Performing Department
Plant Science & Landscape Architecture
Non Technical Summary
Plants recruit beneficial bacteria in the rhizosphere to support growth, health, defense and resilience. These plant growth-promoting bacteria (PGPR), exhibit host-preferences, but the probiotic traits they confer can provide cross-protection against multiple stresses. Whether the probiotic capabilities conferred by PGPR extend to food safety and nutritional quality of crops is not well studied. This grant proposal seeks to characterize a bacterial library of Brassica-derived root and leaf epi- and endophytes for desirable traits that extend beyond the scope of crop yield and disease resistance. The PGPR library will be screened on leafy vegetables for 1) reduced enteric pathogen populations that indicate positive food safety effects, 2) plant growth-regulating capabilities to bolster plant resilience by priming stress responses, and 3) induced plant secondary (specialized) metabolites to boost nutritional quality of crops, in addition to mediating tolerance to biotic and abiotic stresses.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The goal of this project is tocomplete screening of Brassica-derived bacterial isolates for plant growth promotion of one kale and two romaine lettuce cultivars. We will explore growth promoting potential, positive food safety effects and abiotic stress resilience onisolates in our collection, including plant beneficial traits and effects on the plant metabolome, to explore underlying mechanisms that benefit plants. To this end, the objectives of this proposal are:Objective 1: Explore effects of root-inoculated Brassica isolates on lettuce and kale resilience to a) biotic interactions with foodborne enteric pathogens and b) abiotic stress 1a) E. coli O157:H7, Listeria monocytogenes and Salmonella (kale only) applied to leaves.1b) tolerance to heat stress.Objective 2: Evaluate plant growth-promoting traits of Brassica isolates in vitro2a) Screen bacterial library for phosphate solubilization, siderophore production, indole-3-acetic acid (IAA) production. Objective 3: Evaluate the effects of root-inoculation with Brassica isolates on lettuce and kale nutritional quality3a) Screen plants inoculated with Brassica isolates for shifts in phenolic and flavonoid content, and antioxidant capacity.3b) Conduct metabolomic analysis using LC-MS/MS of plants inoculated with Brassica isolates showing shifts in phenolics and flavonoids in objective 3a.
Project Methods
Resilience in lettuce and kale will be evaluated byscreening for PGPR effects on enteric pathogen-plant associations (that would enhance food safety), lettuce and kale plants whose roots are colonized with single species Brassica isolates will in turn be inoculated with enteric pathogens E. coli O157:H7 and Listeria monocytogenes applied to leaves. Kale will also be tested for effects on Salmonella applied to leaves.Enteric pathogen assay: Foodborne illness outbreak strains or environmental strains of E. coli O157:H7, Listeria monocytogenes and Salmonella Typhimurium, all adapted to an antibiotic for selective enumeration, will be used to inocualte plants colonized with the presumptive PGPR strains.Plants will be maintained in the BSL-2 growth chamber for an additional 24 h.Bacterial enumeration will be conducted 24 h post-inoculation using a serial plating technique.Heat tolerance assay: To determine presumptive PGPR effects on plant heat tolerance, plants will be germinated, root inoculated with Brassica isolates and grown for 24 days and heat stress applied by placing pots in a plant growth chamber set at 28°C versus controls kept at 18°C. All other growth parameters will be kept constant as described above. After 4 days, rosette diameter, number of leaves and fresh weight will be recorded. Fresh weight will be measured by clipping the rosette at the stem-soil interface and weighed immediately before any water loss occurs. Plant tissue will be flash frozen in liquid nitrogen than preserved at -80°C for future phytochemical analysis. Plant growth-promotion traits will be measured by in vitro screening of Brassica isolate library. The assays will investigatephosphorus solubilization, siderophore production andIAA production:Nutritional quality effects on lettuce and kaleas affected by root colonization with Brassicaisolateswill be investigated using a phytochemical analysis approach, antioxidant capacity measurements and chemical profiling of plant tissue.Lettuce and kale plants will be inoculated with isolates from the bacterial library and grown under heat stress or no heat stress. At 4 weeks, leaf tissue samples will be collected from each plant and phenolics and flavonoids extracted. Appropriate dilutions will be made, and the absorbances of the extracts will be measured in scan mode between 210 - 400 nm using a UV-Vis spectrophotometer. Absorbance readings at 225 nm for quercetin, 273 nm for gallic acid, 280 nm for catechin, and 339 nm for apigenin will be used to determine the equivalent concentrations of each metabolite in the sample extracts, based on standard curves generated for each compound.Antioxidant capacity of kale and lettuce samples will be measured according to a modified ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) method and an Oxygen Radical Absorbance Capacity (ORAC-FL) assay.Isolates that demonstrate an ability to cause induction of phenolics and flavonoids (objective 3a) will be used to root inoculate fresh seedlings for plant metabolomic analysis of plant tissue. These will be extracted and submitted for analysis using a natural products analysis pipeline by reversed phase liquid chromatography high-resolution tandem mass spectrometry. The relative peak heights and compound identification will give sample profiles of phenolic compounds that can be used to compare against the no-PGPR controls and assign compound identifications to induced peaks. Plants subjected to biotic (enteric pathogens) and abiotic (heat) stress will also be analyzed (objective 1), which will help decipher plant responses to these exposures.