Identification and molecular characterization of Candidatus Phytoplasma mali isolates in north-western Italy

Apple proliferation (AP) is an important disease and is prevalent in several European countries. The causal agent of AP is Candidatus Phytoplasma mali ( Ca. Phytoplasma mali ). In this work, isolates of Ca. Phytoplasma mali were detected and characterized through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of 16S rRNA gene and non-ribosomal DNA fragment. The presence of three AP subtypes (AT-1, AT-2 and AP-15) was identified in 31 symptomatic apple trees and two samples each constituted by a pool of five insects, collected in north-western Italy, where AT-1 is a dominant subtype. Subsequent nucleotide sequence analysis of the PCR-amplified 1.8 kb (P1 ⁄ P7) fragment, containing the 16S rDNA, the 16S–23S intergenic ribosomal region and the 5¢-end of the 23S rDNA, revealed the presence of at least two phytoplasmal genetic lineages within the AT-1 subtype, designed AT-1a and AT-1b. Moreover, in silico single nucleotide polymorphism (SNP) analysis based on 16S rDNA sequence can differentiate AT-1 subtype from AT-2 and AP-15 subtypes. Our data showed a high degree of genetic diversity among Ca. Phytoplasma mali population in north-western Italy and underlined the possible use of the 16S rDNA analysis for the identification and the geographical origin assignation of isolates of AP phytoplasma. Molecular markers on 16S rDNA, here identified, could be useful for studying the epidemiology of AP disease.