Elevated temperatures cause an increase in transpiration as the plant’s mechanism to decrease leaf temperature . However, prolonged transpiration and dry soil conditions may alter a plant’s strategy to mitigate heat stress due to the need to conserve water. Considering that we saw an increase in ABA content with increased CEC concentrations, we speculated that the cucumber plant’s ability to adapt and mitigate heat stress would be further impaired due to exposure to CECs. The ABA content in cucumber tissues followed the same general trend under heat stress conditions as it did under non-heat stressed conditions . No differences in root ABA content due to heat stress were observed within the treatments, however the 1X and 20X CEC treatment with heat stress were significantly decreased from the control with heat stress . Similarly, significant differences in leaf ABA content due to heat stress were not observed within the same CEC treatment, but did show a consistent elevated trend across the range of CEC treatment levels when the plant was also subjected to heat stress. It must be noted that only a 4 d heat stress with somewhat moderate, but realistic, temperature regimes was examined in this study. If this observed trend were to continue, it could have a significant effect over the long-term health and development of the plant. It is possible that prolonged exposure to heat stress and the resulting increase in abscisic acid content and anti-transpiration activity could ultimately affect biomass production and water use efficiency.The virus is a leading cause of food borne illness in the United States,25 liter pot causing nearly 58% of foodb orne disease . Fresh produce has been identified as a leading cause of food borne illness and is a major high risk food associated with human NoV outbreaks in the US . Vegetable row crops and fruits were responsible for 30% and 21%, respectively, of human NoV food borne outbreaks in the US .
Vegetables and crops can be contaminated with human NoV at any point from farm to fork. Evidence has shown that the source of pre-harvest contamination mainly comes from soil, fertilizer, or irrigation water. Though human NoV is a major contributor to fresh produce associated outbreaks, the modes of contamination and persistence of the virus in vegetables remains poorly understood. Human NoV is a non-enveloped single stranded positive sense RNA virus of the family Caliciviridae . The major challenge in human NoV research is that currently there is no robust cell culture system for the virus. Recently, two cell culture systems were developed for human NoV, but have not yet been optimized to produce high viral titers . Therefore, much of the understanding of human NoV molecular biology, pathogenesis, and environmental stability has come from the study of surrogate viruses . Many viruses within the family Caliciviridae have been utilized as human NoV surrogates, including murine norovirus , feline calicivirus , and Tulane virus . Tulane virus is a newly recognized surrogate for human NoV and is member of the genus Recovirus within Caliciviridae . TV was shown to have similar pH stability to human NoV and other surrogates at ranges from pH 3 to pH 8 . TV causes enteric infection in primates and also recognizes histo-blood group antigens as a cellular attachment receptor, similar to human NoV . Internalization of pathogens in growing produce is considered one of the potential routes for contamination of fresh produce . Bacterial pathogens, such as Salmonella enterica serovar Typhimurium and Esherichia coli O157:H7, have been shown to be internalized in vegetables including lettuce, radishes, alfalfa and green onions . Human NoV has also been shown to be internalized in growing produce such as lettuce, spinach, and green onion . However, few studies exist where multiple types of produce have been evaluated for viral internalization in parallel. In addition, the influence of initial inoculum levels on viral internalization in different types of produce has not been evaluated extensively.
In this study, green onion, radishes, and Romaine lettuce were selected to evaluate the effect of growth matrix, inoculum level, and vegetable type on the internalization of human NoV and TV in fresh produce. Human NoV GII.4 strain 707 was originally isolated from an outbreak of acute gastroenteritis in Ohio. The virus genomic RNA was quantified by reverse transcriptase quantitative PCR and then stored at −80 °C. TV was generously provided by Xi Jiang at Cincinnati Children’s Hospital. TV was propagated in confluent monolayers of the monkey kidney cell line MK2-LLC . MK2-LLC cells were cultured at 37 °C in a 5% CO2 atmosphere in low serum Eagle’s minimum essential medium supplemented with 2% FBS. Before virus infection, MK2-LLC cells were washed with Hanks’ balanced salt solution and subsequently infected with TV at an MOI of 1. After 1 h of incubation at 37 °C, 18 ml of Opti-MEM with 2% FBS was added. The virus was harvested 2 days post infection by three freeze thaw cycles followed by centrifugation at 3000 × g for 10 min to remove cell debris. After centrifugation, the supernatant was collected and virus stocks were stored at −80 °C. TV titer was determined by plaque assay. Seeds of green onion and radish were purchased from Livingston Seed and stored at 4 °C. Before sowing, the seeds were soaked for 1 day at room temperature in a 500 ml beaker of tap water. Subsequently, the water was drained and the seeds were covered with 2 layers of wet Kim wipes for 2 days to accelerate seed germination. After germination, the seeds were planted in 4 inch plastic pots and grown in plant growth chamber under long day conditions in Metro-Mix® 300 soil . Two month old green onions and Romaine lettuce and one month old radishes were used for experiments. For growth in the hydroponic system, plants were removed from soil and roots were washed thoroughly with tap water to remove soil and then placed in racks above feed water. Feed water was aerated using a pump and antibiotics were added to limit microbial growth. The feed water was not replaced during the study period.
After viral inoculation, only the roots contacted the feed water and a plastic barrier separated the aerial plant tissues and feed water to limit cross contamination. For green onion grown hydroponically, TV was diluted in feed water to result in a high titer and low titer virus concentration in the feed water. Human NoV was diluted in the feed water of hydroponically growing green onions to achieve 1.0 × 105 RNA copy/ml. For hydroponically grown radishes, only the high concentration of TV was tested. For each treatment group, a 10 ml sample feed water was collected before plants were inserted and was used a control to determine the stability of TV in feed water without plants. In addition, uninoculated control groups were set up for each treatment group. The total volume of the hydroponic feed water following virus dilution was 400 ml and the initial virus titer was determined using plaque assay or RT-qPCR . Negative control plants were kept in separate tanks and no virus was added. At days 0 , 1, 3, 7, and 14, four plants were harvested for each condition and a sample of the feed water was collected. To minimize cross contamination, the upper most leaves were harvested first, followed by shoots, and roots were harvested last . Harvested tissues were placed in individual sterile plastic bags and each tissue was weighed. Next,25 liter plant pot the surface of each tissue was decontaminated by submersion in 1000 ppm chlorine followed by rinsing with water and inactivation of residual chlorine with 0.25 M sodium thiosulfate. Samples were then homogenized by grinding with mortars and pestles with 5 ml PBS . Sample homogenates were transferred to 15 ml tubes and centrifuged at 3000 × g for 10 min to remove cellular debris and the virus-containing supernatant was then transferred to a new collection tube. No virus concentration step was performed prior to detection. Detection of viruses in plant tissue samples and feed water samples was determined by RT-qPCR and plaque assay . The detection limit for RT-qPCR was determined to be 5 RNA copy/ml using dilution of plasmid standard in each sample matrix. The detection limit for plaque assay was determined to be 10 PFU/ml by diluting TV stock in each sample matrix. Three technical replicates were executed during RT-qPCR analysis and two technical replicates were used for plaque assay for each sample. The consumption of fresh fruits and vegetables continues to increase in many countries and fresh produce is an important vehicle for food borne disease transmission. The outbreaks associated with fresh produce have resulted in considerable public health and economic burdens .
Among the pathogens associated with fresh produce outbreaks, an increased incidence of infections and outbreaks are attributed to food borne viruses, most notably human NoV . It has been demonstrated that various types of plants are susceptible to internalization of human viral pathogens during growth . Various factors have the potential to affect the ability of human pathogens to internalize in growing plants including the growth substrate , plant developmental stage, pathogen type, inoculum level, plant species and cultivar, abiotic and biotic stresses . In this study, different plants types, inoculum levels, and growth matrices were tested to determine their effect on the internalization and dissemination of human NoV. Previous research has shown that the level of pathogen inoculum used to contaminate plants can greatly influence the rate of bacterial internalization and dissemination . For example, in Arabidoposis thaliana, low inoculum levels of Salmonella Typhimurium were able to colonize the root surface but did not invade the lateral root junctions. When the inoculum level was increased to 106 CFU/mL, the bacteria were able to internalize into the root . In this study, two different levels of TV inoculum, high and low , were used to contaminate the feed water of hydroponically growing green onions. TV was detected in roots of green onions in both the high and low inoculum groups. The high inoculum group had infectious virus detected in the aerial portions of the plant while no virus was detected in these tissues when a low virus inoculum was applied. Unlike bacteria that have the ability to replicate in the phyllosphere, viruses are unable to replicate outside of their host cell. However, in general it would be expected that higher inoculum levels would result in higher levels of internalization . This was the conclusion made in another study in which higher inoculums of E. coli O157:H7 in spinach showed higher contamination incidences . In another study, the internalization of Salmonella Typhimurium was highly dependent on the concentration of the pathogen present in irrigation water. Irrigation water containing 5 Log CFU/mL Salmonella Typhimurium resulted in limited incidence of internalization in lettuce, while 8 Log CFU/mL Salmonella Typhimurium in the irrigation water, significantly increased the internalization frequency. Similar to these studies, we found that increased TV contamination levels in green onions lead to increased internalization of the virus in hydroponically grown green onion. In addition, we evaluated the internalization of human NoV in hydroponically growing green onion. We found that high levels of human NoV RNA were detected in all harvested green onion tissues starting on day 1 post inoculation. The levels of RNA decreased over the study period; however RNA was still detectable in all tissues on day 14 post inoculation. The data indicates that human NoV internalized via the root of the green onions and transports to the leaves. We also used RT-qPCR to evaluate the level of internalized TV RNA in this study. We found that high levels of TV RNA were detected starting on day 1 post inoculation and that the level of RNA remained at this level at all subsequent time points. However, this differs from the internalization results obtained for TV using plaque assay as the method of detection, in which lower levels of internalization were detected and there were reductions in the level of virus internalized at later time points. This could be due to the fact that RT-qPCR is more sensitive than plaque assay and that RNA detection alone may not accurately reflect the recovery of infectious viruses in these samples.