Table 1. Primers used for amplification of the tuf DNA barcode.

Primer cocktail	Position in AYWB tuf gene	Primer cocktail components	Primer sequence	Proportionsof each primerin a primercocktail	Notes
Tuf340	157–179	Tuf340a	GCTCCTGAAGAAARAGAACGTGG	1∶1	used as aforward
		Tuf340b	ACTAAAGAAGAAAAAGAACGTGG		primer mixin a directPCR assay
Tuf400	211–236	Tuf400aM13F	GTAAAACGACGGCCAGTGAAACAGAAAAACGTCAYTATGCTCA
		Tuf400bM13F	GTAAAACGACGGCCAGT GAAACTTCTAAAAGACATTACGCTCA		used as aforward
		Tuf400cM13F	GTAAAACGACGGCCAGTGAAACATCAAAAAGACAYTATGCTCA	1∶1:1∶1:1	primer mix ina nested
		Tuf400dM13F	GTAAAACGACGGCCAGTGAAACAGAAAAAAGACAYTATGCTCA		PCR assay
		Tuf400eM13F	GTAAAACGACGGCCAGTCAAACAGCTAAAAGACATTATYCTCA
Tuf835	628–654	Tuf835raT7	TAATACGACTCACTATAGGGAACATCTTCWACHGGCATTAAGAAAGG		used as a reverse
		Tuf835rbT7	TAATACGACTCACTATAGGGAACACCTTCAATAGGCATTAAAAAWGG	1∶1:1	primer mix in a nested
		Tuf835rcT7	TAATACGACTCACTATAGGG AACATCTTCTATAGGTAATAAAAAAGG		PCR assay
Tuf890	685–710	Tuf890ra	ACTTGDCCTCTTTCKACTCTACCAGT		used as a reverse
		Tuf890rb	ATTTGTCCTCTTTCWACACGTCCTGT	1∶1:1	primer mix in a direct PCR assay
		Tuf890rc	ACCATTCCTCTTTCAACACGTCCAGT

Two pairs of primer cocktails were used for universal amplification of the tuf barcode from all phytoplasma strains employed in this study in a nested PCR assay. Tuf 340/Tuf890 and Tuf400/Tuf835 primer cocktails were used in direct and nested PCR respectively. Each primer cocktail contained slightly different variants of the same primer mixed in equimolar amounts. The nucleotide sequences of the general sequencing primers M13F and T7 are underlined with a single and a double line, respectively. Primer positions correspond to the positions in the tuf gene of ‘Ca. P. asteris’ strain AY-WB (Genbank accession number CP000061).