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Phytophthora genus-specific primer pair
Two primer pairs have been developed for amplification of a Phytophthora genus-specific amplicon that differ in specificity.
•• Phy-8b + Phy-10b - This primer pair is used for detection of P. ramorum
? This primer pair is specific for amplification of Phytophthora spp. and has not amplified the plant or Pythium spp. tested thus far (a faint amplicon of 0.4 kb was observed for one isolate of Pythium sylvaticum).
•• They were derived from mitochondrial sequences of the cox II (base 624 to 657) and cox I gene (base 73 to 98), respectively, and the amplicon includes the spacer region between the two genes.
•• The specificity for amplification of Phytophthora spp. and not Pythium spp. is due to primer FMPhy-8b; the terminal base is an A while the corresponding base for Pythium spp. (and the plant sequences examined) is T (sequence alignments: Phy8b, forward primer;Phy10b, reverse primer)
? FMPh-8b (dAAAAGAGAAGGTGTTTTTTATGGA)
? FMPh-10b (dGCAAAAGCACTAAAAATTAAATATAA)
? Amplicon is 457 bp in size for P. ramorum. Since this region does encompass a spacer region there can be some variation in size depending on the species.
? In some amplifications a doublet band may be seen, however, this behaves the same as a single band in subsequent species-specific amplifications.
? Amplification best at 3 mM Mg, very good at 4 mM, none at 1 or 2 mM
? Get amplification at 67 C, but since this primer pair is not a perfect sequence match for all Phytophthora spp. there are some species that amplify poorly at this annealing temperature.
•• The normal annealing temperature is 66 C with 2% glycerol. While most species amplify well under these conditions, P. lateralis and P. sojae only exhibit weak amplification. Lowering the annealing temperature by 1 C or eliminating glycerol in the amplification mixture enhanced amplification of these species.
•• Eliminating glycerol from the amplification mixture reduced the stringency and allowed amplification of Prunus sp., Citrus sp., Quercus agrifolia, Juniperus sp. and Fragaria x ananassa.
? Table of Phytophthora species tested and isolate history
Amplification of various Phytophthora spp. with the Phytophthora genus-specific primers FMPh-8b + FMPh-10b with 2% glycerol in the reaction mixture and an annealing temperature of 66? C with 11 ?l of the amplification mixture 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 Phytophthora genus-specific primers FMPh-8b + FMPh-10b generated amplicons when 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).
Test of amplification of various Pythium spp. with the Phytophthora genus-specific primers FMPh-8b + FMPh-10b with 2% glycerol added to the reaction mixture and an annealing temperature of 66? C. A minimum of 100 ng Pythium total DNA was added to each reaction with 12 ?l of the amplification mixture 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.
Test of amplification of various plant species with the Phytophthora genus-specific primers FMPh-8b + FMPh-10b with 2% glycerol added to the reaction mixture and an annealing temperature of 66? C. 12 ?l of the amplification mixture 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.
This primer pair did not amplify sequences from the following additional plants:
Arbutus unedo - 'Compacta' Cotoneaster apiculata - 'Tom Thumb'
Aucuba japonica - 'variegata' Delphinium elatum - 'Summer Skies'
Avacado - 'Fuerta' Euonymus japonica - 'Chollipo'
Azalea - Geovie Tabor Pink Patio Hedera helix - 'Thorndale'
Azalea - satsuki 'Getsutoku' Hibiscus rosa-sinensis
Azalea - satsuki 'Juko' Ligustrum japonica - 'Korean choice'
Azalea - satsuki 'Kinpai' Lonicera sp. - 'Hummingbird's gold
Azalea - satsuki 'Nuccio's Wild Cherry' Malus sylvestris - 'Granny Smith'
Azalea - 'Girard's Fuchsia' Prunus persica
Azalea - 'Herbert' Prunus sp (plum)
Azalea - 'Hino?Crimson' Pyrus commumis
Azalea - 'Holland Red' Rhaphiolepis indica - 'Ballerina'
Begonia - Tubor hybrid Verbina romania mix
Camelia sp. Viburnum burkwoodii
Camelia - 'Winter's Rose' Viburnum opulus - 'Sterile'
Camelia - 'Winter's Star' Viburnum plicatum tomentosum
Camelia - 'Betty Sette' Viburnum rhytidophylloides - 'Allegheny'
Camelia japonica - 'April remembered' Viburnum tinus - 'Compactum'
Camelia japonica - 'Fumasaka' Vitis vinifera
Clematis sp. - 'Ramona'
.
Alternative primer pair for amplification of Phytophthora amplicon
•• FMPhy-8 + FMPhy-10
? FMPh-8 (dAAGGTGTTTTTTATGGACAATGTA)
? FMPh-10 (dGCAAAAGCACTAAAAATTAAATATAAA)
? This primer pair is more robust than FMPhy-8b + FMPhy-10b under the amplification conditions used and will generate greater amounts of amplified product.
? This primer pair should be used with caution where the presence of a genus-specific band alone is used for diagnostic purposes.
•• While they do not amplify the plant species that have been tested, they will amplify Pythium spp.
•• Since Pythium spp. are found predominantly to cause diseases of below ground plant parts, for detection of foliar diseases caused by Phytophthora spp. they should work well.
? Good amplification at 3 or 4 mM Mg, weaker at 2 mM and none at 1 mM
? Amplification done using the same procedure as FMPhy-8b + 10B except that 9% glycerol was added and the annealing temperature was 64 C.
? Sequence alignment used for primer selection (Phy 8, forward primer, Phy10, reverse primer)
