Molecular typing of ‘Candidatus Phytoplasma mali’ and epidemic history tracing by a combined T-RFLP/VNTR analysis approach

Abstract

Apple proliferation caused by ‘Candidatus Phytoplasma mali’ is a disease of apple trees gaining increasing importance in Europe. The present study describes a high-throughput method for simultaneous typing of ‘Ca. P. mali’ at two genetic loci. This novel approach combines terminal restriction fragment length polymorphism (T-RFLP) analysis of a putative rhodanese-like protein gene and the analysis of the variable number of tandem repeats (VNTR) of the ribosomal protein L22 gene. The typing approach was applied to analyse a collection of DNA isolates from 310 apple trees tested positive for ‘Ca. P. mali’. Samples were taken between 2002 and 2010 in South Tyrol (Northern Italy). In addition, 15 samples of Cacopsylla melanoneura and 19 of C. picta were typed. Seven combined genetic profiles were found in the samples of infected apple trees: AT-2/rpX-A (81.0%), AT-1/rpX-D (8.4%), AT-1/rpX-E (4.2%), AT-1/rpX-A (3.2%), AT-1/rpX-B (1.6%), AT-1/rpX-C (1.0%) and AP/rpX-A (0.3%), and one mixed infection AP + AT-1/rpX-A + rpX-D (0.3%). Subtype rpX-E was discovered for the first time. In C. melanoneura samples the most frequent subtype was AT-1/rpX-E, followed by AT-1/rpX-D and AT-1/rpX-C. All C. picta samples displayed subtype AT-2/rpX-A. Analysis of the temporal distribution of subtype frequencies in apple trees revealed that exclusively subtype AT-1 in combination with four rpX subtypes was present in South Tyrol in the period from 2002 to 2004. From 2006 onwards subtype AT-2/rpX-A became dominant with an average frequency of 90%. The data obtained suggest that there may be a co-adaptation of particular ‘Ca. P. mali’ subtypes with different insect vector species.