Plant Associated and Environmental Microbes Database


How to use PAMDB to identify your unknown isolates

At this point, you can only use PAMDB when you know the genus of your isolate. If you do not know the genus of your isolate, it will first be necessary to either sequence 16s rRNA from your isolate and compare the obtained sequence taking advantage of the 16s rRNA database at the Ribosomal Database Project website, or to determine the genus using non-molecular methods like Biolog, fatty acid analysis, or any other phenotypic test. Once you know the genus of your isolate, and the genus has been included in PAMDB, you can determine to which sub-group within the genus your isolate belongs. At this point, the following plant pathogens have been included in PAMDB: Pseudomonas syringae, Xathomonas sp. and Ralstonia solanacearum. If your isolate belongs to one of these organisms, do the following:
  1. Obtain sequences of one or more loci
    • Extract DNA from your isolate, using for example the Gentra Puregen kit (optional)
    • Choose a locus or several loci of your organisms for which there are sequence data in PAMDB (click on the organism below to download an excel table with all loci and primer sequences)
    • Order primers for the chosen loci
    • Perform PCR using DNA or a bacterial colony as template. When using a colony, only use 1 day-old colonies (touch colony very gently with pipette tip, and immerse tip in PCR mix)
    • Clean PCR products
    • Sequence PCR products at your sequencing center of choice
    • Edit your DNA sequence (optional)
  2. Compare your sequence to the sequences in the database
    • Login
    • Click on the "Search" link
    • Click on the "Blast" button
    • Next to the Database link choose the database of your organism
    • Paste your DNA sequence in the window
    • Click on the "search" button
    • Scroll down the results
As a rule of thumb: Isolates with a 99% to 100% match can be considered to be very closely related to your isolate and may belong to the same pathovar or the same clonal line. Isolates that are between 96% and 99% identical to your isolate are probably of the same subspecies or genomospecies. Isolates with less than 96% identity are only distantly related to your isolate. If the exact geographic location of isolation of any of the closely related isolates is known, you can view that location on a Google map by clicking on the Google Map symbol. If you want to precisely identify your isolate, you need to sequence at least two loci. The best way to do this, is to add your isolate to PAMDB, add the allele data to your isolate, download the sequence of your isolate with all other sequences available in PAMDB and build a phylogenetic tree