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 µl 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).
  
  This primer pair has been validated for use with SYBR green and TaqMan real-time PCR by P. Uribe and F. Martin (manuscript in preparation).
  

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
• This primer pair has also been used in TaqMan real-time PCR (Tooley et al. 2006)
  
  
  
  
  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 µl 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).
  
  This primer pair has been validated for use with SYBR green real-time PCR by P. Uribe and F. Martin (manuscript in preparation).