The number of food borne illnesses associated with the consumption of fresh fruits and vegetables and their minimally processed products (juices) has increased over the past years. Of particular interest is the ability of microbial pathogens to internalize and survive in fresh produce that are commonly used for juices. This research project addresses the issue of the ability of Escherichia coli O157:H7 to internalize and survive in whole apples before and after harvest. Four cultivars of apples, Redfree, Red Delicious, Golden Delicious, and York, were inoculated under field conditions with a surrogate strain of E. coli, Escherichia coli ATCC 25922. The Redfree cultivar was inoculated at the beginning of its growth stage (day 0), and again 30 days later, and sampled for two weeks, until E. coli was not recoverable through microbiological methods after three successive sampling days. Red Delicious, Golden Delicious, and York cultivars were spray inoculated with the surrogate strain two weeks before their anticipated harvest date and sampled every other day until E. coli was not recoverable for three successive sampling days. For each cultivar, the presence of E. coli ATCC 25922 was not detectable after 7 to 9 days. In the laboratory study the Red Delicious, Golden Delicious, Rome, and York cultivars received one of three treatments; unblemished control, bruising, or puncturing. The apples were inoculated by immersion in cold water containing E. coli O157:H7 GFP, incubated for three days then microbiologically analyzed for presence of the bacteria. In all cases, the punctured apples of each cultivar showed the greatest uptake of E. coli O157:H7 GFP. Escherichia coli O157:H7 GFP was visualized in flesh and core sections of untreated, bruised, and punctured apples of all cultivars. The microbe was found in between cells, but not within cells of the apple. Internalization of Escherichia coli in whole apples on the tree is not likely, and leads to the conclusion that internalization is a post-harvest problem. Internalization may occur before pressing or processing of apples, leading to an increased risk of infection with E. coli for consumers of apple products that are not properly treated to destroy pathogens. Internalization does occur when apples are immersed in solutions containing the pathogen Escherichia coli O157:H7, and better post harvest controls need to be implemented in order to prevent this in whole apples that are used for cider and juice production. / Master of Science

Master of Science

This problem purposes not only to show the relationships, if any, that exist between terminal growth and yield, but also to ascertain the statistical methods that may be best adaptable. / Master of Science

The main objective of this project is to gain an understanding of the internalization of Escherichia coli in the tissues of apples. This broad statement includes the rate of internalization in young versus mature apples as well as injured versus non-injured apples. Five apple varieties, Redfree, Red Delicious, Golden Delicious, Rome Beauty and York Imperial, were used to compare differences and similarities in structure and ability to internalize the pathogen. Both the surrogate species, E. coli ATCC 25922, and the pathogen, E. coli O157:H7, were used for field and lab studies, respectively. Internalization of E. coli in apples under natural environmental conditions was addressed in the first study using a controlled outdoor setting. Escherichia coli species (ATCC 25922) was used as an alternative to the pathogenic species. The bacterial culture was applied to topsoil and spread evenly on a 6x6-foot area. Red Delicious, Golden Delicious, and Rome Beauty apples were placed randomly on the soil much like a drop or windfall apple. The position was noted as to whether the apple fell calyx up, down or on its side. Apples were examined for the presence of E. coli and sampled on days 1, 3, 8, and 10. Skin, flesh, inner, and outer core samples were plated on MacConkey agar supplemented with cycloheximide and MUG to ease in identification. Escherichia coli was found in the inner core and flesh samples of all apple varieties, indicating the potential for infiltration by the organism outside laboratory conditions. The second study determined the rate of internalization in immature apples. Redfree was used in a long-term study in which individual apples were spray inoculated at the beginning of the growing season with E. coli ATCC 25922 at 104 cfu/apple. The apples were picked on days 1, 30 and 60, and sectioned into skin, flesh, inner and outer cores. The remaining four apples species were used in an intensive, two-week study. In the long-term study, apples were inoculated two weeks prior to harvest and picked every other day until harvest. The surrogate E. coli was not found in the apples after day 1. Other coliforms, such as E. vulneris, Klebsiella pneumoniae and Kl. ozaenae were present in each pick. The two-week study showed higher rates of internalization in Red and Golden Delicious than in Rome and York, with the E. coli present in all four sections of the apples. Red Delicious apples showed a trend of increasing counts of bacteria over the two-week period with initial counts ranging from less than one cfu/ml to final counts as high as 2.64±1.90 log cfu/ml. Again Klebsiella species and E. vulneris were found in the apples. Microscopy was used for imaging of the apples tissues. Morphological differences were found in the skin, where lenticel presence or absence may affect internalization. Differences were also shown in the flesh where cell wall thickness was shown to vary depending on variety. Imaging thick sections of skin showed cuticle cracks and thickness, which also vary depending on the apple variety. This study indicates that internalization occurs at a high degree in drop apples and to a limited extent in tree apples. However, with the low infective dose required for illness, it is necessary to instate strict regulations to ensure safety. The most effective treatment involves the inclusion of a five-log reduction of the target organism, E. coli O157:H7. This reduction can be obtained through one step or the combination of two or more steps. / Master of Science

The US cider market has expanded in recent years, but limited research-based information is available on fermentation management. This study investigates how apple cultivar and yeast inoculation affect the chemical and sensory properties of cider. Four ciders were produced in triplicate using combinations of two different apple cultivars - Harrison, a cider cultivar and GoldRush, a dessert cultivar - and two fermentation strategies - inoculated with dry active yeast EC1118 or Pied de Cuvé ambient fermentation. Ciders were analyzed for alcohol content, free/total SO2, titratable acidity, volatile acidity, malic acid, pH, and residual sugar. Sensory evaluation was conducted using Descriptive Analysis with trained panelists. Results were analyzed via ANOVA and Principal Component Analysis. Apple cultivar and fermentation method resulted in significant differences for chemistry and sensory parameters. Malic acid concentration was greater in the control ciders while concentrations of both residual sugar and volatile acidity were higher in the PDC ciders. The interactions effect of cultivar*fermentation method influenced both malic acid and residual sugar concentrations, where concentration differences between control and ambient ciders is smaller for GoldRush than for Harrison, showing that fermentation style produces different results across cultivars. Volatile acidity produced opposite interaction effects as differences between fermentation styles was larger for GoldRush. For sensory attributes, Harrison ciders produced high intensities for multiple attributes, but also higher variability. Multiple sensory descriptors displayed interaction effects as the fermentation method produced different results in different cultivars. This study demonstrates that increasingly popular practices in the industry can produce significantly different ciders. / Master of Science in Life Sciences / The US cider market has grown rapidly in recent years, with many new products entering the market. However, there is limited research-based information available on cider fermentation management. This study investigates how production variables, namely apple cultivar and yeast inoculation, affect the chemical and sensory properties of the cider created. The overall goal of this project is to assess the chemical and sensory characteristics that come from cider production treatments. In this study, four experimental ciders were produced using combinations of two different apple cultivars – Harrison, a cider cultivar and GoldRush, a dessert/fresh market cultivar – and two fermentation management strategies – inoculated with dry active yeast strain EC1118 or indirect inoculation through a natural fermentation method. Ciders were analyzed for alcohol content, free and total SO2, titratable acidity, volatile acidity, malic acid, pH and residual sugar. Sensory evaluation was conducted by a trained panel providing descriptive terms and intensities for each sample. Both chemical and sensory results were analyzed to reveal significant differences in samples based on not only apple type and inoculation method, but also the interactions between those two variables. This study demonstrates that increasingly popular practices in the cider industry – like natural fermentation or the use of cider-apple varieties – can produce significantly different ciders. This highlights the idea that producers need to treat each apple cultivar differently, as they behave differently throughout production.

The apple growers and packaging houses are interested in methods that can evaluate the quality of apples non-destructively. Harvested fruits are a mixture of immature, mature, and over mature fruits, thereby posing a great problem in deciding their end use and storage time. It is expected that the technique developed from the present project could be effectively used to classify the harvested fruit into immature, mature and over mature apples, rapidly and non-destructively. It would also help the growers to predict the optimum dates to harvest the fruits. York and Gala were the varieties of apples that were used in this study and were obtained from Virginia Tech College of Agriculture and Life Sciences Kentland Farm. Apples were harvested at different times resulting in different maturity groups (immature, mature and ripe). Gala apples were harvested on three dates with an interval of 10 days, while York apples were harvested on four dates with an interval of 14 days. They were stored at 0oC until sampled. For each harvest date, the experiments were conducted in two sets (10 each) on two consecutive days. First the ethylene levels were measured, followed by gas chromatograph and electronic nose. Then the maturity indices were measured. Three maturity indices, starch index, firmness and soluble solids were used as the three variables for the statistical analysis to identify and categorize the fruits into three maturity categories referred as immature, mature and over mature fruits. Apples were also categorized into three maturity groups based on the emanation levels of the aroma compounds evolved from the fruits. Then electronic nose sensor responses were categorized into the above maturity categories, and their effectiveness was determined using a statistical procedure called Discriminant Analysis (DA). From the DA cross validation results the correct classification percentage for Gala and York apples into maturity groups was 95%. The Electronic nose sensor's effectiveness to categorize the same observations based on sensor responses in to the above classified maturity categories was 83% correct in case Gala apples and 69% for York apples. The EN sensors response data were analyzed by the EN system software and the correct classification percentage for Gala was 83% and for York was 81%. Aroma-based categorization for Gala apples was 100% correct, while the electronic nose for the same analysis was 80%. Based on the three physical parameters, an objective evaluation of maturity could be accomplished. Principal Component Analysis, Canonical Discriminant Analysis and DA results demonstrated that the electronic nose could be used to classify apples into three identified maturity-based groups. The EN sensors (Gala apples), could also classify the apples into aroma-based categories. Thus, it can be concluded that the EN system holds promise as non-destructive evaluation technique to determine the maturity of an apple. / Master of Science

Two experiments were conducted with field- and container-grown 'Delicious' apple trees to evaluate the effect of nitrogen fertilization on the physiology of apple leaves subjected to European red mite feeding, and the subsequent recovery of leaf function following mite removal. Nitrogen (N) fertilization affected the visual damage, and the photosynthetic response of 'Imperial Delicious' apple leaves to mite feeding. Visual damage occurred first, and remained greatest on the low N treatment throughout the experiment. High N trees exhibited a linear decline in Pn with increasing MD, in contrast to the quadratic decline found under low N treatment. With equal mite populations, high N trees retained higher Pn rates. Transpiration, dark respiration, relative water content, leaf nitrogen, and total chlorophyll were negatively related to MD, but specific leaf weight was positively related to MD. No interaction was found between N treatment and mite feeding for these parameters. Following removal of ERM populations, 'Redchief Delicious' leaves subjected to greater than 4000 MD exhibited recovery in Pn by 28 days after mite removal. Leaves receiving less MD (1500-3000) demonstrated an inconsistent response over time. Transpiration and diffusive resistance showed no recovery. Relative water content, specific leaf weight, leaf nitrogen, and total chlorophyll linearly declined with mite feeding, and were unaffected by foliar urea treatment. / Master of Science

The effects of pheromone trap placement on male moth catches of both species were studied. For P. flavedana, traps hung at 2.1 and 3.0 meters captured the greatest number of moths. Trap heights of 1.2, 2.1, and 3.0 meters caught the greatest number of P. idaeusalis moths. The outside-the-canopy trap position captured more P. flavedana moths, while the within-canopy trap location caught the greatest number of P. idaeusalis moths. Traps placed in the west portion of the tree captured the greatest number of P. flavedana moths. P. idaeusalis moth catches were not influenced by compass quadrants. Trap design and pheromone dispenser and rate influenced trap catches of P. flavedana. Development of P. flavedana and P. idaeusalis on a meridic diet was observed at constant temperatures in the laboratory. Lower developmental threshold values for egg, larval, and pupal stages of P. flavedana were: 10.6, 8.6, 9.0°C, respectively. Lower developmental threshold values of 9.7, 7.0, and 8.5°C were estimated for P. idaeusalis egg, larval, and pupal stages, respectively. An average of 101.5 °D_10.6’, 379.6 °D_8.6’, and 126.0 °D_9.0’ were required for development of egg, larval, and pupal stages of P. flavedana, respectively. P. idaeusalis required 104.7 °D_9.7’, 442.7 °D_7.0’, and 132.2 °D_8.5’ to complete development in the egg, larval, and pupal stages, respectively. Differences in rate of development were observed between food sources for both species. Within-tree spatial distribution of egg masses and fruit damage resulting from larval feeding for both species was investigated. P. flavedana egg masses were mostly found in the southern portion of the tree below 1.8 meters. Egg masses of P. idaeusalis were observed in greatest numbers in the southern and eastern quadrants of the tree below 2.8 meters. Fruit damage caused by larvae of both species was greatest in the lower portion of the tree. Wind dispersal of first-instar larvae between trees is believed to have influenced fruit damage distribution. The seasonal activity of P. flavedana and P. idaeusalis was monitored. Degree-day accumulations for first moth catch, first and peak egg deposition, and first and peak egg hatch of both generations are presented. / Ph. D.

In a series of related experiment: some aspects of the histology and physiology of the disorder bitter pit in apple (Malus domestica Borkh.) were studied. A two year field study was conducted to induce consistent bitter pit development in type ’Delicious’ (D) and ’Golden Delicious’ (GD) apple fruit. Multiple spray treatments of CaCl₂ and MgCl₂, combined with paper bag fruit covers, were applied and subsequent bitter pit development examined. The main effect of bags in both cultivars was increased pit development, decreased Ca in fruit and increased fruit K. CaCl₂ sprays resulted in less pit development, increased Ca in fruit, and less fruit Mg. MgCl₂ sprays resulted in increased bitter pit, decreased fruit Ca, and increased fruit Mg. Overall, field treatments provided a wide range of bitter pit incidence after storage, from 100% (bag and MgCl₂ spray combined) to 3% (CaCl₂ spray alone) in both D and GD. The cellular morphology of pitted apple fruit from field trees was examined using transmission and scanning electron microscopy. The overall tissue morphology of both cultivars was similar, but in pitted tissues differences were observed in tannin localization, starch hydrolysis, and cell wall morphology. Cation levels in the tissues were examined using X-ray microanalysis. High Mg levels were localized in pit cells, while K levels were similar in both healthy and pitted cells. Ca levels in both tissue types were too low to be detected by this method. Using ’Golden Delicious’ fruit from the field study, the relationship between pyruvate kinase activity, fruit cation concentration and bitter pit was investigated. Pyruvate kinase activity during early fruit growth was higher in fruit which developed 100% bitter pit after storage (MgCl, spray + bag), than in fruit that developed 3% bitter pit (CaCl₂ spray). Fruit with a high bitter pit incidence had a lower Ca: Mg + K ratio than fruit with a low level of the disorder. There was a strong positive correlation between enzyme activity early in the season and bitter pit incidence alter storage. An assay for pyruvate kinase may be valuable for early prediction of postharvest bitter pit development. Finally, the qualitative electrophoretic patterns of soluble fruit proteins from each treatment were examined starting early in the season and continuing until termination of fruit storage. Patterns from all treatments were almost identical throughout the season. / Ph. D.

Crown gall disease is an agricultural problem caused by the soil-borne bacterium, Agrobacterium tumefaciens. A. tumefaciens oncogenes cause transformed plant cells to overproduce the hormones, auxin and cytokinin. High hormone levels cause unorganized plant cell growth resulting in a gall. Control of crown gall disease is difficult because after plant cell transformation has occurred, the bacterium is no longer required for the disease to progress. Apple trees engineered to express double-stranded RNA of two A. tumefaciens oncogenes, ipt and iaaM, silenced the expression of the wild-type oncogenes and prevented crown gall disease. Only the iaaM oncogene was targeted for posttranscriptional gene silencing (PTGS) as measured by biological assays and by quantitative reverse-transcription polymerase chain reaction (q-RTPCR) on transgenic tissue. However, if the translation initiation sequence of the iaaM construction was eliminated, gall formation was not prevented, indicating that translatable RNA initiates silencing whereas untranslatable RNA does not. Other data indicate that the Arabidopsis thaliana micro-RNA pathway gene is involved in A. tumefaciens-mediated tumorigenesis. A. thaliana plants with a mutation in HEN1, a gene required for micro-RNA maturation, demonstrated a tenfold reduction in tumorigenesis upon A. tumefaciens infection compared to wild-type. The same plant line showed no difference in T-DNA transfer and nuclear uptake. / Graduation date: 2006