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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZATION OF THE PHYTOPLASMA ASSOCIATED WITH ALMOND BROWN LINE DISEASE AND DEVELOPMENT OF A MOLECULAR ASSAY FOR ITS DETECTION
2012 Annual Report


1a.Objectives (from AD-416):
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.


1b.Approach (from AD-416):
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 season 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.


3.Progress Report:

The agreement was established in support of Objective 1 of the in-house project, which is to characterize the etiology, biology, and ecology of key phytopathogenic agents and their interactions with economically important tree and grapevine species. The goal of this project is to characterize the phytoplasma associated with Almond Brown Line Disease and to develop a molecular assay for it's detection. Investigations were conducted on an outbreak of almond brown line (ABL) disease in a 2nd leaf stage almond orchard in Sutter County to determine the etiology of the pathogen and to develop a molecular assay to detect the agent. The outbreak lasted from 2nd leaf (year 2008) to 4th leaf (2010) with most of the trees showing disease in the 2nd and 3rd leaf stage. Annual surveys for symptomatic trees in 2011 indicated there were no new infections in the orchard and symptomatic trees from previous years had died. Polymerase chain reaction (PCR) assays using phytoplasma specific general primers P1 and Tint, and primers specific to 16S-23S rDNA regions of peach yellow leafroll phytoplasma (PYLR-P) were able to amplify products of 1.6 kb and 530 bp, respectively, from nucleic acid extracts (NAs) from diseased trees. However, primers specific to non-rDNA regions of PYLR-P failed to amplify a product of expected size from NA extracted from almond trees with ABL symptoms. It appears that the phytoplasma in question is related to PYLR-P in the rDNA region only, but not elsewhere in the genome. A quantitative PCR (qPCR) for real-time detection of the phytoplasmas: PYLR-P, PD-P, and ABL-P, has been optimized to detect these phytoplasmas in peach, almond and pear samples, respectively.

Bottle grafts from almond trees showing ABL symptoms in 2008 and 2009 failed to reproduce ABL symptoms on almond (cultivar Peerless) test plants on Marianna 2624 following bottlegraft inoculations. In February 2011, we received 60 trees of cultivar Winters on plum rootstock Marianna 2624 suspected to be having ABL from a nursery. Four of these trees showed ABL symptoms in fall. However, PCR analysis failed to reveal the presence of a phytoplasma in these trees. Healthy almond trees of cultivar Mission on peach (Lovell) rootstock were planted close to two almond trees showing ABL symptoms and approach grafted. Symptomatic trees on Marianna 2624 had recovered from ABL symptoms. It is believed that the biological agent responsible for ABL has been retained in these trees. We anticipate that these trees will provide: (1) inoculum for performing critical graft experiments and (2) nucleic acid extracts for future genetic analysis by deep sequencing to determine the nature of the biological agent responsible for ABL.


Last Modified: 12/25/2014
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