in exudates collected from regular watering kale plants inoculated with P. putida S4 was significantly higher than regular watering kale plants inoculated with P. putida S2.
This project aims to evaluate the effects of Pseudomonas putida strains on the promotion of kale growth, nutritional value and resilience to drought, salinity and the human pathogen S. enterica under restricted water and high salt conditions. Specifically, this study proposes to investigate how kale plants can mitigate abiotic (drought and salinity) and biotic (S. enterica) stresses in an age-dependent manner in the presence or absence of PGPR strains of P. putida strains S2 and S4 colonizing roots. The objectives of this study are to address three aspects of crop production, growth promotion, nutritional quality, and microbial safety.
The specific aims are described below:
1) Promoting Growth: Investigate the effect of root colonization of kale plants with P. putida on plant shoot and root biomass in drought-affected, salt-affected and regularly watered plants. Assessment of biomass accumulation and leaf chlorophyll content using kale plants from different treatments will determine if P. putida associating with kale roots enhance root and shoot biomass accumulation and promoting plant growth under regular watering conditions and protecting kale plants from drought and salt stress without reducing biomass.
2) Improving Nutritional Quality: Investigate the effect of root colonization of kale plants with P. putida on the major health-beneficial bioactive substances of kale plants under regular watering, irrigation-withholding conditions, and salt stress. Assessment of kale nutritional value under different treatments will determine if P. putida association with roots induces the synthesis of bioactive compounds under regular watering conditions and protecting kale plants from stress while increasing nutritional value.
3) Ensuring Microbial Safety: Assess the effect of root colonization of kale plants with P. putida on the metabolomic and exo-metabolomic (surface) profiles of leaf tissue and exudates and leaf surface survival of S. enterica Newport under regular watering, irrigation withholding conditions, and salt stress. This measurement will reveal if P. putida create a less favorable surface environment for S. Newport under different treatments and change the phytonutrient landscape for bacteria.
4) Providing Educational Opportunities: Promote kale farming and the use of P. putida and other PGPR strains with growers in a sustainable and safe manner. I will provide information on kale production to Maryland growers and point out the need to increase kale farming. I will attend meetings with local growers, present my research and disseminate factsheets summarizing key findings. These seminars aim to promote kale farming and the use of PGPR among growers while considering agricultural and environmental sustainability.
The purpose of this project is to support the agricultural production of safe and nutritious kale in Maryland in a way that sustains soil health, improves productivity and enhances resilience to abiotic stresses such as drought and high soil salinity. This project proposes to investigate PGPR as a way to mitigate abiotic (drought and salinity) and biotic (human pathogen) stresses. The key themes in sustainable agriculture being addressed are “reduction of environmental and health risks in agriculture, and improved productivity and the reduction of costs”. Kale is one of the ten most economically important vegetables in the global agriculture markets. Yet, in Maryland, kale production only fulfilled 20.9% of the total amount consumed in 2012. Environmental stress can greatly hamper productivity and quality. A 0.6 to 1.1℃ rise in temperature and intensive drought stress were observed in the last century in Maryland. Moreover, one of Maryland’s most important agricultural regions, the Eastern Shore, is experiencing a high sea-level rise, and coupled with subsidence and groundwater extraction is exacerbating saltwater intrusion into aquifers and soil that is affecting agriculture. Drought stress and high salinity reduce the leaf area, fresh and dry weight of kale plants. Thickening and whitening of cuticles were reported under drought, which is detrimental to the texture of kale. Additionally, the closure of stomata under drought and high salinity limits the photosynthesis of kale and affects the accumulation of bioactive substances, and in turn, may impact the nutritional value of kale.
Bacteria colonizing roots and the rhizosphere, the component of soil under the influence of roots, can enhance the growth of plants and are called plant growth-promoting rhizobacteria or PGPR. PGPR have been widely recognized as biofertilizers. Higher yields have been recorded in potatoes, radishes, rice, tomato and apples as a result of PGPR inoculation. When plants are under drought and high salinity stress, PGPR are capable of protecting plants by adjusting osmolyte and reducing oxidative damage. Plant metabolism may be altered following the inoculation of PGPR strains, which may impact the nutritional value of crops, but data are lacking in this area and how plant nutritional value is affected by PGPR is not well-studied. In-depth research is required to investigate the impact of PGPR not only on crop productivity but also on plant stress mitigation and nutritional value, especially for frequently consumed leafy greens.
Aside from productivity, food safety is another important aspect affecting the economic viability of agricultural crops that are consumed raw. The presence of human pathogens such as Salmonella enterica on produce can cause foodborne illness outbreaks, recalls and crop culling, which has enormous public health and economic burden on communities and growers. Foodborne pathogens can be introduced to fields by wildlife, or through contaminated irrigation water or fertilizer. Once human pathogens such as Salmonella enterica associated with plants, the availability of nutrients on plant surfaces is a major determinant of successful colonization of human pathogens. This study will assess the relationship between abiotic stresses and plant metabolome.
In this proposal, I aim to study the beneficial effects of PGPR strains of Pseudomonas putida on growth, nutritional and food safety aspects of kale plants under drought and high salinity stresses. I will disseminate data to growers through local grower meetings. Deliverables from this study will augment current knowledge of PGPR in sustainable agriculture and ways to enhance kale production, nutritional value, safety, and resilience.
Plant and Bacterial Preparation:
PGPR inocula: Two rifampicin (rif) resistant Pseudomonas putida strains S2 and S4 (Dr. Brain Klubek (Southern Illinois University Carbondale) have been previously in our lab . Frozen stock of P. putida strains will be streaked on Trypticase Soy Agar (TSA) plates and incubated at 28°C for 48 hours. Single colonies will be transferred to Trypticase Soy Broth (TSB) and grow to late-log phase. The suspension will be pelletized at 10,000 rpm for 15 min and diluted with 0.1% peptone water (PW) to OD600=0.8.
PGPR inoculation: Kale plants will receive 2 mL of 108 CFU/mL bacterial suspension or 0.1% PW (uninoculated control) at the base of stems. Two separate root inoculations will be carried out 2-days and 9-days post-germination. Successful root colonization with each strain of P. putida will be confirmed by colony enumeration of root rinsates on TSA-rif plates upon completion of experiments.
Plant preparation: Kale cultivar ‘Improved Dwarf’ seeds will be sowed in plastic pots filled with Sunshine Professional Growing Mix #1 LC1 (Sungro Horticulture, Agawam, MA) and transferred to single pots after germination, to be grown in a growth chamber (16 hours light at 23°C/8 hours dark at 18°C in 50% relative humidity). Regular watering will follow until drought or salinity stress is imposed.
Drought treatment: Two weeks post-germination, kale plants will be subjected to irrigation-withholding for 6 days or watered regularly.
1 Growth Promoting:
Biomass measurement: After drought treatment, the PGPR-treated and uninoculated kale plants will be clipped at the base, put in a pre-weighed foil tray and baked in an oven at 70°C for 48 h for shoot dry weight measurements.
2 Microbial Safety:
Salmonella Newport inocula: The S. enterica Newport strain to be used is a tomato outbreak strain  adapted for rifampicin resistance. Single colony of S. Newport grown on TSA with 50 mg/mL rifampin will be streaked on fresh TSA-rif plates and grown at 35°C overnight. Inocula will be made by suspending bacterial colonies in 0.1% PW to an OD600=0.5, to obtain a cell density of ~109 CFU/mL. A 100-fold dilution of this suspension will be used for leaf inoculation.
S. Newport inoculation: An aliquot of 100 ml of Newport inoula will be pipetted onto the adaxial side of the marked third or fourth true leaf of each plant. Final concentration of bacteria applied will be around 106 CFU/mL. The actual inoculum level will be determined on TSA-rif plates.
S. Newport retrieval from inoculated leaves: Inoculated leaves will be harvested 24 h post-inoculation, by clipping leaves off the stem aseptically and placed in a sterile bag, immersed in 30 mL of 0.1% PW. Bag will be hand massaged for 30 s, sonicated for 1 min to dislodge attached Salmonella cells and shaken at 150 rpm for 10 min, Serial dilutions will be prepared from leaf rinsates for S. Newport enumeration on TSA-rif plates.
Exudates collection: Whole plants will be immersed in 30 mL 5% methanol and shaken at 150 rpm for 24 h at room temperature to collect exudates. The solution will be filtered through 0.45 m sterile syringe filters and lyophilized in a freeze dryer. The dried powder will be collected for exo-metabolomic profiling.
1 Growth Promoting: Biomass measurement results:
As for now, the results showed that shoot biomass accumulation of either PGPR Pseudomonas putida S2 or S4 inoculated kale plants did not significantly increase after 20 days.
This result does not match our hypothesis. However, given 20 days might not be long enough for kale plants to go through the whole vegetative stage. We decided to measure the shoot biomass after 59-day of kale plant growth when all the kale plants are ready to sell.
2 Microbial Safety:
Surface inoculation with Salmonella Newport:
The surface survival of Salmonella Newport on the P. putida S4-treated regularly watering 20-day-ola kale plants was significantly lower than negative control kale plants (p< 0.05). However, there was no difference regarding the survival of S. Newport on 6-days drought-treated negative control and PGPR-treated kale plants.
Growth Curve of Salmonella Newport in plant exudates
There were no significant differences regarding the retrieval of Salmonella within 24 hours in exudates from all plants under different treatments.
The lower retrieval of Salmonella Newport was found on P. putida S4 inoculated regularly watered 20-day-old kale plants surface. However, the retrieval of S. Newport in exudates using the same P. ptuida S4-treated plants did not show any differences within 24 hours compared to any other treatments. This might indicate that more replicates are needed to make sure that the results are solid. In the meantime, these results will push us to do chemical analyses and HPLC-MS analyses on exudates from all treatments.
As for now, our conclusion is that plant growth-promoting rhizobacteria Pseudomonas putida S4-treated regularly watered young plants created a more resistant environment to human pathogen Salmonella on their surfaces.
Education & Outreach Activities and Participation Summary
I am working on a factsheet as an introduction to growers in Maryland with three other Ph.D. students at the University of Maryland. This factsheet will be submitted to the UMD Extension.
To date, research results indicate that after 20-days, kale plants inoculated with Pseudomonas putida S2 and S4 appeared to not show plant growth-promoting effects. For all regularly watered plants, the PGPR inoculated plants showed significantly lower biomass accumulation after 20 days. Drought-subjected kale plants that were inoculated with P. putida S2 accumulated less biomass than drought-subjected plants without any PGPR inoculation. This might be due to the inoculation of PGPR, which impacts the physiology of kale plants, affecting the biomass accumulation of kale plants. It may also be attributed to the juvenile growth stage analyzed. These experiments are being repeated for different developmental stages. In this case, we decided to extend the plant growth time until 59-days when kale plants are ready for sale. If we could choose the correct PGPR that can increase the nutritional values, yield, and microbial safety of kale and other leafy greens, we then could promote the use of these PGPR as commercial inoculants for growers not only in the northeast but also across the state.
Biochemical measurements have been conducted to identify the impact of PGPR on improving nutritional quality. For kale plants under regular watering condition, PGPR inoculated kale plants showed no difference in antioxidant capacity compared to non-PGPR inoculated negative plants. A similar trend was found when comparing the antioxidant capacity among drought-treated kale plants; PGPR inoculated plants showed no significant difference with non-PGPR inoculated negative plants. However, for P. putida S2 or S4 inoculated kale plants, the antioxidant capacity of drought-treated plants was significantly higher than regular watering plants. Similarly, drought-treated non-PGPR negative kale plants showed higher antioxidant capacity than regularly watered kale plants. The inoculation of PGPR did not cause any difference in the content of flavonoids among all kale plants under both watering regimes. Drought-treated kale plants inoculated with P. putida S4 showed a significantly higher accumulation of total phenolics compared to regularly watered kale plants inoculated with P. putida S4. Similar results were reported for PGPR-negative plants, drought-induced the accumulation of total phenolics. However, for all kale plants under both regular watering and drought, the inoculation of PGPR did not cause a change in total phenolics. The inoculation of both P.putida S2 and S4 significantly increased the accumulation of glucosinolates in drought-treated plants compared to regularly watered plants. However, the inoculation of PGPR did not induce an increase in the accumulation of glucosinolates among all kale plants under the same watering regime.
The inoculation of P. putida S4 reduced the surface survival of S. Newport compared to PGPR-negative plants, for kale plants under regular watering conditions. However, under the influence of drought treatment, the impact of PGPR was not detected anymore. The growth of Salmonella Newport after 24 hours in exudate collected from regular watering plants showed that exudates from P. putida S4 inoculated plants supported higher growth than exudates from P. putida S2 inoculated plants. After 24 hours, for all pair-wise comparisons, the growth of S. Newport in exudate collected from regularly watered plants was significantly higher than in exudate from drought-treated plants for both PGPR inoculated and PGPR-negative kale plants.
In this project, I continued to learn about plant growth-promoting rhizobacteria (PGPR) and made a factsheet regarding PGPR with three other teammates at the University of Maryland. These experiences educated me as a better researcher, writer, and communicator. It also gave me the opportunity to translate the research to a stakeholder audience.
We had one more collaborator, Dr. Yue Li, from the Department of Chemistry and Biochemistry at the University of Maryland, who worked with us for High-Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS). For now, we have plant samples ready. The analyses will be processed in Spring 2020.
A no-cost extension was granted for one year since the research was severely disrupted by COVID measures.