1a.Objectives (from AD-416):
1. Characterize microbial communities associated with tissue necrosis during the terminal stage of lethal yellowing disease of palms.
2. Determine genetic variability/diversity of phytoplasmas affecting potato and other crops.
The ultimate goal of the project is to gain new knowledge of ecology, niche adaptation and pathogenicity evolution of phytoplasmas, causal agents of economically important plant diseases worldwide.
1b.Approach (from AD-416):
For Objective 1, necrotic tissues will be harvested from lethal yellows (LY) disease-affected palm trees and total DNA will be extracted from selected tissues. Microbial populations will be characterized using 454 pyrosequencing technology with primers designed to target V3 and V6 hypervariable regions of bacterial 16S rRNA genes. For Objective 2, total DNA will be extracted from symptomatic tissues of phytoplasma-infected plants, and phytoplasma DNA will be enriched through cesium chloride gradient centrifugation. Phytoplasma genome survey sequencing will be conducted using 454 pyrosequencing technology using coded primers. For both Objectives, the sequence data obtained will be analyzed and annotated using a suite of bioinformatic tools.
This cooperative research was initiated through the use of genome survey sequencing and metagenomic sequencing approaches. The overall goal of the project is to acquire new knowledge of ecology, niche adaptation and pathogenicity evolution of phytoplasmas, cell wall-less bacteria that cause diseases in ecologically and economically important plants worldwide. In this reporting period, metagenomic data from studies of the microbial community associated with tissue necrosis during the terminal stage of lethal yellowing disease of palms were analyzed. Results gave no indication that microbes other than phytoplasma were involved in the necrosis of the tissue. Thus, the hypothesis that non-phytoplasma microbes were mainly if not completely responsible for the observed necrosis was not supported by the findings. Instead, the results have compelled the construction of a new hypothesis, that the phytoplasma genome encodes as yet unidentified factors that interact with host plant components and induce the lethal decline of palm. In pursuing the second objective, nucleotide sequence data from survey genome sequencing of two potato-infecting phytoplasmas were analyzed. Additional samples were prepared for further sequencing of the two phytoplasma genomes. Since the potato phytoplasmas represent two closely-related but distinctly different lineages, the new information gained is providing bases for enhanced understanding of phytoplasma pathogens affecting potato, tomato and other crops.