DETERMINING THE POTENTIAL MITIGATION OF TRICLOSAN ACCUMULATION IN COMMERCIAL ONION PLANTS USING PLANT-GROWTH PROMOTING RHIZOBACTERIA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1001121
• Grant No.: 2014-67020-21617
• Project No.: TEXW-2013-02707
• Proposal No.: 2013-02707
• Program Code: A1401
• Project Start Date: Jan 1, 2014
• Project End Date: Dec 31, 2018
• Grant Year: 2014

Project Director
Mendez, M. O.

Recipient Organization
TEXAS A&M INTERNATIONAL UNIVERSITY FOUNDATION, INC.
5201 UNIVERSITY BLVD
LAREDO,TX 780411920

Performing Department
Biology & Chemistry

Non Technical Summary
Triclosan, a broad-spectrum antimicrobial, is one of the seven most prevalent wastewater contaminants found in freshwater streams in the United States. Due its ubiquitous nature and potential impact on human and environmental health, including endocrine disruption activity and biodiversity loss in aquatic systems, Triclosan is considered a chemical of emerging concern. Triclosan is the active ingredient in detergents, various personal hygiene products, textiles and plastics as well as food contact materials and is therefore classified as one of the pharmaceutical and personal care products (PPCP). Even with high removal rates in wastewater treatment facilities, Triclosan primarily reaches the environment via treated wastewater effluent (recycled waters). Most agricultural regions use waters from potentially contaminated streams for irrigation and as a result are exposing crops to chronic low-levels of contamination for much of the growing season. Our previous studies indicate that Triclosan may reduce the overall biomass in onion plants, but most importantly, it can be found in all edible portions of onion plants. Additionally, the abundance of the associated microbial community is reduced when irrigated at environmentally relevant concentrations. There is concern that irrigation using recycled waters will not only affect profitability of commercial crops but also increase food contamination. In this project, we intend to determine the use of Triclosan-resistant plant growth-promoting rhizobacteria (PGPRs) in order to 1) reduce the effects of Triclosan on onion plant productivity and 2) decrease the uptake of Triclosan by onion plants. Triclosan-resistant PGPRs will be evaluated for the use of Triclosan as a primary carbon source and the degradation of Triclosan when bacteria cultures are applied to soils. Based on concentrations of Triclosan and its metabolites (methyl-Triclosan and chlorophenols) remaining in soils, those PGPRs that increase Triclosan degradation will be used in a greenhouse study to evaluate effects of these PGPRs on plant growth and Triclosan uptake by plants. Both soils and plant tissues will be evaluated for Triclosan concentrations, along with its metabolites, in order to determine the PGPRs with optimum Triclosan degradation activity. The outcomes of this project will allow us to understand the fate and transport of Triclosan in agroecosystems, particularly when applied via irrigation waters. This investigation will be used as a model for the use of PGPRs in preventing contamination of crops, while sustainably enhancing agroecosystems. Additionally, we can begin to better understand the interaction between Triclosan and the plant-microbe relationships that are important in promoting microbial diversity, plant health, and environmental sustainability.

Animal Health Component

0%

Research Effort Categories

Basic

40%

Applied

60%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	1451	1150	50%
102	0110	1060	50%

Knowledge Area
133 - Pollution Prevention and Mitigation; 102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
0110 - Soil; 1451 - Onion, garlic, leek, shallot;

Field Of Science
1060 - Biology (whole systems); 1150 - Toxicology;

Keywords

triclosan

degradation

onions

plant growth-promoting rhizobacteria

recycled irrigation water

Goals / Objectives
In order to evaluate the efficacy of using plant growth-promoting rhizobacteria (PGPR) for reducing Triclosan accumulation, while simultaneously increasing productivity in onion plants, the objectives of this study are as follows: To identify PGPRs capable of using Triclosan as a carbon source. Previously isolated PGPRs that are Triclosan resistant and demonstrate PGPR activity (phosphate solubilization, siderophore production, and IAA production) will be examined to determine their ability to utilize Triclosan as a sole-carbon source in addition to degradation activity in situ. The effect of PGPR inoculation on concentrations of Triclosan and its metabolites (methyl-Triclosan and chlorophenols) in soils will be determined. To determine the effect of Triclosan-degrading PGPRs on productivity of onion plants. Individual Triclosan-degrading PGPRs and a PGPR consortium will be evaluated as an inoculum for ameliorating Triclosan-induced inhibition of plant productivity. To examine the impact of Triclosan-degrading PGPRs on Triclosan uptake by onion plants. Assessment of the concentrations of Triclosan (and its metabolites) in soils and plant tissues will determine the potential use of Triclosan-degrading PGPRs to prevent accumulation of Triclosan from contaminated irrigation waters. Overall, the goal of this project is to enhance understanding of the role of PGPRs on the impact of Triclosan, a chemical of emerging concern, by mimicking a natural transmission route via repeated application of contaminated irrigation water. The use of PGPRs in preventing losses in plant productivity and quality through degradation of an organic contaminant found in recycled irrigation water will improve the long-term sustainability of managed agroecosystems. This investigation will provide a model for the use of PGPRs in preventing contamination of crops and will serve as a critical first step towards sustainable practices decreasing potential contamination of foodstuffs via irrigation using recycled waters. Additionally, we can begin to elucidate the interaction between Triclosan and the plant-microbe relationships that are important in promoting microbial diversity, plant health, and environmental sustainability.

Project Methods
Approach: Seven previously isolated rhizobacteria that are resistant to Triclosan (> 1,500 µg/l) with an optimal diversity of PGPR activity (siderophore production, phosphate solubilization, IAA production, and ACC-deaminase activity) will serve as inocula in an initial study to determine Triclosan degradation (Exp #1 & 2) and in a greenhouse study to determine onion productivity and Triclosan uptake by onions (Exp #3) when irrigated with Triclosan. In the Triclosan degradation studies (Exp #1 & 2), the utilization of Triclosan as a sole carbon source and the enhancement of Triclosan degradation, in situ by these PGPRs, will be determined. In the greenhouse study (Exp #3), onion plants will be grown in a combination of Triclosan and inoculation treatments in order to determine 1) the impact of Triclosan-degrading PGPRs (TD-PGPRs) on onion plant productivity; and 2) the concentration of Triclosan and its metabolites in soils and onion plants. Individual isolates will be tested along with a consortium of selected strains to determine if the effects are enhanced by cooperative PGPR activity. As an indicator of overall soil health, soils will be examined for numbers of heterotrophic bacteria (total and Triclosan-resistant). Additionally, concentrations of Triclosan and its metabolites (methyl-Triclosan and chlorophenols) will be assessed. Experiment #1. Triclosan as a sole carbon source. Each isolate will be incubated on chloride-free minimal salts medium (MSM) amended with glucose (2 g/l) as a control (MSMG) and Triclosan (2 g/l) as a treatment (MSMT) in triplicate. Based on growth and clearing, isolates positive for Triclosan use on MSMT will be transferred onto fresh MSMT plates to confirm results. Analysis and Evaluation: Positive results on MSMT will be based on ranking (none, minimal, intermediate, or high) of growth and clearing observed. The level of diffuse clearing observed will be directly related to Triclosan degradation. Based on at least a minimal ranking for both factors, those strains will be further examined in Exp #2. Strains with the highest rankings and diversity of PGPR activity will be included in the consortium inoculum. Experiment #2. Triclosan degradation in soils. All isolates confirmed as TD-PGPRs from Exp #1 will then be tested (both individually and as a consortium), in triplicate. Prior to inoculation, soils will be characterized for physicochemical properties and trace concentrations of Triclosan. Each sterilized mesocosm will contain 300 g of soil inoculated by irrigating with each culture (equivalent to 106 CFU/ml) prior to mixing soils, and then irrigated with 0 and 1.5 µg/l Triclosan for 30 days. Analyses: Soils will be subsampled at days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 to determine Triclosan and metabolite concentrations (methyl-Triclosan and chlorophenols) using a modification of previously used methods to simultaneously extract the chlorophenols (2,4DCP and 2,4,6-TCP). Methyl-triclosan will be extracted separately with a 1:1 water:acetonitrile and 5 mM ammonium acetate. All extracts will be derivatized using N-Methyl-N-(t-butyldimethylsilyl) trifluoroacetamide prior to analysis by GC-MS. Initial and final heterotrophic plate counts of the total (R2A) and Triclosan-resistant (R2AT, amended with 1.5 µg/l Triclosan) populations will be determined. Evaluation: Concentrations of Triclosan and metabolites over time will be used to develop decay curves. For chemical extractions, quality control measures will be monitored throughout the process to ensure the target chemical has been evaluated. A comparison of Triclosan degradation trends among the inocula, in addition to diverse PGPR activity, will determine the inocula to be used in Exp #3. Additionally, a method for determining methyl-Triclosan in soils will be optimized. Although transformation of Triclosan to chlorophenols generally occurs in anaerobic conditions, it will still be assessed based on its classification as a priority pollutant. If chlorophenols and/or methyl-Triclosan are not detected, we will eliminate them from the greenhouse study analyses. Experiment #3. Greenhouse study. Plants will be grown in a 2 x 5 factorial experiment with five replicates per treatment. Two Triclosan treatments (0.0 and 1.5 µg/l) and five inoculation treatments (absence of TD-PGPRs, three individual TD-PGPRs, and a consortium) will be tested. Seeds will be inoculated prior to germination and planting. Soils will also be inoculated with TD-PGPRs by irrigating soils prior to mixing. An initial germination study using petri dishes will precede the plant growth experiment. In the greenhouse, inoculated onion seeds will be germinated directly into amended soils and irrigated with Triclosan for 16 weeks with one plant per pot (five pots per treatment). Analysis of Plant Productivity: Germination, height, and biomass will be compared. Root length, volume, and surface area will also be determined using the digitized root system analysis in a WinRHIZO PRO. Initial and final heterotrophic plate counts for both the total (R2A) and the Triclosan resistant (R2AT, amended with 1.5 µg/l Triclosan) populations will also be determined. Analysis of Triclosan Concentrations: Using the plant tissues and soils collected from the greenhouse experiment, Triclosan and its metabolites (methyl-Triclosan and chlorophenols) will be extracted as previously optimized for Exp #2. Plants will be sectioned into bulb (including the roots and basal plate) and leaves. Evaluation: Plant growth and Triclosan concentrations (including metabolites) will be compared to determine the optimum inocula for both productivity enhancement and reduction of Triclosan accumulation. Optimal Triclosan-degrading PGPRs (TD-PGPRs) used in this study will have mineralized Triclosan and possess a diversity of PGPR activity. Efforts: Results of this project will be communicated to the scientific community at national scientific meetings and in peer-reviewed scientific journals in order to contribute to the understanding of chemical cycling of Triclosan, particularly in agroecosystems, and its metabolites in soils and plant tissues. As a model for the use of PGPRs in preventing crop contamination via recycled waters, findings will be reported to the Texas A&M AgriLife Extension. Several minority students (undergraduate and graduate) will receive training in laboratory practices (experimental design, data analysis, literature research, and scientific writing) and protocols within the fields of plant science, environmental science, chemistry, and microbiology. Students will also have an opportunity to present data at a national scientific meeting and network with the scientific community. Evaluation: Increase in understanding the degradation of Triclosan: Observations of MSMT agar assay to determine the use of Triclosan as a sole-carbon source by PGPRs. Identification of TD-PGPRs based on soil Triclosan and metabolite concentrations along with accompanying decay curves. Assessment of overall soil health by comparison of total (R2A) and Triclosan-resistant (R2AT) heterotrophic bacteria numbers. Increase in productivity of onion plants by using TD-PGPRs: Comparison of height, biomass, and root architecture among treatments. Assessment of overall soil health by comparison of the total (R2A) and Triclosan-resistant (R2AT) heterotrophic bacteria numbers. Identification of TD-PGPRs and the PGPR activities that contribute to ameliorating Triclosan-induced inhibition of plant growth. Decreased uptake of Triclosan in onion plants by using TD-PGPRs: Identification of TD-PGPRs to be commercially developed for mitigating Triclosan accumulation in onion plant tissues by comparison of concentrations of Triclosan and its metabolites. Cumulative Evaluation: Examination of our model as we consult with Texas A&M AgriLife Extension for broader use with organic contaminants in the field.

Progress 01/01/18 to 12/31/18

Outputs
Target Audience:Target audiences included students at Texas A&M International University, Laredo College, and research scientists attending local and national scientific engagements. A total of 15 Hispanic students (11 females and 4 males) were targeted for training in agricultural sciences to complete laboratory experiments, data analysis and perform field work. Of the 15 students, two were paid as undergraduate research assistants, 11 students obtained academic credits for our undergraduate research course, one graduate student was funded as a graduate research assistant and one graduate student was completing her thesis research. Data collected during the 2018 reporting period was presented to a wide scope of scientists and research students involved in STEM fields at our university-wide research conference (Lamar Bruni Vergara Student Research Conference) and at the USDA NIFA and NSF Water and Soils Meeting. A group of 20 faculty and staff members at Texas A&M International University were introduced to chemistry in agricultural science. Other target audiences included members of the Rio Grande International Study Center, extension specialists, soil scientists, and other principal investigators and graduate students in soil science. Changes/Problems:Although not initially proposed in the request for funding, a field sampling of commercial onion farms was completed during the onion growing season this year to determine if data collected from the greenhouse studies are applicable to trends from field data collections. Final analyses of the triclosan and methyl triclosan concentrations in soils and plant tissues collected during both the greenhouse study (Objectives 2 & 3) and the farm study (Objectives 2 & 3) were delayed due to auto-sampler repairs needed in Spring 2018, modifications of GC-MS protocols for plant tissues in Fall 2018, and most recently, a repair needed for the internal MS unit. Final GC-MS analysis of the farm samples and confirmations for selected samples will continue through Spring 2019 which has delayed completion of the manuscript. Our new timeline includes manuscript preparation and submission for the phylogenetic analysis of triclosan resistant and triclosan-degrading rhizobacteria (Objective 1) and for the chemical analysis and evaluation of the soil degradation study (Objective 1) during Spring 2019. Chemical analysis of the farm study (Objectives 2 & 3) will be completed by the end of Spring 2019. Evaluation of the farm data and comparisons to the greenhouse study will be completed during Spring 2019 to prepare for manuscript submission in Summer 2019. Undergraduate manuscripts are currently being submitted. A total of at least 21 genome annotations (funded by the NH-INBRE/NIH IDeA programs) of the triclosan-resistant rhizobacteria isolated from the greenhouse study are currently being submitted with final submissions to be completed by Summer 2019. What opportunities for training and professional development has the project provided?The primary trainees served under the activities (8 Hispanic female undergraduates, 3 Hispanic male undergraduates, 2 Hispanic female graduate students, and 1 Hispanic male graduate student; 14 Hispanic students in total) were trained in microbial culturing, soil and plant chemical extractions, microbial analysis, and field sampling. Trainees are more competitive for national summer research programs and admission into PhD programs, specifically in agricultural science or a related field. One Hispanic female graduate student won first place in the graduate student competition for Biology & Chemistry at our local research conference and grand prize (graduate) for the university. Two other students were awarded for their research presentations at our local research conference: one Hispanic male graduate student won third place in the graduate student competition for Biology & Chemistry and one Hispanic female undergraduate student won third place in the undergraduate student competition for Biology & Chemistry. One Hispanic female graduate student was awarded 2nd place in the 2019 Caminos Thesis Competition in Food and Agricultural Sciences, a national master's thesis competition supported by a USDA-AFRI grant. She will be provided travel funds to attend professional workshops developed by the program next year. Three Hispanic females and one male will be primary and co-authors on two manuscripts in preparation. How have the results been disseminated to communities of interest?Data was disseminated to a wide audience of students, scientists, and professionals. Trainee presentations were at a local conference, TAMIU's LBV Student Research Conference for TAMIU and Laredo College students. Professionals from NRCS and Texas Parks & Wildlife were invited to the research conference along with members of a local water conservation organization. Two project directors' meetings, the USDA NIFA and NSF Water and Soils Meetings served to disseminate information to program directors and national program leaders with backgrounds in soil and crop science, and environmental chemistry. Trainees communicated with farmers and extension service personnel on their agricultural research related to the supported objectives. There was a total of five presentations for this reporting period. What do you plan to do during the next reporting period to accomplish the goals?Within the next six months, manuscripts will be submitted for the phylogenetic analysis of triclosan resistant and triclosan-degrading rhizobacteria by Spring 2019 (Objective 1) and the soil degradation study by Summer 2019 (Objective 1). Completion of the manuscript and a thesis on the effect of triclosan-degrading rhizobacteria on onion productivity (Objective 2) and mitigation of triclosan accumulation in soils and onion tissues (Objective 3) will be completed Summer 2019. Data will be presented at a national meeting. Further, soils, DNA extracts, and a culture library of triclosan-resistant bacteria from the greenhouse study and commercial onion farms will be further analyzed for effects of triclosan on the microbial community, influence of triclosan on antimicrobial resistance development, and microbial community changes affecting triclosan levels in soils. Data collections will be processed with analyses that support an additional two thesis students along with multiple publications. Genome annotations of the culture library collected from studies here will be published in 2019. These additional studies not supported by this grant were funded by the TAMIU University Research Grant program for proposals funded in 2017 and 2018.

Impacts
What was accomplished under these goals? Impact Statement: Triclosan, an antimicrobial, is one of seven most prevalent wastewater contaminants in U.S. freshwater streams. Due to its prevalence and potential impact on human and environmental health, triclosan is a chemical of emerging concern. Triclosan reaches the environment via treated wastewater effluent (recycled waters). Waters from these receiving streams are used for irrigation, resulting in chronic low-level exposure to crops. Triclosan levels in the Rio Grande, a receiving stream for the U.S.-Mexico border region, are higher than national levels. Our study focuses on decreasing levels of triclosan in soils and onion plants, a major South Texas crop, using root bacteria. Three root bacteria capable of using triclosan as a food source and possessing plant growth-promoting activities were added to pots in a soil study (no plants) and an onion study (with plants). Bacteria were added individually and in a mixture. The soil study analysis showed that by adding the selected bacteria individually, the levels of triclosan decreased. However, the mixture of all three bacteria was most effective, decreasing triclosan levels by 45% compared to the control (no bacteria added). Levels of methyl triclosan (the initial breakdown product by bacteria) increased over time, indicating that triclosan degradation is still occurring even in the control soils. Methyl triclosan levels were lowest in the soils with the mixture of bacteria. Analysis of the onion study showed that adding bacteria individually or as a mixture decreased levels of triclosan in soils and onion plants compared to the control (no bacteria added). Triclosan decreased by up to 84% in soils, 95% in onion bulbs, and 88% in leaves when soils were inoculated with bacteria. Numbers of triclosan-resistant bacteria also increased in inoculated treatments within soils and the root zone. Our study shows that bacteria in mixtures were better at breaking down triclosan and reducing levels in plant tissues, but methyl triclosan will remain in soils at low concentrations, especially when crops are not present. Also, some bacteria will be negatively affected by triclosan in irrigation waters, but we do not yet know which species of beneficial bacteria are impacted. This investigation will be used as a model for the use of beneficial bacteria in preventing contamination of crops, while sustainably enhancing agroecosystems. 1) Major activities completed/experiments conducted Objective 1 Analysis of data collected from the soil irrigation study (1.5 µg/100 g dry soil every other day for 30 days) was completed. Levels of triclosan and methyl triclosan were compared among inoculation treatments: control (no inoculum), OT1-11C2, OT2-03A, OT2-17 and OTMIX. An additional 6-day study was performed to determine the transformation rate of triclosan to methyl triclosan by spiking soils once with 3.0 µg triclosan. Objective 2 Greenhouse study (91 days) evaluating the effect of triclosan-degrading plant growth-promoting rhizobacteria on onion productivity: Bacterial data collected from soils and rhizosphere material (roots, bulbs, and attached soils) was analyzed. Field sampling of South Texas onion farms: Soil (within the onion root zone, 0-10 cm; and below, 10-30 cm), water and onions were collected from three farms irrigated with Rio Grande waters to compare data collected in greenhouse study. Objective 3 Chemical extracts of soils and onion tissues from the greenhouse study described in Objective 2 were analyzed for triclosan and methyl triclosan. 2) Data collected Objective 1 Chemical data (triclosan and methyl triclosan concentrations) from soils in the 30-day triclosan degradation study were analyzed. Chemical data (triclosan and methyl triclosan concentrations) was collected from the 6-day soil study. Objective 2 Total/triclosan-resistant rhizobacterial counts and total/triclosan-resistant soil bacterial counts data collected were 1) comparisons between inoculation treatments for the greenhouse study; and 2) a collection of bacteria from the farm samples and comparisons between soils and rhizosphere samples. Objective 3 Chemical extractants from soils and plant tissue (bulbs and leaves) samples from the 91-day greenhouse onion study were analyzed by GC-MS for triclosan and methyl triclosan concentrations. 3) Summary statistics and discussion of results Objective 1 Compared to control soils, OTMIX (a mixture of all three bacteria) decreased triclosan levels in soils by 45%. Methyl triclosan levels increased and did not plateau during the study. The control soil and OTMIX had the lowest levels of methyl triclosan, 27% lower than when adding individual bacteria. The OTMIX treatment could be breaking down triclosan completely or to a by-product not analyzed in this study. In the 6-day study, triclosan transformation to methyl triclosan was 6.3 days. We can now use this to model triclosan decay in soils. Objective 2 In the greenhouse study, triclosan-resistant bacteria increased in soils and onion rhizosphere samples when OT2-17 and the OTMIX treatment (all 3 bacteria together) were added to pots. This may be increasing the resiliency of the microbial communities from the negative impacts of antimicrobial exposure. From the farm samples, triclosan-resistant bacterial counts were greater in the top layer (0-10 cm) of soils, within the onion root zone. Triclosan resistant root-associated bacteria was as high as 56% of the total bacteria, which was lower than the 98% of total bacteria found in onion roots from the greenhouse study. Seventy-five onion rhizobacteria were collected from farm samples for further study. Identification of the beneficial soil bacteria affected by triclosan in irrigation waters will be determined using funds from the Texas A&M International University Research Grant. Objective 3 Triclosan-degrading rhizobacteria decreased triclosan by up to 84% in soils, 95% in onion bulbs, and 88% in leaves compared to the control. Methyl triclosan in soils was higher with inoculation when triclosan was irrigated with molecular water, but levels were lower (up to 55% with bacteria added) when triclosan was irrigated with simulated river water. Despite water type, inoculation decreased methyl triclosan in bulbs but increased levels in leaves. Overall, OT2-17 alone or OTMIX (all three rhizobacteria) were the most effective at decreasing levels of triclosan. Methyl triclosan levels were minimal (< 2 ng/g) and not likely a concern for human consumption. 4) Key outcomes or other accomplishments realized Objective 1: Change in knowledge: a mechanism to model expected conversion of triclosan to methyl triclosan by bacteria to compare mitigation efforts. Change in action: a graduate trainee mentoring four undergraduates in the development and analysis of a small degradation study. Change in condition: one Hispanic male undergraduate able to pursue research in biodegradation of pollutants in soils and three rhizobacteria reduced triclosan levels by 45% in unplanted soils. Objective 2: Changes in knowledge: four Hispanic undergraduates trained in field sampling, triclosan-resistant bacteria are higher in triclosan-irrigated rhizospheres than in bulk soils, irrigation with triclosan in the field may not affect bacteria below the onion root zone. Change in action: an onion farm has been identified for future studies. A change in condition is that seven Hispanic trainees will be able to pursue field research in agricultural science. Objective 3: Change in knowledge: plant growth-promoting rhizobacteria that decrease triclosan in onions were identified, methyl triclosan does not accumulate at high levels in plant tissues but is at higher concentrations in field water, and one Hispanic female undergraduate with technical expertise in chemical extractions. Change in condition: inoculation with triclosan-degrading rhizobacteria mitigated triclosan uptake in onions by up to 95%.

Publications

• Type: Theses/Dissertations Status: Accepted Year Published: 2018 Citation: Garcia, Ashley M. 2018. Effect of triclosan-tolerant plant growth-promoting rhizobacteria on triclosan degradation in soils. Masters Thesis, Texas A&M International University, Laredo, TX.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Gonzalez, O.D., A.M. Garcia, A. Addo-Mensah, and M.O. Mendez. 2018. Influence of inoculation with plant growth-promoting rhizobacteria to reduce triclosan levels in onions and soils. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Apr 25-26. *Third place in graduate student competition for Biology & Chemistry
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Carlo, P. A.M. Garcia, J.I. Cohen and M.O. Mendez. 2018. Phylogenetic analysis of triclosan exposed onion rhizobacteria. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Apr 25-26. *First place in graduate student competition for Biology & Chemistry *Grand Prize Winner (Graduate)
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ortiz, A., F. Rayo, and M.O. Mendez. 2018. Mechanisms of triclosan-resistance of plant growth-promoting rhizobacteria isolated from onions. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Apr 25-26. *Third place in undergraduate student competition for Biology & Chemistry
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mendez, M.O., A.M. Garcia, O.D. Gonzalez, and A. Addo-Mensah. 2018. Determining the potential mitigation of triclosan accumulation in commercial onion plants using plant-growth promoting rhizobacteria. USDA NIFA and NSF Water and Soils Meeting, USDA, Newark, DE, Oct 2-3.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mendez, M.O., A.M. Garcia, O.D. Gonzalez, and A. Addo-Mensah. 2018. Determining the potential mitigation of triclosan accumulation in commercial onion plants using plant-growth promoting rhizobacteria. USDA NIFA and NSF Water and Soils Meeting, USDA, Washington, D.C., Jan 29-31.

Progress 01/01/14 to 12/31/18

Outputs
Target Audience:Target audiences included students at Texas A&M International University, Laredo College, high school students and research scientists attending local and national scientific engagements. A total of 56 Hispanic students (40 females and 16 males) and 2 White non-Hispanic females were targeted for training in agricultural sciences to complete laboratory experiments, data analysis and perform field work. Twenty-two of the undergraduate students and one graduate student were trained through a laboratory module for my Environmental Microbiology course. Of the other 35 students, four were paid as undergraduate research assistants, 23 students obtained academic credits for our undergraduate research course, three students produced an honors thesis, one student received our ACT on IDEAs undergraduate research fellowship, two graduate students were funded as graduate research assistants and two graduate students will be using collected materials for their graduate theses. Data collected during the project funding period (2014-2018) was presented to a wide scope of scientists and research students involved in STEM fields at six regional research conferences (TAMIU's Lamar Bruni Vergara Student Research Conference - 2015, 2016, 2017, 2018; Texas Undergraduate Research Day - 2015; and Texas A&M University System's Pathways Conference - 2015), four national society meetings (American Society of Microbiology 115th General Meeting; Annual Society for Advancing Chicanos/Native Americans in Science National Conference - 2015, 2016; and ASA/CSA/SSA 2017 Annual Meeting) and five project director's meetings (NIWQP and AFRI Project Director's Meeting - 2014, 2015, 2016; USDA NIFA and NSF Water and Soils Meeting - Jan 2018, Oct 2018). A group of 360 primarily Hispanic high school students were introduced to agricultural science research at the Interdisciplinary Science Research Conference (TAMIU, Laredo, TX) two years in a row (2016 and 2017). High school students (40 Hispanic students) were introduced to the connection between environmental pollution and food security at the J.B. Alexander High School Career Day 2017. A group of 20 faculty and staff members at Texas A&M International University in 2018 were introduced to chemistry in agricultural science. Other target audiences included members of the Rio Grande International Study Center, extension specialists, agricultural engineers, soil scientists, and other principal investigators and graduate students involved in geochemical, geophysical, and microbial analysis of agroecosystems. Changes/Problems:Repairs and maintenance issues for the GC-MS significantly impacted our schedule. This included two auto-sampler repairs and an internal MS repair. Analyses of soil and plant tissue samples were delayed and QA/QC protocols were also reconfigured throughout the funding period. In addition, our greenhouse where the soil degradation study took place was demolished to build a new science building in its place. The inoculation onion study was delayed by 5 months due to the construction of a new greenhouse on campus. Therefore, the data collection and analysis were delayed. Final analyses of the triclosan and methyl triclosan concentrations in soils and plant tissues collected during the greenhouse study and the added farm sampling study were delayed due to GC-MS repairs and modifications of plant tissue protocols. Final GC-MS analysis of the farm samples and confirmations for selected samples will continue through Spring 2019 which has delayed completion of the manuscript. Our new timeline includes manuscript preparation and submission for the phylogenetic analysis of triclosan resistant and triclosan-degrading rhizobacteria (Objective 1) and for the chemical analysis and evaluation of the soil degradation study (Objective 1) during Spring 2019. Chemical analysis of the farm study (Objectives 2 & 3) will be completed by the end of Spring 2019. Evaluation of the farm data and comparisons to the greenhouse study will be completed during Spring 2019 to prepare for manuscript submission in Summer 2019. Undergraduate manuscripts are currently being submitted. A total of at least 21 genome annotations (funded by the NH-INBRE/NIH IDeA programs) of the triclosan-resistant rhizobacteria isolated from the greenhouse study are currently being submitted with final submissions to be completed by Summer 2019. What opportunities for training and professional development has the project provided?The primary trainees served under the activities (24 Hispanic female undergraduates, 1 White non-Hispanic female undergraduate, 6 Hispanic male undergraduates, 3 Hispanic female graduate students, and 1 Hispanic male graduate student; 34 Hispanic students in total) were trained in microbial culturing, soil and plant chemical extractions, microbial analysis, greenhouse management, soil physicochemical analysis, and field sampling. Students gained professional development in analyzing, interpreting, and presenting data, a tool that can be translated to other research projects. Trainees are more competitive for national summer research programs and admission into PhD programs, specifically in agricultural science or a related field. As a direct result of their participation in the research supported here, students received several recognitions for their work. This included professional workshops and awards for their research participation. In 2016, a Hispanic female graduate student trained under this grant received a competitive opportunity to attend the 2016 USDA CULTIVAR Graduate Fellow Career Preparation Institute and the 2016 Global Marine Sustainability Workshop Arctic Seas and Gulf of Mexico in Oban, Scotland. In 2017, a Hispanic female graduate student won the NRCS Laredo Resource Team's Conservation Teacher of the Year for her extension work and first place for the graduate student competition for Biology & Chemistry at TAMIU's LBV Student Research Conference. A Hispanic male graduate student and a Hispanic female undergraduate attended the Analysis of Repeated Measures Workshop at the ASA/CSSA/SSSA Annual Meeting in Tampa, FL.In 2018, three students received four awards for their research presentations at TAMIU's LBV Student Research Conference: one Hispanic female graduate student won first place in the graduate student competition for Biology & Chemistry and grand prize (graduate) for the university; one Hispanic male graduate student won third place in the graduate student competition for Biology & Chemistry; one Hispanic female undergraduate student won third place in the undergraduate student competition for Biology & Chemistry. In addition, a Hispanic female graduate student was awarded 2nd place in the 2019 Caminos Thesis Competition in Food and Agricultural Sciences, a national master's thesis competition supported by a USDA-AFRI grant. She will be provided travel funds to attend professional workshops developed by the program next year. Two undergraduate Hispanic females and one graduate Hispanic female were co-authors on a publication in the Journal of Environmental Quality. Three Hispanic females and one Hispanic male will be primary and co-authors on three manuscripts in preparation. Students trained as part of this funding in my Environmental Microbiology laboratory course (21 undergraduates and one graduate student) in isolation techniques for bacterial identification and screening for both plant growth promoting activities and use of triclosan as a carbon source using rhizobacterial isolates from the inoculation greenhouse study. They will also be co-authors on genome annotations of these bacteria. How have the results been disseminated to communities of interest?Data was disseminated to a wide audience of students, scientists, and professionals. Trainee presentations were at six regional research conferences: TAMIU's LBV Student Research Conference (2015, 2016, 2017, 2018) for TAMIU and Laredo College students; Texas Undergraduate Research Day - 2015 for Texas policymakers, scientists, and undergraduate students; and Texas A&M University System's Pathways Conference (2015) for Texas A&M systemwide researchers, graduate students, and undergraduates. Professionals from NRCS and Texas Parks & Wildlife were invited to the regional research conferences along with members of a local water conservation organization. Trainees presented to soil scientists, environmental microbiologists, hydrologists, and crop scientists at four national society meetings: American Society of Microbiology 115th General Meeting; Annual Society for Advancing Chicanos/Native Americans in Science National Conference - 2015, 2016; and ASA/CSA/SSA 2017 Annual Meeting. Annual research progress has been presented at five project director's meetings: NIWQP and AFRI Project Director's Meeting - 2014, 2015, 2016; USDA NIFA and NSF Water and Soils Meeting - Jan 2018, Oct 2018. Agricultural science research and the impact of recycled waters on crops was presented to high school students at the Interdisciplinary Science Research Conference (TAMIU, Laredo, TX) two years in a row (2016 and 2017) and at the J.B. Alexander High School Career Day in 2017. A group of 20 faculty and staff members at Texas A&M International University in 2017 and 2018 were introduced to chemistry in agricultural science. Seven trainees communicated with farmers and extension service personnel on their agricultural research related to the supported objectives. There was a total of 9 regional research conference presentations, 7 national research conference presentations, 5 research presentations at project director's meetings, 3 outreach presentations, and 3 informal outreach events. Trainees were co-authors on a publication in the Journal of Environmental Quality. Research supported here was featured in the American Society of Agronomy's News Release (May 25, 2016), in Farm World (June 29, 2016) and in TAMIU's 2016 Annual Report. Additionally, two honor's theses and one master's thesis were produced. What do you plan to do during the next reporting period to accomplish the goals?Within the next six months, manuscripts will be submitted for the phylogenetic analysis of triclosan resistant and triclosan-degrading rhizobacteria by Spring 2019 (Objective 1) and the soil degradation study by Summer 2019 (Objective 1). Completion of the manuscript and a thesis on the effect of triclosan-degrading rhizobacteria on onion productivity (Objective 2) and mitigation of triclosan accumulation in soils and onion tissues (Objective 3) will be completed Summer 2019. Data will be presented at a national meeting. Further, soils, DNA extracts, and a culture library of triclosan-resistant bacteria from the greenhouse study and commercial onion farms will be further analyzed for effects of triclosan on the microbial community, influence of triclosan on antimicrobial resistance development, and microbial community changes affecting triclosan levels in soils. Data collections will be processed with analyses that support an additional two thesis students along with multiple publications. Genome annotations of the culture library collected from studies here will be published in 2019. These additional studies not supported by this grant were funded by the TAMIU University Research Grant program for proposals funded in 2017 and 2018.

Impacts
What was accomplished under these goals? Impact Statement: Triclosan, an antimicrobial, is one of seven most prevalent wastewater contaminants in U.S. freshwater streams. Due to its prevalence and potential impact on human and environmental health, triclosan is a chemical of emerging concern. Triclosan reaches the environment via treated wastewater effluent (recycled waters). Waters from receiving streams are used for irrigation, resulting in chronic low-level exposure to crops. Triclosan levels in the Rio Grande, a receiving stream for the U.S.-Mexico border region, are higher than national levels. Our study focuses on decreasing levels of triclosan in soils and onion plants, a major South Texas crop, using root bacteria. We have determined that 29% of the triclosan-resistant bacteria from onion roots were capable of using triclosan as a food source, indicating their potential for breaking down triclosan in the environment. These bacteria can also be beneficial to plants by increasing nutrient availability and regulating hormones for plant growth. To test our hypothesis, three of the root bacteria were inoculated individually and as a mixture (all three) to pots in a soil study (no plants) and an onion study (with plants). All pots were irrigated with triclosan every other day at environmentally relevant levels. In the soil study, triclosan exposure decreased bacterial numbers when bacteria were not added. The mixture of all three bacteria increased numbers of total and triclosan-resistant soil bacteria ten times more than adding each species alone. Triclosan levels decreased by 45% and methyl triclosan (the initial breakdown product of triclosan) levels decreased by 22% when bacteria were added as a mixture. In the greenhouse study, two species of bacteria were identified that will increase onion productivity with or without triclosan present. The ability of the bacteria to break down triclosan and methyl triclosan was more pronounced when plants were present. Triclosan decreased by up to 84% in soils, 95% in onion bulbs, and 88% in leaves when bacteria were inoculated into soils. Numbers of triclosan-resistant bacteria also increased in the inoculated treatments within soils and the root zone. Our study shows that the cooperative metabolism of multiple bacteria is most effective in breaking down triclosan and reducing levels in plant tissues. Methyl triclosan will remain in soils at low concentrations, particularly in unplanted soils. Also, beneficial soil bacteria will be negatively affected by triclosan, and likely other antimicrobials, in irrigation waters; we do not yet know which species are impacted and its effect on agricultural productivity. Outcomes of this study will be used as a model for using beneficial bacteria in preventing contamination of crops, while sustainably enhancing agroecosystems. 1) Major activities completed/experiments conducted Objective 1 Twenty-one triclosan-resistant rhizobacteria (root-associated bacteria) determined to be plant growth-promoting (PGPRs) were tested on agar plate assays (2 g/L triclosan) to determine if triclosan could be used as a sole carbon source. Isolates were identified by the 16S rRNA gene. Degradation of triclosan by three triclosan-degrading PGPRs (individually and in a mixture, OTMIX) was determined in a 30-day soil irrigation study (no plants; 1.5 µg triclosan every other day). Levels of methyl triclosan, the initial biodegradation product, was also determined. Objective 2 A 91-day greenhouse study evaluated the effect of the same three triclosan-degrading PGPRs from the soil study on onion productivity. Soils were irrigated with 1.5 µg/L triclosan using molecular water (ddH2O) and simulated river water. Bacterial data was collected from soils and rhizosphere material. Twenty-three triclosan-resistant rhizobacteria were isolated to screen for plant growth-promoting activity and the use of triclosan as a sole carbon source. Soil (within the onion root zone, 0-10cm; and below, 10-30cm), water and onions were collected from three South Texas farms irrigated with Rio Grande waters to compare data collected in the greenhouse study. Objective 3 Chemical extracts of soils and onion tissues from the greenhouse study (Objective 2) were analyzed for triclosan and methyl triclosan by GC-MS. 2) Data collected Objective 1 Identifications of triclosan-degrading rhizobacteria were completed. Soil chemical extractants from the 30-day triclosan degradation study were analyzed by GC-MS for triclosan and methyl triclosan. Counts of total and triclosan-resistant heterotrophic bacteria were collected for Days 0, 15 and 30. Objective 2 Onion biomass, total/triclosan-resistant rhizobacterial counts, and total/triclosan-resistant soil bacterial counts were collected from the onion greenhouse study and the commercial onion farms. The identity, plant growth-promoting activity and use of triclosan as a sole carbon source was determined for newly isolated rhizobacteria. Objective 3 Levels of triclosan and methyl triclosan from soils and plant tissue (bulbs and leaves) from the 91-day greenhouse onion study were analyzed. 3) Summary statistics and discussion of results Objective 1 Six isolates were able to use triclosan as a sole carbon source. Data indicates that elevated levels of triclosan pre-exposure is influencing adaptations of rhizobacteria in soils. Compared to control soils, OTMIX (a mixture of all three bacteria) decreased triclosan levels in soils by 45%. and methyl triclosan by 27%. The OTMIX treatment could be breaking down triclosan completely or to a by-product not analyzed in this study. Objective 2 Triclosan increased onion biomass by 20 -50% in the control and OT2-03A inoculation treatments. Without triclosan, OT1-11C2 increased onion biomass. Triclosan irrigation enhanced triclosan resistant (and likely triclosan-degrading) populations when OT2-17 and OTMIX were inoculated. Four isolated rhizobacteria were capable of using triclosan as a sole carbon source and possessed at least one plant growth-promoting activity. Triclosan-resistant bacterial counts were greater in the top layer (0-10 cm) of farm soils, within the onion root zone. Seventy-five onion rhizobacteria were collected for further study. Objective 3 Triclosan-degrading rhizobacteria decreased triclosan by up to 84% in soils, 95% in onion bulbs, and 88% in leaves compared to the control. Methyl triclosan in soils and tissues were minimal (< 2 ng/g) and not likely a concern for human consumption. Overall, OT2-17 alone or OTMIX (all three rhizobacteria) were the most effective at decreasing levels of triclosan. 4) Key outcomes or other accomplishments realized Objective 1: Changes in knowledge: a mechanism to model conversion of triclosan to methyl triclosan by bacteria; 29 students knowledgeable in microbial isolation and identification. Changes in action: a protocol for determining triclosan and methyl triclosan levels in soils and onions; two graduate trainees using QA/QC protocols. Change in condition: three rhizobacteria identified as reducing triclosan levels by 45% in unplanted soils. Objective 2: Changes in knowledge: two rhizobacteria increased onion productivity; triclosan-resistant bacteria are higher in the rhizosphere than in soils; irrigation with triclosan in farms may not affect bacteria below the onion root zone; 19 students knowledgeable in greenhouse study implementation and analyses. Change in action: an onion farm has been identified for future studies. Change in condition: seven Hispanic trainees can pursue field research in agricultural science. Objective 3: Changes in knowledge: a mixture of bacteria is most effective at decreasing triclosan in onions, methyl triclosan is negligible in plant tissues but may be significant in field waters, and 9 students with technical expertise in chemical extractions. Change in condition: triclosan-degrading rhizobacteria mitigated triclosan uptake in onions by up to 95%.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Mendez, M.O., Valdez, E., Martinez, E., Saucedo M., and Wilson, B. 2016. Fate of triclosan in irrigated soil: degradation in soil and translocation into onion and tomato. Journal of Environmental Quality. 45(3):1029-1035. doi: 10.2134/jeq2015.07.0386.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Garcia, Ashley M. 2014. Triclosan resistance of onion rhizobacteria. Senior Honors Thesis, Texas A&M International University, Laredo, TX.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Martinez, Eileen M. 2014. Uptake of triclosan in tomato plants. Senior Honors Thesis, Texas A&M International University, Laredo, TX.
• Type: Theses/Dissertations Status: Accepted Year Published: 2018 Citation: Garcia, Ashley M. 2018. Effect of triclosan-tolerant plant growth-promoting rhizobacteria on triclosan degradation in soils. Masters Thesis, Texas A&M International University, Laredo, TX.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Mendez, M. O. 2014. Determining the potential mitigation of triclosan accumulation in commercial onions plants using plant-growth promoting rhizobacteria. 2014 NIWQP and AFRI Project Directors Meeting, USDA, Washington, DC, Oct 28-29.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Chapa, M.L., A.M. Garcia, J.I. Cohen, and M.O. Mendez. 2015. The effect of triclosan on growth of a plant growth-promoting rhizobacterium. American Society for Microbiology 115th General Meeting, New Orleans, LA, May 30June 2.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Delgado, D., P. Carlo, M. Zuniga, A.M. Garcia, B.A. Wilson, and M.O. Mendez. 2015. Assessing changes in the populations of total and triclosan-tolerant heterotrophic bacteria over time in soils irrigated with triclosan. American Society for Microbiology 115th General Meeting, New Orleans, LA, May 30June 2.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Mendez, M. O. and A. Addo-Mensah. 2015. Determining the potential mitigation of triclosan accumulation in commercial onion plants using plant-growth promoting rhizobacteria. 2015 AFRI and NIWQP Annual Project Directors Meeting, USDA, Greensboro, NC, July 28-29.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Garcia, A., M. Chapa, B.A. Wilson, and M.O. Mendez. 2015. Triclosan Degradation by Triclosan Resistant Onion Rhizobacteria. Texas A&M University System 12th Annual Pathways Student Research Symposium, Corpus Christi, TX, Oct 22-23.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Chapa, M., A.M. Garcia, and M.O. Mendez. 2015. The effect of triclosan on the growth of a plant growth-promoting rhizobacteria. Texas A&M University System 12th Annual Pathways Student Research Symposium, Corpus Christi, TX, Oct 22-23. Third Place in Environmental Science, Undergraduate.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Delgado, D., M. Zuniga, P. Carlo, A.M. Garcia, B.A. Wilson, and M.O. Mendez.2015. Dynamics of populations of total and triclosan-tolerant heterotrophic bacterial populations in soils irrigated with triclosan. Texas A&M University System 12th Annual Pathways Student Research Symposium, Corpus Christi, TX, Oct 22-23.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Garcia, A.M. and M.O. Mendez. 2015. Triclosan degradation in soils inoculated with triclosan resistant plant growth-promoting rhizobacteria. Annual Society for Advancing Chicanos/Native Americans in Science National Conference, Washington, DC, Oct. 29-31.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Garcia, A., M. Chapa, B.A. Wilson, and M.O. Mendez. 2015. Utilization and degradation of triclosan by triclosan-resistant onion rhizobacteria. American Society for Microbiology 115th General Meeting, New Orleans, LA, May 30June 2.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Garcia, A.M., M. Chapa, B. Wilson, A. Addo-Mensah, and M.O. Mendez. 2016. Effect of triclosan-resistant plant growth-promoting rhizobacteria on triclosan degradation in soils. 2016 Society for Advancing Chicanos/Hispanics & Native Americans in Science National Conference, Long Beach, CA, Oct 13-15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Garcia, A.M. and M.O. Mendez. 2016. Triclosan degradation. Lamar Bruni Vergara Conference, Texas A&M International University, Laredo, TX, Apr 1-2.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mendez, M.O. 2016. Research in environmental science: from the field to the lab. Interdisciplinary Science Research Conference, Texas A&M International University, Laredo, TX, Sep 7.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mendez, M.O., A.M. Garcia, A. Addo-Mensah. 2016. Determining the potential mitigation of triclosan accumulation in commercial onion plants using plant-growth promoting rhizobacteria. 2016 AFRI and NIWQP Annual Project Directors Meeting, Washington, DC.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Gonzalez, O.D., A.M. Garcia, A. Addo-Mensah, and M.O. Mendez. 2017. Effects of the accumulation of triclosan and its by-products on soil levels and plant uptake. ASA/CSSA/SSSA Annual Meeting, Tampa, FL, Oct 22-25.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Carlo, P., A.M. Garcia, K. Vallejo, J. Cohen and M.O. Mendez. 2017. Phylogenetic analysis of triclosan-sensitive and triclosan-tolerant onion rhizobacteria. ASA/CSSA/SSSA Annual Meeting, Tampa, FL, Oct 22-25.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Garcia, A.M., A. Addo-Mensah, and M.O. Mendez. 2017. Effect of triclosan resistant plant growth promoting rhizobacteria on triclosan degradation in soils. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Mar 30-31. *First place in graduate student competition for Biology & Chemistry
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Cantu, L., P. Carlo, A.M. Garcia, A. Addo-Mensah, and M. O. Mendez. 2017. Comparison of TCS accumulation in onions using spiked molecular water, tap water and simulated river water. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Mar 30-31.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Mendez, M.O. 2017. Research in Environmental Science: From the Field to the Lab, Interdisciplinary Science Research Conference, Laredo, TX, Sep 06.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Mendez, M.O. 2017. Restoring our environment for human health. J.B. Alexander High School Career Day, Laredo, TX, Dec 15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Gonzalez, O.D., A.M. Garcia, A. Addo-Mensah, and M.O. Mendez. 2018. Influence of inoculation with plant growth-promoting rhizobacteria to reduce triclosan levels in onions and soils. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Apr 25-26. *Third place in graduate student competition for Biology & Chemistry
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Carlo, P. A.M. Garcia, J.I. Cohen and M.O. Mendez. 2018. Phylogenetic analysis of triclosan exposed onion rhizobacteria. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Apr 25-26. *First place in graduate student competition for Biology & Chemistry *Grand Prize Winner (Graduate)
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ortiz, A., F. Rayo, and M.O. Mendez. 2018. Mechanisms of triclosan-resistance of plant growth-promoting rhizobacteria isolated from onions. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Apr 25-26. *Third place in undergraduate student competition for Biology & Chemistry
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mendez, M.O., A.M. Garcia, O.D. Gonzalez, and A. Addo-Mensah. 2018. Determining the potential mitigation of triclosan accumulation in commercial onion plants using plant-growth promoting rhizobacteria. USDA NIFA and NSF Water and Soils Meeting, USDA, Newark, DE, Oct 2-3.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mendez, M.O., A.M. Garcia, O.D. Gonzalez, and A. Addo-Mensah. 2018. Determining the potential mitigation of triclosan accumulation in commercial onion plants using plant-growth promoting rhizobacteria. USDA NIFA and NSF Water and Soils Meeting, USDA, Washington, D.C., Jan 29-31.

Progress 01/01/17 to 12/31/17

Outputs
Target Audience:Target audiences included students at Texas A&M International University as well as high school students and research scientists attending local and national scientific engagements. A total of 34 Hispanic students (22 females and 12 males) and one White non-Hispanic female student were targeted for training. Twenty-two of the undergraduate students and one graduate student were trained through a laboratory module for my Environmental Microbiology course. Of the other 12 students, two were paid as undergraduate research assistants, eight students obtained academic credit for our undergraduate research course, one graduate student was funded as a graduate research assistant and one graduate student was completing her thesis research. Data collected during the 2017 reporting period was presented to a wide scope of scientists and research students involved in STEM fields at our university-wide research conference (Lamar Bruni Vergara Student Research Conference) and at a national meeting (ASA/CSA/SSSA 2017 Annual Meeting). A group of 180 primarily Hispanic high school students were introduced to agricultural science research at the Interdisciplinary Science Research Conference (TAMIU, Laredo, TX). A group of 40 primarily Hispanic high school students were also introduced to the connection between environmental pollution and food security at the J.B. Alexander High School Career Day. Other target audiences included members of the Rio Grande International Study Center, extension specialists, high school science teachers, high school students, soil scientists, other principal investigators and graduate students in soil science. Changes/Problems:During the end of Fall 2017, the greenhouse where the soil degradation study was performed was replaced to build a new science building. The inoculation greenhouse study was delayed by five months due to the construction of a new greenhouse on campus. Therefore, the data collection for the greenhouse study to complete the last two objectives was delayed. In addition, there was a delay in acquiring a technician for a preventive maintenance and GCMS auto-sampler repair for 3 months following the end of the greenhouse study. Our new timeline includes manuscript preparation and submission for the phylogenetic analysis of triclosan resistant and triclosan-degrading rhizobacteria (Objective 1) during Spring 2018 and for the chemical analysis and evaluation of the soil degradation study (Objective 1) by Summer 2018. Chemical analysis of the final greenhouse study (Objectives 2 & 3) will be completed by the end of Spring 2018. Evaluation of the experiments for Objectives 2 & 3 will be completed during Summer 2018. Manuscript preparation for the greenhouse study will be initiated at the end of Summer 2018 and completed during Fall 2018. What opportunities for training and professional development has the project provided?The primary trainees served under the activities (10 undergraduate and two graduate students) were trained in greenhouse experimental design, inoculation of soils and plants, microbial culturing, soil chemical extractions and microbial analysis. They are more competitive for national summer research programs and admission into PhD programs. One Hispanic female graduate student won third place in the Biology & Chemistry division of our local research conference and the NRCS Laredo Resource Team's Conservation Teacher of the Year for 2017 for her extension work. One male graduate student and one female undergraduate student presented research at the ASA/CSSA/SSA meeting and attended a SAS workshop. In my Environmental Microbiology laboratory course, 22 undergraduate and one graduate student were trained in isolation techniques for bacterial identification and screening for both plant growth promoting activities and use of triclosan as a carbon source using rhizobacterial isolates from the inoculation greenhouse study. They will also be co-authors on genome annotations of these bacteria. How have the results been disseminated to communities of interest?Data was disseminated to a wide audience of students, scientists, and professionals. Presentations at two local conferences, TAMIU's LBV Student Research Conference for TAMIU and Laredo Community College students, the Interdisciplinary Science Research Conference for primarily Hispanic high school students, and the J.B. Alexander High School Career Day. Professionals from NRCS and Texas Parks & Wildlife were invited to the research conference along with members of a local water conservation organization. A national conference (the ASA/CSSA/SSSA annual meeting) served to disseminate information to a broad range of those in soil and crop science. There were a total of six presentations for this reporting period. What do you plan to do during the next reporting period to accomplish the goals?Within the next period, manuscripts will be submitted for the phylogenetic analysis of triclosan resistant and triclosan-degrading rhizobacteria by Spring 2018 (Objective 1) and the soil degradation study by Summer 2018 (Objective 1). Completion of the GC-MS analysis of soil and plant tissues (Objective 3) will be completed within Spring 2018 so that we can analyze the data and prepare a manuscript by the end of the funding period to complete Objectives 2 & 3. Data will be presented at a national meeting and to local soil and water conservation agencies.

Impacts
What was accomplished under these goals? Impact Statement: Triclosan, an antimicrobial, is one of seven most prevalent wastewater contaminants in U.S. freshwater streams. Due to its prevalence and potential impact on human and environmental health, triclosan is a chemical of emerging concern. Triclosan reaches the environment via treated wastewater effluent (recycled waters). Waters from potentially contaminated streams are used for irrigation, resulting in chronic low-level exposure to crops. Our study focuses on decreasing levels of triclosan in soils and onion plants using root bacteria. We have determined that approximately 29% of the triclosan-resistant bacteria from onion roots were capable of using triclosan as a food source. These bacteria are also beneficial to plant growth; they can breakdown triclosan in soils to reduce the triclosan levels in plant tissues. Three of the individual bacteria and a mixture of all three were added to soils to determine the amount of triclosan removed after irrigating soils with triclosan. The mixture of all three bacteria increased numbers of soil bacteria ten times more than adding each species alone; however, decreased triclosan levels were more pronounced (up to 40%) when bacteria were added separately to the soils. Two different species of bacteria that will increase onion productivity with or without triclosan were identified. A mixture of bacteria decreased triclosan levels in soil by 26% and in onions by up to 36%. Findings suggest that triclosan-contaminated irrigation waters could encourage the presence of bacteria capable of breaking down other soil pollutants, that bacteria are better at reducing triclosan when present individually but perhaps act together when plants are present. Another important finding is that some methyl triclosan (an important break down product of triclosan) may remain in soils, especially when crops are not present. The outcomes of this project will allow us to understand where and how much of triclosan and its breakdown products end up in agroecosystems when irrigation waters are contaminated. This investigation will be used as a model for the use of beneficial bacteria in preventing contamination of crops, while sustainably enhancing agroecosystems. 1) Major activities completed/experiments conducted Objective 1 A phylogenetic analysis of the triclosan-resistant onion rhizobacteria and those that degrade triclosan was completed. Soils irrigated with triclosan (1.5 µg/100 g dry soil) every other day for 30 days during the soil study were evaluated for levels of triclosan and methyl triclosan. Levels of chemical data were compared among inoculation treatments: control (no inoculum), OT1-11C2, OT2-03A, OT2-17 and OTMIX. Objective 2 Biomass analysis for preliminary irrigation study: Biomass was compared when onions were irrigated for 6 weeks in the absence and presence of triclosan, 0 and 1.5 µg/l triclosan, in three media: ddH2O, tap water and simulated river water. Greenhouse study (91 days) evaluating the effect of triclosan-degrading plant growth-promoting rhizobacteria on onion productivity: A 2 x 5 factorial experiment with five replicates per treatment was completed: two triclosan treatments (0.0 and 1.5 µg/l) and five inoculation treatments (control, no inoculum; OT1-11C2; OT2-03A; OT2-17 and OTMIX). Two water types were used: ddH2O and simulated river water. Twenty-three triclosan-resistant rhizobacteria were isolated to screen for plant growth-promoting activity and the use of triclosan as a sole carbon source. Objective 3 Extraction of triclosan and methyl triclosan from soils and onion tissues: Soils and onion tissues from the greenhouse study described in Objective 2 were collected, dried and extracted for triclosan and methyl triclosan. 2) Data collected Objective 1 Final identifications of triclosan-degrading rhizobacteria was completed. Chemical extractants from soils in the 30-day triclosan degradation study were analyzed by GC-MS for triclosan and methyl triclosan. Objective 2 Onion biomass, total/triclosan-resistant rhizobacterial counts, and total/triclosan-resistant soil bacterial counts were collected. The identity, plant growth-promoting activity and capability of newly isolated rhizobacteria to use triclosan as a sole carbon source was determined. Objective 3 Chemical extractants from soils and plant tissue samples from the 91-day greenhouse onion study were collected. GC-MS analysis of triclosan and methyl triclosan is ongoing. 3) Summary statistics and discussion of results Objective 1 Two strains identifying as the same species showed sensitivity to triclosan at a pre-exposure level of 0.15 µg/l triclosan but not at 1.5 µg/l. Elevated levels of triclosan pre-exposure is likely influencing adaptations of rhizobacteria in soils. Inoculation of soil by individual isolates decreased triclosan levels up to 40% and methyl triclosan levels by 15-30% compared to the control or OTMIX. The OT2-17 isolate may be competing with other inocula. This is being investigated in a metagenomics study funded by a TAMIU University Research grant. Objective 2 In the 6-week preliminary irrigation study, triclosan decreased biomass by 50% in ddH2O and 30% in tap water; triclosan in simulated river water increased onion biomass by 60%. Simulated river water and ddH20 were then used in the greenhouse inoculation study. In the greenhouse inoculation study, triclosan increased onion biomass by 20 to 50% in the control and OT2-03A inoculation treatments when triclosan was applied, despite water type. In all other inoculation treatments, onion biomass decreased by 20 to 40% in the presence of triclosan. In the absence of triclosan, OT1-11C2 increased onion biomass. Triclosan irrigation enhanced triclosan resistant (and likely triclosan-degrading) populations. Further analysis will allow us to observe relationships between culturable counts and concentrations of targeted analytes. Four newly isolated rhizobacteria were capable of using triclosan as a sole carbon source and possessed at least one plant growth-promoting activity. Objective 3 Concentrations of triclosan and methyl triclosan in soils and onion tissues decreased by 25 to 57%% and 36 to 62%, respectively, in the OTMIX inoculation compared to the control. Analysis of triclosan concentrations is ongoing. 4) Key outcomes or other accomplishments realized Objective 1: Changes in knowledge include: justification for examining a genetic mechanism for pre-exposure of triclosan influencing resistance and long-term studies to understand methyl triclosan accumulation; and one trainee with knowledge in bioinformatics. Changes in action include a graduate trainee mentoring peers in developing QA/QC protocols for the GC-MS. Changes in condition include a reduction in soil methyl triclosan levels by three inoculum treatments. Objective 2: Changes in knowledge include: triclosan may enhance growth, depending on the inoculum and medium of exposure; 23 laboratory course students knowledgeable in bacterial isolation and screening for plant growth promoting activity and triclosan degradation using agar assays; 10 trainees experienced in setting up an inoculation study and harvesting plants/soils. A change in action is that irrigation studies with simulated river water will be used in future studies. A change in condition is that four female and two male Hispanic undergraduate students will be able to pursue research within the areas of crop and soil microbial productivity. Objective 3: A change in knowledge is that inoculation with plant growth-promoting rhizobacteria decrease triclosan in onions and one Hispanic male trainee with technical expertise in operating GC-MS and executing QA/QC protocols. Expected outcomes will be based on linking differences in soil/rhizosphere bacterial counts with contaminant levels in tissues.

Publications

• Type: Other Status: Published Year Published: 2017 Citation: Gonzalez, O.D., A.M. Garcia, A. Addo-Mensah, and M.O. Mendez. 2017. Effects of the accumulation of triclosan and its by-products on soil levels and plant uptake. ASA/CSSA/SSSA Annual Meeting, Tampa, FL, Oct 22-25.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Carlo, P., A.M. Garcia, K. Vallejo, J. Cohen and M.O. Mendez. 2017. Phylogenetic analysis of triclosan-sensitive and triclosan-tolerant onion rhizobacteria. ASA/CSSA/SSSA Annual Meeting, Tampa, FL, Oct 22-25.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Garcia, A.M., A. Addo-Mensah, and M.O. Mendez. 2017. Effect of triclosan resistant plant growth promoting rhizobacteria on triclosan degradation in soils. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Mar 30-31. *First place in graduate student competition for Biology & Chemistry
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Cantu, L., P. Carlo, A.M. Garcia, A. Addo-Mensah, and M. O. Mendez. 2017. Comparison of TCS accumulation in onions using spiked molecular water, tap water and simulated river water. Lamar Bruni Vergara/Guillermo Benavides Z. Academic Conference, Laredo, TX, Mar 30-31.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Mendez, M.O. 2017. Research in Environmental Science: From the Field to the Lab, Interdisciplinary Science Research Conference, Laredo, TX, Sep 06.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Mendez, M.O. 2017. Restoring our environment for human health. J.B. Alexander High School Career Day, Laredo, TX, Dec 15.

Progress 01/01/16 to 12/31/16

Outputs
Target Audience:Target audiences included students at Texas A&M International University as well as high school students and research scientists attending local and national scientific engagements. A total of 8 Hispanic students (one male and seven females) and one White non-Hispanic female student were targeted for training in agricultural sciences. Seven of these students were undergraduates: one student paid as undergraduate research assistants, two students obtained academic credits for our undergraduate research course, two students were working towards an honors thesis, and two additional students were participating in training and data collection. One graduate student was funded as a graduate research assistant and one graduate student has begun training. Data collected during the 2016 reporting period was summarized and presented to a wide scope of scientists and research students involved in STEM fields at our university-wide research conference (Lamar Bruni Vergara Student Research Conference), at a national meeting (Annual Society for Advancing Chicanos/Native Americans in Science National Conference), and at the 2016 AFRI and NIWQP Annual Project Director's Meeting. A group of 180 primarily Hispanic high school students were introduced to agricultural science research at the Interdisciplinary Science Research Conference (TAMIU, Laredo, TX). Other target audiences included members of the Rio Grande International Study Center, extension specialists, high school science teachers, high school students, agricultural engineers, and other principal investigators involved in geochemical, geophysical, and microbial analysis of agroecosystems. Changes/Problems:Previous and ongoing maintenance issues for the GC-MS delayed the analysis of soil samples. However, we have ameliorated these issues. As previously reported, the soil degradation experiment was repeated and new QA/QC protocols were also developed. The project has been delayed for one year. The final greenhouse experiment will be completed during 2017 and finalized by the end of the next grant reporting period. We have requested a one year no-cost extension to complete the objectives and activities of this grant. Our new timeline includes the completion of chemical analysis and evaluation of the soil degradation study (Objective 1) by the beginning of 2017. The final greenhouse study (Objectives 2 & 3) and all chemical extractions of materials will be completed by the end of Summer 2017. Evaluation of the experiments to complete Objectives 2 & 3 will be completed during Fall 2017. Manuscript preparation for two publications for Objective 1 are in progress and will be completed during Spring 2017, while the publication for Objective 2 & 3 will be completed at the end of the no-cost extension period. The preliminary soil degradation studies for optimization of protocols designed to extract and maintain QA/QC during the study resulted in an unexpected outcome. Irrigation with 1.5 µg/l triclosan (environmentally relevant) resulted in detection of methyl triclosan below the 1 ng/g detection limits. Since this was not feasible to understand the impact of soil inoculation with triclosan-degrading bacteria on levels of methyl triclosan, the experimental design for the soil degradation study was adjusted so that a total mass of 1.5 µg triclosan dissolved in irrigation waters was added to soils at each watering. This allowed for the observation of methyl triclosan during the 30 day study. Further, the same trends in bacterial numbers when triclosan was added to soils were observed in both the first soil degradation study and the second soil degradation study. In order to maintain relevancy to the soil data obtained during this grant period, the greenhouse study will include two irrigation treatments of triclosan, the 1.5 µg/l triclosan treatments as proposed and an additional treatment of 1.5 µg triclosan per watering. What opportunities for training and professional development has the project provided?Trainees served under the activities of this grant included: five undergraduates (three Hispanic females, one Hispanic male and one White non-Hispanic female) and two graduate students (one Hispanic male and one Hispanic female). These students are trained in agricultural science, including greenhouse experimental design, soil chemical extractions and microbial analysis. They are more competitive for national summer research programs and admission into PhD programs. As a result of her research experience, a Hispanic female graduate student trained under this grant received a competitive opportunity to attend the 2016 USDA CULTIVAR Graduate Fellow Career Preparation Institute and the 2016 Global Marine Sustainability Workshop Arctic Seas and Gulf of Mexico in Oban, Scotland. How have the results been disseminated to communities of interest?Data was disseminated to a wide audience of students, scientists, and professionals. Presentations at two local conferences, TAMIU's LBV Student Research Conference for TAMIU and Laredo Community College students and the Interdisciplinary Science Research Conference for primarily Hispanic high school students. Professionals from NRCS and Texas Parks & Wildlife were invited to the research conference along with members of a local water conservation organization. A national conference (the SACNAS annual meeting) served to disseminate information to a broad range of those in STEM fields. There were a total of four presentations for this reporting period. One scientific article was published during this funding period. Partial funding provided for the completion of GC-MS analysis of soil and plant triclosan concentrations for a previous study of onion and tomato plants. This data was published in the Journal of Environmental Quality, targeting agricultural and soil scientists. What do you plan to do during the next reporting period to accomplish the goals?Due to GC-MS equipment issues, project activities have been delayed and a one year no-cost extension has been requested. Our revised timeline will allow us to complete the final greenhouse study during the extension period. Completion of chemical analysis and evaluation of the soil degradation study (Experiment 2) to complete Objective 1 will be accomplished by the beginning of 2017. The final greenhouse study (Objectives 2 & 3) will be completed during Spring 2017, including the optimization of plant chemical extractions, the tissue harvest and soil collection. Chemical extractions of all materials will be performed at the end of Spring 2017 and will be completed by the end of Summer 2017. Evaluation of the experiments to complete Objectives 2 & 3 will be completed during Fall 2017. Manuscript preparation for two publications for Objective 1 are in progress and will be completed during Spring 2017, while the publication for Objective 2 & 3 will be completed at the end of the no-cost extension period. Data will be presented at a national meeting and to local soil and water conservation agencies.

Impacts
What was accomplished under these goals? Impact Statement: Triclosan, an antimicrobial, is one of seven most prevalent wastewater contaminants in U.S. freshwater streams. Due to its prevalence and potential impact on human and environmental health, triclosan is a chemical of emerging concern. Triclosan reaches the environment via treated wastewater effluent (recycled waters). Waters from potentially contaminated streams are used for irrigation, resulting in chronic low-level exposure to crops. Our study focuses on decreasing levels of triclosan in soils and onion plants using root bacteria. We have determined that approximately 27% of the triclosan-resistant bacteria from onion roots were capable of using triclosan as a food source. These bacteria are also beneficial to plant growth; they can breakdown triclosan in soils to reduce the triclosan levels in plant tissues. Three of the individual bacteria and a mixture of all three were added to soils to determine the amount of triclosan removed after irrigating soils with triclosan. The mixture of all three bacteria increased numbers of soil bacteria ten times more than adding each species alone. This same mixture of bacteria decreased triclosan levels 30% more than when no bacteria were added. Findings suggest that triclosan-contaminated irrigation waters could encourage the presence of bacteria capable of breaking down other soil pollutants, that bacteria are better at reducing triclosan when several bacteria are present and that some methyl triclosan (an important breakdown product of triclosan) may remain in soils. The outcomes of this project will allow us to understand where and how much of triclosan and its breakdown products end up in agroecosystems when irrigation waters are contaminated. This investigation will be used as a model for the use of beneficial bacteria in preventing contamination of crops, while sustainably enhancing agroecosystems. 1) Major activities completed/experiments conducted Objective 1 Optimization of Triclosan Irrigation and Analyte Recovery: Two optimization studies were conducted to determine the detection of methyl triclosan and trichlorophenol: a 15-d study using 1.5 µg/l triclosan irrigation treatments; a 6-d study using two concentrations of triclosan treatments to achieve a total addition of 1.5 µg triclosan in soils at each irrigation time point. A protocol for determining recovery of target analytes from soil samples was developed using 13C-labeled triclosan, methyl triclosan and trichlorophenol. Determination of Soil Triclosan Degradation: Soils were irrigated with triclosan (1.5 µg/100 g dry soil) every other day for 30 days. Soils were inoculated with five treatments: control (no inoculum), OT1-11C2, OT2-03A, OT2-17 and OTMIX. Soils were extracted for triclosan, methyl triclosan, and trichlorophenol. Total and triclosan tolerant heterotrophic bacterial populations were enumerated using three agars: R2A, R2AT1.5 (amended with 1.5 µg/l triclosan), and R2AT150 (amended with 150 µg/l triclosan). Objective 2 Optimization of irrigation solution for onion study: A 6-wk experiment was completed to determine the differences in onion productivity when irrigated in the absence and presence of triclosan, 0 and 1.5 µg/l triclosan, in three media: ddH2O, tap water and simulated river water. Total heterotrophic (R2A) and triclosan tolerant (R2A amended with 1.5 µg/l triclosan) bacterial counts were determined. Objective 3 Optimization of chemical extraction from onion tissues: A 6-wk experiment was completed to develop protocols for extraction of triclosan and by-products in soils irrigated with 1.5 µg/l triclosan. 2) Data collected Objective 1 Chemical extractants from soils in optimization studies and the 30-day triclosan degradation study were collected. This included GC-MS analysis of triclosan, methyl triclosan and 2,4,6-trichlorophenol. Using the soil samples collected during the 30-day degradation study, plate counts of total heterotrophic bacteria and triclosan-tolerant heterotrophic bacteria were collected for Days 0, 15 and 30. Objective 2 Onion height and final biomass (shoots and roots) was obtained. Total and triclosan tolerant heterotrophic bacterial numbers were also determined. Objective 3 Plant tissues and soils were extracted for triclosan, methyl-triclosan and 2,4,6-trichlorophenol. 3) Summary statistics and discussion of results Objective 1 A protocol was designed and implemented for spiking control soils for determination of recovery during the 30-day degradation study. Recovery of 13C-labeled targets was determined to be between 95 - 97%. To maintain a 30% gravimetric moisture content, soils were irrigated with 10 to 20 ml. Optimization studies showed that the 1.5 µg/l triclosan irrigation treatments were insufficient (below 1 ng/g detection limit) to be able to observe methyl triclosan in soils without plants. Therefore, the irrigation treatments for the degradation study was changed to 1.5 µg triclosan at each irrigation time point. Triclosan accumulation has shown to plateau after Day 9, while methyl triclosan accumulated until Day 15 after which fluctuations of 20 to 30 ng/g were observed until Day 30. Compared to the control, OTMIX decreased triclosan concentrations (30% less than controls) and maintained low levels of methyl triclosan. Approximately > 92% of triclosan was degraded by three inoculum treatment. Trichlorophenol was not detected in samples. Irrigation with triclosan immediately decreased total bacterial populations in control soils. Triclosan addition enhanced the total population in the OTMIX inoculum only, while inoculation with OTMIX and OT1-11C2 soils increased triclosan tolerant population numbers. On Day 15, OTMIX continued to enhance total and triclosan tolerant populations by a 1-log difference, while OT1-11C2 inoculation treatments enhanced total and triclosan tolerant populations on Day 30. Further analysis will allow us to observe relationships between culturable counts and concentrations of targeted analytes. Objective 2 Root biomass decreased by 10-30% with triclosan irrigation; however, there was a slight increase in simulated river water amended with triclosan. Data analysis of plant growth data and microbial counts is ongoing. Objective 3 Concentrations of methyl triclosan were higher in root tissues, approximately 8- to 10-fold higher than that detected in shoots. Analysis of triclosan concentrations is ongoing. An additional optimization study will be repeated prior to the final greenhouse study. 4) Key outcomes or other accomplishments realized Objective 1: Changes in knowledge include: a justification for re-examining the importance of methyl triclosan as a by-product in soils alone; a collection of bacteria that decreased triclosan concentrations in soils; and one Hispanic female trainee with technical expertise in operating GC-MS and executing QA/QC protocols. Changes in action included a protocol for preparing samples to accurately determine the recovery of target analytes and a new method for irrigating soils to evaluate triclosan by-products. Changes in condition include a reduction in soil triclosan levels by two inoculum treatments. Objective 2: A change in knowledge is that triclosan-contaminated irrigation waters may enhance growth, depending on the medium. An expected change in action is that further analysis of the data will determine the irrigation water to be used for the final greenhouse study. A change in condition is the ability of three female Hispanic undergraduate students to pursue research in agricultural science within the areas of crop and soil microbial productivity. Objective 3: A change in knowledge is that methyl triclosan should be monitored and mitigated during production of bulb crops. Expected key outcomes for the next grant period are identifying rhizobacteria capable of reducing accumulation of triclosan and methyl triclosan in onion tissues.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Mendez, M.O., Valdez, E., Martinez, E., Saucedo M., and Wilson, B. 2016. Fate of triclosan in irrigated soil: degradation in soil and translocation into onion and tomato. Journal of Environmental Quality. 45(3):1029-1035. doi: 10.2134/jeq2015.07.0386.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Garcia, A.M., M. Chapa, B. Wilson, A. Addo-Mensah, and M.O. Mendez. 2016. Effect of triclosan-resistant plant growth-promoting rhizobacteria on triclosan degradation in soils. 2016 Society for Advancing Chicanos/Hispanics & Native Americans in Science National Conference, Long Beach, CA, Oct 13-15.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Garcia, A.M. and M.O. Mendez. 2016. Triclosan degradation. Lamar Bruni Vergara Conference, Texas A&M International University, Laredo, TX, Apr 1-2.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Mendez, M.O. 2016. Research in environmental science: from the field to the lab. Interdisciplinary Science Research Conference, Texas A&M International University, Laredo, TX, Sep 7.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Mendez, M.O., A.M. Garcia, A. Addo-Mensah. 2016. Determining the potential mitigation of triclosan accumulation in commercial onion plants using plant-growth promoting rhizobacteria. 2016 AFRI and NIWQP Annual Project Directors Meeting, Washington, DC.

Progress 01/01/15 to 12/31/15

Outputs
Target Audience:Target audiences included students at Texas A&M International University as well as students and research scientists attending regional and national scientific meetings. A total of 7 Hispanic students were targeted for training in agricultural sciences. Six of these students were undergraduates: two students paid as undergraduate research assistants, one student obtained academic credits for our undergraduate research course, and three other students were participating in training and data collection. One graduate student is currently being funded as a graduate research assistant. Data collected during the 2015 reporting period was summarized and presented to a wide scope of scientists and research students involved in STEM fields at regional conferences (Texas Undergraduate Research Day, TAMIU's LBV Student Conference, and Texas A&M University System's Pathways Conference), environmental microbiology at a national meeting (American Society for Microbiology 115th General Meeting), STEM fields at a national meeting (Annual Society for Advancing Chicanos/Native Americans in Science National Conference), and water issues in agroecosystems at the 2015 AFRI and NIWQP Annual Project Director's Meeting. Target audience included members of the Rio Grande International Study Center, extension specialists, science educators, agricultural engineers, and other principal investigators involved in geochemical, geophysical, and microbial analysis of agroecosystems. Changes/Problems:Quality assurance and quality control for the GC-MS analysis after several maintenance issues for the GC-MS delayed the analysis of soil samples. Timely analysis of soil samples collected from the degradation study has been impeded. Therefore, the soil degradation experiment will be repeated within the next reporting period using the newly developed protocols and QA/QC procedures. This will delay the project by at least six months. Preliminary experiments and optimization for the final greenhouse study has been delayed a by six months due to the absence of final data from the soil degradation study. Therefore, the greenhouse data analysis will not be completed during the 2016 reporting period. We will likely be requesting a one-year no cost extension at the end of 2016 in order to complete the experiments and data analysis. What opportunities for training and professional development has the project provided?Six undergraduates and one graduate received scientific training in the agricultural sciences. Student training included best safety practices, hazardous materials handling, solution chemistry, and aseptic techniques. Specific to the project, students were trained in GC-MS instrumentation analyses, microbial culture techniques, culture library management, greenhouse management, soil physicochemical analysis, and batch culture assays. Students gained professional development in analyzing, interpreting, and presenting data, a tool that can be translated to other research projects. How have the results been disseminated to communities of interest?During this reporting period, data was disseminated to a wide audience of students, scientists, and professionals. Presentations at one local (TAMIU's LBV Student Conference) and two regional conferences (TX Undergraduate Research Day and TAMU Pathways Conference) served to inform those involved in a broad area of STEM fields within Texas, including environmental science, agricultural science, and water conservation. A national conference (ASM's annual meeting) served to disseminate information to environmental microbiologists and a broad range of students involved in microbiological research. An additional national conference, the SACNAS annual meeting, targeted those in STEM fields. There were a total of ten presentations for this reporting period. One scientific article was published during this funding period. Partial funding provided for the completion of GC-MS analysis of soil and plant triclosan concentrations for a previous study of onion and tomato plants. This data was published in the Journal of Environmental Quality, targeting agricultural and soil scientists. What do you plan to do during the next reporting period to accomplish the goals?Due to GC-MS equipment issues, the in situ study will be repeated using the new QA/QC protocols developed during this reporting period. Soil extractions for this project will be completed by summer of 2016 in order to proceed with the plant growth experiment in fall of 2016. Results will be used to design the plant growth experiment. Protocols for onion seed inoculation will be developed and completed prior to the fall 2016 plant growth experiment using triclosan-contaminated irrigation waters. Data will be presented at a national meeting, and the manuscript characterizing the inocula used in these studies will be submitted to a peer-reviewed scientific journal for publication during the reporting period.

Impacts
What was accomplished under these goals? Impact Statement: Triclosan, a broad-spectrum antimicrobial, is one of the seven most prevalent wastewater contaminants found in freshwater streams in the United States. Due to its ubiquitous nature and potential impact on human and environmental health, triclosan is considered a chemical of emerging concern. Triclosan primarily reaches the environment via treated wastewater effluent (recycled waters). Most agricultural regions use waters from potentially contaminated streams for irrigation and as a result are exposing crops to chronic low-levels of contamination for much of the growing season. In this project, we have determined that approximately 27% of the triclosan-resistant bacteria from onion roots (rhizobacteria) tested were capable of using only triclosan as a food source. These bacteria are also plant growth-promoting rhizobacteria (PGPRs); therefore, they have the potential to breakdown triclosan in soils to reduce accumulation of triclosan in plant tissues. Four of these bacteria were used in a greenhouse study to determine the amount of triclosan degraded by the bacteria when inoculated into soils. Two of these bacteria enhanced the abundance of bacteria when soils were irrigated with triclosan; however, a bacterial consortium had the most influence on decreasing triclosan levels in soils. Findings suggest that triclosan contaminated irrigation waters could encourage the abundance of bacteria that are capable of degrading soil contaminants. The outcomes of this project will allow us to understand the fate and transport of triclosan in agroecosystems, particularly when applied via irrigation waters. Further, this investigation will be used as a model for the use of PGPRs in preventing contamination of crops, while sustainably enhancing agroecosystems. 1) Major activities completed/experiments conducted Objective 1 Characterization of Triclosan-utilizing PGPRs: Selected isolates for the project were analyzed for differential growth in 1.5 µg/l triclosan as compared to a control medium in batch culture assays. Growth curves were developed for the selected isolates. Optimization of GC-MS Analysis: The Gas-Chromatography Mass Spectrometer was calibrated for triclosan and by-product analysis. Standard solutions of triclosan, methyl-triclosan, 2,4-dichlorophenol, and 2,4,6-trichlorophenol were used to develop a standard curve. For QA/QC, recovery of target analytes was performed by spiking soils with known concentrations of each analyte. Determination of Soil Triclosan Degradation: Soils from the degradation study performed in 2014 were extracted for triclosan, methyl-triclosan, and 2,4,6-trichlorophenol. For soil bacterial enumeration, soils were serially diluted to obtain heterotrophic plate counts on both R2A and R2AT (amended with 1.5 µg/l triclosan) to determine the total heterotrophic population and the triclosan tolerant population, respectively. Objective 2 Optimization of seed and soil inoculation: To determine the potential effect of triclosan -utilizing PGPRs on onion seed germination and growth, seeds were sterilized and inoculated with five inoculation treatments (OT1-09, OT1-11C2, OT2-03A, OT2-17, and OT-MIX) along with a control (uninoculated). Seed germination was performed in sterile petri dishes and in sterilized soil. Onion plant growth was determined using height measurements. Objective 3 No activities completed. 2) Data collected Objective 1 Absorbance readings of batch cultures of selected isolates was collected over a 36 hour period to determine differential growth patterns when exposed to triclosan. Data collected for optimization and QA/QC of GC-MS analysis of triclosan and its by-products included standard curves and recovery of target analytes from spiked soils. Using soils collected during the 30-day degradation study, plate counts of total heterotrophic bacteria and triclosan-tolerant heterotrophic bacteria were collected. Objective 2 Germination rates of onion seeds inoculated with triclosan-utilizing PGPRs was collected over a three week period in sterile petri dishes and over a 5 week period in soil. Onion heights were also collected during a five week period following the soil germination study. Objective 3 No data collected. 3) Summary statistics and discussion of results Objective 1 Isolates Pantoea sp. OT1-11C2 and Paenibacillus polymyxa OT2-17 were determined to not be impacted by triclosan at 1.5 µg/l as compared to a control broth. Therefore, these isolates do not grow differently in the absence or presence of triclosan concentrations examined in these studies. For QA/QC, GC-MS protocols for analysis were examined for triclosan, methyl-triclosan, and 2,4,6-trichlorophenol. A new standard curve for each of these chemicals was developed using optimized GC-MS operation procedures in EI-SIM mode with an r-squared greater than 0.96 for all analytes examined, recovery rates greater than 98%, and detection limits between 1-5 ng/µl. The half-life of life of triclosan was lower (< 15 d) when soil was inoculated with a consortium of TD-PGPRs as compared to the control soil (uninoculated). With inoculation, triclosan concentrations were reduced by 4-fold as compared to the control. However, triclosan concentrations were similar at the end of the study, indicating that triclosan accumulated in soils despite degradation. Further, 2,4,6-trichlorophenol concentrations increased, indicating the photolysis occurred. Irrigation with triclosan immediately decreased total bacterial populations; however, no effect on triclosan tolerant populations was observed even when soils were inoculated with TD-PGPRs. Inoculation with a consortium of TD-PGPRs (OTMIX) increased total bacterial population numbers and triclosan tolerant bacterial counts which was maintained throughout the study. Objective 2 Onion seed germination was similar among treatments in sterile plates. However, inoculation using OT2-03A and OT1-11C2 enhanced seed germination in the sterile soil germination study. Mean onion heights were similar in the control and OT2-17 inoculum treatment. Onion growth appears to be inhibited by OT1-09. Plant data confirms the plant growth-promoting activity of OT1-11C2, OT2-03A, and OT2-17. Objective 3 No results to report. 4) Key outcomes or other accomplishments realized Objective 1: Batch culture studies examining the effect of triclosan on growth of the isolates in a low nutrient broth confirmed that triclosan at environmentally relevant concentrations does not inhibit growth of the selected isolates. We have developed new QA/QC protocols and run conditions for GC-MS analysis that will be used in the soil degradation study to be repeated at the beginning of the next grant period. The bacterial consortium treatment appears to be the most promising in decreasing triclosan concentrations. Research activities performed allowed for the training of five students in agricultural science, including soil chemical extractions and microbial analysis. Three of these students presented their research at one local, one regional, and two national conferences. Due to these research and professional experiences, students are more competitive for national summer research program for undergraduates and admission into PhD programs. Objective 2: Based on the germination studies and preliminary growth study, OT1-09 will likely be removed from the consortium mix in the next experiments. Another set of preliminary growth studies will be performed to evaluate the removal of OT1-09 from the inoculated consortium treatment (OTMIX). Two undergraduate students were trained in techniques within the agricultural sciences, including soil analysis, microbial culture techniques, and plant growth analysis. Objective 3: For this objective, there are yet no major activities or experiments to report. This analysis will be conducted during 2016. Therefore, there is no collected data, summary statistics, and key outcomes to report.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Mendez, M.O., Valdez, E., Martinez, E., Saucedo M., and Wilson, B. 2015. Fate of triclosan in irrigated soil: degradation in soil and translocation into onion and tomato. Journal of Environmental Quality. doi: 10.2134/jeq2015.07.0386.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Mendez, M. O. and A. Addo-Mensah. 2015. Determining the potential mitigation of triclosan accumulation in commercial onion plants using plant-growth promoting rhizobacteria. 2015 AFRI and NIWQP Annual Project Directors Meeting, USDA, Greensboro, NC, July 28-29.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Chapa, M.L., A.M. Garcia, J.I. Cohen, and M.O. Mendez. 2015. The effect of triclosan on growth of a plant growth-promoting rhizobacterium. American Society for Microbiology 115th General Meeting, New Orleans, LA, May 30June 2.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Delgado, D., P. Carlo, M. Zuniga, A.M. Garcia, B.A. Wilson, and M.O. Mendez. 2015. Assessing changes in the populations of total and triclosan-tolerant heterotrophic bacteria over time in soils irrigated with triclosan. American Society for Microbiology 115th General Meeting, New Orleans, LA, May 30June 2.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Garcia, A., M. Chapa, B.A. Wilson, and M.O. Mendez. 2015. Utilization and degradation of triclosan by triclosan-resistant onion rhizobacteria. American Society for Microbiology 115th General Meeting, New Orleans, LA, May 30June 2.

Progress 01/01/14 to 12/31/14

Outputs
Target Audience: Target audiences included students at Texas A&M International University and scientists attending the 2014 NIWQP and AFRI Project Director's Meeting. A total of 8 Hispanic undergraduate students were targeted for training in agricultural sciences. Two of these students were graduating seniors and six of the students had a junior standing during the reporting period. One of the graduating senior students is now a graduate student, currently being funded as a graduate research assistant through this grant funding. Two were paid participants, four students obtained academic credits for our undergraduate research course, and two students were voluntarily participating in the training and data collection. Data collected during the 2014 reporting period was summarized and presented to fellow scientists studying water issues in agroecosystems. This included extension specialists, science educators, agricultural engineers, and other principal investigators involved in geochemical, geophysical, and microbial analysis of agroecosystems. Changes/Problems: The GC-MS has recently been serviced and re-tuned. This caused a delay in the analysis for triclosan and its by-products. Re-optimization will be performed again prior to soil analyses to ensure quality control and assurance for ion selectivity and sensitivity. We are in the process of recalibrating the standard curves. This has delayed the timeline by a few months. It is not expected that this will cause a significant delay in the overall timeline of the project. What opportunities for training and professional development has the project provided? Eight students, seven undergraduates and one graduate, received scientific training in the agricultural sciences. General student training included best safety practices, hazardous materials handling, solution chemistry, and aseptic techniques. Specific to the project, students were trained in microbial culture techniques, culture library management, greenhouse management, soil physicochemical analysis, and bacterial identification by 16S rDNA-PCR. Students have gained professional development in analyzing and interpreting data, a tool that can be translated to other research projects. How have the results been disseminated to communities of interest? Preliminary data from the project was presented at a sustainability conference sponsored by the University College of Texas A&M International University in Laredo, TX. The audience was primarily high school students participating in the conference. Students were introduced to water quality issues related to food contamination and how microbes can be used to improve agricultural sustainability. What do you plan to do during the next reporting period to accomplish the goals? Finalize re-calibration of GC-MS for detection of triclosan, methyl-triclosan, and chlorophenols. Complete analysis of soils for concentrations of triclosan and its by-products. In addition, the microbial counts will be completed. These two sets of data will be used to determine inocula to include in the onion growth study. During summer and fall of the 2015 reporting year, a greenhouse study will be conducted that examines the effect of the inocula tested during the reporting period of 2014 on onion growth and accumulation of triclosan in onions. Data will be presented at the American Society for Microbiology annual conference in May 2015. A graduate and one undergraduate student will be presenting a poster at the conference.

Impacts
What was accomplished under these goals? Impact Statement: Triclosan, a broad-spectrum antimicrobial, is one of the seven most prevalent wastewater contaminants found in freshwater streams in the United States. Due its ubiquitous nature and potential impact on human and environmental health, triclosan is considered a chemical of emerging concern. Triclosan primarily reaches the environment via treated wastewater effluent (recycled waters). Most agricultural regions use waters from potentially contaminated streams for irrigation and as a result are exposing crops to chronic low-levels of contamination for much of the growing season. In this project, we have determined that approximately 27% of the triclosan-resistant bacteria from onion roots (rhizobacteria) tested were capable of using only triclosan as a food source. These bacteria are also plant growth-promoting rhizobacteria (PGPRs); therefore, they have the potential to breakdown triclosan in soils to reduce accumulation of triclosan in plant tissues. Four of these bacteria were used in a greenhouse study to determine the amount of triclosan degraded by the bacteria when inoculated into soils. Findings suggest that triclosan contaminated irrigation waters could encourage the abundance of bacteria that are capable of degrading soil contaminants. The outcomes of this project will allow us to understand the fate and transport of triclosan in agroecosystems, particularly when applied via irrigation waters. Further, this investigation will be used as a model for the use of PGPRs in preventing contamination of crops, while sustainably enhancing agroecosystems. Objective 1 1) Major activities completed/experiments conducted Utilization of Triclosan as a Carbon Source: To determine if triclosan can serve as a sole carbon source, 24 triclosan-resistant isolates (resistant to 1,500 µg/l triclosan by disk diffusion assay) were plated onto a chloride-free minimal salts medium (MSM) amended with glucose (2 g/l) as a control (MSMG) and amended with triclosan (2 g/l) as a treatment (MSMT) in triplicate. Identification of Isolates: Isolates demonstrating utilization of triclosan as a carbon source were selected for sequencing and identification. The 16S rRNA gene was amplified by PCR from isolate lysates using universal bacterial primers. All PCR products were submitted to the University of Arizona Genetics Core for DNA sequencing. Soil characterization: A fine sandy loam, Copita series, collected from a previously undeveloped area was used for the greenhouse experiment and mixed with potting soil to achieve a 1:9 ratio of potting soil to native soil. Prior to use, the soil mixture was analyzed for soil physicochemical characteristics and triclosan. Optimization of GC-MS Analysis: The Gas-Chromatography Mass Spectrometer was calibrated for triclosan and by-product analysis. Standard solutions of triclosan, methyl-triclosan, 2,4-dichlorophenol, and 2,4,6-trichlorophenol were used to develop a standard curve. Determination of Soil Triclosan Degradation: Triclosan-utilizing PGPRs from the MSMG/MSMT growth assay were used for a 2 x 5 factorial greenhouse experiment with two triclosan irrigation treatments (0 and 1.5 µg/l triclosan) and five inoculation treatments (OT1-09, OT1-11C2, OT2-03A, OT2-17, and OT-MIX). The OT-MIX treatment was a consortium of all four rhizobacteria. Soils were serially diluted to obtain heterotrophic plate counts on both R2A and R2AT (amended with 1.5 µg/l triclosan) to determine the total heterotrophic population and the triclosan tolerant population. 2) Data collected Bacterial isolates which successfully grew on chloride-free minimal salts medium amended with glucose (MSMG) and triclosan (MSMT) after 14 days of incubation were observed for growth. Plate cultures were categorized as minimal, intermediate, moderate, or high growth to determine isolates to be used in the greenhouse study. A soil characterization was completed prior to the greenhouse experiment. During the greenhouse experiment, soils were subsampled every 3 days from Day 0 to 30. Data collected for optimization included concentrations of standard solutions as detected by the GC-MS for development of a standard curve to be used for future GC-MS analysis. Ions specific to the targeted compounds were identified. 3) Summary statistics and discussion of results From twenty-four isolates classified as triclosan resistant, six isolates tested positive for triclosan utilization, four of which had minimal growth and two with moderate growth along the streak. Results indicated that pre-exposure to triclosan is required for potential utilization of triclosan as a carbon source. Isolates previously found to degrade xenobiotics were identified: Stenotrophomonas spp., Pantoea sp., Microbacterium oxydans, and Paenibacillus polymyxa. Based on the known metabolism of these genera, irrigation with triclosan may encourage bacterial populations capable of xenobiotic metabolism. The mixture of a 1:9 potting to native soil used in the soil study was a silt loam and found to be typical of semi-arid with a pH 8, EC of 0.396 dS/m, l6 mg/kg P, and a C:N ratio of 7. Therefore, the low organic carbon content would likely favor microbial metabolism of organic contaminants. No triclosan was detected in the soil. Prior to the greenhouse study, the GC-MS was optimized for analysis of triclosan and its by-products. Unique peaks have been identified for all chemicals to be examined: triclosan (m/z 347), methyl-triclosan (m/z 413), and the chlorophenols (2,4-dichlorophenol and 2,4,6-trichlorophenols; m/z 353). Recovery rates were above 97%. The presence of triclosan and its by-products were not analyzed during the reporting period due to recalibration of the GC-MS after it was serviced. This has required the development of a new standard curve for all chemicals. Based on heterotrophic plate counts, the initial data indicates that the effects of triclosan irrigation were dependent on the sampling time along with the inoculum used in the soil, and control soils generally contained less triclosan tolerant bacteria. 4) Key outcomes or other accomplishments realized Pre-exposure to triclosan by irrigation with recycled waters impacts the potential metabolism of bacteria by increasing the number of rhizobacteria capable of using triclosan as a carbon source. We now have evidence that inoculation with triclosan-degrading plant growth-promoting rhizobacteria may prevent the loss in bacterial populations from triclosan exposure. However, this needs to be studied further. Further, the research activities performed in 2014 allowed for the training of eight students in agricultural science, including soil analysis, microbial analysis, and greenhouse protocols. Due to this research experience, students have become more competitive for national research experiences for undergraduates and PhD programs. Objective 2: For this objective, there are yet no major activities or experiments to report. This experiment will be conducted in Fall 2015 through Spring 2016. Therefore, there is no collected data, summary statistics, and key outcomes to report. Objective 3: For this objective, there are yet no major activities or experiments to report. This analysis will be conducted from Spring 2016 to Summer 2016. Therefore, there is no collected data, summary statistics, and key outcomes to report.

Publications

• Type: Other Status: Other Year Published: 2014 Citation: Garcia, Ashley M. 2014. Triclosan resistance of onion rhizobacteria. Senior Honors Thesis, Texas A&M International University, Laredo, TX.
• Type: Other Status: Other Year Published: 2014 Citation: Martinez, Eileen M. 2014. Uptake of triclosan in tomato plants. Senior Honors Thesis, Texas A&M International University, Laredo, TX.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Mendez, M. O. 2014. Determining the potential mitigation of triclosan accumulation in commercial onions plants using plant-growth promoting rhizobacteria. 2014 NIWQP and AFRI Project Directors Meeting, USDA, Washington, DC, Oct 28-29.