Improving the Safety and Quality of Organic Leafy Greens: Assessment of Good Production Practices Along the Farm to Fork Continuum

<p>NON-TECHNICAL SUMMARY:<br/>

Consumers today are aware of the health benefits of consuming fresh produce. Many consumers prefer organic fresh produce over conventional due to the risks associated with the presence of pesticides or other chemical residues on conventionally grown fresh produce. Also, due to the foodborne illness outbreaks associated with fresh produce in recent years, safety of fresh produce has also become a cause for concern. In the proposed integrated project, scientists from the academia and the US Department of Agriculture, in conjunction with extension specialists, growers and processors will work collaboratively to assess the role of organic production practices in safety and quality of organic leafy greens. The mechanisms of attachment and possible internalization of microorganisms on organic leafy greens, and attachment on harvesting equipment will
be investigated. Environmental factors and agricultural practices that can affect organic leafy greens safety and quality will be determined. Irrigation canal water and sediments will be tested for foodborne bacteria and viruses in summer and winter including rainy and non-rainy days. The effects of organic certified fertilizers such as compost teas on plant growth and on the microbiological safety of leafy green crops will be assessed. Post-harvest interventions (organic plant derived antimicrobials, organic sanitizers and plant antimicrobial incorporated fruit or vegetable based edible films) that can prevent spoilage and pathogenic microbial (bacteria and viruses) growth in organic leafy greens will be evaluated. Natural plant based antimicrobials and organic sanitizers will be applied as rinses, and antimicrobial edible films will be included in packaged leafy greens. The effects of
recycling of antimicrobials and organic sanitizers on their efficacy will be studied. The most effective interventions will be validated on a semi commercial scale. Sensory analysis will be carried out on uninoculated antimicrobial treated leafy greens. An aggressive and extensive outreach program and a follow-up evaluation program for different sectors of the society including agricultural professionals, industry personnel and consumers will be developed and implemented. This program will be shared with other states throughout the nation.

<p>APPROACH:<br/>Attachment and Internalization: The attachment and biofilm forming properties of Escherichia coli O157:H7 and Salmonella enterica on leafy greens will be determined. Bacteria labeled with green fluorescent protein genes will be evaluated for internalization in leafy greens. Surface charge and hydrophobicity measurements will be done and attachment will be viewed under confocal scanning laser microscope (CSLM). Field studies will be conducted using nonpathogenic surrogates, and attachment assessed on coring tool and lettuce. Attachment strength will be calculated. Environmental Factors and Agricultural Practices: Irrigation canal water and sediments will be tested for the presence of foodborne bacteria and viruses using standard procedures during both summer and winter months. If problems exist in the concentration of pathogens from water due to sediment
clogging of the 0.45 micron membrane, micro fiber glass filters or immunomagnetic separation will be employed. Rainy days and non-rainy days will be included. Quantitative microbial risk assessment will be used to model microbial risks from contaminated irrigation water used for leafy greens production. The microbial quality of commercial compost teas will be determined. Crops will be subject to common production and handling practices and effects of compost teas on microbiological safety of leafy green crops will be assessed. Leafy greens will be grown with different input of organic fertilizers to determine any benefits to plant growth. Pre- and Post-harvest Interventions: The efficacy of various plant antimicrobials and organic sanitizers against bacteria (both spoilage and pathogenic) and viruses will be tested on coring equipment and on organic leafy greens (14 day storage). The
re-usability of plant antimicrobials and organic sanitizers for washing leafy greens will be evaluated. Effectiveness of antimicrobial edible films in packaged organic leafy greens (10 day storage) against spoilage and pathogenic bacteria will be tested. The most effective treatments will be validated on a semi commercial scale. Sensory analysis will be done on uninoculated treated samples. Outreach/Education Activities: The results of research will be used to educate the organic agricultural professionals and industry personnel via training workshops, in service training, traditional contact, and consumers via mass media, on practices that improve the safety and quality of organic leafy greens. An Organic Leafy Greens Awareness Website will be created Data Analysis: Data will be analyzed using ANOVA or other appropriate statistical methods such as LSD or Tukeys (SAS software).
Evaluation of Outputs: Evaluation of goals 1, 2 will be based on results obtained from laboratory and field studies. Evaluation of goal 3 will be based on finding effective alternative interventions for organic leafy greens. The findings from all 3 goals will be used in developing the outreach/education program. The evaluation of goal 4 will be based on successful implementation of safe alternative practices by the organic leafy greens growers and industry. The presentations and publications resulting from the project will be enumerated.
<p>PROGRESS: 2012/09 TO 2013/08<p>Target Audience: <br/>Target Audiences The target audiences for this project include: growers, producers, and processors of organic leafy greens; manufacturers of natural antimicrobials and organic sanitizers; and consumers of organic leafy greens. Leafy green producers and manufacturers of natural antimicrobials and organic sanitizers have been involved in this project by providing in-kind support to the project. They have been informed periodically on the results of this project through various forums such as seminars, talks and demonstrations. Dr. Ravishankar visited fresh produce growers and industry personnel in Goodyear, AZ and Yuma, AZ in May 2012 and December 2012, respectively, to discuss research needs. She was invited as the featured speaker at the Yuma Fresh Vegetable Association Annual Meeting in December 2012 in which she
provided an overview of the organic leafy greens project and an update on the project progress to the attendees that included fresh produce professionals. Dr. Ravishankar attended the Food Safety Research Update Meeting with the Yuma Fresh Produce Growers in September 2013 and gave the most recent update on the results from this project. Information about the results from the project was also shared with stakeholders at the Food Safety Consortium at the University of Arizona in October 2013, where posters on efficacy of plant antimicrobial combinations, biofilm formation by E. coli on produce wash water contact surfaces and attachment and cross transfer of Salmonella from soil to produce surfaces were presented. The audience in these meeting included produce growers, students and other faculty involved in food safety research. Dr. Ravishankar also presented the research results generated
from this project at the Annual Meetings of the International Association for Food Protection (IAFP; Charlotte, NC) and Institute of Food Technologists (IFT; Chicago, IL) in 2013 in the form of posters. The audiences at these conference included food safety professionals, produce growers, produce industry executives and students. Dr. Ravishankar's research team conducted food safety demonstrations about natural antimicrobial/organic sanitizers for washing produce, safe produce handling and cross contamination for the local Tucson community at the Science City, Tucson Festival of Books in March 2013 and for the local school students at the Youth Biotechnology Career Expo in October 2013, hosted by the BIO5 Institute, University of Arizona. Dr. Ravishankar's postdoc, Dr. Dev Kumar presented the work on biofilm based attachment of E. coli to washing equipment surfaces in seminar
presentations at the University of Arizona (October 2013) and the Arizona Iceberg Lettuce Research Council (AILRC) Quarterly meeting attended by produce growers (November 2013) in Yuma, AZ. Dr. Ravishankar's graduate student presented a poster on the efficacy of plant antimicrobial combinations at the University of Arizona Student Poster Competition in November 2013 to inform the university community about the efforts associated with making produce safer and the novel strategies implemented to achieve this goal. Dr. Nolte of the Yuma Agricultural Center has used multiple communication strategies towards knowledge dissemination. His team has developed a website, uploaded YouTube videos, developed training DVDs and has made information accessible over facebook.com, Pintrest and Twitter. Apart from improving the accessibility to the latest research in food safety and good
agricultural practices he has also conducted seminars and training series in different parts of Arizona addressing pressing issues in food safety. A unique feature of his efforts has been the bilingual mode of instruction in order to reach a larger target audience. Dr. Nolte's team presented posters related to this project at the 2013 Annual Meeting of the American Society for Horticultural Sciences in Palm Desert, CA. At the University of Oklahoma, Dr. Brandenberger has given four talks to several fresh produce farmers in Oklahoma, related to fresh produce safety. Dr. William McGlynn has incorporated the results of the studies carried out in Dr. Jaroni's lab in five workshops. He also attended the Center for Produce Safety Produce Research Symposium in June 25 and 26 in Rochester, NY where he participated in discussions and planning sessions related to implementing Good
Agricultural Practice (GAP) programs designed to allow smaller-scale, local fresh produce growers and processors to safely supply produce to local and regional retail outlets. He also gathered information about emerging microbial threats and potential mitigation strategies. The Principal Investigators from Oklahoma State University have also established a website related to fresh produce safety. Dr. Jaroni and her graduate students presented the research results generated from this project at the 2013 Annual Meeting of the International Association for Food Protection (IAFP) in Charlotte, NC in the form of posters. Dr. Friedman of the USDA and Dr. Ravishankar participated with Marcia Wood (ARS) in drafting an article for April 2013 issue of Agricultural Research. The article and press release resulted in numerous exchanges of information with reporters of health magazines. Dr. Friedman
is also a member of the W3122 Multistate Research Project entitled "Beneficial and Adverse Effects of Natural Chemicals on Human Health and Food Safety" for which he exchanged ideas with members (university professors and ARS scientists) to facilitate progress associated with food safety and human health. Dr. Jitu Patel of the USDA-ARS shared the results of his research on internalization and natural antimicrobials with the largest organic growers in the mid-Atlantic region. All post-doctoral fellows, technicians and students hired to work on this project have received training on good laboratory practices, including pathogen safety precautions, media preparation, product sampling, field sampling and other laboratory activities related to the project. Dr. Dev Kumar received training in PCR and use of confocal scanning laser microscopy to study the attachment/internalization of bacteria
on/in organic leafy greens from the Palanivelu lab, School of Plant Sciences, University of Arizona, Tucson. Over the past year numerous media outlets have provided coverage/stories on the organic leafy greens research project (see publication list) to reach a wide variety of audience.

Changes/Problems:Nothing Reported

<p>What opportunities for training and professional development has the project provided? All post-doctoral fellows, technicians and students hired to work on this project have received training on good laboratory practices, including pathogen safety precautions, media preparation, product sampling, field sampling and other laboratory activities related to the project. Dr. Sadhana Ravishankar's lab currently has 6 undergraduate students, 3 graduate students and a postdoctoral research associate involved with organic leafy greens research. Undergraduate and graduate
students have been trained in bacterial inoculation & enumeration, environmental isolation of pathogens, soil evaluation and have been involved with conducting experiments under the guidance of Drs. Ravishankar and Dev Kumar. Undergraduate and graduate students have been trained to analyze and present data and have presented research posters at the Food Safety Conference in the University of Arizona. Dr. Sadhana Ravishankar also trains high school students in microbiological techniques and allows them to work on produce safety projects using non-pathogenic bacterial strains. She currently has 2 high school students conducting their science fair projects in her lab. The use of mass media for the propagation of food safety information to stakeholders has given students and other researchers in the Ravishankar lab and at the Yuma Agricultural Center an opportunity to participate in
interviews about their research for youtube videos. The new forum helps students and researchers present their findings in a succinct and easily accessible format. Dr. Dev Kumar received training in PCR and use of confocal scanning laser microscopy to study the attachment/internalization of bacteria on/into organic leafy greens from the Palanivelu lab, School of Plant Sciences, University of Arizona, Tucson. Dr. Dev Kumar presented the work on biofilm based attachment of E. coli to washing equipment surfaces in seminar presentations at the University of Arizona (October 2013) and the Arizona Iceberg Lettuce Research Council (AILRC) Quarterly meeting attended by produce growers (November 2013) in Yuma, AZ. Dr. Ravishankar's graduate student presented a poster on the efficacy of plant antimicrobial combinations at the University of Arizona Student Poster Competition in November 2013
to inform the university community about the efforts associated with making produce safer and the novel strategies implemented to achieve this goal. Drs. Ravishankar, Jaroni, Gerba, Bright, Patel and Nolte's students have had the opportunity to present their research findings at international and state level conferences such as the Annual Meetings of the International Association for Food Protection (IAFP), the Institute of Food Technologists (IFT) and the American Society for Horticultural Sciences (ASHS), as well as the Annual Food Safety Conference in Tucson, AZ, through posters.

<p>How have the results been disseminated to communities of interest? The extension teams at the Yuma Agricultural Center, Oklahoma State University and Dr. Ravishankar's laboratory groups have been involved with the dissemination of information to communities of interest. Seminars, training
sessions, conferences, demonstrations, videos, internet based communication and print media have all been used. Workshops have been conducted throughout the state to provide content detailed training involving the safe production of organically grown leafy greens in Flagstaff (December, 2012), 35 participants; Phoenix (January, 2013), 18 participants; Maricopa (February, 2013), 22 participants; Flagstaff (March, 2013), 28 participants; Snowflake (April, 2013), 22 participants; Yuma (May, 2013), 7 participants; and Nogales (October, 2013), 18 participants. Extension Videos were developed covering the following topics: Edible Films, Microbial Internalization & Surface Adhesion in Leafy Greens, Organic Sanitizers, Compost Teas, Reducing Contamination in Irrigation Water, Safe Management Practices for Using Composted Manures. A 4-part, video series of field worker training modules (English and Spanish) was developed. Bilingual production of training modules has provided an additional avenue of outreach that includes a clientele base that could not necessarily be otherwise reached. While total viewership has reached 2,100 views (February 2012) since the release of the Fresh Produce Safety Channel in April, 2013, user testimonials have been extremely positive. These training segments cover topical areas that include: Hand Washing, Field Sanitation, Bodily Fluid Discharge, and Field Etiquette. Extension also includes programming that uses a dedicated web interface, Facebook, Twitter, and Pinterest. Since their release the sites have generated over 3,300 client interactions (February 2012) which continue to grow daily. Details Fresh Produce Safety Website (2,700 views). A Safe Organic Training website has been created for producers, and progress is being made to integrate this into eXtension. The site contains key information about the USDA Good Handling and Agricultural Practices (GAP/GHP) program, as well as the online, bilingual version of the training materials (http://cals.arizona.edu/fps/). Facebook (102 fans). The Fresh Produce Safety Facebook page provides networked follower's tips, trends and key information about the practices involving fresh produce safety (https://www.facebook.com/FoodSafety101). Twitter (377 followers). Followers receive instant updates related to fresh produce safety production practices, research developments, workshops, and demonstrations. Allows a seamless way to keep updated (https://twitter.com/FreshFoodSafety). Pinterest (56 subscribers). Allows fresh produce safety materials to be shared with others, a leader in creating referral traffic (http://www.pinterest.com/producesafety). Our team from the
Oklahoma state University also participated in various talks, presentations and has developed a website for information dissemination. Dr. Brandenberger has carried out four talks to several fresh produce farmers in Oklahoma, related to fresh produce safety. Dr. William McGlynn has incorporated the results of the studies carried out in Dr. Jaroni's lab in five workshops. Presentations McGlynn, W.G. & Brandenberger, L. 2013. Food Safety 101, presentation given on March 12 at: Oklahoma Market Gardening School. Oklahoma Cooperative Extension Offices, Tulsa, OK. McGlynn, W.G. 2013. Good Agricultural Practices (GAPs): an Introduction. Oral presentation, handouts, given on March 13 to the Otoe-Missouria tribes / Ponca as part of a Plasticulture Market Garden demonstration project. McGlynn, W.G. 2013. Good Agricultural Practices (GAPs): an Introduction. Oral presentation, handouts, given on April 9 to the Otoe-Missouria tribes / Ponca as part of a Plasticulture Market Garden demonstration project. Brandenberger, L.; McGlynn, W.G. 2013. Basic Good Agricultural Practices for Fresh Produce. Oral presentation, handouts, and hands-on demonstration given on May 8 to the Hmong people as part of a Plasticulture Market Garden demonstration project. Brandenberger, L. 2013. Safe Use of Pesticides for Produce Production. Oral presentation, handouts, and hands-on demonstration given on July 2 to the Hmong people as part of a Plasticulture Market Garden demonstration project. Brandenberger, L.; McGlynn, W.G. 2013. Basic Good Agricultural Practices for Fresh Produce. Oral presentation, handouts, and hands-on demonstration given on July 24 to the Otoe-Missouria / Ponca tribes as part of a Plasticulture Market Garden demonstration project. The PIs from Oklahoma State University
have also established a website related to fresh produce safety titled: Fresh Produce Safety (www.freshproducesafety.okstate.edu). Dr. Jaroni and her graduate students presented the research results generated from this project at the 2013 Annual Meeting of the International Association for Food Protection (IAFP) in Charlotte, NC in the form of posters. Dr. Jitu Patel of the USDA-ARS shared the results of his research on internalization and natural antimicrobials with the largest organic growers in the mid-Atlantic region. Dr. Sadhana Ravishankar has also been involved with multiple demonstration based activities to educate the public about plant antimicrobials, safe produce handling and the risk of cross contamination in the kitchen. She and her team conducted Food Safety Demonstrations on these topics for the local Tucson community at the Science City, Tucson Festival of Books; for the
local school students at the Youth Biotechnology Career Expo, hosted by the BIO5 Institute, University of Arizona. Models were created to demonstrate cross contamination from cutting board to produce surfaces. Biophotonic images of pathogens on produce surfaces before and after antimicrobial treatments were also used to demonstrate the effectiveness of plant based antimicrobials. Dr. Ravishankar was invited as the featured speaker at the Yuma Fresh Vegetable Association Annual Meeting in December 2012 in which she provided an overview of the organic leafy greens project and an update on the project progress to the attendees that included fresh produce professionals. She attended the Food Safety Research Update Meeting with the Yuma Fresh Produce Growers in September 2013 and gave the most recent update on the results from this project. Information about the results from the project was also shared with stakeholders at the Food Safety Consortium at the University of Arizona in October 2013, where posters on efficacy of plant antimicrobial combinations, biofilm formation by E. coli on produce wash water contact surfaces and attachment and cross transfer of Salmonella from soil to produce surfaces were presented. The audience in these meetings included produce growers, students and other faculty involved in food safety research. Dr. Ravishankar also presented the research results generated from this project at the Annual Meetings of the International Association for Food Protection (IAFP; Charlotte, NC) and Institute of Food Technologists (IFT; Chicago, IL) in 2013 in the form of posters. The audiences at these conference included food safety professionals, produce growers, produce industry executives and students. Dr. Dev Kumar presented the work on biofilm based attachment of E. coli to washing equipment surfaces in seminar presentations at the University of Arizona (October 2013) and the Arizona Iceberg Lettuce Research Council (AILRC) Quarterly meeting attended by produce growers (November 2013) in Yuma, AZ. Dr. Ravishankar's graduate student presented a poster on the efficacy of plant antimicrobial combinations at the University of Arizona Student Poster Competition in November 2013 to inform the university community about the efforts associated with making produce safer and the novel strategies implemented to achieve this goal.

<p>What do you plan to do during the next reporting period to accomplish the goals? Objective 1 To study the fate of Salmonella on organic leafy greens we plan to grow spinach sprouts that have been challenged with the pathogen through Salmonella contaminated seed or contaminated water in organic soil. Once the plant reaches the 4 leaf stage, the leaves, stem and roots will be sampled for the presence of surface attached and internalized Salmonella to understand distribution of pathogen after contamination event during germination. To understand the effect of soil contamination by Salmonella on organic leafy greens, we plan to grow spinach in soil contaminated by the pathogen. We will also study fate of pathogen on organic leafy greens when irrigation water is contaminated. The effect of these conditions when root damage occurs will be studied. Both surface contamination and possible internalization by the pathogen will be accounted for. Objective 2 Studies to understand the impact of compost teas on the survival of non-pathogenic Escherichia coli on plants and the benefits of the application of compost teas on plant yield will continue. Survival times and safest methods of compost tea application will be evaluated. Studies on the survival of E. coli in composts will continue to determine raw materials most suitable and safe for composting. Dr. Bright will continue her survey of Arizona irrigation water and sediments for norovirus and hepatitis A virus. A microbial risk assessment of irrigation water will be done. Objective 3 Efficacy of combinations of plant based antimicrobials will be evaluated against Salmonella on organic leafy greens to gauge antimicrobial activity of the combinations at the lowest concentrations. The effectiveness of edible film based antimicrobial delivery in salad packaging in reducing pathogen survival and growth will be evaluated against E. coli O157:H7. The use of plant based antimicrobials to enhance the safe reuse of produce wash water will further be pursued to develop a viable alternative to chlorination for the fresh cut produce industry. Efficacy of antimicrobials and their combinations as determined from our data will be tested in a pilot scale. Our team at the University of Arizona will conduct a sensory evaluation to determine acceptance of organic leafy greens sanitized by plant antimicrobials. Continuation of the antiviral efficacy of plant based antimicrobials will occur to determine most effective concentrations and understand mechanisms of antiviral activity. Our team at Oklahoma state University will complete their evaluation of plant based antimicrobials and commercial sanitizers against spoilage lactic acid bacteria and E. coli O157:H7. Objective 4 Our outreach team will continue their efforts towards education of growers on safe produce handling practices and good agricultural practices. We plan to disseminate the information we have gathered to our stakeholders through conference presentations, meetings, farm visits and consultations. Our efforts to make information more accessible will continue through the development of bilingual training DVD's, youtube videos and classes. Information access through social networking websites has been extremely popular and we will continue to add content to our facebook page, twitter accounts and pinterest. The University of Oklahoma will continue their outreach and extension efforts to inform growers about safe organic leafy green handling and on farm strategies. The team will make information more accessible through the development of their website and conducting training modules and classes for our stakeholders. Dr. Ravishankar and other investigators will be continuing their efforts to provide growers with information and guidance regarding produce safety through seminars, webinars, farm visits and guest lectures. The courses taught by Dr. Ravishankar at the University of Arizona focus on produce safety and information about her research extensively, and are a primer for students interested in food safety and food microbiology. She will also continue conducting her demonstrations on natural interventions, safe produce handling and cross contamination risks at the Science City- Tucson Festival of Books and other educational fairs. All investigators will be presenting their research and findings at various international conferences such as the Annual Meetings of the International Association for Food Protection (IAFP), Institute of Food Technologists (IFT), the American Society for Horticultural Sciences (ASHS) and others.
<p>PROGRESS:2011/09/01 TO 2012/08/31

<p>OUTPUTS:<br/>The potential for internalization and consequential phylloplane transfer of shiga-toxigenic Escherichia coli through the root system of hydroponically grown organic spinach (cultivars Waitiki and Space) was studied. Hydrophobicity and curli production ability of Salmonella Newport were tested along with the proclivity of organic leafy greens wash water in aiding S. enterica biofilm formation. Hydrophobicity of organic leafy green microflora isolated from wash water was measured. Survival of S. Newport and E. coli O157:H7 in 8 different commercial organic composts and that of S. Newport in 7 compost teas was determined. Iceberg and romaine lettuces were irrigated with compost teas with and without E. coli K12 to understand risks associated with compost tea application on organic leafy greens in organic plots at the Yuma Agricultural Center. Effect of 3 coring tool designs was evaluated for risk of contamination during coring of lettuce. Comparison was made with a current tool used commercially in reducing the risk of lettuce contamination in the field. Organic leafy green wash waters were analyzed for changes in pH, turbidity and microbial counts with reuse. Irrigation water and sediments were sampled from Maricopa and Yuma agricultural systems during and after growing season and from Yuma region after rainfall events. These samples were analyzed for Salmonella, and E. coli. These are also being processed for hepatitis A, noroviruses and other viruses that could serve as indicators of fecal contamination of water. Hydrogen peroxide, olive, grapeseed and apple extracts were tested against E. coli O157:H7 on organic romaine and iceberg lettuces and mature and baby spinaches and Pediococcus damnosus on
baby spinach. Hibiscus tea, hydrogen peroxide, carvacrol (CAR), citral, and essential oils of oregano, cinnamon, and lemongrass were evaluated against E. coli O157:H7 on organic leafy greens. Antimicrobial properties of oregano, cinnamon and lemongrass essential oils, CAR, cinnamaldehyde (CIN), hibiscus tea, green tea and grapeseed extract were explored against S. Newport on organic leafy greens. Hibiscus concentrate, olive and apple extracts, oregano, cinnamon and lemongrass oils were tested against Pseudomonas fluorescens on organic iceberg lettuce. Dietary supplements, mushroom extracts and rice hull liquid smoke were tested against S. enterica. Antimicrobial activities of apple skin polyphenols against L. monocytogenes, E. coli O157:H7, and S. enterica and bactericidal activities of 10 nutraceuticals against E. coli O157:H7, S. enterica, L. monocytogenes, and Staphylococcus aureus were tested. Oregano oil, CAR, cinnamon oil, CIN, lemongrass oil, citral, allspice oil and olive extract were tested in vitro against murine norovirus. Three commercial organic sanitizers and calcium hypochlorite were tested on organic leafy greens inoculated with S. Newport. Antibacterial activities of apple-based edible films containing apple polyphenols were evaluated. Apple, carrot, and hibiscus-based edible films with CIN and CAR were tested for physico-chemical properties and ability to inactivate S. Newport and P. fluoresens in organic leafy green salad bags.

<p>PARTICIPANTS:<br/> Project Director: Sadhana Ravishankar, Ph.D. Dept. of Veterinary Science & Microbiology, University of Arizona (UA), Tucson, AZ. Staff in Dr. Ravishankar's group: a post-doctoral fellow, research technician, graduate and 5 undergraduate students. She oversees all microbiological work related to attachment and
internalization of Salmonella enterica on/in leafy greens, and pre- and post-harvest interventions against S.enterica and Pseudomonas spp. on leafy greens. She is involved in education and outreach. The postdoctoral fellow in Dr. Ravishankar's lab received training in sample preparation and use of confocal scanning laser microscopy to study the attachment/internalization of bacteria on/in organic leafy greens from the USDA, ARS, BARC research facility in Beltsville, MD. Dr. Ravishankar has been involved with organic leafy green safety knowledge dispersion in classrooms, conferences, book fairs and symposiums. Co-Project Directors: Charles Gerba, Ph.D. & Kelly Bright, Ph.D. Dept. of Soil, Water & Environmental Sciences, UA. Staff in their group: 3 grad. students and one research technician. Dr. Gerba assists with data analysis and performs quantitative microbial risk assessment for test
pathogens. Dr. Bright conducts field sampling and laboratory testing of irrigation water samples for bacterial and viral pathogens and evaluates post-harvest interventions against viruses. Jorge Fonseca, Ph.D. Vegetable & Post-harvest Specialist, Yuma Agricultural Center, UA-Yuma, AZ. Dr. Fonseca has 2 research technicians and plans to hire a post-doctoral fellow. Dr. Fonseca investigates attachment of non-pathogenic E.coli on harvesting equipment, and conducts assessment of compost teas/biostimulants and their effects on nonpathogenic E.coli in organic leafy green fields. He is involved in outreach. Kurt Nolte, Ph.D. Director, Yuma County Coop. Extension, Yuma, AZ. Dr. Nolte has a student worker to assist with extension and outreach. He is responsible for all extension activities of this project. Divya Jaroni, Ph.D. Department of Animal Science, Oklahoma State University. Dr. Jaroni has 1 graduate student and one postdoctoral fellow. Dr. Jaroni oversees all microbiological work related to interventions for E.coli O157:H7 and Lactococcus spp. She is involved in education and outreach along with Drs. McGlynn and Brandenberger. Dr. Jitendra Patel, Ph.D. Lead Scientist, Environmental Microbiology & Food Safety Laboratory, USDA-ARS, Beltsville, MD. Dr. Patel has a post-doctoral fellow and 1 research technician. Dr. Patel investigates attachment and internalization of E.coli O157:H7 on/in organic leafy greens. Mendel Friedman, Ph.D. USDA-ARS WRRC, Produce Safety & Microbiology Research, Albany, CA. Dr. Friedman is a consultant, providing expertise on plant antimicrobials. JV Farms, Yuma, AZ provided organic leafy greens. CreAgri Inc., Hayward, CA and Apple Poly, LLC., Morrill, NE provided antimicrobial plant extracts. The following companies provided organic sanitizers: GTX Technologies, Amarillo, TX; E3 Organics, Inc., CA; Summerdale, Inc., Verona, WI; ASC Agrecom, Inc., Tucson, AZ; Arch Chemicals, Inc., Charleston, TN; and BioSafe Systems, East Hartford, CT. TARGET AUDIENCES: Target audiences for these projects include: growers, producers, and processors of organic leafy greens; manufacturers of natural antimicrobials and organic sanitizers; and consumers of organic leafy greens. Leafy green producers and manufacturers of natural antimicrobials and organic sanitizers have been involved in this project by providing in-kind support to the project (see participants). They have been informed periodically on the results of this project and Drs Nolte and Ravishankar have been involved with creating online and mobile application versions of Fresh Organic Produce safety training, aimed towards meeting needs of commercial producers and direct marketers. Drs. Bright, Gerba and Ravishankar have had multiple interactions with growers and producers in the Yuma, AZ region. Discussion with growers and food safety managers (JV Farms, Dole Fresh Produce, Pasquinelli Produce Company, Fresh Express, Taylor Farms, Duncan Family Farms and Foxy Fresh Produce) were conducted to understand current issues associated with safety of organic leafy greens. Drs. Bright, Ravishankar and Gerba attended the Food Safety Summit at the Yuma Agricultural Center (attended by 65 industry personnel, researchers from University of Arizona, UA Cooperative Extension). Dr. Bright traveled to Yuma, AZ to meet growers and discuss current irrigation water sampling methods and Drs. Bright, Gerba and Ravishankar attended the Yuma Ag Summit that included a panel discussion entitled, "Quality Standards and Sampling of Irrigation Water for Food Safety". Drs. Bright, Gerba, Meer and Ravishankar attended a meeting with Dr. Michelle Russell from U.C. Davis and the President and Food Safety Managers of JV Farms in Yuma, AZ to discuss research needs and possible research collaborations with UC Davis scientists. Drs. Bright, Dev Kumar, Ravishankar and Nolte toured and visited Duncan Family Farms in Goodyear, AZ to discuss food safety concerns and best management practices. Drs. Bright, Ravishankar, Nolte and Gerba currently have ongoing research projects in the Yuma, AZ region. Many of these involve cooperation with stakeholders. Dr. Nolte was involved in conducting Arizona outreach food safety workshops with a goal to increase the applied knowledge in specific areas within the production of safe organically grown leafy greens. Dr. Ravishankar and Dr. Dev Kumar conducted a seminar for local high school students about approaches for antimicrobial applications on leafy greens and laboratory techniques involved. Demonstration of safe produce handling practices was performed at the University of Arizona book fair by Drs. Ravishankar and Dev Kumar. Dr. Ravishankar gave a seminar on natural antimicrobials and organic leafy greens to undergraduate microbiology majors. Drs. Ravishankar and Nolte conducted a seminar for students in the University of Arizona on organic leafy green contamination, issues for farmers and steps taken towards contamination mitigation. Dr. Jaroni spoke at workshops held in Opelousas, LA and focus groups in Baton Rouge, LA for fresh produce farmers. These activities were designed to increase food safety knowledge of organic fresh produce farmers in LA. PROJECT MODIFICATIONS: Dr. Divya Jaroni transferred from the Southern University Agricultural Research & Extension Center (SUAREC), Baton Rouge, Louisiana, to Oklahoma State University, Stillwater, Oklahoma. She is currently an Assistant professor in the Department of Animal Sciences at the Oklahoma State University, Stillwater, Oklahoma. She has included Dr. William McGlynn, Horticultural Products Processing Specialist, and Dr. Lynn Brandenberger, Professor, Department of Horticulture & Landscape Architecture at the Oklahoma State University to conduct the extension and outreach activities of this project. Project funds have been transferred to the Oklahoma State University for Dr. Jaroni's work in the project. No significant project modifications with regard to the goals of the project have been made.
<p>PROGRESS: 2010/09/01 TO 2011/08/31<br/>
<p>OUTPUTS: <br/>Attachment of Escherichia coli O157:H7 isolates to organic romaine and iceberg lettuce and green cabbage was studied. Curli expression, hydrophobicity, and biofilm formation by these strains were evaluated as potential virulence factors. Persistence of wild type and curli and celluose mutant E.coli O157:H7 strains on 4 organic spinach cultivars (Lazio, Waitiki, Emilia, and Space) for 14 days was studied. Effect of natural hormones (growth regulators) on the ability of E.coli to form biofilms and have better survival opportunities in field conditions was investigated. Irrigation water sampling from Yuma, AZ; 15 samples/month are collected and analyzed for indicator organisms, quality parameters, and E.coli O157:H7, Salmonella, hepatitis A virus, and norovirus surveillance. Presumptive positives are analyzed for confirmation. Romaine and iceberg lettuce were planted in semi-commercial fields in Yuma, AZ. One field was planted in October and harvested in January, the other in December and harvested in March, respectively. Both fields are part of the Organic Parcel of the Yuma Agricultural Center managed by Dr.Fonseca, and are used for quality and microbial risk tests of foliar application of organic biostimulants and compost teas. Parallel to first phase of these trials, the survival of E.coli in different compost teas and biostimulants was studied in the lab and in the field being incorporated through foliar application using different spray systems (providing different solution drop sizes). Studies were conducted to determine the risk impact of using a contaminated tool to core iceberg lettuce destined to the fresh-cut industry. Efficacy of low levels of chlorine washes to prevent cross-contamination from field-contaminated lettuce to non-contaminated lettuce was evaluated. Plant compounds (green tea and olive extracts, cinnamaldehyde, citral, and oils of allspice, clove. lemongrass, cinnamon) were tested in vitro against E.coli. Cinnamaldehyde, carvacrol and grapeseed extracts were tested for their activity in vitro against S.typhimurium and E.coli O157:H7. Effects of hibiscus concentrate and tea, olive extract, carvacrol, cinnamldehyde, citral and oils of cinnamon, clovebud, allspice and lemongrass were evaluated on MS-2 bacteriophage. The bactericidal effects of aqueous extracts of roselle calyces and leaves against E.coli O157:H7, Salmonella enterica and Listeria monocytogenes were evaluated. Roselle calyx concentrate and tea were also tested against E.coli O157:H7 on romaine lettuce and Salmonella on sprouts. The antimicrobial effects of plant extracts (olive, apple, 1-5%; hibiscus, 10-30%) and five organic sanitizers (fulvic acid-based, 1-3%; citric acid-based, 0.05%) against S.Newport on organic romaine and iceberg lettuce, and adult and baby spinach stored at 4C for 3 days were investigated. Plant extracts were also tested against background flora of all 4 organic leafy greens. Effects of carvacrol and cinnmaldehyde (1.5 and 3%) containing edible apple, carrot and hibiscus films against S.Newport in bagged iceberg lettuce, baby spinach and spring mix, and against E.coli O157:H7 in bagged spinach stored at 4C for 7 days were evaluated. PARTICIPANTS: Project Director: Sadhana Ravishankar, Ph.D. Dept. of Veterinary Science & Microbiology, University of Arizona, Tucson, AZ. Staff in Dr. Ravishankar's group involved in this project include: 1 post-doctoral fellow, 1 technician, 1 grad. Student and 2 undergrad. students. She oversees all microbiological work related to attachment and internalization of Salmonella enterica on/in leafy greens, and pre- and post-harvest interventions against S.enterica and Pseudomonas spp. on leafy greens. She is involved in education and outreach. Co-Project Directors: Charles Gerba, Ph.D. & Kelly Bright, Ph.D. Dept. of Soil, Water & Environmental Sciences, University of Arizona, Tucson, AZ. Staff in their group involved in this project include: 3 grad. students and one research technician. Dr. Gerba assists with data analysis and performs quantitative microbial risk assessment for test pathogens. Dr. Bright conducts field sampling and laboratory testing of irrigation water samples for bacterial and viral pathogens, and evaluates post-harvest interventions against hepatitis A and murine norovirus. Jorge Fonseca, Ph.D. Vegetable & Post-harvest Specialist. Yuma Agricultural Center, University of Arizona-Yuma, AZ. Dr. Fonseca has 2 research technicians involved in this project and plans to hire a post-doctoral fellow. Dr. Fonseca investigates the attachment of non-pathogenic Escherichia coli on harvesting equipment, and conducts assessment of compost teas/biostimulants and their effects on non pathogenic E.coli in organic leafy green fields. He is involved in outreach activities. Kurt Nolte, Ph.D. Director, Yuma County Coop. Extension, Yuma, AZ. Dr. Nolte expects to hire a grad. student this summer to assist with extension and outreach. He is responsible for all extension activities of this project. Divya Jaroni, Ph.D. Southern University Agricultural Research & Extension Center (SUAREC), Baton Rouge, LA. Dr. Jaroni has 1 grad. student involved in the project and plans to hire a post- doctoral fellow. Dr. Jaroni oversees all microbiological work related to interventions for E.coli O157:H7 and Lactococcus spp. She is involved in education and outreach. Jitendra Patel, Ph.D. Lead Scientist, Environmental Microbiology & Food Safety Laboratory, USDA-ARS, Beltsville, MD. Dr. Patel has a post-doctoral fellow and 1 research technician involved in the project. Dr. Patel investigates the attachment and internalization of E.coli O157:H7 on/in organic leafy greens. Mendel Friedman, Ph.D. USDA-ARS Western Regional Research Center, Produce Safety & Microbiology Research, Albany, CA. Dr. Friedman is a consultant, providing expertise on plant antimicrobials and is involved in education and outreach. JV Farms, Yuma, AZ provides in-kind support in the form of organic leafy greens. CreAgri Inc., Hayward, CA and Apple Poly, LLC., Morrill, NE provide in-kind support in the form of antimicrobial plant extracts. The following companies provide in-kind support in the form of organic sanitizers. GTX Technologies, Amarillo, TX.; E3 Organics, Inc., CA; Summerdale, Inc., Verona, WI.; ASC Agrecom, Inc., Tucson, AZ; Arch Chemicals, Inc., Charleston, TN; and BioSafe Systems, East Hartford, CT. TARGET AUDIENCES: The target audiences for this project include: growers, producers, and processors of organic leafy greens; manufacturers of natural antimicrobials and organic sanitizers; and consumers of organic leafy greens. Leafy green producers and manufacturers of natural antimicrobials and organic sanitizers have been involved in this project by providing in-kind support to the project (see participants). They have been informed periodically on the results of this project. Drs. Ravishankar, Gerba and Bright attended the Arizona Produce Research Priorities meeting organized by the Center for Produce Safety, September 2010 to discuss our fresh produce/leafy greens research. They also visited Yuma in December 2010 to meet leafy greens producers, discuss their research needs and provide an introduction to the organic leafy greens grant. Dr. Ravishankar presented an overview of the grant and some results at the Food Safety Summit in Yuma, January 2011, attended by 65 fresh produce industry professionals. In March 2011, Drs. Ravishankar, Bright, and Jaroni traveled to Yuma, Arizona to attend the Southwest Agricultural Summit. They along with Drs. Nolte and Fonseca discussed this project with agricultural professionals attending the meeting. In April 2011, Drs. Ravishankar, Bright, Gerba, and Meer traveled to Yuma to meet with growers and University of California, Davis researchers, to discuss the research needs of the producers and to develop future plans, including additional collaborations between the growers and researchers at the University of Arizona, Tucson, AZ, and the University of California, Davis, CA. In microbiology classes, at the University of Arizona, for undergraduate and graduate students Dr. Ravishankar has incorporated laboratory exercises to isolate the background microflora, spoilage and pathogenic microorganisms from organic leafy greens. In collaboration with the Joint Institute of Food Safety and Applied Nutrition, Dr. Fonseca offered 2 courses on Good Agricultural Practices at Costa Rica and Mexico for produce exported to the U.S. He plans to offer an on-line course this summer on post-harvest physiology, technology and produce safety for undergrad./grad. students and industry professionals at the University of Arizona. Dr. Ravishankar will offer a course on food microbiology and biotechnology in spring 2012 which will cover organic leafy greens safety and quality aspects. Information generated from this project was/will be used in all these courses. All post-doctoral fellows, technicians and students hired to work on this project have received training on good laboratory practices, including pathogen safety precautions, media preparation, product sampling, field sampling and other laboratory activities related to the project. Dr. Meer received training in sample preparation and use of confocal scanning laser microscopy to study the attachment/internalization of bacteria on/in organic leafy greens from the USDA, ARS, BARC research facility in Beltsville, MD. Over the past year numerous media outlets have provided coverage/stories on the organic leafy greens research project (see publication list) to reach a wide variety of audience. PROJECT MODIFICATIONS: We have not made any significant project modifications with regard to the goals of the project. However, we intend to request a no-cost extension for the first year of this project since there was a significant delay in the transfer of funds to the subcontracts, and the process of hiring technical assistance (e.g., graduate and undergraduate students, postdoctoral fellows, research technicians, etc.) took longer than anticipated.</p>