Specifity of multiplex PCR in the detection of Xanthomas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in rice (Oryza sativa L. ) seeds

Da-Young Lee

Date

2011

Degree

Bachelor of Science in Biology

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Bernadette C. Mendoza

Co-adviser

Casiana M. Vera Cruz

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Abstract

A broad collection of 109 unidentified yellow colony-forming bacterial isolates, similar in colony appearance to Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), from the Seed Health Unit (SHU) of IRRI was used in this study. The colonies of the isolates on Suwa’s medium or modified Wakimoto’s Medium Without Potato (WF-P) varied in size but all were yellow-pigmented, had a circular and entire margin, with some being mucoidal, like the colonies of Xoo and Xoc. All 109 unidentified bactria were analyzed using BOX-PCR to determine their genetic diversity, specifically in relation to Xoo and Xoc. A 49% similarity coefficient was used to group the isolates, based on the dendrogram clusters formed for Xoo and Xoc generated using the Unweighted Pair Group Method with Arithmetic averages (UPGMA) and Jaccard’s correlation coefficient. A total of 51 isolates, consisting of 41 isolates representing each dendrogram cluster and 10 isolates genotypically closest to Xoo and Xoc on the dendrogram, were selected for multiplex PCR analysis. Gram staining of these

isolates revealed that all were rod-shaped and that the majority (37 out of 51) were Gram- negative; a few (10 out of 51) were Gram-positive, three had mixed Gram reactions and

one was Gram stain-resistant. The 51 isolates selected and three control Xanthomonas strains (Xoo, Xoc and Xanthomonas campestris pv. dieffenbachia) were used to determine the specificity of the multiplex PCR for Xoo and Xoc detection developed by Lang and co-workers (2010) and optimized by IRRI. The multiplex PCR products were resolved into the following fragments depending on the organism: one fragment (162 bp) exclusive for Xoo strains, two fragments (691 bp and 945 bp) for Xoc strains and a 331 bp-fragment common to both Xoo and Xoc. The expected fragments were amplified for the control strains while no band amplification was observed for the 51 unidentified SHU isolates. The results indicated that the multiplex PCR used in this study is robust in detecting and differentiating Xoo and Xoc strains from non-pathogenic, rice seed-associated, yellow colony-forming bacteria.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Document Type

Thesis

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