Location: National Germplasm Resources Laboratory
Project Number: 8042-22000-302-005-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2018
End Date: Sep 30, 2019
The goal of the proposed research is to elucidate the causal agent(s) of Rapid Apple Decline (RAD), a recently emerging biotic problem of apple trees in the mid-Atlantic and northeastern U.S. The objective is to analyze additional RAD-affected samples to better understand the pathogen(s) involved in the decline. Results from this collaboration will provide information about RAD etiology, and deliver sensitive, robust and high-throughput disease diagnostics. These diagnostic tools will improve current testing methods and help determine the incidence of the pathogens from growing-season samplings. The project will accelerate the development of new approaches to disease management, and promote the use of clean plant materials (especially with new orchards) and elimination of pathogens.
1. Conduct virosome analysis: To search for the pathogens of RAD, two hundred samples from RAD-affected orchards in different location of PA and NY will be collected from orchards during the mid-growing season. Ten individual samples will be pooled into a single sample, and a total of 20 pooled samples will be subject to Illumina RNA sequencing (SeqMatic, LLC., San Francisco, CA). Sequence analysis for pathogen identification will be performed using the CLC Genomics Workbench 10.2 platform (Qiagen, Redwood City, CA) and Blaxt searches to identify both known and unknown pathogens including viruses and viroids. Presence of any known viruses identified by RNA sequencing will be confirmed by RT-PCR. To obtain the genomic sequence of any previously undescribed pathogen (virus and/or viroid), specific primer pairs will be designed according to contig sequence/sequences obtained in this study and used to amplify the overlapping fragments of the virus/viroids for PCR cloning and Sanger sequencing (MCLAB, CA). The genomic sequence will be analyzed by BLAST search and various analysis tools. All complete genomic sequences will be deposited into the NCBI GenBank. 2. Develop rapid, sensitive and reliable RT-PCR assays for the detection of ALV-1 and other new pathogens: To develop a qRT-PCR assay, sequences of different isolates of ALV-1 as well as other new virus/agent will be aligned, and several virus-specific primer and probe sets will be designed to anneal the conserved region to ensure the amplification of broad range of genetic variants. The qRT-PCR will be conducted using a QuantiTect Multiplex RT-PCR Kit (Qiagen, Germantown; MD) in a Bio-Rad C1000 Thermal Cycler. The primers/probe sets will be compared as described by Lin et al. , and the most efficient set will be selected for further evaluation. The sensitivity of the qRT-PCR will be compared with that of the conventional RT-PCR. The qRT-PCR protocol will be validated using orchard samples at the Pennsylvania Department of Agriculture lab on Quant Studio 3 ABI real-time PCR platform using TaqMan® Fast Advanced Master Mix (Invitrogen). For the detection of any new virus and viroid, specific primers will be designed based on the genome sequence of the pathogen(s) and tested in RT-PCR/PCR. Sensitivity and efficiency of an optimized protocol will be evaluated by orchard samples. 3. Initiate the correlation study of ALV-1/new agent and RAD: Budwood from PA21, a Fuji apple tree infected only with ALV-1 (as determined by HTS), will be used as the scion source for grafting experiments on M9 rootstock. The grafted trees (80) will be kept in screen house for one year, transferred to an isolated field plot, and observed for RAD symptoms.