1a.Objectives (from AD-416)
The proposed research will have four objectives: (1) Repeat the earlier demonstration that Xfp can be transmitted through contaminated seed to seedlings in the greenhouse with the production of symptoms of CVC in the seedlings. (2) Provide additional evidence of the contamination of the seed and seedlings with Xfp by electron microscopy of PCR positive seed and plant tissues to provide insight into the sites and mechanisms of colonization of citrus seed and seedlings by Xfp. (3) The earlier report of transmission of Xfp to sweet orange seedlings through contaminated seed harvested from extremely symptomatic fruit begs the question of transmission of Xfp through seed collected from healthy appearing fruit harvested from CVC-diseased trees. This idea will be tested. (4) The presence of Xfp in citrus seed and fruit imported into the U.S.A. would pose an obvious risk to the citrus industry. A simple therapeutic approach to ameliorate this risk would be helpful. X. fastidiosa is known to be sensitive to low temperatures. Therefore refrigeration of CVC-symptomatic fruit will be tested to see if Xfp can be killed without damaging the fruit. This experiment will require the development and implementation of live/dead PCR protocols to document the presence of Xfp in the seed and distinguish viable from nonviable bacteria.

1b.Approach (from AD-416)
Sweet orange seed infested with Xylella fastidiosa subsp. pauca, the pathogen that causes citrus variegated chlorosis disease will be collected in Sao Paulo, Brazil and sent to Beltsville under permit. Samples of seed will be tested for the presence of the pathogen by PCR and isolation in vitro and electron microscopy. The remaining seed will be germinated and grown in the containment greenhouse under permit from USDA, APHIS. Seedlings will be tested for the presence of the pathogen by PCR and electron microscopy and held for the development of symptoms. Tests will be carried out to determine if refrigeration can kill the pathogen. This will require the development of methods for PCR testing that distinguish between living and dead bacteria.

3.Progress Report

This project is funded by USDA APHIS Center for Plant Health Science and Technology (CPHST). The cooperator is interested in earlier work that showed that Xylella fastidiosa is transmitted through sweet orange seed to seedlings. This was an unexpected result and important to control the global dissemination of this pathogen. We have recruited and hired a visiting scientist to take the lead in the work. We have also prepared laboratory supplies needed for the project. A special transfer permit has been obtained from USDA APHIS to allow the shipment of citrus seed infected with Xylella fastidiosa to us for our experiments. This seed is coming from Brazil. We have developed quantitative PCR assays specific for the citrus strain of Xylella fastidiosa which will be used in this project. We have also set up an SCA with the University of Florida who will collaborate in this work by performing electron microscopy and serological assays on materials provided by ARS.