Thesis
Assessment of chickpea seed disinfestation procedures and detection of Ascochyta rabiei in chickpea seed using quantitative-polymerase chain reaction
Washington State University
Master of Science (MS), Washington State University
2011
Handle:
https://hdl.handle.net/2376/102633
Abstract
Ascochyta blight is a devastating disease of chickpea caused by the fungal pathogen Ascochyta rabiei (teleomorph; Didymella rabiei). Attempts at limiting the dispersal and destructive potential of A. rabiei have focused on early detection of this pathogen in seed through routine testing and seed certification programs. The absence of internationally accepted standards for assaying chickpea seed has made it difficult to predict Ascochyta blight epidemics based on seed infection/infestation rates. Additionally, disinfestant treatments used in screening chickpea seed for the presence of A. rabiei vary, affecting pathogen recovery rates detected by conventional assays. In this study, ethanol, sodium hypochlorite and water were compared as seed disinfestant treatments and rinses and evaluated for their effectiveness in recovering A. rabiei and other fungi from chickpea seed assayed using a conventional agar plate assay. Results suggest that the incidence of A. rabiei recovery was not significantly affected by surface disinfestant treatment used; however, the recovery rates of other chickpea seed mycoflora were significantly influenced by disinfestant treatments involving sodium hypochlorite and/or ethanol solutions. Rinsing seeds in a solution of 0.5% sodium hypochlorite for 2 minutes without any ethanol pre-treatment was the most effective seed treatment for A. rabiei recovery and suppression of other mycoflora, that can interfere with pathogen detection. A quantitative polymerase chain reaction assay incorporating TaqMan-MGB fluorescent chemistries was developed for the rapid detection of A. rabiei in chickpea seed. A. rabiei-specific primers and a fluorescent probe were developed using sequence data from the intergenic spacer (IGS) region of the 28s-18s rDNA genes. The specificity of this molecular assay was demonstrated in PCR and qPCR using purified genomic DNA of A. rabiei, Ascochyta relatives and other fungi isolated from chickpea seed. This qPCR assay was shown to be sensitive enough to detect 1 pg to 100 fg of purified A. rabiei DNA, with PCR efficiency of 104%. Refinement of the conventional seed plating assay and development of a quantitative PCR assay will provide additional insight into the epidemiological implications associated with seed-borne transmission of A. rabiei, and will improve the management of Ascochyta blight.
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Details
- Title
- Assessment of chickpea seed disinfestation procedures and detection of Ascochyta rabiei in chickpea seed using quantitative-polymerase chain reaction
- Creators
- Christian Grace Aguilar
- Contributors
- Tobin L. Peever (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Plant Pathology, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; Pullman, Wash. :
- Identifiers
- 99900525300501842
- Language
- English
- Resource Type
- Thesis