RAPID AND ACCURATE DIAGNOSTIC DETECTION/IDENTIFICATION OF EXOTIC PHYTOPLASMAS INFECTING GRAPEVINE, APPLE, AND STONE FRUITS
Project Number: 1245-22000-282-14
Start Date: Aug 01, 2012
End Date: Sep 30, 2013
Phytoplasmas cause important diseases in grapevines and fruit trees such as apple stone fruits such as peach, and they adversely affect trade when a country is positive for a specific phytoplasma. Currently there are several phytoplasmas on the Cooperative Agricultural Pest Survey (CAPS) national survey target priority list; australian grapevine yellows, apple proliferation, and European stone fruit yellows phytoplasmas. These phytoplasmas are known to be exotic to the United States including the continental United States, the Pacific region (Hawaii, Guam, and American Samoa), and the Caribbean region (Puerto Rico and the U.S. Virgin Islands). In 2013/2014, a commodity-based survey for these phytoplasmas is planned. Due to the difficulties in achieving sensitive, rapid, and accurate diagnostic detection and identification of phytoplasmas, they are rarely intercepted at ports of entry. Early detection surveys are vital to find these pathogens before they become established and while eradication is possible. Accurate and sensitive diagnostics are also necessary to establish control schemes once an exotic phytoplasma has been found. The objectives of this project are to develop PCR assays for the aforementioned exotic phytoplasmas and conduct confirmatory PCR detection tests and nucleoc acid sequencing to provide species level identification for each sample. Species-specific PCR primers will also be designed and evaluated to examine their specificity and applicability as a diagnostic method for the phytoplasmas, so that introduced exotic phytoplasmas can be efficiently detected and distinguished. ARS will transfer the technology for detection of the exotic phytoplasmas to the Cooperator. The Cooperator has expertise on extraction protocols for rapid and reliable production of total nucleic acids from plant tissues and methods to dry/freeze plant tissue for storage prior to extraction of nucleic acids.
ARS and the Cooperator will select the best methods to use for plant leaf storage and for rapid and reliable extraction protocols for nucleic acids from plant tissues. ARS will develop PCR assays for grapevine-, apple-, and stone fruit- infecting phytoplasmas not indigenous to the U.S., and conduct confirmatory PCR detection tests and nucleotide sequencing to provide species level identification for each phytoplasma-positive sample. Species-specific PCR primers will also be designed and evaluated to examine their specificity and applicability as a diagnostic method for the aforementioned phytoplasmas, so that introduced phytoplasmas can be efficiently detected and distinguished. ARS will transfer the technology for detection of the phytoplasmas the Cooperator.