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United States Department of Agriculture

Agricultural Research Service

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Research Project: CHARACTERIZATION OF THE PHYTOPLASMA ASSOCIATED WITH ALMOND BROWN LINE DISEASE AND DEVELOPMENT OF A MOLECULAR ASSAY FOR ITS DETECTION

Location: Crops Pathology and Genetics Research

Project Number: 5306-22000-015-07
Project Type: Reimbursable

Start Date: Jul 01, 2011
End Date: Oct 31, 2012

Objective:
1) Survey the orchard for symptomatic trees in spring, summer and fall season. 2) Graft chip buds from symptomatic trees on to peaches and almonds on Marianna 2624 rootstock 3) Conduct PCR-based assays on leaf and petioles and developing green almonds from 'Winters' and 'Sonora' trees for the presence of PYLR phytoplasma. 4) Develop specific primers for quantitative real time PCR and monitor the titer of PYLR phytoplasma in peach and almond trees maintained at UC Davis orchard.

Approach:
Surveying the orchard for symptomatic trees: We have conducted two surveys, one in each of the last two years, in the almond orchard with ABL in Sutter County. We will continue monitoring new infections in the orchard the next two years and surveys will be conducted during spring, summer and fall, to study the incidence of the disease. We propose to sample developing nuts during spring, and leaf samples during spring, summer and fall for laboratory analyses. Nucleic acid extracts will be made from the samples using commercial kits, and PCR reactions will be performed using P1 and Tint primers which amplify the 16S-23S spacer region of PYLR-phytoplasma and X disease phytoplasma. Amplified products will be sequenced directly and the sequence will be compared with the sequences of PYLR-phytoplasma and other phytoplasma in the public domain database. Bioassays-We plan to graft bud chips collected from symptomatic trees positive for phytoplasma in PCR and graft them on almonds growing on Marianna 2624 rootstock. Grafted plants will be monitored for the development of foliar symptoms and ABL development. Results of these experiments will have to wait for at least a year after completion of grafting. All symptomatic trees will be tested by PCR for confirmation. The sequence information obtained from PCR products generated from nucleic extracts from different samples will be aligned and primers specific to the agent will be developed for real-time quantitative PCR. We will run quantitative PCR assays in the following seaseon along with primers designed for Cherry Western X disease and PYLR phytoplasma to determine the titer of these infectious agents in samples taken during spring, summer and fall. We have almond trees infected with Western X and PYLR phytoplasma in our experimental orchard to provide positive controls.

Last Modified: 10/1/2014
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