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Location: Floral and Nursery Plants Research

Title: A computer program for fast and easy typing of partial endoglucanase gene sequence into phylotypes and sequevars 1&2 (select agents) of Ralstonia solanacearum

item STULBERG, MICHAEL - Orise Fellow
item Huang, Qi

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2016
Publication Date: 2/11/2016
Citation: Stalberg, M., Huang, Q. 2016. A computer program for fast and easy typing of partial endoglucanase gene sequence into phylotypes and sequevars 1&2 (select agents) of Ralstonia solanacearum. Journal of Microbiological Methods. 123:101-107.

Interpretive Summary: A bacterium, Ralstonia solancearum, causes a serious brown rot disease in potato and is therefore not allowed to enter the U. S., Canada, and Europe. To determine if this harmful bacterium is present in imported plants, a fast and easy method is needed to screen plants at entry points. We wrote a computer program that can quickly and easily classify whether the bacterium in question belongs to the highly regulated bacterial group based on a part of the bacterium’s gene sequence. Our computer program will be useful to regulatory officials by helping them make timely decisions to exclude this highly regulated bacterium from entering and/or becoming established in the countries where the bacterium is banned.

Technical Abstract: The phytopathogen Ralstonia solanacearum is a species complex that contains a subset of strains that are quarantined or select agent pathogens. An unidentified R. solanacearum strain is considered a select agent in the US until proven otherwise, which can be done by phylogenetic analysis of a partial endoglucanase (egl) sequence approximately 700-bp in length. Phylogenetic analysis, however, requires expert knowledge to properly trim the sequence, include the correct reference strains, and interpret the results. By comparing GenBank egl sequences of representative R. solanacearum strains, we identified phylotype- and sequevar 1 and 2-specific single nucleotide polymorphisms (SNPs). We also designed primers to amplify a shorter, 526-bp, egl fragment from R. solanacearum strains for easy sequencing of the amplicon, and to facilitate direct and specific amplification of egl from R. solanacearum-infected plant samples without the need to isolate bacteria. We wrote a computer program (Ralstonia solanacearum typing program) that analyzes a minimum 400-bp user-input egl sequence from a R. solanacearum strain for egl homology and SNP content to determine 1) whether it belongs to the R. solanacearum species complex, 2) if so, to which phylotype, and 3) whether it is of the sequevar type (sequevars 1 and 2) associated with the select agent/quarantined R. solanacearum strain. When tested against 371 egl sequences from R. solanacearum strains with known phylotypes and sequevars, obtained either from GenBank or through personal communication, our program correctly typed all 371 strains. Additionally, our program successfully typed 25 R. solanacearum strains in our collection with no prior sequevar information, as well as 4 strains in infected plant samples, using their partial egl sequences amplified and sequenced with primers designed in this study. Our program was also used to type 53 partially characterized Brazilian R. solanacearum strains using their egl sequences deposited in GenBank, since these strains included a high diversity of sequevars 1 and 2 and their closely related strains. The Ralstonia solanacearum typing program does not require expertise or specific knowledge to use, gives results in seconds, and provides data interpretation for the user. The program and primers can help expert or non-expert users to quickly type an unknown R. solanacearum strain to determine whether it is a highly regulated R. solanacearum strain.