Source: OHIO STATE UNIVERSITY submitted to
PLANT-MICROBIOME NETWORKS IMPACT PLANT PRODUCTIVITY AND MITIGATE PLANT DISEASE AND FOOD SAFETY RISKS IN HYDROPONIC PRODUCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022834
Grant No.
2020-67017-31798
Project No.
OHO03062-CG
Proposal No.
2019-08318
Multistate No.
(N/A)
Program Code
A1402
Project Start Date
Aug 15, 2020
Project End Date
Aug 14, 2024
Grant Year
2020
Project Director
Benitez Ponce, M. S.
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Plant Pathology
Non Technical Summary
Much of the microbiome research in agriculture has been focused on soil and soil-based systems. One of the challenges of microbiome research is to develop predictions of crop productivity from microbiome information and manipulation. We propose that hydroponic production systems offer a closed, low diversity, model to test and predict the environmental impact on microbiome dynamics. In hydroponics, plant roots are suspended in a nutrient solution and are housed in enclosed systems where the environment of the root and shoot zones are tightly controlled. In addition, in this low-diversity system, we expect to increase our ability to use microbiomes to predict the impact on plant growth, plant health, and pathogen persistence. We aim to characterize the functionality of microorganisms in hydroponically grown plants, as well as on changes in microbiome functionality in relation to the hydroponic system setup. In this proposal, we focus on lettuce due to its market and food safety relevance. In addition, its short production cycle allows for robust and replicable manipulation required for the functional characterization of lettuce microbiome interactions. We manipulate specific abiotic and biotic variables to optimize production practices in order to promote a microbiome that enhances plant growth. Furthermore, we will test the hydroponic microbiome contributions to food safety and plant diseases. At the end of this project, we will develop a working knowledge on production parameters that could be used to predict the functional diversity of hydroponic lettuce microbiomes and the major microbiome contributions to plant productivity and human safety.
Animal Health Component
0%
Research Effort Categories
Basic
85%
Applied
15%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2051430110060%
2161430110220%
7121430110320%
Knowledge Area
205 - Plant Management Systems; 216 - Integrated Pest Management Systems; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1430 - Greens and leafy vegetables;

Field Of Science
1103 - Other microbiology; 1100 - Bacteriology; 1102 - Mycology;
Goals / Objectives
Our long-term goal is to understand how to manipulate, in a predictable manner, agricultural microbiomes within hydroponic systems to enhance system resiliency, improve plant health, and reduce human pathogen persistence. Using lettuce hydroponic systems as a production model we will study the root, water and surface microbiomes and strategically manipulate abiotic and biotic variables to identify and optimize production practices that will improve plant health and reduce food safety and plant disease risks. This proposed work will improve our understanding of how microbiomes contribute to and shape measures of plant productivity and health and can serve as a model system for testable microbiome studies. We predict that validation of the use of hydroponic system as a model for the study of microbiome dynamics will enhance our ability to better understand more biologically complex microbiome systems such as those found in soils that are inherently harder to study and predictably manipulate. For this, we have assembled a team of researchers that uniquely complement expertise in hydroponic plant production, plant microbiome research, biological control, and food safety; as well as have access to a network of stakeholders in Ohio and surrounding states, and other resources for successful completion of the proposed experiments. The specific objectives of this proposal are:Objective 1. Determine the diversity and abundance of the resident microbiome in commercial nutrient film technique (NFT) and deep water flow (DWF)lettuce hydroponic operations. We hypothesize that system setup characteristics, including water source and design (DWC vs. NFT), are the greatest drivers of commercial production microbiome differences. Specifically, DWC systems will have a more stable and diverse microbial community than NFT and a greater abundance of beneficial microorganisms due to the lower frequency of water change and lower water flow.Objective 2: Determine system management parameters that promote greater functional diversity of the resident microbiome in hydroponic lettuce production. We hypothesize that nutrient solution conditions which promote plant growth will also promote microbial growth and diversity. However, changes in nutrient conditions or concentrations that lead to less-than-optimal growth will likely lead to an increase in overall microbial abundance, but a decrease in microbial diversity and function, which could in turn increase pathogen persistence.Objective 3: Evaluate hydroponic system resilience to plant and human pathogens, as a function of system management. We hypothesize that system management parameters that promote both plant growth and microbial functional diversity (as determined in objectives 1 and 2) will result in lower human and plant pathogen survival and colonization in the system.
Project Methods
Research Objective 1. Determine the differences in diversity and abundance of the resident microbiome of commercial lettuce hydroponic production with differing systems.We have identified hydroponic leafy green production greenhouses that utilize NFT and/or DWC as collaborators. We will use a stratified sampling design to evaluate the impact of water source, system setup design, sampling time, and sampling location (within the hydroponic system) on the diversity of the active microbiome of hydroponic lettuce. For this, we will survey the bacterial, fungal, and protist composition of water (at its source), nutrient solution, equipment surfaces, root systems, and leaf tissue from the selected farms. To ensure that plant genotype and sampling will be consistent across farms, we will provide seed of our focal crop, butterhead lettuce, to growers, create planting plans, assist in planting, and take all samples at the same phenological stage. For each site, replicate samples will be taken at three time points (spring, summer and fall) in the first year of the project following a standardized protocol.For microbiome analysis, plants will be harvested by removal from the system, cutting at the base of the shoot, and washing roots free of growth media. Shoots and roots will be weighed and stored separately. Nutrient solution will be collected in sterile bottles and filtered through sterile 0.2 mm bottle-top filters, and filters (containing microorganisms in solution) will be stored. Equipment swabs will be also collected. To target the active component of the microbiome, total RNA will be extracted using established methods. cDNA synthesis will then be performed using random primers or target-specific primers and amplicon metabarcoding libraries will be prepared for sequencing on Illumina MiSeq. Community data will be analyzed using PerMANOVA, random effects models, and network analysis to determine strength of response to water source and system design; b) individual members of the community consistently associated with sampling site and time of the year and c) unique and shared microbial assemblages across systems and sites.Research Objective 2. Determine abiotic system management parameters that promote greater functional diversity of the resident microbiome of hydroponic lettuce production.We will examine the influence of water source and system setup (municipal vs. rainwater in NFT and/or DWC), nutrient solution characteristics (pH, EC), and plant and human pathogen management treatments (sanitation and biologicals) on microbiome composition and function in our systems. Two replicated commercial systems are contained in a certified BSL2 facility to allow for research with non-bloodborne human pathogens. In addition, one DWC system is currently available for research use. In order to also determine responses to system setup (NFT vs. DWC) and achieve enough replication we propose to setup one additional DWC research unit in the BSL2 facility. Manipulation experiments include 1) Lettuce microbiome responses to hydroponic setup and water source: we will test the three main water sources municipal water, collected rainwater, and well water and a combination (1:1:1 vol) all with recirculation in our experimental hydroponic NFT and DWC systems. 2) Lettuce microbiome responses to nutrient solution characteristics in NFT systems. Here we will test two pH (5.0 and 5.8) and three EC (1.8, 1.5 and 1.0 mS/Cm) levels and their combinations. We expect to observe interactions between these levels and their impact in microbiome community composition and potential functionality. 3) Lettuce microbiome responses to plant and human pathogen management treatments in NFT systems. Here we will specifically test the effects of biocidal products (chlorine and Green-Shield) at recommended manufacturer rates, and biocontrol Bacillus and Pseudomonas strains available from our extensive collections of biocontrol organisms. System sanitation will be performed prior to transferring the seedlings into the NFT channels. Biological inoculants will be applied at the time of planting to maximize establishment within the root system. For all three experiments a parallel standard setup will be run as control. Both plant and microbial traits will be measured for all experiments as a function of system management at the time of harvest. Root and shoot samples will be processed separately for biomass measurements, and foliar chlorophyll readings and measure of "greenness". Shoots will also be visually evaluated on a five-point ordinal scale for any symptoms of phytotoxicity. Roots will be examined and scored for discoloration based on determined boundaries of the expected color range and intensity for this particular variety. In addition, marketable yield will be determined. For microbiome analysis, four biological replicates per treatment will be collected from root and nutrient solution samples to characterize the bacterial, fungal and protist communities. To help identify changes in functionality of the microbiome within each of experiment we will use shotgun metagenomics to characterize changes in gene content in the system. We will use an in-situ culturing approach, based on an Ichip, to culture bacteria from our test hydroponic systems and perform genomic analysis. Individual genomes of up to twenty bacterial isolates will be obtained using MiSeq technology.Research Objective 3. Evaluate hydroponic system resilience to plant and human pathogens, as a function of system management.For this study we will use conditions identified in Objectives 1 and 2 that consistently result in both greater productivity and microbiome functional diversity in lettuce hydroponics. A subset of conditions identified from Objective 2, will be used to test our system responses to inoculation with plant and human pathogens, compared to the standard recommended parameters. The overall goal is to successfully replicate the microbiome composition, based on known and predicted responses to system parameters, as tested in Objective 2, and determine the impact of system and microbiome management in plant and human pathogen colonization and activity. Four experiments will be performed, each involving inoculations with a single plant or human pathogen, or a combination of them. Controlled inoculations with the plant, human pathogen or combinations will be applied to the water source. Prior to set up all the components (water, water reservoirs, tubing and germination substrate) of the control (uninoculated) and treatment systems will be treated with bleach or autoclaved. The water source will then be inoculated with a microbial slurry, generated from samples of a high diversity system obtained from Objective 2. To generate the inoculum, we will use a series of water filtrates, as well as the contents of the Ichip design. Pathogens will be applied two weeks after lettuce seedlings have been transplanted to the hydroponic system to allow for both seedling and microbiome establishment. After two weeks in the production systems, plants will be harvested and evaluated for quality measurements as described in Objective 2 (biomass, marketable yield, foliar chlorophyll and color, root discoloration and necrosis, phytotoxicity symptoms). The percentage of root area with necrotic lesions (severity) and the percentage of leaf area covered with lesions (severity) will be rated on a scale of 0-100%. Standard laboratory methods will be used to isolate each pathogen from the root, rockwool, and shoot (human pathogens only). Pathogen load in the recirculated nutrient solution at the time of harvest will also be measured. A subset of nutrient solution and roots will be harvested before and after inoculation events to confirm expected microbiome composition based on the results of Objective 2 and compare to composition of the original microbial slurry applied.

Progress 08/15/22 to 08/14/23

Outputs
Target Audience:During the 2022-2023 period, we reached different audiences with this project, specifically leafy green hydroponic producers, and students and researchers in the areas of controlled environment agriculture, plant pathology, microbiology, and food safety. Specifically, during this period members of the team performed visits to four hydroponic leafy green facilities in the state. These visits were focused on sampling nutrient solutions, which were used as a starter material to seed greenhouse experiments focused on microbiome establishment. Our results have been shared with the research community through different venues, including invited seminars and webinars at various academic institutions and scientific societies, targeting audiences of undergraduate and graduate students, as well as faculty, other researchers, and producers. For example, Dr. Benitez Ponce was invited to apply to join the Phytobiomes Alliance and industry-academia collaborative effort in system-level research in agriculture. As part of this, Dr. Benitez Ponce presented a webinar, with international reach, and is currently the co-chair of Phytobiome Alliance's coordinating committee of the Controlled Environment Agriculture Working Group. We provided training in hydroponic production to a first-year undergraduate student researcher (OSU Agricultural Technological Institute, Plant Pathology major) and a visiting scholar, and continued the training of four graduate students (Plant Pathology and Human Nutrition), a postdoctoral researcher, and two research technicians. Finally, we are using material from our lettuce experiments as the source of root material for bacterial isolation and characterization in the Advanced Plant Pathology laboratory course (PP6002.01, Department of Plant Pathology at OSU). Changes/Problems:We continue to encounter delays in the shipment and availability of reagents and other consumables necessary for this research. We accommodate different reagents and consumables when it will not compromise the reproducibility of experiments. These also affect the turn-around time on services, including sequencing facilities. What opportunities for training and professional development has the project provided?Graduate student, Fiama Guevara attended and presented a poster at the 2023 MidWest Microbiome Symposium (May 2023), PI Soledad Benitez Ponce also attended the meeting. Several members of the team (Fiama Guevara, Christopher Taylor, Uttara Samarakoon) participated in the Ohio Controlled Environment Agriculture Center, annual meeting held July 2023. Graduate students, Fiama Guevara, Edwin Navarro, and Gustavo Garay attended and presented (G Garay oral, F Guevara, E Navarro, poster) at the 2023 Department of Plant Pathology Spring Symposium (May 2023). Graduate student Gustavo Garay attended and presented (oral) at the 2023 OSU Plant Science Symposium (Feb 2023), and will attend and present (poster) at the 2023 Plant Health Meeting (August 2023). Graduate student Abigail Mensa attended and presented at the 2023 meetings of the International Association of Food Protection and RKS 2023 How have the results been disseminated to communities of interest?The results have been disseminated to both the scientific community and hydroponic producers. As part of this, several seminars at academic institutions and scientific meetings, as well as poster presentations and short talks are listed under Products. In addition, our team continues to develop online and printed resources about hydroponic production and food safety management in leafy green hydroponics, which are available through the team's websites: https://u.osu.edu/benitezponce-1/links/ and https://producesafety.osu.edu/ What do you plan to do during the next reporting period to accomplish the goals?Activities to be completed in the next reporting period are indicated below by objective. Objective 1: Determine the differences in diversity and abundance of the resident microbiome of commercial lettuce hydroponic production with differing growing system setups. The team will continue with data analysis and manuscript preparation. Objective 2: Determine system management parameters that promote greater functional diversity of the resident microbiome of hydroponic lettuce production The team will generate and retrieve sequencing data associated with experiments of manipulation of nutrient solution (EC, pH, water source, and nutrient solution seeding). The data will then be analyzed and prepared for manuscript publication. We will complete the comparative genomic analysis of bacterial isolates. The whole genome sequencing information will then be correlated with phenotypic tests, as well as characteristics of the origin of each isolate. The data will then be prepared for manuscripts for publication. Objective 3: Evaluate hydroponic system resilience to plant and human pathogens, as a function of system management. Complete experiments with plant and human pathogens, and interactions with system management and resident members of the microbial community.

Impacts
What was accomplished under these goals? Accomplishments during the 2022-2023 are listed below by objective. Objective 1: Determine the differences in diversity and abundance of the resident microbiome of commercial lettuce hydroponic production with differing growing system setups. To characterize the diversity and relative abundance of bacteria and fungi associated with leafy green hydroponic production, we completed the library preparation, sequencing, and descriptive statistics of 736 samples obtained from ten commercial facilities in Ohio. Sample types included surface swabs, growing media, nutrient solution, lettuce leaves, and roots. Samples originated from facilities that varied in size (23-13150m2), system setup (Nutrient Film Technique (NFT), Deep Water Culture (DWC), Ebb & flow (EF), or Vertical Drip (VD)), a water source used for nutrient solution preparation, as well as pH and EC parameters of nutrient solution. We applied amplicon metabarcoding of the ribosomal markers, 16S rRNA, and ITS1 to survey bacteria and fungi, respectively. For roots, leaves, and nutrient solution, we recovered 16,963 and 3,533 taxa (or amplicon sequence variants (ASVs)) for bacteria and fungi, respectively. Community composition analysis indicated that the bacterial and fungal communities' structure differed between habitats (leaves, roots, and nutrient solution). Bacterial and fungal richness and evenness also showed differences between habitats, with roots and nutrient solution showing higher richness than leaves. However, bacterial evenness was significantly higher in leaves, whereas fungal evenness was higher in leaves and nutrient solution, compared to roots. Further comparison of root samples between production systems showed higher bacterial richness, but lower evenness in DWC systems, whereas greater fungal richness and evenness were observed in EF and NFT systems. Lastly, communities from nutrient solution were less variable across facilities, compared to other sample types. The most prevalent bacterial genus in root samples was Flavobacterium; whereas in leaves, an unclassified Comamonadaceae and Anhiella were prevalent. Additionally, fungal genera Olpidium and Golovinomyces were prevalent in both leaves and roots. And, consistent groups of bacteria were recovered from the nutrient solution (through amplicon metabarcoding and culturing), including the genera Bosea, Sphingomonas, and Sphingobium. Objective 2: Determine system management parameters that promote greater functional diversity of the resident microbiome of hydroponic lettuce production Our research focused on greenhouse experiments manipulation of two characteristics, nutrient solution management, and system setup (DWC vs NFT and associated surface materials). For the nutrient solution management experiments, we manipulated nutrient content, through electrical conductivity (EC), pH, and the water source. Experiments for EC and pH manipulation were completed in both experimental DWC and NFT; whereas water source experiments were completed in a small-scale, 5-gallon DWC setup. As indicated in the literature, greater lettuce vigor was observed under recommended conditions of 1.8 EC and pH of 5.8; however, we recovered more culturable bacteria (colony-forming units) from roots of lettuce grown at an EC of 2.5. From our pH experiments, we determined that maintaining a consistently low pH (5.0) in a production system is challenging and is related to changes in the nutrient solution as the lettuce head begins to form. This resulted in inconclusive data on pH effects on bacterial abundance in roots. Lastly, lettuce vigor, when grown in a nutrient solution prepared with rainwater (RW), was higher, than when grown in a nutrient solution prepared with reverse osmosis-filtered municipal water. In addition, a subset of roots from lettuce grown in RW nutrient solution harbored diverse bacterial communities, which differed in dominant bacterial taxa, compared to municipal water. Plant and growth media samples from these experiments (nutrient solution management) have been processed for nucleic acid extraction and microbial communities will be further characterized through amplicon metabarcoding. In addition to testing parameters of nutrient solution characteristics, we also tested the effect of spent, or reused nutrient solution, and its community composition, on subsequent lettuce growth cycles. This experiment allowed us to determine if: a) nutrient solution can be used to seed, or inoculate, a leafy green hydroponic setup and b) the degree of contribution of the past nutrient solution, compared to the current setup, on hydroponic system microbial community composition. From two experimental runs, which included seeding with nutrient solution from active, commercial hydroponic facilities, no significant effect on lettuce biomass was observed, regardless of nutrient solution origin and the richness of bacteria recovered from the nutrient solution was lower at the end of the experiment, compared to the original "seed". Analysis of the effect of nutrient solution seed in lettuce root microbial communities is ongoing. For system type (DWC vs NFT) characteristics, we focused on the impact of surface materials (i.e. polyethylene liner and PVC, for DWC and NFT, respectively) on the microbial buildup of biofilms and the diversity of bacteria from surface swabs. Our results indicated that even though the DWC liner results in greater microbial buildup, the diversity of bacteria recovered was greater from PVC channels. In addition, the surfaces within an NFT channel accumulated different amounts of bacteria, than the surface of the nutrient tank, and the nutrient solution. To evaluate functional diversity in the microbial communities of hydroponic system production, we are characterizing the activity and genome composition of isolates recovered from nutrient solution and surface biofilms. A total of 287 isolates have been classified into 67 genera, with the most abundant being the genus Pseudomonas. Other dominant genera differed between samples, with Bacillus being predominant in surface samples. A subset of isolates produced siderophores, can solubilize phosphorous, inhibit pathogen growth in vitro and in plants, and show the potential to fix nitrogen. 72 of these isolates have been selected for whole genome sequencing. Objective 3: Evaluate hydroponic system resilience to plant and human pathogens, as a function of system management. For this objective, in the current reporting period, we first tested the efficacy of inoculation protocols for the application of the leaf and root pathogens in hydroponically grown lettuce (deep water cultures). As a result of this, we developed protocols that result in consistent infection of Pythium root rot, powdery mildew, and Xanthomonas sp. These protocols were then used to set up experiments to evaluate the effect of pathogen introduction in the resident microbial communities in lettuce roots and leaves when grown in DWC. In addition, experiments were run to determine the impact of potential biological control agents on Pythium disease in hydroponic lettuce production. Lastly, for experiments involving multiple microorganisms, we currently have available GFP-transformed isolates adapted to hydroponic systems. The team also evaluated the effect of pH-manipulation of nutrient solution and the application of sanitizers to the nutrient solution (SaniDate 12.0, 200 ppm) in eliminatingthe human pathogen Salmonella Typhimuriumfrom NFT hydroponic systems. The treatments tested (pH-modification or SaniDate) did not eliminate Salmonella. Further, Salmonella was transferred, or moved, from the nutrient solution to the leaves even after sanitizer application. The tested pH and sanitizer treatments did not impact the concentration of bioactive compounds in hydroponic lettuce, but the 200 ppm application of SaniDate in nutrient solution resulted in lower biomass than non-treated controls.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Benitez Ponce MS, Guevara F, Frey T, Taylor L. System design, surface characteristics, and recirculation influence microbial communities in hydroponic leafy green production. International Phytobiomes Conference, 2022. Abstract. Accepted for presentation but presentation canceled due to illness.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Benitez Ponce MS, Guevara F, Frey T, Garay G, Taylor L. Plant and Animal Genome Meeting. January 2023. Exploring Phytobiomes Workshop speaker. Title: System Design, Surface Characteristics, and Recirculation Influence Microbial Communities in Hydroponic Leafy Green Production.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Garay G, Benitez Ponce MS. Impact of aeration and water source on yield and bacterial abundance in deep water culture hydroponics. Plant Science Symposium, The Ohio State University. February 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Mensah, AA., Melanie. ILL., TM., Ilic, S. (2023) Mitigation of Salmonella Typhimurium in Nutrient Film Technique (NFT) hydroponic system for improved food safety and nutrition.RKS 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Rivas, G., Mensah, AA., Collin B., Melanie. ILL., Ilic, S. (2023) Food Safety of Hydroponic Fresh Produce: A Scoping Review, IAFP 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Mensah, AA., Melanie. ILL., TM., Ilic, S. (2023) Eliminating Salmonella Typhimurium from lettuce grown in NFT hydroponic system for improved food safety and nutrition, IAFP 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Guevara F, Navarro E, Benitez Ponce, MS. Functional characterization of Bacteria associated with lettuce hydroponic production systems in Ohio. Midwest Microbiome Symposium. May 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Garay G. Comparison of different water sources for hydroponic lettuce production. Department of Plant Pathology, The Ohio State University, Spring Symposium. May 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Guevara F, Frey T, Benitez Ponce, MS. Unraveling microbial communities of commercial lettuce hydroponic production systems in Ohio. 4th Plant Microbiome Symposium, Quito-Ecuador, 2023. Abstract. Accepted for presentation but presentation canceled due to travel cancelations.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2023 Citation: Garay G, Benitez Ponce MS. 2023. Water Source influence on lettuce on controlled environment system. Plant Health 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Navarro, E. Uncovering T6SSs in a collection of Pseudomonas spp. Department of Plant Pathology, The Ohio State University, Spring Symposium, poster presentation. May 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Guevara-Guillen F. Bacterial communities in lettuce hydroponic systems. Department of Plant Pathology, The Ohio State University, Spring Symposium, poster presentation. May 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Benitez Ponce MS. Drivers of Microbial Community Composition in Hydroponic Leafy Green Production. Phytobiomes Alliance, Webinar Series. May 24, 2023. https://youtu.be/uNCAOyxsN2A
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Benitez Ponce MS. Spatial distribution of microbial communities in agricultural systems. Michigan State University, Department of Plant, Soil and Microbial Sciences. Invited Seminar Speaker. Oct 12, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Navarro E. El uso de la microbiolog�a en la producci�n agr�cola: �nfasis en el uso de Pseudomonas como control biol�gico Sociedad Estudiantil de Microbiolog�a Industrial- Universidad de Puerto Rico, Mayag�ez.February 9, 2023


Progress 08/15/21 to 08/14/22

Outputs
Target Audience:During the 2021-2022 period we reached different audiences with this project. Our efforts were targeted toward leafy green hydroponic producers in the state of Ohio, and students and researchers that work in different aspects of plant production, controlled environment agriculture, plant pathology, microbiology, and food safety. Specifically, in 2021-2022 we performed two visits to leafy green growers in Ohio state to sample from their hydroponic facilities andassess microbes in their production system. This amounts to a total of 10 leafy green hydroponic facilities sampled in this project.Additional visits were performed to support individual growers' needs, in particular, to discuss specific questions on food safety in leafy-green hydroponic production. As part of the efforts on food safety, updates on good agriculture practices to promote food safety in hydroponic productions were published, and an online self-paced course was developed for producers. We also provided training in hydroponic production to three undergraduate student researchers, and continued the training of two graduate students, two postdoctoral researchers, and a research technician. Our results have been shared with the research community through different venues, including invited seminars at various academic institutions and scientific conferences. Finally, we are incorporating small experiments in our hydroponic setup into hands-on laboratory courses (Department of Plant Pathology at OSU), focused on plant-microbial interactions. Changes/Problems:During this reporting period, we encountered delays due to the availability of reagents and other consumables necessary for nucleic acid extractions. To solve this challenge, we had to modify protocols to use materials more readily available, but this has resulted in delays in the sequencing process. What opportunities for training and professional development has the project provided?Graduate student, Fiama Guevara attended the in person meeting of the American Phytopathological Society in August 2022. Several members of the teamparticipated in different roles at the Ohio Controlled Environment Agriculture Center, annual meeting held July 2022. Among the organizers of the 2022 meeting, Dr. Christopher Taylor is a co-PI on this project. The topic of this annual meeting was "Advancement of Microbial Technologies for Controlled Environment Agriculture", and we had two presentations by members of our team (Frey et al and Ilic et al). In addition, undergraduate students, graduate students, postdocs and researchers from multiple labs attended this meeting. Graduate student, Fiama Guevara attended the Microbiome Informatics Workshop, organized for OSU Centers of Microbiome Science, Fall 2021. Postdoctoral researcher Timothy Frey, graduate student Fiama Guevara, and undergraduate student Gustavo Garay participated in OSU's Code Club during the period Fall 2021-Summer 2022. How have the results been disseminated to communities of interest?The results have been disseminated to both the scientific community and hydroponic producers. As part of this, several seminars at academic institutions and scientific meetings, as well as poster presentations and short talks are listed under Products. In addition, our team prepared printed material, online resources, and performed grower visits to contribute to food safety management in leafy green hydroponics. What do you plan to do during the next reporting period to accomplish the goals?Activities to be completed in the next reporting period are shown below by objective. Objective 1: Determine the differences in diversity and abundance of the resident microbiome of commercial lettuce hydroponic production with differing growing system setup. Retrieve the sequence data from samples of all commercial facilities. Complete the data analysis and publication of the microbial community characterization from the commercial hydroponic facilities visited. Prepare data to share with individual growers Objective 2: Determine system management parameters that promote greater functional diversity of the resident microbiome of hydroponic lettuce production. Repeat experiments on the influence of pH of the nutrient solution on lettuce health and associated microbial communities in DWC and NFT. Complete experiments on the water source as a determinant of microbial community composition in hydroponic lettuce roots. Setup, run experiments, and evaluate the effect of biological inoculants on microbial community composition in hydroponic lettuce. Perform comparative genomic analysis of bacterial isolates obtained from diverse types of hydroponic systems. Analyze data and prepare manuscripts for publication. Objective 3: Evaluate hydroponic system resilience to plant and human pathogens, as a system management function. Setup and run experiments to evaluate hydroponic system response, including microbial community dynamics, to inoculation with individual plant or human pathogens, or a combination thereof. Analyze data and prepare manuscripts and other materials toshare our results with stakeholders.

Impacts
What was accomplished under these goals? Accomplishments during 2021-2022 are listed below by objective. Objective 1: Determine the differences in diversity and abundance of the resident microbiome of commercial lettuce hydroponic production with differing growing system setups. Established contact, visited, and sampled two additional commercial leafy green hydroponic facilities in Ohio. This sums to a total of 10 commercial facilities visited (and sampled) throughout the duration of this project. One facility, out of the 10, was selected to perform multiple samplings during this project. During 2021-2022 we completed the second sampling visit. Completed nucleic acid extraction from plant material, nutrient solution, and system surface samples obtained from all commercial facilities and time points sampled. Submitted nucleic acids for amplicon sequencing of bacterial and fungal ribosomal gene markers and sequencing is currently in progress. Objective 2: Determine system management parameters that promote greater functional diversity of the resident microbiome of hydroponic lettuce production. Completed two experiments on the influence of nutrient solution electrical conductivity on lettuce growth and associated microbial communities in research DWC. Completed an experiment on the influence of pH of nutrient solution on lettuce health and associated microbial communities in each DWC and NFT systems. Designed and set up experiments on water source influence in lettuce roots and associated microbial communities in DWC. Completed experiments to evaluate different types of sanitizers on NFT surfaces. Determined the effects of system surface characteristics (PVC for NFT and pool liner for DWC) on bacterial biofilm formation and composition. Performed experiments to evaluate differences in bacterial biofilm formation and composition concerning location in a leafy green hydroponic system (i.e. water tank, proximal to the Rockwool plug, root tip, end of the NFT channel). Objective 3: Evaluate hydroponic system resilience to plant and human pathogens, as a function of system management. Tested inoculation methods for consistent Pythium infection in lettuce DWC. Tested for the survival of human pathogens in leafy green NFT and DWC.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Malacrino A; Abdelfattah A; Berg G; Benitez MS; Bennett AE; B�ttner L; Xu S; Schena L. 2022. Exploring microbiomes for plant disease management. Biological Control. 169: 104890. doi.org/10.1016/j.biocontrol.2022.104890.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Guevara F, Frey T, Taylor L, Benitez Ponce MS. Isolation and identification of bacteria associated with Nutrient Film Technique (NFT) hydroponic production systems in Ohio. Plant Health 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Benitez Ponce MS. V International Phytopathology Symposium. Universidad San Francisco, Quito-Ecuador. Sep 15, 2021. Keynote presentation. Title: Din�mica de comunidades microbianas en ecosistemas agr�colas.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Guevara F. Understanding the role of microbial communities in lettuce hydroponic production systems. Department of Plant Pathology, OSU, Spring Symposium. May 9, 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Frey T, Guevara F, Taylor L, Benitez Ponce S. Microbial communities and biofilms associated with hydroponic lettuce production systems in Ohio. Ohio Controlled Environment Agriculture Center Conference. July 20, 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Benitez Ponce MS. Millersville University, Department of Biology Colloquim Speaker. Oct 20, 2021. Title: Microbial community dynamics in agricultural systems.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mensah, Abigail Aba, et al. "Mitigation of Human Pathogen Contamination in Lettuce and Basil Produced in NFT Hydroponic System." Current Developments in Nutrition 6.Supplement_1 (2022): 522-522.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Ilic S. Human pathogens in plant production systems. Master in Plant Health Management Program, Seminar Series, The Ohio State University, Department of Plant Pathology. March 28, 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Ilic S, Ivey M. Survival of Foodborne Bacterial Pathogens in Leafy Green Hydroponic Production Systems. Ohio Controlled Environment Agriculture Center Conference. July 20, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Sirsat S, Dong M, Ilic S. Mixed Methods Approaches to Investigating Microbial Produce Safety Hazards and Mitigation in Hydroponic and Aquaponic Operations. International Association of Food Protection. August 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mensah, AA, Ivey ML, Miller TM, Ilic S. Impact of Sanitizers on Nutrient Film Technique (NFT) Grown Lettuce and Basil. International Association of Food Protection. August 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Moodispaw MR, Ivey ML, Ilic S. Effectiveness of Surface Sanitizers Against Salmonella Typhimurium in Hydroponic Lettuce System. International Association of Food Protection. August 2022.


Progress 08/15/20 to 08/14/21

Outputs
Target Audience:Our efforts were targeted to reach leafy green hydroponic producers in the state of Ohio. We contacted, visited, and/or communicated with 9 production facilities. The facilities ranged in size, number of heads produced, and system design. In addition, we worked with graduate students, research technicians, and postdoctoral researchers. Changes/Problems:The greatest challenge we encountered during this reporting period was the difficulty to visit and have access to production facilities due to the COVID-19 pandemic. In addition, the pandemic resulted in delays in recruiting both graduate students and postdoctoral researchers. The team is currently complete and many of the restrictions to visit facilities have been lifted. What opportunities for training and professional development has the project provided?As part of our project, and to train project participants (post-doctoral researcher, research technician, and graduate students) we developed a 6-hour workshop on experimental design considerations and data analysis of amplicon sequencing experiments for plant microbiome analysis. This workshop was imparted to a total of 12 participants. How have the results been disseminated to communities of interest?We have had direct communication with hydroponic producers. The goal of this has been to inform our project objectives and engage a greater number of facilities to participate. In addition, the scope and goals of this project, as well as preliminary was shared with the scientific community through a poster presentation at the Virtual Conference of the American Phytopathological Society (Plant Health 2021) and as part of a virtua seminar series at the University of California, Santa Barbara. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will complete the sampling, sequencing, and data analysis of the microbial community surveys of the facilities we have identified, lined up and visited during the first year of this project. In addition, experiments will be established in the greenhouse to test specific management conditions that influence microbiota and plant health in DWC and NFT. Conditions to be managed include source or water, nutrient solution characteristics (pH, EC), sanitation strategies (UV, bleach), and biological inoculants (for plant growth promotion/biological control). For these experiments, microbial communities will be assessed using different methods, including culturing, amplicon sequencing, and metagenomic approaches.

Impacts
What was accomplished under these goals? Microbial communities of plants grown in hydroponic systems have not been thoroughly studied in contrast with the efforts in soils. In hydroponics, plant roots are suspended in a nutrient solution, and the complexity of the ecosystem is lower. Two production systems are most common for leafy greens, Nutrient Film Technique (NFT) and Deep Water Culture (DWC). These two systems differ in the delivery mode of nutrients. In NFT roots grow in a thin layer of a nutrient solution which flows at a constant rate through PVC channels. Whereas in DWC, roots are completely immersed in a pool of aerated nutrient solution. In our preliminary studies, we showed that the diversity of microorganisms associated with lettuce roots and nutrient solution is related to the hydroponic system setup. In this project we aim to characterize the microbial communities in commercial hydroponic lettuce production; and, taking advantage of the environmental control systems, use this information to develop models of microbial community management and contributions to plant health. We will further manipulate the conditions of the nutrient solution in our research greenhouses and test the effect of the resident hydroponic microbiota on plant pathogen and human pathogen spread and establishment. We have established collaborations with nine commercial leafy green production facilities across Ohio. These facilities range in size (from 23 to 13150 m2) and production capacity (up to a million heads per year). Based on the characteristics of the production facilities, we have identified a common set of samples to be collected, which will allow for comparisons across locations. The sampling design targets plant tissue (shoot and root), nutrient solution, and water sources, as well as operational surfaces. We have worked on the standardization and reproducibility of techniques used for sampling, nucleic acid extraction, and recovery of microorganisms from water, nutrient solution, system surfaces, and other facility zones. Our sampling strategy also incorporates environmental sampling zones to monitor members of the hydroponic microbiome that could represent a potential food safety concern. In addition, in collaboration with one of our production facilities, we designed a DWC system to be used for experimentation in our research greenhouses. We currently have the capacity to set up replicated experiments with nine independent DWC, each holding 56 lettuce heads. Together with the already available NFT systems on campus, we plan to manipulate growing conditions to compare and contrast microbiome and their manipulation across hydroponic systems.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: F. E. Guevara, T. Frey, A. Malacrin�, Maria-Soledad Benitez. 2021. Understanding microbial communities composition for management practice improvement in lettuce hydroponic systems in Ohio. APS Plant Health. Virtual poster presentation. August 2-5 2021.