Location: Crops Pathology and Genetics Research
Project Number: 2032-22000-016-39-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2018
End Date: Mar 31, 2020
1. Conduct surveys of cherry orchards for stem pitting disease. 2. Analyze RNA profile of cambial scrapings from pitted trunks of rootstock and scion portions of diseased cherry trees by Illumina sequencing. 3. Find potential pathogens by bioinformatics. 4. Design primers for potential etiological agents and conduct RT-PCR tests.
Survey of Orchard for Diseased Specimens: Beginning in summer 2018, three orchards in San Joaquin valley planted with Coral on Mahaelb rootstock will be surveyed for disease symptoms and the spatial patterns over years will be assessed. Scion and roostock portions of the trunks from declining trees will be collected and brought to laboratory for analyzing RNA profile. RNA extraction, deep sequencing and bioinformatics: After removing the bark, cambial scraping from the trunks showing stem pitting symptoms will be removed and used for RNA extraction using MirVana kit (Life Technologies, dsRNA is extracted as per standard procedures. Samples from a control cherry tree not showing stem pitting symptoms will be used as a control. Extraction protocols will not use DNAse treatment to include the genomes of infectious agents with DNA genomes. The RNA portion of the nucleic acid extract is subjected to depletion of ribosomal RNA and further fractionated into polyA tagged mRNA and small RNA, converted into cDNA, amplified, and subjected to deep sequencing using Illumina sequencing technology at the UC-Davis DNA core facilities. The sequence reads will be trimmed to remove bar code and the reads are subjected to de novo assembly to generate contigs using CLC Bio Genomics workbench. The contigs will be subjected to BLAST searches to determine putative infectious agents. PCR analysis for putative viruses and virus-like agents: For all putative pathogens, primers will be designed and RT-PCR assays will be performed on nucleic acid fraction obtained from symptomatic trees. Healthy tees from orchards not showing CSP will be used as controls. Pitfalls: Examination of mRNA and small RNA are normally good enough to reveal viruses with RNA and DNA genomes as well as viroids. However, samples from one location or one year may not good enough to correlate symptoms with associated viruses especially when the disease is a result of synergy/interaction between different viruses. A multi-year survey is expected to overcome this pitfall.