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

Approximately 2000 sweet orange seed, some infected with Xylella fastidiosa and other not infected have been germinated and grown to seedlings in our greenhouse. Samples from these seedlings have been tested for the presence of Xylella fastidiosa. A replication of the experiment is underway. A new batch of citrus seed was received from Brazil in July, 2011. This seed has been divided for PCR based testing for the presence of Xylella fastidiosa, and for sowing in the greenhouse. Seedlings will be tested for the presence of the pathogen as they emerge. Budwood from the trees that were the source of the Xylella fastidiosa were also obtained and used to graft inoculate sweet orange trees in the greenhouse to replenish our collection.