Biological Control of Brazilian Pepper
by José Pacheco, Kateel G. Shetty, K. Jayachandran

   
INTRODUCTION

     I come from Puerto Rico. I am a Biology undergraduate student at Florida International University. I was accepted in the Agroecology Certificate Program by Dr. Jayachandran and Dr. Mahadev G. Bhat (Department of Environmental Studies). I am interested in learning the various positive links between the preservation of natural ecosystems and the practice of sustainable agriculture and how I can be a part of this process. For me the most important aspect of protecting the environment is to find new management practices that can help reduce the contamination of soil and water from pesticides or herbicides usage in agriculture. The toxic chemicals through the food chain will ultimately end up inside animals and humans. My Internship is with Dr. Jayachandran, I chose to work on biological control of Brazilian pepper for my Independent Study project and I have been working under the supervision of Dr. Kateel Shetty in Dr. Jayachandran’s lab. The Brazilian pepper (Schinus terebinthifolis) is an exotic invasive species and it was introduced from Brazil and Argentina in the 1850’s as ornamental plant and now it has become a serious problem in the Everglades National Park. Because of its invasive nature Brazilian pepper has become a threat to the Florida native species in Scrub, Pine Flatwoods and Mangrove communities.

METHODS

     In the beginning I spent time in the lab learning basic microbiological and plant pathological techniques such as clean laboratory practices: use of autoclave to sterilize media and glassware, filter sterilization technique for heat labile compounds, preparation of culture media for fungi and bacteria, use of Biological safety cabinet, culturing and sub-culturing bacterial and fungal isolates, handling stereo and compound microscope, Koch’s postulate steps for isolation of plant pathogens from diseased plant tissue and inoculation procedures.
To collect diseased Brazilian pepper leaves I visited the Nature Preserve at the FIU campus. The leaves with blight or leaf spot symptoms were brought to the laboratory and washed using a mild soap solution to removing surface contaminants. Later the leaves were surface sterilized for 0, 3, 9, 12 and 16 minutes in 10% bleach solution. All the leaves were washed 3 times in deionized water to remove residual bleach. Using sterile scalpel approximately 5 mm2 pieces were made from the diseased spot on the leaf and the leaf pieces were placed on appropriate agar media plate with 3 replications for each sample. The media used were DRBC agar (Dichloran-rose bengal-chloranphenicol), Potato dextrose agar (PDA) for isolation of fungi and Tryptic soy agar (TSA) with anti-fungal antibiotic cycloheximide for isolation of bacteria. The TSA plates were incubated at 28o C and the DRBC and PDA plates were kept at room temperature (approximately 26o C). The bacterial growth observed on the infected leaf tissue was transferred on to fresh TSA plates (supplemented with yeast extract). Agar plugs from the outer most edges of fungal growth from the diseased tissue were transferred to fresh PDA plates.
     Three of the bacterial isolates were used for inoculation study. The inoculum suspension was prepared using 24 hours bacterial culture from TSA plates. Three potted Brazilian pepper seedlings (3 months old) were used for inoculation in the greenhouse. Each isolate was inoculated on 3 leaves by first dipping a 2 cm2 sand paper in the inoculum solution and then gently rubbing it on the leaf surface. The entire plant was covered with polythene bag for 72 hours and was observed for symptom development. The same three bacterial isolates were also inoculated by dipping the Brazilian pepper leaves into a beaker containing bacterial inoculum solution and covering it with polythene bag for 72 hours.
Three of the fungal isolates that were selected from the 12 and 16 minutes bleach (10%) treatment plates did not produce spores on PDA. Without spores it is not possible to do a good inoculation study. The three fungal cultures were grown on Malt agar, Oatmeal agar, Tomato juice agar, Fungal sporulation agar and incubated at room temperature with and without light. No spore production was observed after 6 weeks of incubation. A small piece of agar plug with mycelial growth was taken out from each of the fungal isolates on PDA agar plate and was used as inoculum to test the fungal isolates. The mycelial agar plugs were placed in contact with the Brazilian pepper leaf and was gently position in place using parafilm. The inoculated plants were covered with polythene bag to maintain high humidity for 24 hours.

RESULTS

     I had to make repeated efforts to learn the clean lab procedures and to understand how critical it is to avoid contamination while conducting microbiological work.
     Among the 3 bacterial isolates tested on Brazilian pepper seedling one of the isolate produced disease symptoms (chlorosis) only when inoculated by means of surface injury (sand paper) and it did not produce any disease symptom when inoculated through leaf dipping.
     One fungal isolate inoculated using agar plug contact method produced disease symptoms (necrotic spots, blight and chlorosis) after one week under the greenhouse conditions. The same fungal isolate was again inoculated using the agar plug method and the leaves were observed under stereomicroscope at various time intervals. Under stereomicroscope various stages of disease development could be observed. At the contact site aggregates of many black specks were observed, the specks our transferred to a clean slide and observed under compound microscope. The black specks were found to be fungal conidia and it was tentatively identified as Pestalotiopsis spp.

CONCLUSION

     I will be continuing the research project and I am planning to test whether incorporation extracts of Brazilian pepper leaves in the fungal media will have any beneficial effect on fungal sporulation.
     It’s been a continuing process of learning since the summer of 2006. My Internship in this new Agroecology program has helped me understand the basic principles involved in conducting research and has broadened my educational experience. Through educational trips and workshops I was exposed to new ideas and concepts from peoples who are actively involved in the preservation of environmental health and sustainable agriculture practices in the South Florida. In addition with the help of this program, I was able to make co-operative interactions with other students and expand my knowledge. I am in the process of improving my English speaking and writing skills, my active involvement in the Agroecology program was very helpful in improving my language skill. I am confident that internship project that I am doing will be useful in my overall goal to work in the farms and incorporate sustainable agriculture practices. The techniques and ideas that I have learnt from this project have applications in agriculture, use of biological control methods have a strong potential in the management of weeds and there by help reduce the herbicides related contamination problems.

ACKNOWLEDGEMENTS

This project has been made possible by a funding support under the USDA CSREES HSI Higher Education Grant Program.USDA-CSREES Grant Number 2005-36422-15940.