Characterization of phytoplasma strains associated with coffee crispiness and nogal cafetero witches broom in Colombia.

Coffee (Coffea arabica L.) is among the most important cultivated crops in Colombia, where it is frequently shaded with nogal cafetero [Cordia alliodora (Ruiz & Pavón) Oken], a tree species also used for timber and reforestation programs. Since 1998 severe cases of phytoplasma-related symptoms were observed in coffee plantations, and starting on 2002 also in nogal cafetero; both epidemics are concentrated to limited areas dispersed in the main coffee growing region, but with incidences and severities above 90%, resulting in total yield losses. Coffee crispiness was recently associated to 16SrIII phytoplasma infection, and PCR analyses allow the identification of phytoplasmas of the same group in nogal cafetero trees showing witches broom, branch malformation and abnormal shoot sprouting. Further molecular characterization was therefore performed towards epidemiological studies to prevent disease spreading. One phytoplasma strain from coffee and one from nogal cafetero were amplified and sequenced on the 16Sr gene and/or spacer region obtaining sequences of 1,356 and 1,487 bp respectively. The two sequences showed 99% of homology between them and to several 16SrIII strains in the GenBank. In particular only two SNPs were present between sequences from coffee and nogal cafetero strains. Phylogenetic analyses and real and virtual RFLP profiles obtained on R16F2/R2 amplicons (1,240 bp) with HhaI and HpaII allow to tentatively classify this phytoplasma in a new subgroup 16SrIII-U. However the real and virtual RFLP profiles obtained with TruI did not match to each other indicating a possible presence of mixed infection in phytoplasmas detected in coffee and in some other nogal cafetero samples. Ribosomal protein (rp) genes of (about 1,100 bp) of some nogal cafetero samples were sequenced after nested amplification and comparison of real and virtual RFLP profiles obtained with TruI, and Tsp509I indicates the presence of two rp differentiable strains in the coffee sample, one of which is the prevalent in nogal cafetero samples. These results indicate a complex disease aetiology involving more phytoplasma strains in the two studied plant species.