Progress 10/01/11 to 09/30/15

Outputs
Target Audience:Our findings from these experiments were disseminated at several stakeholder meetings, including the American Phytopathological Society's annual meeting (2012-2015), Food Research Institute annual meeting in Madison, WI, (2014), and the Hemipteran-Plant Interactions Symposium (HPIS) 2014. HPIS is an interdisciplinary symposium bringing together researches who study phytophagous piercing/sucking insects, their complex interactions with their host plants, and transmission of plant pathogens.This project was featured in GROW magazine published by the University of Wisconsin-Madison, College of Agricultural and Life Sciences (CALS). They report the most significant aspects of CALS research. This publication is published three times each year and mailed to all CALS alumni, faculty and staff, graduate students, and leaders in agriculture, natural resources, and biosciences communities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Both Dr. Groves, co-PI, and Dr. Barak have been involved in mentoring of the graduate student, Jose Pablo Dundore-Arias (formerly Soto-Arias), who has carried out all the experiments to date. Under our mentorship, Jose Pablo was chosen as a Borlaug Summer Institute on Global Food Security Fellow (2012), Next Generation of Global Agricultural Leaders (2013), national finalist for a Howard Hughes Medical Institute International Student research fellow (2013), and American Phytopathological Society's Public Policy Intern. How have the results been disseminated to communities of interest?Results have been disseminated by publications in scientific journals, conference proceedings, university magazines, and news organizations and talks at scientific meetings, university symposia and seminars, and grower meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This research has produced a change in knowledge concerning the potential of phytophageous insects to act as food safety risk factors for fresh produce. This knowledge is necessary to guide future research and direct governmental regulation to insure a safe food supply with the new knowledge that specific phytophagous hemipterans, green peach aphids and Aster leafhoppers, influence the dispersal of Salmonella enterica in edible leaves. In addition, we identified agricultural inputs which affect E. coli and Salmonella differently. Phytophagous insects feed frequently and recurrently in plant tissues that could be contaminated with S. enterica, suggesting they represent an ecological risk for human illness, as they may repeatedly encounter and interact with enteric pathogens on leaves. Under objective 1, we have found that S. enterica grows significantly more on lettuce leaves damaged by insects (thrips), compared with mechanically damaged or undamaged plants. Our acquisition studies show that thrips acquire S. enterica from contaminated produce, and the pathogen persist on or in thrips for extended periods. With fluorescent microscopy we have seen S. enterica adhering to the integument of both thrips and aphids. We have shown that plants acquired S. enterica in areas damaged by contaminated thrips. We also have found that S. enterica is retained and transmitted by phytophagous hemipterans. We found that both Aster leafhoppers and green peach aphids ingested S. enterica from either contaminated leaves or artificial diet. Higher populations of S. enterica were recovered from insects that fed on contaminated leaves, presumably because insects became externally contaminated during feeding. S. enterica was recovered from the honeydew of both insect species after feeding on contaminated sources, but a higher proportion of honeydew samples from Aster leafhoppers compared to green peach aphid were positive for S. enterica. S. enterica was recovered from non-inoculated leaves or liquid diets post-feeding demonstrating that both species of insects were capable of transmitting the bacteria in ways that are not limited to mechanical transmission. These results suggest that phytophagous insects may serve as risk factors for dispersal of S. enterica within and among crop plants. Phytophagous insects can encounter Salmonella enterica on contaminated plant surfaces due to their frequent and recurrent feeding. We have previously shown that phytophagous Hemipterans transmit externally adhered S. enterica and internalized bacteria on and among leaves; suggesting they represent an ecological risk for human illness. We previously reported on the excretion of ingested S. enterica by the leafhopper Macrosteles quadrilineatus; however, the sites of persistence of ingested bacteria remain undetermined. For Objective 3, fluorescence microscopy revealed the presence and persistence of S. enterica in various organs of M. quadrilineatus fed an inoculated diet and then moved to two consecutive noninoculated diets. Ingested S. enterica was predominantly observed in the filter chamber, midgut, and Malpighian tubules of M. quadrilineatus dissected immediately after acquisition and at 24- and 48-h post-acquisition access periods (post-AAPs). Additionally, we examined the potential roles of the Salmonella pathogenicity island 1 (SPI-1) and SPI-2 type III secretion systems (T3SSs) in the persistence and excretion of ingested S. enterica. In competition assays, a prgH mutant lacking a functional SPI-1 T3SS was recovered at significantly lower levels than the WT in insect homogenates, and complementation with prgH restored S. enterica persistence. This study provides novel insight into the presence and persistence of S. enterica inside M. quadrilineatus and demonstrates that the SPI-1 T3SS influences the persistence of the pathogen in the gut of a potential vector. To combat bacterial leaf diseases of tomato, plant foliage is sprayed with copper-containing bactericides every 5-7 days repeatedly throughout the growing season. Unlike E. coli, S. enterica has two MerR-type copper response systems - Cue and Gol. The Cue system in S. enterica is functionally similar to that in E. coli; however, the Gol system is a cytoplasmic detoxifying apparatus that is unique to Salmonella. The causal agents of bacterial spot and speck of tomato have chromosomal and/or plasmid-borne resistance to copper. We found, under objective 2, that the chromosomal resistance to copper in Xanthomonas vesicatoria is homologous to the Gol system of S. enterica. A S. enterica golS mutant is more sensitive to copper in planta and in vitro. We have also shown that S. enterica is more resistant to copper than environmental E. coli and clinical E. coli O157:H7 strains. Our results suggest S. enterica has a fitness factor which allows persistence on tomato plants during normal agricultural practices, such as pesticide sprays, increasing the likelihood to contaminate fruit and cause human illness. To control human pathogen contamination of fresh produce, it is imperative to identify the genes and mechanisms used by these pathogens during their life cycle on/in plants. Growth is a commonly overlooked, yet essential step during niche colonization. Screening deletion mutants (under Objective 3) for plant colonization defects led to the discovery that an aroA mutant replicated poorly in alfalfa root exudates. AroA is part of the chorismic acid biosynthesis pathway, a central metabolic node involved in aromatic amino acid and siderophore production. Addition of ferrous sulfate restored growth of the aroA mutant in root exudates, as well as root colonization. Tryptophan and phenylalanine auxotrophs had minor plant colonization defects suggesting that sub-optimal concentrations of these amino acids in root exudates occur, but are not major limiting factors for Salmonella replication and colonization. An entB mutant defective in siderophore biosynthesis had similar colonization and growth defects as the aroA mutant and the defective phenotype was complemented with addition of ferrous sulfate or by entB. We found biosynthetic genes of each Salmonella siderophore, enterobactin and salmochelin, were up-regulated in alfalfa root exudates; yet, salmochelin was not required for plant survival and persistence. Enterobactin biosynthesis is only the second mechanism identified that is required for both plant and animal host survival. These results support the conclusion that there is no appreciable overlap between required mechanisms for S. enterica colonization and survival in both environments.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Dundore-Arias, J.P., Groves, R.L. and Barak, J.D. 2015. Influence of prgH on the persistence of ingested Salmonella enterica in the leafhopper Macrosteles quadrilineatus. Appl. Environ. Microbiol. 81 (18): 6345-6354.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dundore-Arias, J. P., Groves, R. L. and Barak, J.D. 2015. Phytophagous hemipteran insect honeydew as growth medium for plant-associated Salmonella enterica. Phytobiomes 2015: Designing a New Paradigm for Crop Improvement.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dundore-Arias, J. P., Groves, R. L. and Barak, J.D. 2015. Presence of Salmonella enterica in the alimentary canal of Macrosteles quadrilineatus, and role of prgH in persistence of the ingested bacteria. Phytopathol. 105. American Phytopathological Society Annual Meeting
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Soto-Arias, J. P., Groves, R. L. and Barak, J.D. 2013. Phytophagous insects, Salmonella enterica, fresh produce: a tri-trophic interaction that can make you sick. Phytopathol. 103: S2.171.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Soto-Arias, J. P., Groves, R., and Barak, J. 2012. Potential for Western flower thrips (Frankliniella occidentalis) transmission of Salmonella enterica to crop plants. Phytopathol. 102: S4.112

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Our findings from these experiments were disseminated at several stakeholder meetings, including a the 2014 American Phytopathological Society's annual meeting in Minneapolis, MN, Food Research Institute annual meeting in Madison, WI, and Hemipteran-Plant Interactions Symposium (HPIS). HPIS is an interdisciplinary symposium bringing together researchers who study phytophagous piercing/sucking insects, their complex interactions with their host plants, and transmission of plant pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Both Dr. Groves, co-PI, and Dr. Barak have been involved in mentoring of the graduate student, Jose Pablo Soto-Arias, who has carried out all the experiments to date. How have the results been disseminated to communities of interest? This project was featured in GROW magazine (fall 2014) published by the University of Wisconsin-Madison, College of Agricultural and Life Sciences (CALS). They report the most significant aspects of CALS research. This publication is published three times each year and mailed to all CALS alumni, faculty and staff, graduate students, and leaders in agriculture, natural resources, and biosciences communities. What do you plan to do during the next reporting period to accomplish the goals? Our plan is to identify bacterial factors required for S. enterica survival within the Aster leafhopper, our model for enteric human pathogen - phytophagous hemipteran interactions.

Impacts
What was accomplished under these goals? This research has produced a change in knowledge concerning the potential of phytophageous insects to act as food safety risk factors for fresh produce. This knowledge is necessary to guide future research and direct governmental regulation to insure a safe food supply with the new knowledge that specific phytophagous hemipterans, green peach aphids and Aster leafhoppers, influence the dispersal of Salmonella enterica in edible leaves. For objective 1, we found that both Aster leafhoppers and green peach aphids ingested S. enterica from either contaminated leaves or artificial diet. Higher populations of S. enterica were recovered from insects that fed on contaminated leaves, presumably because insects became externally contaminated during feeding. S. enterica was recovered from the honeydew of both species after feeding on contaminated sources, but a higher proportion of honeydew samples from Aster leafhoppers compared to green peach aphid were positive for S. enterica. S. enterica was recovered from non-inoculated leaves or liquid diets. Post-feeding demonstrated that both species of insects were capable of transmitting the bacteria in ways that are not limited to mechanical transmission. These results suggest that phytophagous insects may serve as risk factors for dispersal of S. enterica within and among crop plants. For objective 3, we have begun to investigate bacterial factors that facilitate persistence within the insect. We have found that S. enterica can persist in the alimentary canal of the Aster leafhopper. This persistence is not reliant on biofilm formation or exopolysaccharide production by S. enterica. Research for objective 3 is ongoing.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Soto-Arias, J.P., Groves, R.L. and Barak, J.D. 2014. Transmission and retention of Salmonella enterica by phytophagous hemipterans insects. Applied Environmental Microbiology 80(17): 5447-5456.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Soto-Arias, J. P., Groves, R. L. and Barak, J.D. 2014. Biological factors that increase human pathogen survival in produce. Food Research Institute Annual Meeting, Madison, WI
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Soto-Arias, J. P., Groves, R. L. and Barak, J.D. 2014. Hemipteran-Plant Interactions Symposium. University of California  Riverside
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Soto-Arias, J. P., Groves, R. L. and Barak, J.D. 2014. Phytophagous hemipterans as vectors of Salmonella enterica. Phytopathology 104: S3.112

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Both Dr. Groves, co-PI, and Dr. Barak have been involved in mentoring of the graduate student, Jose Pablo Soto-Arias, who has carried out all the experiments to date. Jose Pablo and Dr. Barak co-organized a special symposium at the American Phytopatholocial Society's annual meeting on the reserach topic specific to this project. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This research has produced a change in knowledge concerning the potential of phytophageous insects to act as food safety risk factors for fresh produce. In addition, this research has produced a change in knowledge concerning the effect of possible agricultural inputs on human pathogen populations on plants. This knowledge is necessary to guide future research and direct governmental regulation to insure a safe food supply with the new knowledge that specific phytophageous insects, western flower thrips and Aster leafhoppers, influence the survival of Salmonella enterica in edible leaves. For objective 1, we examined the effect of feeding by phytophagous insects on survival of Salmonella on lettuce. Larger Salmonella populations were found on leaves infested with Aster leafhoppers. Higher Salmonella populations were also found on leaves with areas with Western flower thrips feeding damage compared to areas without. These results suggest the possibility of some specificity with select insects and the persistence of Salmonella. We also demonstrated the potential for phytophagous insects to become contaminated with Salmonella from contaminated plant material. Salmonella was detected in approximately 50% of all Aster leafhoppers, Western flower thrips, and green peach aphids after 24 h exposure to Salmonella-contaminated leaves. Particularly, 17% of Western flower thrips, the smallest of the insects tested, harbored more than 100 Salmonella per insect. Our results show that phytophagous insects may influence the population size of Salmonella in agricultural crops. This study provides evidence of a human bacterial pathogen interacting with phytophagous insect during plant infestation. For objective 2, we have begun to examine the metabolic state of Salmonella in association with plants. We found that addition of iron, a possible agricultural input as a plant micronutrient, increased Salmonella populations. Curtailing enteric human pathogen growth in plants below a human infective dose, through understanding Salmonella metabolism in this environment, is a novel food safety intervention strategy. For objective 3, we have begun to investigate plant survival factors among enteric human pathogens. Research for objective 2 and 3 are ongoing.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Soto-Arias, J.P., Groves, R.L. and Barak, J.D. Benefits of piercing and sucking, enhanced persistence of Salmonella enterica on plants due to phytophagous hemipterans. PLoS ONE. 8(10): E79404

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

Outputs
OUTPUTS: We conducted experiments to determine whether phytophageous insects play a role in human pathogen persistence in lettuce. These experiments targeted the interactions between Salmonella and western flower thrips. We also examined the capacity for acquisition of the human pathogen by both western flower thrips and aphids. We analyzed our results from experiments that examined the vectoring of S. enterica by both thrips and aphids to lettuce and tomato plants. Finally, we initiated experiments to examine the antbiosis of a common Erwinia sp. found in the gut of thrips. Our findings from these experiments were disseminated at several stakeholder meetings, including the 2012 American Phytopathological Society's annual meeting. PARTICIPANTS: Dr. Barak has been the principal investigator of the project. Both Dr. Groves, co-PI, and Dr. Barak have been involved in the experimental design and data analysis for all experiments and mentoring of the graduate student, Jose Pablo Soto-Arias, who has carried out all the experiments to date. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This research has produced a change in knowledge concerning the potential vectoring of human pathogens via phytophageous insects to plants. This knowledge is necessary to guide future research and direct governmental regulation to insure a safe food supply with the new knowledge of phytophageous insects acting as a risk factor for human pathogen contamination of fresh produce.

Publications

• No publications reported this period

Progress 09/01/11 to 12/31/11

Outputs
OUTPUTS: We conducted experiments to determine whether phytophageous insects play a role in human pathogen persistence in lettuce. These experiments targeted the interactions between Salmonella and western flower thrips. We also examined the capacity for acquisition of the human pathogen by both western flower thrips and aphids. PARTICIPANTS: Dr. Barak has been the principal investigator of the project. Both Dr. Groves, co-PI, and Dr. Barak have been involved in the experimental design and data analysis for all experiments and mentoring of the graduate student, Jose Pablo Soto-Arias, who has carried out all the experiments to date. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This research has the potential to produce a change in knowledge concerning the potential biological multiplication of human pathogens by phytophageous insects. This knowledge is necessary to guide future research and direct governmental regulation to insure a safe food supply.

Publications

• No publications reported this period