ADDITIONAL NESTED SPECIES-SPECIFIC PRIMER PAIRS

 P. nemorosa species-specific primer pair 5/1/04

P. pseudosyringae species-specific primer pair. 5/1/04

Species-specific primers have been developed for these additional species and will be posted on the website in the near future

Phytophthora citricola

Phytophthora fragariae

Phytophthora lateralis

Phytophthora nicotianae

Phytophthora syringae

Species-specific primers are currently being tested for:

Phytophthora cactorum

Phytophthora palmivora

Phytophthora kernoviae

If there are other species that you would like species-specific primers for please contact me

Sequence alignments of thePhytophthora genus-specific amplicons will be completed soon and posted to the web site.

P. nemorosa species-specific primer pair

Nested within the genus-specific amplicon

••         FMnem-1 + FMnem-2

?         FMnem-1 (dAATAAAATTAATTTTAATATATAATTAG)

?    FMnem-3 (dTATGTTTAATATCTGTAAATAATAG)

?    100 bp in size (sequence for genus specific amplicon)

?    Good amplification at 4 mM Mg, no amplification observed at lower concentrations

?    Uses an annealing temperature of 61 C and the same second round amplification parameters as the P.         ramorum species-specific primers (Phytophthora genus-specific primers were omitted).

?    Table of Phytophthora species tested and isolate history

Test for species specificity of amplification with the P. nemorosa species-specific primer pair (FMnem-1 + FMnem-2) with a 1:100 dilution of products of amplification with the Phytophthora genus-specific primers FMPh-8b + FMPh-10b observed in Figure 2 as the target DNA and an annealing temperature of 61? C.  A total of 12 ?l of the amplification mixture was loaded into each well of a 3% NuSieve 3:1 agarose gel.    The molecular size marker is a 100-bp ladder from New England BioLabs.

In addition to the Phytophthora spp. noted above the P. nemorosa species-specific primers did not amplify bands when FMPh-8b + FMPh-10b amplicons from the following species were used as a template DNA: P. clandestine, P. humicola, P. ideai, P. iranica, P. katsurae, P. medii, P. melonis, P. inflata, P. primulae, P. richardiae, P. porri, P. quercina, P. tentaculata, or isolates from three of the tentative species groupings described by Brasier et al. (2003)(P. taxon Raspberry, P. taxon Pgchlamydo, or Phytophthora sp. "O" group).

P. pseudosyringae species-specific primer pair

Nested within the genus-specific amplicon

••         FMPps-1 + FMPps-2 

?    FMPps-1 (dCAGTTTCATTAGAAGATTATTTAC

?    FMPps-2 (dAAAATTGTTTGATTTTATTAAGTATC)

?    158 bp in size (sequence for genus specific amplicon)

?    Good amplification at 1,2, 3 or 4 mM Mg, optimum at 3 mM

?    Uses an annealing temperature of 65 C and the same second round amplification parameters as the P. ramorum species-specific primers (Phytophthora genus-specific primers were omitted).

?    Table of Phytophthora species tested and isolate history

Test for species specificity of amplification with the P. pseudosyringae species-specific primer pair (FMPsy-1 + FMPsy-2) with a 1:100 dilution of products of amplification with the Phytophthora genus-specific primers FMPh-8b + FMPh-10b observed in Figure 2 as the target DNA and an annealing temperature of 65? C.  A total of 12 ?l of the amplification mixture was loaded into each well of a 3% NuSieve 3:1 agarose gel.    The molecular size marker is a 100-bp ladder from New England BioLabs.

In addition to the Phytophthora spp. noted above the P. pseudosyringae species-specific primers did not amplify bands when FMPh-8b + FMPh-10b amplicons from the following species were used as a template DNA: P. clandestine, P. humicola, P. ideai, P. iranica, P. katsurae, P. medii, P. melonis, P. inflata, P. primulae, P. richardiae, P. porri, P. quercina, P. tentaculata, or isolates from three of the tentative species groupings described by Brasier et al. (2003)(P. taxon Raspberry, P. taxon Pgchlamydo, or Phytophthora sp. "O" group).