Research Projects Introduction Activities of the Georgia Envirotron facility include interdisciplinary researches in Environmental Science, Food Safety, Crop and Soil Sciences, Entomology, Plant Pathology, Urban Agriculture etc. Research summaries of leading scientists, graduate, undergraduate and Young Scholars Internship students are presented below. Ongoing and completed research projects at the Envirotron (selected) Development of an efficient method for screening of sorghum phosphorus efficiency and evaluation of root morphology and architecture under controlled p-concentration Viktor Tishchenko (Univestity of Georgia), Ming Li Wang (USDA – PGRCU), Daniel Sabo (Georgia Institute of Technology) Phenotype screening and selection for tolerance to low-phosphorus stress conditions was developed at the Georgia Envirotron. Methodology that uses P-loaded alumina as a phosphorus buffer in the quarts sand culture was used to perform efficient, economical, high throughput sorghum screening for P-efficiency under controlled nutrition conditions. Solid-phase sand-alumina culture system provided stable, diffusion-limited, slow-release conditions with varying P availability to plants. This technique provided better media conditions control and reproducibility compared to complex soil systems and, at the same time, better mimicked natural conditions compared to hydroponic cultures. Sorghum was selected due to its wide range adaptability to abiotic stress (such as drought and barren soil). Significant genetic variation in tolerance to abiotic stress exists in sorghum germplasm and cultivars. In situ electrical capacitance tomography (ETC) method was used for monitoring root development with greater accuracy due to media and plants consistency. The identified materials may be used in plant breeding programs for development of cultivars with high phosphorus use efficiency. Phenotyping for Gummy Stem Blight Resistance in Watermelon Winnie Gimode and Cecilia McGregor, University of Georgia Institute of Plant Breeding, Genetics and Genomics, University of Georgia. Gummy stem blight (GSB) is a major fungal disease of field-grown watermelons in southeastern US, caused by Stagonosporopsis pathogen. This pathogen thrives in warm and humid conditions that is conducive for germination of the spores. Infection is optimum at temperatures between 21°C-26°C, and high moisture levels (above 90% relative humidity), accompanied by leaf wetness. The impact of GSB on watermelon production can be severe, resulting in huge yield losses. Resistance to fungicides poses a major challenge in management of GSB. In addition, the repeated use of fungicides may have a negative impact on the environment, particularly if residues persist in the soil. Using cultivars resistant to GSB would be a cheaper, more environmentally sustainable option for disease management. Currently, no commercial watermelon cultivars are resistant to GSB. This study therefore aims to identify quantitative trait loci (QTL) associated with gummy stem blight resistance in watermelon and further introgress the loci into watermelon cultivars. In order to achieve this, we are utilizing the growth chambers for phenotyping purposes, since the temperature, light and humidity settings can be controlled. Phenotyping for GSB involves screening watermelon seedlings by inoculating them with the fungus (Stagonosporopsis citrulli) through spraying. Due to the high humidity and consistent temperature levels required for development of this disease, the growth chambers have been ideal for these purposes, and we are getting relatively consistent results! Heat/cold tolerance studies of cotton genotypes differing in the degree of fatty acid saturation. Dr. John L. Snider. University of Georgia Heat or cold tolerance can be influenced by fatty acid saturation levels, where plants having cellular membranes with a greater degree of fatty acid saturation have lower membrane fluidity at cool temperatures, which makes them more prone to chilling injury and likely limits seedling vigor under cool conditions. The study currently being conducted at the Envirotron facility assesses seedling vigor and cold tolerance for cotton genotypes differing in the degree of fatty acid saturation. We will be measuring seedling growth characteristics (nodal development, leaf area development, fresh weight, dry weight) and photosynthetic efficiency under contrasting temperature regimes. Application of root demand irrigation approach on turfgrass (Seashore Paspalum). Dr. Paul L. Raymer (University of Georgia), Dr. Viktor Tishchenko (University of Georgia) Root Demand Irrigation (RDI) – innovative, low-pressure, energy-efficient, plant-driven subsurface irrigation. Porous subsurface tubes are used which serve as water reservoir releasing water in response to plant exudates when it’s needed. RDI is claiming to improve water-use efficiency by delivering water – and potentially nutrients – directly to the root zone. It uses low-pressure (about 2 psi or less) that requires less energy and allows to use natural flow (without pumping) from collected and stored water in uplifted containers. RDI requires minimal filtration and can use ground water, ponds, and canals as well as recycled or treated water without expensive treatment because water doesn’t reach the surface. Here, turfgrass subsoil irrigation is tested and optimized on the example of Cecil soils of the Piedmont plateau. RDI tubes installed at 12, 18, and 24 in. intervals and 6 and 12 in. depths (see the graph). Soil moisture is measured with Decagon soil moisture sensors (GS1) installed between lines at the depth of 3 in. together with water flow meter, solenoid valves and sensors are connected to a timer (Toro) and a data logger (Campbell Scientific, CR1000) to continuously track water use and plant response. Different subsurface irrigation timing and pressure modes together with installation spacing and depths are compared to conventional sprinkle irrigation. Eschericha coli O157:H7 pre-harvest contamination of ready-to-eat produce. Dr. Marilyn Erickson. Center for Food Safety, University of Georgia. Over the past six years, growth chambers at the Envirotron have been in use by Dr. Marilyn Erickson from the Center for Food Safety to explore a number of issues associated with pre-harvest contamination of ready-to-eat produce. As a source of contamination of enteric pathogens (Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes), animal manure-based soil amendments are of concern for their potential to introduce and contaminate fields and crops grown in those fields. Aerobic composting of animal manures is one process that may be used to reduce pathogen levels; however, insufficient heat generated at the surface of compost heaps may arise leading to pathogen survival. Although turning of compost piles is advocated to facilitate exposure of all material to heat generated from microbial breakdown of compost materials, Dr. Erickson’s lab has also been investigating nonthermal parameters that affect pathogen inactivation at surface sites of composting piles by holding compost mixtures in small trays or containers within the growth chambers under defined temperature, humidity, and light conditions. Results from several of these studies are highlighted below: Using “fresh” chicken manure, both Salmonella and Listeria monocytogenes survived for longer periods at 25°C in mixtures formulated with wheat straw to a carbon:nitrogen ratio of 40:1 compared to formulations of 30:1 and 20:1. Holding or aging of chicken manure for 3 weeks prior to mixing with carbon amendments had a detrimental effect on pathogen inactivation. No decreases in initial Salmonella levels (~5 log CFU/g) were noted in compost systems formulated with aged chicken manure and stored for 4 weeks at 25°C whereas the pathogen was not detected in compost mixtures prepared with “fresh” chicken manure and stored for 1 week at 25°C. Inactivation of Salmonella was also significantly impacted by the carbon amendment used in compost formulations with decreased survival occurring in wheat straw systems compared to pine needle systems. Another focus of research that has required our use of controlled and contained conditions in growth chambers is to ascertain whether lettuce-infesting insects could influence the fate of either surface or internalized populations of E. coli O157:H7. Brief exposure (~18 h) of lettuce leaves to insects (5 cabbage loopers, 10 thrips, or 10 aphids) prior to inoculation of plants with E. coli O157:H7 resulted in significantly reduced internalized populations of the pathogens within these leaves after approximately 2 weeks, as compared with leaves not exposed to insects. These results suggest that internalization of E. coli O157:H7 may be minimized by plant defenses that are induced in response to intrusive insect activities. A third area of research employing leafy green plants cultivated in growth chambers has been to examine the potential for internalization of enteric pathogens through roots or leaf stomata. E. coli O157:H7 in contaminated water was applied by application to the soil or by spraying the leaves of the plants. Uptake of pathogen into roots occurred when soil contained 7 log CFU/g. Greater internalization of E. coli O157:H7 occurred in lettuce and parsley roots surrounded by saturated soil compared to moist soil. Uptake into roots occurred later for parsley than lettuce or spinach under saturated conditions. Exposure of the leaf surface to 7 log CFU/ml spray led to internalization of the pathogen into leaves of mature lettuce and spinach plants. When sprayed at this concentration, no differences in the degree of internalization occurred for virulent and non-virulent surrogate strains of E. coli O157:H7. Internalized populations of E. coli O157:H7 were greater in spinach compared to lettuce one week after the spray event implying differences in the level of defenses activated by these leafy greens. Currently, growth chambers are being used to explore the short-term survival of E. coliO157:H7 sprayed on five different cultivars of mature cabbage plants. Pathogen survival on these cultivars will be compared to the plant’s expression of several plant defense protein genes and to the plant’s constitutive levels of total phenols and antioxidant capacity. In addition, chemical treatments that have previously been shown to alter phytochemical levels in leafy greens will be applied to one cultivar one week prior to pathogen exposure. Subsequently, treated and untreated plants will be applied to determine if pathogen survival has been impacted. Interspecific Arachis Breeding Project. Noelle Barkley, Roy Pittman, Angie Lewis. USDA The growth chambers are being utilized to grow four wild Arachis species which are being used as female parents for interspecific crosses. The males Arachis species are being grown in our greenhouse facility. The use of the growth chambers allow us to synchronize pollinations by controlling light and temperature so that the male flowers can be collected and immediately used to pollinate the females. In the greenhouse, male flowers are collected in the morning and then used in the evening to pollinate the female plants. Each day flowers from the male plants are harvested and transported to the growth chamber to apply pollen to the stigma. This process is repeated each day flowers are available to help increase the success of obtaining a true hybrid since Arachis species are vigorous self pollinating plants. The resulting hybrid F1’s will be used to create synthetic allotetraploids by treatment with colchine which doubles the chromosomes producing a tetraploid from a diploid. (The majority of Arachis species are diploid whereas cultivated peanuts are tetraploid). The resulting synthetic allotetraploids will be crossed with cultivated peanuts to create mapping populations and introgress disease resistance traits into cultivated peanuts. Morphological Characterizations of Subterranean Clover for Determination of Genetic Redundancy. Brad Morris. Plant Genetics Resources Conservation Unit. Genetic redundancy is of prime concern for curation of crop species. The USDA, ARS, PGRCU Trifolium subterranean collection is suspect of having genetic redundant accessions. Our goal was to determine whether or not genetic redundant accessions do in fact exist within the U.S. subterranean clover collection. Subclover seed were planted in potting soil within each of five4″ plastic pots. A sub-sample of 90 subclover accessions were tested. Soon after seed germination, each pot utilized in the test were moved to growth chambers at the Georgia Envirotron. Plants were grown in a 16 hour photoperiod regime with a 27°C / 17 °C day/night temperature setting. Successful morphological characterizations were recorded for leaf marking, flower color, and stipule color. Combined Effects of Elevated Carbon Dioxide Levels and Temperature on the Biology of the Mealybug Phenacoccus madeirensis Green (Homoptera: Pseudococcidae) Juang-Horng Chong1, Marc W. Van Iersel2, and Ron D. Oetting2 1 Department of Horticulture, Sunchon National University, South Korea, 2 Department of Horticulture The combined effects of elevated CO2 levels (400 and 700 µL/L) and temperatures (20, 25 and 30 0C) on the development, survival and reproduction of two generations of the mealybug Phenacoccus madeirensis were investigated. Mealybugs were reared on chrysanthemums grown in growth chambers set at a specific CO2 level and temperature. The duration to egg hatching and to adulthood of the mealybugs was recorded by examining the mealybug cohorts daily. Hatching rates of eggs and survival rate to adulthood were determined by recording the number of individuals that successfully molted into the next developmental stage. The proportion of females in the population was determined by fractioning the number of females over the total number of adults at the end of the experiment. Adult females were isolated in leaf cages and their eggs were collected daily to determine fecundity. The nutritional status (carbon concentration, nitrogen concentration, and the relative water content of leaves) of chrysanthemum were also studied to interpret the performance of mealybugs at elevated CO2 level and temperature. The development of mealybug is temperature-dependent. Duration of development did not differ among different CO2 level treatments and generations. A female completed its development in about 20 days at 30 0C, 28 days at 25 0C, and 47 days at 20 0C. Males have longer duration of development than females. Survival rates, proportion of females, fecundity, duration of reproduction, and the parameters of host plant nutritional status did not differ significantly among temperature and CO2 level treatments and between generations. Fate of Eschericha coli O157:H7 in Manure Compost Applied to Soil to Grow Vegetables in an Envirotron Growth Chamber. Mahbub Islam1, Jennie Morgan1, Michael P. Doyle1 and Xuiping Jiang2. 1Center for Food Safety, 2University of Clemson. Animal waste in the form of raw manure or composted manure is routinely applied to the land as a crop fertilizer and/or soil amendment. A potential risk arising from the disposal of animal waste of fecal origin is the spread of enteric pathogens. Many outbreaks or cases of E. coliO157:H7 infection have been associated with water or food directly or indirectly contaminated with animal manure. Cross-contamination of produce from manure or improperly composted manure used on the farm can be a source of pathogen contamination during preharvest. Although competition with soil microorganisms and adverse environmental conditions can reduce pathogens, there is little information regarding the ability of E. coliO157:H7 to survive in manure-amended soils. In this study, our objective was to determine the fate of E. coli O157:H7 in soil and on vegetables in a controlled and contained plant growth chamber environment. A five-strain mixture of green fluorescent protein (GFP)-expressing i O157:H7 was prepared and inoculated at 107 CFU/g into the compost. The inoculated compost was mixed with Tifton clay soil at a ratio of 1: 100. Twenty horticultural pots for each of baby carrot and green onion plants were filled with inoculated and fertilized soil (ca.5000 g). Three healthy transplants of each plant were planted into each pot 100 mm apart from each other, and then irrigated with city tap water. The pots were placed in the Envirotron with control of light, temperature, and CO2 levels. Special air filters was installed to prevent pathogens from spreading to the environment. Plants were irrigated every other day, and fertilized with soluble fertilizer (Sam’s Choice Deep Feeding All purpose Food) every two weeks. Soil samples from around the plant (Soil), plant leaves and stem samples (Plant), and soil samples just under the roots (S/p) in triplicate were analyzed for E. coli O157:H7 at approximately weekly intervals for the first four weeks, and every 2 weeks for the rest of plant growth cycle (up to 3 months). Soil moisture content and pH were also determined. Over a period of 64 days in onion, the population of GFP-expressing E. coli O157:H7 in soil and soil under roots samples was steadily reduced by 3 log , whereas in plant samples was reduced by 2. With carrot, it took 84 days to achieve a reduction of 2.3 log in soil. Seventy days were needed to get a reduction of 1.7 log in carrot plant. Image analysis for non-destructive and non-invasive quantification of root growth and soil water content in rhizotrons. Rolf O. Kuchenbuch1 and Keith T. Ingram2. 1Center for Agricultural Landscape and Land Use Research, Muncheberg, Germany, 2Department of Crop and Soil Sciences. Studies aiming at quantification of roots growing in soil are often constrained by the lack of suitable methods for continuous, nondestructive measurements. A system is presented in which maize (Zea mays L.) seedlings were grown in acrylic containers – rhizotrons – in a soil layer 6-mm thick. These thin-layer soil rhizotrons facilitate homogeneous soil preparation and nondestructive observation of root growth. Rhizotrons with plants were placed in an Envirotron CG72 growth chamber, on a rack slanted to a 45o angle to promote growth of roots along the transparent acrylic sheet. At 2- to 3-day intervals, rhizotrons were placed on a flatbed scanner to collect digital images from which root length and root diameters were measured using RMS software. Images taken during the course of the experiment were also analyzed with QUACOS software that measures average pixel color values. Color readings obtained were converted to soil water content using images of reference soils of known soil water contents. To verify that roots observed at the surface of the rhizotrons were representative of the total root system in the rhizotrons, they were compared with destructive samples of roots that were carefully washed from soil and analyzed for total root length and root diameter. A significant positive relation was found between visible and washed out roots. However, the influence of soil water content and soil bulk density was reflected on seminal roots rather than first order laterals that are responsible for more than 80% of the total root length. Changes in soil water content during plant growth could be quantified in the range of 0.04 to .26 cm3 cm–3 if image areas of 500 x 500 pixel were analyzed and averaged. With spatial resolution of 12 x 12 pixel, however, soil water contents could only be discriminated below 0.09 cm3 cm-3 due to the spatial variation of color readings. Results show that this thin-layer soil rhizotron system allows researchers to observe and quantify simultaneously the time courses of seedling root development and soil water content without disturbance to the soil or roots. Refereed Publications Georgia Cotton Research And Extension Report. Annual Publication 108-3, University of Georgia, College of Agriculture and Environmental Sciences, July 2015. Wang M.L., Tonnis B., Pinnow D., Tishchenko V., Pederson G.A. 2015. Newly Identified Natural High Oleate Mutant from Arachis hypogaea L. (in press). Snider J.L., Chapman K.D., Tishchenko V.T. 2015. The Effect of Delta-12 Fatty Acid Desaturase (FAD2) Dene Expression on Seedling Vigor Under Cool Temperatures (in press). Erickson, Marilyn C, Jean Liao, Alison S Payton, Cathy C Webb, Li Ma, Guodong Zhang, Ian Flitcroft, Michael P Doyle, and Larry R Beuchat. 2013. “Fate of Escherichia coli O157:H7 and Salmonella in soil and lettuce roots as affected by potential home gardening practices.” Journal Of The Science Of Food And Agriculture 93, no. 15: 3841-3849. Erickson, M.C., J. Liao, A.S. Payton, D.G. Riley, C.C. Webb, L.E. Davey, S. Kimbrel, G. Zhang, I. Flitcroft, M.P. Doyle, and L.R. Beuchat. 2010. Preharvest internalization of Escherichia coliO157:H7 into lettuce leaves as affected by insect and physical damage. J. Food Prot. 73:1809-1816. Yoon, S.T.; Hoogenboom, Gerrit; Flitcroft, Ian; Bannayan, Mohammad. 2009. Growth and development of cotton (Gossypium hirsutum L.) in response to CO2 enrichment under two different temperature regimes. Environmental & Experimental Botany, 67, 1, pp. 178-187. Bannayan, M., Tojo Soler, C. M., Garcia y. Garcia, A., Guerra, L. C.; Hoogenboom, G. 2009. Interactive effects of elevated [CO2] and temperature on growth and development of a short- and long-season peanut cultivar. Climatic Change, 93, Issue 3-4, pp. 389-406. Zhang, G., L. Ma, L.R. Beuchat, M.C. Erickson, V.H. Phelan, and M.P. Doyle. 2009. Heat and drought stress during growth of lettuce (Lactuca sativa L.) does not promote internalization of Escherichia coli O157:H7. J. Food Prot. In Press. Zhang, G., L. Ma, L.R. Beuchat, M.C. Erickson, V.H. Phelan, and M.P. Doyle. 2009. Lack of internalization of Escherichia coli O157:H7 in lettuce (Lactuca sativa L.) after leaf surface and soil inoculation. J. Food Prot. 72:2028-2037. Zhang, G., L. Ma, L.R. Beuchat, M.C. Erickson, V.H. Phelan, and M.P. Doyle. 2009. Evaluation of treatments for elimination of foodborne pathogens on the surface of leaves and roots of lettuce (Lactuca sativa L.). J. Food Prot. 72:228-234. Beuchat, L. R., A. J. Scouten, R. I. Allen, and R. S. Hussey. 2003. Potential of a plant-parasitic nematode to facilitate internal contamination of tomato plants by Salmonella. J. Food Prot. Chong, J.-H, M.W. van Iersel, and R.D. Oetting. 2003. Effects of elevated carbon dioxide levels and temperature on the life history of the Madeira Mealybug, Phenacoccus madeirensis Green (Hemiptera: Pseudococcidae). Journal of Entomological Sciences. Kuchenbuch, R.O., Ingram,K.T. 2002: Image analysis for non-destructive and non-invasive quantification of root growth and soil water content in rhizotrons. Journal of Plant Nutrition and Seil Science 165, 573-581 Schwarz, D., Van Iersel, M.W., Ingram, K.T., and Kläring, H.-P. 2002. Growth and photosynthetic response of tomato to nutrient solution concentration at two light levels. Journal of the American Society of Horticultural Science, 127, 6, 984-990. Guo, X., J. Chen, R. E. Brackett, and L. R. Beuchat. 2001. Survival of salmonellae on and in tomato plants from the time of inoculation at flowering and early stages of fruit development through fruit ripening. Appl. Environ. Microbiol. 67:4760-4764. Kang J.G. and M.W. van Iersel. 2001. Interactions between temperature and fertilizer concentration affect growth of subirrigated petunias. Journal of Plant Nutrition 24:753-765. Schwarz, D., Ingram, K.T. Hung, Y.-K., and Kläring, H.-P. 2001. Effect of model based control of nutrient solution concentration on tomato growth and fruit quality. Journal of the American Society of Horticultural Science 126, 6, 778-784. Ingram, K.T., G. Hoogenboom, and L. Liu. 1998. The Georgia Envirotron – A multidisciplinary research facility for the study of interacting environmental stresses on plants. ASAE Paper 98-4151, American Society of Agricultural Engineers, St. Joseph, Michigan. Abstracts, Book Chapters, Reviewed Conference Proceedings Mahbub Islam, Jennie Morgan, Michael P. Doyle, and Xiuping Jiang. 2003. Fate of Escherichia coli O157:H7 in manure compost applied to soil to grow vegetables in a growth chamber. Accepted for poster presentation at the International Association for Food Protection Annual Meeting (IAFP 2003), August 10-13, 2003, in New Orleans, Louisiana. Guo, X., J. Chen, R. E. Brackett, and L. R. Beuchat. 2001. Survival of Salmonella on and in tomato plants from the time of inoculation at flowering through fruit ripening. USDA, Coop. Regional Res. Proj. S-294, Postharvest Quality and Safety of Vegetables and Fruits, 8 April, Phoenix, AZ, p. 3-4. Kuchenbuch, R. O., K. T. Ingram 2001: Quantification of root growth of young corn plants (Zea mays L) at a transparent surface under the influence of soil bulk density. – In: Horst, W. J. [Hrsg.]: Plant Nutrition: Food security and sustainability of agro-ecosystems through basic and applied research; XIV International Plant Nutrition Colloquium: 584-585; Dordrecht (Kluwer Academic Publishers) Developments in plant and soil sciences, 92. Schwarz, D., M.W. Van Iersel, K.T. Ingram and H.-P. Kläring, 2001: Nutrient solution concentration effects on growth and photosynthesis of tomato grown hydroponically. In: Plant nutrition. Food security and sustainability of agro-ecosystems through basic and applied research. Eds.: W.J Horst et al., Developments in Plant and Soil Sciences. 92, 432-433. Kluwer Academic Publishers. Schwarz, D. K.T. Ingram, Y.-K. Hung and H.-P. Kläring 2000: Tomato growth and fruit quality affected by model based control of nutrient solution concentration. Griffin, May 2000. Submitted or in Preparation Erickson, M.C., J. Liao, A.S. Payton, C.C. Webb, L. Ma, G. Zhang, I. Flitcroft, M.P. Doyle, and L.R. Beuchat. 2013. Fate of Escherichia coli O157:H7 and Salmonella in soil and lettuce roots as affected by potential home gardening practices. J. Food Sci. Agric. In Press.