Development of a PCR-Based Diagnostic Assay and Sampling Protocol for the Detection of Aphelenchoides fragariae in Ornamental Host Crops

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Title: Development of a PCR-Based Diagnostic Assay and Sampling Protocol for the Detection of Aphelenchoides fragariae in Ornamental Host Crops
Author: McCuiston, Jamie Leigh
Advisors: Dr. Christine A. Casey, Committee Member
Dr. Eric L. Davis, Committee Co-Chair
Dr. Colleen Y. Warfield, Committee Chair
Abstract: The objective of this study was to develop a PCR technique for the detection of Aphelenchoides fragariae, a common pathogen in nurseries and greenhouses. The standard test currently used to detect the nematode relies on the ability of the nematode to crawl or swim out of a leaf submerged in water, as well as the ability of a person to recognize specific small morphological characteristics. This method is both time consuming and often not reliable because of contamination from other saprophytic nematodes and the minute differences in characteristics of other non-pathogenic Aphelenchoides species. In order to replace the current standard method, the PCR detection method needs to be specific, rapid, and adaptable to routine analysis. The ITS1 sequence for A. fragariae were used to develop sequence-specific primers that were specific to only A. fragariae. The sequences of seven A. fragariae, isolated from different geographical areas throughout North Carolina, showed that within the area amplified by the species-specific primers developed was 100% homologous to the sequence that the primers were developed from as well as from a known culture of A. fragariae (Figure 3). The ITS1 region of a recent comparison with sequences recently added to the GenBank database since the time this study was originally initiated, show 80% sequence homology within the amplified region of the ITS1 region with A. fragariae in the Netherlands. (EF213107, Pham et al., 2007) From this information the ITS1 region appears to be mainly conserved throughout this region. Specimens of Aphelenchoides spp. were recovered from 121 plant samples. Based on morphology, 89 of these isolates were identified as A. fragariae, the nematodes from the remaining 32 plant samples either were not identified or did not swim out of the plant tissue but was detected using the PCR-assay. Genomic DNA containing both plant and nematode DNA produced a 169-bp fragment when PCR-amplified with primers FragF1 and FragR1 for 100 of the isolates identified as A. fragariae (Table 1). By using several isolates collected in NC, we demonstrated the utility of this newly developed primer pair for discriminating A. fragariae. The ITS1 sequence for A. besseyi and A. ritzemabosi were obtained from Sergei Subbotin at the University of California. Dr. Subbotin also provided DNA of Aphelenchoides besseyi for the use in testing the specificity of the selected primers. Based on BLASTn comparison of the A. fragariae sequence with both the A. ritzemabosi and A. besseyi ITS1 sequence, the primers would not amplify either of these species. The DNA was used in the PCR-diagnostic assay did not produce an amplicon, demonstrating the specificity to only the A. fragariae species of foliar nematode. A. fragariae cohabitate with other saprophytic and pathogenic organisms. These organisms cannot interfere with the validity of the assay in order for it to be effective. After performing the assay on the most common saprophytic and pathogenic organisms of greenhouses and nurseries, it was found that the assay has no cross-reactivity to any of these organism.
Date: 2008-04-30
Degree: MS
Discipline: Plant Pathology
URI: http://www.lib.ncsu.edu/resolver/1840.16/1151


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