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:
- 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)
- 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 
