CSI: “The Leaf Dilemma” Krissya Carranza and Sandra Westmoreland, Phd Department of Biology Master of Arts in Teaching ABSTRACT RESULTS Plants are susceptible to a multitude of different pathogens, including bacteria, fungi, and viruses. Therefore, they have developed highly complex and effective immune systems to protect them against infections caused by pathogens. In addition, biotic and abiotic environmental factors create stress for plants, thereby, affecting a plant’s growth rate, root to shoot ratio, reproductive production, and increase susceptibility to bacterial and fungal infection (Boyer 1982). Therefore, the purpose of this investigation was to determine whether the cause of damaged tissue on a Primula vulgaris plant was due to a pathogen infection or result of environmental stress. In this study, a scanning electron microscope was used to investigate how P. vulgaris damaged leaf tissue compared to healthy leaf tissue. Digital images were taken of leaf samples from three healthy leaves and three leaves exhibiting damaged tissue. Digital images of damaged and healthy tissue were taken at same magnification and surface view of leaf.   P. vulgaris growth   On day one of investigation, P. vulgaris plant exhibited signs of disease. Three leaves showed yellowing, wilting, and spotting. On day 15th, the flowers showed signs of discoloration and wilting. Six leaves showed yellowing, wilting, and discoloration. Some leaves showed purple spots at base of leaf. On day 22nd, the flowers wilted and only three leaves exhibiting healthy tissue were left. On day 30th, all leaves showed signs of discoloration, yellowing, and wilting. Some leaves began to decay and white webbing was present at the base of the stems. Figure 3: Scanning electron micrograph (4000X) showing non-infected stomata. Figure 1: Image of P. vulgaris on day one. Figure 2: Scanning electron micrograph (600X) showing healthy leaf epidermis. REFERENCES INTRODUCTION    Boyer JS. 1982. Plant Productivity and Environment. Science. 218 (4571): 443-448. Hall CR, Hodges AW, Haydu J. 2006. The economic impact of the green industry in the United States. Horttechnology. 16(2). 345-353.   Majid A, Hassan S, Hussain W, Khan A, Hassan A, Khan A, Khan T, Ahmad T, Rehman M. 2014. In vitro approaches of Primula vulgaris leaves and roots extraction against human pathogenic bacterial strains. World Appl Sci J. 30 (5): 575-580. Stewart-Wade SM. 2011. Plant pathogens in recycled irrigation water in commercial plant nurseries and greenhouses: their detection and management. Irrig Sci. 29. 267-297. Vidaver AK, Lambrecht PA. 2004. Bacteria as plant pathogens. Plant Health Instructor [Internet]. [cited 31 Mar 2017]. Available from http://www.apsnet.org/edcenter/intropp/pathogengroups/pages/bacteria.aspx   The United States ornamental horticulture industry is composed of floriculture, the culture of flowers, and landscape horticulture, the production and use of plants for landscape architecture. Ornamental horticulture is a multibillion dollar industry, and one of the fastest growing sectors of the nation’s agricultural economy (Hall et al. 2006). Plants are susceptible to a multitude of different pathogens, including bacteria, fungi, and viruses, which can cause great damage and have an economic cost. Therefore, poor horticulture practices can increase the dispersal of plant pathogens, increase the susceptibility to bacterial and fungal infection, and increase the use of pesticides to control disease outbreaks (Stewart-Wade 2011). Thus, exercising good horticulture practices is essential for disease management and prevention. In agricultural landscape, Primula vulgaris, is a long-lived perennial herb that is intensively used. Primula is a low emerging plant with bright color flowers and broad leaves. The Primula has been cultivated for ornamental purposes and used in herbal medicine (Majid et al. 2014). Primula plants have extensively been used in medicine to treat conditions involving cramps, spams, paralysis, and rheumatic pains (Majid et al. 2014). Primula plant contains saponins, which has an expectorant effect and salicylates which has an anti-inflammatory effect (Majid et al. 2014). In addition, saponin form primula has antimicrobial properties which makes P. vulgaris remarkably useful in the treatment of pathogenic bacteria strains, and aid in the study of new classes of drugs for bacterial infections (Majid et al. 2014). The purpose of this descriptive study was to determine whether the cause of damaged tissue on a P. vulgaris plant was due to a pathogen infection or result of environmental stress. Figure 6: Scanning electron micrograph (3000X) showing bacteria around stomata. Figure 5: Scanning electron micrograph (600x) showing bacteria on leaf epidermis. Figure 4: Image of P. vulgaris exhibiting signs of infection on day 15. CONCLUSION MATERIALS AND METHODS   Plant Sample Collection A Primula vulgaris plant showing signs of disease was obtained from a commercial nursery. The plant was watered every two days over the span of three weeks. The plant was stored at room temperature and ambient sunlight. All leaf samples were collected in zip-bloc bags and brought to the laboratory and used within two hours of collection. Sample preparation for scanning electron microscopy The Hitachi tabletop scanning electron microscope (TM-100) was used to observe the leaf epidermis of P. vulgaris. Three samples of healthy leaf tissue and three samples of damaged leaf tissue was collected from a P. vulgaris plant. Fresh healthy and damaged leaf tissue was cut into approximately 1-2 cm and placed unto stubs. Digital images of P. vulgaris damaged and healthy leaf tissue were taken at same magnification and surface view of leaf. The scanning electron micrographs showed the presence of bacteria (cocci) in the leaf epidermis of damaged leaf tissue (Figure 5 & 6). Therefore, it can be concluded that the signs of damaged tissue on the P. vulgaris plant was due to a bacterial infection. However, in order to identify which bacterial strain was the cause of infection a tissue culture must be performed. Because the plant was obtained from a commercial nursery, it is not known how the plant became infected with the pathogen. However, it most likely was due to bacteria entering through the stomata, abrasions or wounds on leaves, stems or roots, or through placement by feeding insects (Vidaver and Lambrecht 2004). In addition, nutrient conditions in the plant may favor multiplication in different parts (Vidaver and Lambrecht 2004). Another possibility is the horticulture practices of the nursery, recycling irrigation water in commercial plant nurseries is a common practice, which acts as a primary inoculum source and effective inoculum dispersal mechanism (Stewart-Wade 2011). Plant pathogens may be present in initial water source, may accumulate and disperse from various points throughout the irrigation system, thereby, increasing the irrigated plant’s risk of disease (Stewart-Wade 2011). Therefore, good horticulture practices are essential in the prevention of disease. Poor horticulture practices of commercial nurseries can increase the risk of disease and plant death; therefore, increasing the number of unsaleable plants and economic cost. ACKNOWLEDGEMENTS I would like to thank Dr. Sandra Westmoreland for mentoring me throughout this project and providing a inquiry based learning experience. Also, I would like to thank Dr. Camelia Maier for her assistance in identifying the cause of disease.