Final report for GNE19-207
Project Information
Climate change is increasing the frequency of unfavorable weather events for agriculture, subjecting crops to more prolonged abiotic stresses such as drought and increased salinity in the soil. Kale is a popular and nutritious commodity that can be marketed as a value-added product. This study assessed the effects of a plant growth-promoting rhizobacterium (PGPR) Pseudomonas spp. on the growth and nutritional value of kale plants, as well as resilience to abiotic stresses and association with the human foodborne pathogen Salmonella enterica. Kale was evaluated under regular watering and irrigation-withholding conditions, as well as under salt stress, at different marketable developmental stages (baby and mature leaves). Phytocompounds measured were phenolics, flavonoids, and glucosinolates. At different plant growth stages, the effect of the physiological changes induced by abiotic stresses and bacterial colonization on nutritional content and food safety risk were explored.
Drought and salinity stress reduced the biomass of kale plants and the inoculation with PGPR did not promote plant growth. However, the biomass of mature kale plants inoculated with PGPR was not further reduced by drought, suggesting that PGPR had a protective effect on plant growth under stress. Mature plants grown under regular watering conditions (control) accumulated more bioactive compounds than young plants (baby leaf stage) and could therefore be considered of higher nutritional quality. PGPR-inoculated, drought-subjected, and salinity-affected baby kale plants had higher levels of bioactive compounds than regularly watered plants not colonized by PGPR. Our findings showed that regulated abiotic stresses (drought and salinity) and biotic interactions (PGPR root colonization) can enhance the nutritional quality of baby kale. Moreover, at both baby and mature kale stages, PGPR-inoculated, drought- and salinity-subjected kale plant surfaces were less favorable to Salmonella. Abiotic stresses and PGPR, therefore, had a positive effect on food safety outcomes. This enhanced resistance to Salmonella also occurred as kale matured when plants were grown under regular conditions. Application of regulated stresses or inoculation with PGPR to baby kale are promising strategies to enhance the nutrition value and food safety of this popular green.
This project aimed to evaluate the effects of Pseudomonas spp. S4 on the promotion of kale growth, nutritional value, and resilience to drought, salinity, and the human pathogen Salmonella enterica under restricted water and various salt conditions. Specifically, this study proposed to investigate how kale plants could mitigate abiotic (drought and salinity) and biotic (Salmonella enterica) stresses in an age-dependent manner in the presence or absence of PGPR strain of Pseudomonas spp. S4 colonizing roots. The objectives of this study were to address three aspects of crop production, nutritional quality, and microbial safety.
The specific aims were described below:
1) Promoting Growth: Investigation of the effect of root colonization of kale plants with Pseudomonas spp. S4 on plant shoot biomass in drought-affected, salt-affected, and regularly watered plants. Assessment of biomass accumulation using kale plants from different treatments could determine if Pseudomonas spp. S4 associated with kale roots could enhance shoot biomass accumulation, promote plant growth under regular watering conditions, and protect kale plants from drought and salt stress without reducing biomass.
2) Improving Nutritional Quality: Determination of the effect of root colonization of kale plants with Pseudomonas spp. S4 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 could determine if Pseudomonas spp. association with roots could induce the synthesis of bioactive compounds under regular watering conditions and protect kale plants from stress while increasing nutritional value.
3) Ensuring Microbial Safety: Assessment of the effect of root colonization of kale plants with Pseudomonas 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 could reveal if Pseudomonas spp. could create a less favorable surface environment for S. Newport under different treatments and change the phytonutrient landscape for bacteria.
The purpose of this project was to support the agricultural production of safe and nutritious kale in Maryland in a way that could sustain soil health, improve productivity, and enhance resilience to abiotic stresses such as drought and high soil salinity. This project proposed to investigate PGPR to mitigate abiotic (drought and salinity) and biotic (human pathogen) stresses. The key themes in sustainable agriculture being addressed were “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 could 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 could reduce the leaf area, fresh and dry weight of kale plants. Thickening and whitening of cuticles were reported under drought, which was detrimental to the texture of kale. Additionally, the closure of stomata under drought and high salinity could limit 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, could enhance the growth of plants and are called plant growth-promoting rhizobacteria or PGPR. PGPR have been widely recognized as biofertilizers. When plants are under drought and high salinity stress, PGPR protect plants by adjusting osmolyte and reducing oxidative damage. Our study found a positive effect of PGPR when kale was under stress. Plant metabolism may be altered following the inoculation of PGPR strains, which may impact the nutritional value of crops, but data were lacking in this area and how plant nutritional value is affected by PGPR was not well-studied. Our study showed that PGPR enhanced the levels of certain bioactive compounds in baby kale. This study contributed new data about the impact of PGPR not only on crop productivity but also plant stress mitigation and nutritional value for an understudied leafy green whose consumption is on the increase.
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 could cause foodborne illness outbreaks, recalls, and crop culling, which have 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 are associated with plants, the availability of nutrients on plant surfaces is a major determinant of the successful colonization of human pathogens. This study assessed the relationship between abiotic stresses and the plant metabolome.
In this proposal, I studied the beneficial effects of PGPR strains of Pseudomonas spp. on the plant growth, nutritional, and food safety aspects of kale plants under drought and high salinity stresses. I disseminated data through a factsheet and at scientific meetings and prepared our findings for publication in peer-reviewed journals. Deliverables from this study augmented current knowledge of PGPR in sustainable agriculture and ways to enhance kale production, nutritional value, safety, and resilience.
Research
This study assessed 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. spp. were assessed by measuring plant biomass accumulation. Levels of bioactive substances and antioxidant capacity were measured in comparison between plants colonized with PGPR and uninoculated plants. Untargeted metabolome profiling and quantification of phenolics, flavonoids, and glucosinolates in leaf tissue and exudates helped to understand the interaction between physiological changes induced by abiotic stresses, PGPR, and S. enterica colonization.
Plant and Bacterial Preparation:
PGPR inocula: The rifampicin (rif) resistant Pseudomonas spp. strains S4 (Dr. Brain Klubek (Southern Illinois University Carbondale) has been previously in our lab. Frozen stock of P. spp. strains was streaked on Trypticase Soy Agar (TSA) plates and incubated at 28°C for 48 hours. A single colony was transferred to Trypticase Soy Broth (TSB) and grow to the late-log phase. The suspension was pelletized at 10,000 rpm for 15 min and diluted with 0.1% peptone water (PW) to OD600=0.8.
PGPR inoculation: Kale plants received 2 mL of 108 CFU/mL bacterial suspension or 0.1% PW (PGPR-negative) at the base of stems. Two separate root inoculations were carried out 2-days and 9-days post-germination.
Plant preparation: Kale cultivar ‘Improved Dwarf’ seeds were 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 was followed until drought or salinity stress was imposed.
Baby kale and drought treatment: Two weeks post-germination, kale plants were subjected to irrigation-withholding for 6 days or watered regularly (control).
Mature kale and drought treatment: Eight weeks post-germination, kale plants were subjected to irrigation withholding for 3 days or watered regularly (control).
Salinity treatment: Sodium Chlorine (NaCl) was used to create salinity stress for kale plants. Three weeks post-germination, kale plants were subjected to high salt condition (15 g/L, HS), medium salt (5 g/L, MS) for 5 days, or watered regularly (negative control, NC).
Objective 1: Promoting Growth
Biomass measurement: After drought or salinity treatment, the PGPR-treated and PGPR-negative kale plants were 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.
Objective 2: Improving Nutritional Quality
Leaf sample preparation: Kale leaves were flash-frozen and ground into powder using liquid nitrogen and 200 mg of powder was extracted with 1.5 mL 70% methanol/0.5% formic acid. Samples were fully vortexed and allowed to stand overnight at room temperature, followed by centrifugation at 5,000 g for 10 min. The supernatants were used for nutritional quality measurements.
Antioxidant capacity: To prepare the reagent, 31.7 mg 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) (TCI, OR, USA) and 8.6 mg potassium persulfate (Aldon Corp Avon, NY, USA) were dissolved in 10 ml water and allowed to stand in darkness at room temperature for 16 h to form a stable radical, then diluted to absorption of ~1.3 at 734 nm. Twenty μL sample extracts were added to 200 μL ABTS solution and allowed to stand for 15 min, then absorption was measured at 734 nm. A standard curve from the blank-corrected absorption at 734 nm of Trolox (TCI) standards was plotted. Antioxidant capacity was calculated as Trolox equivalent using the standard curve.
Total flavonoids: One hundred μL kale leaf extract were added to 20 μL 5% NaNO2 (Avantor, PA, USA). After 5 min, 20 μL 10% AlCl3 (Alfa Aesar, MA, USA) was added followed by 100 μL 1 mol/L NaOH (VWR Chemicals, BDH) 1 min later. The absorption of the mixture was measured at 510 nm on the microplate reader. A standard curve from the blank-corrected absorption at 510 nm of the catechin standards (Enzo Life Sciences, NY, USA) was plotted. Antioxidant capacity was calculated as catechin equivalent using the standard curve.
Total phenolic content: Eighty μL kale leaf extract were mixed with 900 μL 10% Folin Ciocalteu reagent (MP Biomedicals, CA, USA), then 400 μL 700 mM Na2CO3 (VWR Chemicals, BDH, Radnor, PA, USA) were added in a 96-well plate. All samples were fully vortexed and left to stand for 1 h in a microplate reader at 25℃. Absorption measurements were taken at 765 nm. A standard curve from the blank-corrected absorption values at 765 nm of the gallic acid (Acros Organics Fair Lawn, NJ, USA) standards was plotted. Total phenolics were calculated as gallic acid equivalents using the standard curve.
Estimated glucosinolate content: One hundred μL kale leaf extract was added to 300 μL 2 mM sodium tetrachloropalladate (Acros Organics). The samples were incubated at room temperature for 1 h and absorption was measured at 425 nm. Water was then used as a blank. Total glucosinolates were calculated by putting the OD425 of each sample into the predicted formula: y=1.40+ 118.86*A425.
Objective 3: Ensuring Microbial Safety
Salmonella enterica inocula: Two strains of Salmonella enterica were used in this project. The S. enterica Newport strain used was a tomato outbreak strain adapted for rifampicin resistance. Salmonella Javiana was isolated from pond water and adapted to rifampicin resistance. A single colony of Salmonella grown on TSA with 50 mg/mL rifampin was streaked on fresh TSA-rif plates and grown at 35°C overnight. Inocula were 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 was used for leaf inoculation. S. Newport was used to assess the microbial safety of plants under drought and salinity stress and regular watering condition. S. Javiana was used only to assess the microbial safety of kale plants under salinity stress or regular watering condition.
S. enterica leaf inoculation: An aliquot of 100 ml of Newport or Javiana inoula was pipetted onto the adaxial side of the marked third or fourth true leaf of each plant. Final concentration of bacteria applied was around 106 CFU/mL. The actual inoculum level was determined on TSA-rif plates. Leaf inoculation with Salmonella Newport was conducted on drought-treated, salinity-stressed, and regular watering kale plants. Inoculation of Salmonella Javiana was conducted only on salinity-stressed regular watering kale plants.
S. enterica retrieval from inoculated leaves: Inoculated leaves were 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. The bag was hand massaged for 30 s, sonicated for 1 min to dislodge attached Salmonella cells, and shaken at 150 rpm for 10 min, Serial dilutions were prepared from leaf rinsates for S. Newport or Javiana enumeration on TSA-rif plates.
Exudates collection for Salmonella Newport growth assessment: Whole plants were immersed in 30 mL 5% methanol and shaken at 150 rpm for 24 h at room temperature to collect exudates. The solution was filtered through 0.45 m sterile syringe filters. Two ml of exudate aliquots were used for further analysis. Exudates were collected from baby and mature kale under drought stress or regular watering condition.
S. enterica exudate inoculation and retrieval: Twenty μl of Newport inoculum were added to 2 ml exudate solution at an initial concentration of ~106 CFU/mL and incubated at 35 ℃ with shaking at 150 rpm. S. Newport cell counts were monitored after 24 hours post-inoculation. Serial dilutions were plated on TSA-rif plates for enumeration. Two ml of sterile 5% methanol inoculated with 20 μl of 106 CFU/ml S. Newport were used as a negative control. The growth of Salmonella Newport in plant exudates was measured only in drought-treated and regular watering kale groups.
Exudates powder preparation: Whole plants were immersed in 30 mL 5% methanol and shaken at 150 rpm for 24 h at room temperature to collect exudates. The solution was filtered through 0.45 m sterile syringe filters and lyophilized in a freeze dryer. The dried powder was collected for phytochemical profiling. Exudate powder was collected from baby and mature kale under drought stress and regular watering condition.
Leaf powder collection: One ml aliquots of kale leaf extract supernatants (as described in objective 2) were lyophilized into powder in a freeze dryer. The dried powder was collected for phytochemical profiling. The leaf powder was collected from baby and mature kale under drought stress and regular watering condition.
Phytochemical profiling using electrospray ionization-mass spectrometry (ESI-MS): Plant tissue powder and exudate powder were resuspended in 100 μl 70% methanol with 0.5% formic acid. To ensure all compounds were fully dissolved in the extraction liquid, all tubes were sonicated in a water bath for 2 h at maximum intensity. The remaining sample extract was then transferred into vial inserts for ESI-MS measurements.
A Time-of-Flight mass spectrometer (AccuTOF, JEOL, USA, Inc.) equipped with electrospray ionization (ESI) ion source was used in the mass spectrometric analysis of the extracts. Mass spectra were acquired in the positive and negative modes, respectively, at a rate of one spectrum per second with the m/z range of 50–800 Da. Every mass spectrum was averaged over about one minute. The AccuTOF mass spectrometer settings were as follows: needle voltage = 2100 V, orifice 1 temperature = 100°C, orifice 1 = 30 V, orifice 2 = 5 V, ring = 10 V. The desolvating chamber temperature was set at 250°C, and the flow rates of the nebulizing and desolvating gases were 0.6 and 3.0 L/min, respectively. The sample injection volume was 10 μl and the flow rate was set to 0.25 ml/min. Before and after each sample was analyzed, 10 μl methanol were injected and measured to monitor any possible contaminants or carryover. Calibration for exact mass measurements was accomplished using 5 mM cesium iodide as the internal standard. ESI-MS was conducted for baby and mature kale under drought stress and regular watering condition.
For all the samples, the measurements were repeated at least twice, and the experimental results showed excellent reproducibility.
Pseudomonas spp. application to kale roots had a protective effect on kale plants but this varied with respect to plant developmental stage and type of stress. Kale biomass was lower when plants were under drought and high salt stress. Compared to control plants, the biomass of baby kale was reduced by at least 23% under drought, while the biomass reduction of mature kale was at most 11% under drought. These findings suggested that baby kale plants were more vulnerable to stress. Root inoculation of Pseudomonas spp. protected plant biomass of mature kale from drought stress. These results suggested that applying Pseudomonas spp. to plant roots could protect plant growth under climate change and severe abiotic stresses.
Plant developmental stage and stress had an impact on the nutritional profile of kale plants. Regarding plants under regular watering conditions, mature kale accumulated more bioactive compounds, including flavonoids and phenolics than baby kale, indicating higher nutritional values of mature kale. With the protection provided by Pseudomonas spp., differences in bioactive compound contents of drought-treated baby kale plants were reduced, suggesting that PGPR lessened the nutritional quality variations between plants under stress. Baby kale plants under drought stress or inoculation of PGPR showed a higher accumulation of bioactive compounds, including flavonoids, phenolics, and glucosinolates, compared to regularly watered or PGPR-negative plants. PGPR-inoculated-baby kale plants showed the highest levels of flavonoids and phenolics accumulation. For PGPR-negative mature kale groups, the content of phenolics increased under drought stress. For mature plants under regular watering condition, PGPR-inoculated plants showed a higher accumulation of phenolics. Pseudomonas-inoculated kale plants under high-salt condition accumulated more phenolics and glucosinolates than regularly watered kale plants. PGPR-inoculated kale under high salt condition showed higher levels of glucosinolates. These results suggested that the nutritional values of plants could be enhanced under regulated environments.
Salmonella differentially survived on leaf surfaces with respect to stress and root inoculation of PGPR. Comparing surface survival of Salmonella after 24 hours inoculation, PGPR-negative baby kale plants under drought supported lower levels of Salmonella population; root inoculation of Pseudomonas spp. limited the surface survival of both baby and mature kale under regular watering condition. Exudates collected from regularly watered plants supported higher levels of Salmonella except for PGPR-negative mature kale groups. Regularly watered kale plants showed less retrieval of Salmonella with the protection provided by Pseudomonas spp. Finally, PGPR-negative plants showed lower retrieval of Salmonella under medium salt than in plants under regular watering condition. These findings indicated that stress and PGPR application could possibly create a less favorable environment for Salmonella, thus enhancing food safety.
Drought and salinity stress reduced the biomass of kale plants and the inoculation with PGPR did not promote plant growth. However, the biomass of mature kale plants inoculated with PGPR was not further reduced by drought, suggesting that PGPR had a protective effect on plant growth under stress. PGPR-inoculated, drought-subjected, and salinity-affected baby kale plants had higher levels of bioactive compounds than regularly watered plants not colonized by PGPR. Mature plants grown under regular watering conditions (control) accumulated more bioactive compounds than young plants (baby leaf stage) and could therefore be considered of higher nutritional quality. Our findings showed that regulated abiotic stresses (drought and salinity) and biotic interactions (PGPR root colonization) can enhance the nutritional quality of baby kale. Moreover, at both baby and mature kale stages, PGPR-inoculated, drought- and salinity-subjected kale plant surfaces were less favorable to Salmonella. Abiotic stresses and PGPR, therefore, had a positive effect on food safety outcomes. This enhanced resistance to Salmonella also occurred as kale matured when plants were grown under regular conditions. Application of regulated stresses or inoculation with PGPR to baby kale are promising strategies to enhance the nutrition value and food safety of this popular green.
Education & Outreach Activities and Participation Summary
Participation Summary:
Journal articles under manuscript:
1. Liu X, Li Y, Micallef SA. Kale leaf tissue and leaf surface metabolomes shift with plant development and in response to drought, affecting Salmonella enterica leaf association.
2. Liu X, Li Y, Micallef SA. Plant Growth-Promoting Rhizobacterium Pseudomonas spp. Shift Plant Phytochemical Profiles, Affecting Salmonella enterica Association with Baby Kale leaves.
Journal articles in preparation:
1. Liu X, Li Y, Micallef SA. Kale Leaf Phytochemical Profiles Differ by Plant Growth-Promoting Rhizobacteria Pseudomonas strains and Shift in Response to Salinity Stress, impacting the association with Salmonella enterica.
Presentations:
1. Micallef SA. Antimicrobial Resistance (AMR) E-Seminar Series: Salmonella-plant interactions: bacterial genetic responses, plant responses, the favourability of the plant niche to the enteric pathogen. AMR E-seminars, 5th May 2021, School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, United Kingdom.
2. Micallef SA. Exploring plant metabolite traits that restrict enteric pathogens in fruit and vegetables. In Symposium: Breeding Crops for Enhanced Food Safety. International Association for Food Protection (IAFP) Annual Meeting, August 2-5, 2020 Cleveland, OH. (Symposium organizer: Dr. Max Teplitski and Dr. Isabel Walls, USDA, NIFA) Invited [changed to virtual meeting Oct 26-28, 2020]
3. Liu X, Hsu CK, Micallef SA. Plant growth-promoting rhizobacteria Pseudomonas strains as possible agents to enhance food safety by limiting Salmonella enterica association with kale. International Association for Food Protection (IAFP) Annual Meeting, Hybrid, July 18-21, 2021
4. Liu X, Li Y, Micallef SA. Drought stress affects kale leaf phytochemical profiles and Salmonella enterica leaf association. International Association for Food Protection (IAFP) Annual Meeting, Virtual, Oct 26-28, 2020.
5. Liu X, Bollinger C, Micallef SA. Leaf phytochemical profiles differ by lettuce variety and shift in response to water stress, impacting the association with Salmonella enterica. International Association for Food Protection (IAFP) Annual Meeting. Hybrid, July 18-21, 2021.
6. Liu X, Li Y and Micallef SA, Drought stress shifts the exometabolome profile of leaves in juvenile kale and affects Salmonella enterica growth in leaf exudates. International Association for Food Protection (IAFP) Virtual Annual Meeting, Virtual, Oct 26-28, 2020.
7. Liu X, Li, Y, Micallef SA. Kale Leaf Metabolome and Exometabolome Profile Shifts in Response to drought affects Salmonella enterica-plant association. ASM Microbe 2020 Online. [changed to Virtual meeting due to Covid-19].
8. Liu X, Micallef SA. Increasing accumulation of antioxidant compounds induced by drought stress limit surface colonization of Salmonella enterica on leafy greens. PB19 Plant Biology Aug 3-7, 2019, San Jose, CA.
9. Liu X, Micallef SA. Salmonella enterica colonization of kale leaves is age- and drought stress-dependent. International Association for Food Protection (IAFP) Annual Meeting, 21-24 July 2019, Louisville, KY
Submitted for upcoming International Association for Food Protection (IAFP) Annual Meeting :
1. Liu X, Li Y, Micallef SA. Kale Leaf Phytochemical Profiles Differ by Plant Growth-Promoting Rhizobacteria Pseudomonas strains and Shift in Response to Salinity Stress, impacting the association with Salmonella enterica.
2. Liu X, Li Y, Micallef SA. Plant Growth-Promoting Rhizobacterium Pseudomonas spp. Shift Plant Phytochemical Profiles, Affecting Salmonella enterica Association with Baby Kale leaves
Factsheet:
1. Liu X, Panthi S, Schmidt DE, Solaiman S. Application of Plant Growth Promoting Rhizobacteria (PGPR) for Sustainable Agriculture
Project Outcomes
Pseudomonas spp. application to kale roots had a protective effect on kale plants but this varied with respect to plant developmental stage and type of stress. Kale biomass was lower when plants were under drought and high salt stress. Compared to control plants, the biomass of baby kale was reduced by at least 23% under drought, while the biomass reduction of mature kale was at most 11% under drought. These findings suggested that baby kale plants were more vulnerable to stress. Root inoculation of Pseudomonas spp. protected plant biomass of mature kale from drought stress. These results suggested that applying Pseudomonas spp. to plant roots could protect plant growth under climate change and severe abiotic stresses.
Plant developmental stage and stress had an impact on the nutritional profile of kale plants. Regarding plants under regular watering condition, mature kale accumulated more bioactive compounds, including flavonoids and phenolics than baby kale, indicating higher nutritional values of mature kale. With the protection provided by Pseudomonas spp., differences in bioactive compound contents of drought-treated baby kale plants were reduced, suggesting that PGPR lessened the nutritional quality variations between plants under stress. Baby kale plants under drought stress or inoculation of PGPR showed a higher accumulation of bioactive compounds, including flavonoids, phenolics, and glucosinolates, compared to regularly watered or PGPR-negative plants. PGPR-inoculated-baby kale plants showed the highest levels of flavonoids and phenolics accumulation. For PGPR-negative mature kale groups, the content of phenolics increased under drought stress. For mature plants under regular watering condition, PGPR-inoculated plants showed a higher accumulation of phenolics. Pseudomonas-inoculated kale plants under high-salt condition accumulated more phenolics and glucosinolates than regularly watered kale plants. PGPR-inoculated kale under high salt condition showed higher levels of glucosinolates. These results suggested that the nutritional values of plants could be enhanced under regulated environments.
Salmonella differentially survived on leaf surfaces with respect to stress and root inoculation of PGPR. Comparing surface survival of Salmonella after 24 hours inoculation, PGPR-negative baby kale plants under drought supported lower levels of Salmonella population; root inoculation of Pseudomonas spp. limited the surface survival of both baby and mature kale under regular watering condition. Leaf washes collected from regularly watered plants supported higher levels of Salmonella except for PGPR-negative mature kale groups. Regularly watered kale plants showed less retrieval of Salmonella with the protection provided by Pseudomonas spp. Finally, PGPR-negative plants showed lower retrieval of Salmonella under medium salt than in plants under regular watering condition. These findings indicated that stress and PGPR application could possibly create a less favorable environment for Salmonella, thus enhancing food safety.
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 allowed me to translate the research to a stakeholder audience. Results of this research stated the impact of PGPR on plants under abiotic stresses, and how physiological changes of plants can in turn affect the plant microbiota. These research results can lead to future research in stress application to achieve desired nutritional values and food safety outcomes. In the future, I will continue paying attention to research related to sustainable agriculture and help more people in understanding the benefits of sustainable agriculture.
We had one new 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 and QTOF-LC-MS/MS).
A no-cost extension was granted for one year since the research was severely disrupted by COVID measures.
Although our findings showed that the plant growth-promoting rhizobacterium Pseudomonas spp. S4 had a positive effect on plants under stress, it could not promote the growth of kale plants, as had been previously recorded for spinach, tomato, and lettuce plants. In the future, we plan to test more known PGPR strains to evaluate the growth-promoting effects on various produce including kale.
We reported a positive food safety outcome when plants were subjected to abiotic stresses. Based on the non-targeted metabolome analyses and phytochemical profile data we generated, it was identified that stearidonic acid and 12-oxo-phytodienoic acid (12-OPDA) levels in kale leaves were the major contributors to the differences in the phytochemical profiles under drought stress and varied PGPR inoculation groups compared to the control groups. In the future, more experiments could be conducted to test if stearidonic acid and 12-oxo-phytodienoic acid in plant exudates have an impact on S. enterica growth. In the meantime, similar systems could be applied to other produce and more phytochemicals could be identified as Salmonella growth-limiting compounds. Ultimately, we could grow more fresh produce that accumulates higher levels of bioactive and human pathogen growth-limiting compounds, thus improving food nutritional values and enhancing food safety.