Complex phytoplasma infection in declining liquidambar trees in Colombia.

Liquidambar stiracyflua, introduced to Bogota 10 years ago as a street plant, is showing symptoms possibly associated to phytoplasmas. Symptoms include changes in the architecture of the crown due to unevenly spaced internodes, deliquescent branching, and presence of small and large leaves in branches. Liquidambar trees are located in the same streets in which Fraxinus with yellows phytoplasma, have been described. DNA samples extracted from leaf midribs of four plants were tested to verify phytoplasma presence by amplification of the16SrDNA gene. Direct PCR assays with primers P1/P7 did not provided amplification from any sample. Nested-PCR assays with primers R16F2/R2 produced bands of the expected length (1.2 kb) from all samples tested. RFLP analyses of amplicons with selected restriction enzymes provided restriction profiles referable to Ash yellows phytoplasmas (‘Candidatus Phytoplasma fraxini’ 16SrVII-A), while in two samples L2 and L4 beside the profiles referable to Ash yellows phytoplasmas profiles corresponding to 16SrI and 16SrXII-A ribosomal groups were detected, respectively indicating the presence of multiple phytoplasma infection. Further amplification with primers R16mF1/R16mR2 on P1/P7 amplicons of samples L1 and L3 allowed amplification of nearly full 16S ribosomal gene (about 1,400 nt). Direct sequencing with R16mF1, M1, F3 and R16mR2 primers generated two fragments of 1,240 and 1,078 nt, respectively (GU810150 and GU810151) showing 100% homology between them for overlapping part (937 bp); this part was also identical those of several ‘Ca. P. fraxini’ strains available in Genbank (16SrVII-A subgroup). Some SNPs of both L1 and L3 strains with ‘Ca. P. fraxini’ strains were observed in the non overlapping parts of the sequences.