In July to October of 2013-15, more than 40 blocks of Pennsylvania apple orchards from nine counties were surveyed for phytoplasma. Out of 218 apple samples submitted to Pennsylvania Department of Agriculture Plant Diagnostic Lab (PDA PDL), three mature trees from two farms in Adams County were found to be positive for phytoplasma. Positive trees belonging to three different varieties showed abnormally small fruits, clumps of small leaves, leaf curl, and premature reddening, with the best symptoms showing in August to October. PDA PDL extracted total DNA from leaf midveins with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. Positive results were obtained when screened for presence of phytoplasmal DNA using a real-time PCR assay (Hodgetts et al. 2009). For analysis of the 16S rDNA genomic region, nested PCR was carried out with primer pair P1/P7 followed by R16F2n/R16R2 or P1A/P7A (Lee et al. 2004). For analysis of ribosomal protein genes, DNA was amplified in nested PCRs primed by rpL2F3/rp(I)R1A followed by rp(III)F1/rp(III)R1 (Davis et al. 2013). For analysis of secY genomic regions from 16SrIII group phytoplasmas, DNA was amplified in nested PCRs by using primers L15F1A(III)/MapR1A(III), followed by secYF1(III)/secYR1(III) (Lee et al. 2010). PCR products were purified with QIAquick PCR Kit (Qiagen) and sequenced on an Applied Biosystems 3730XL by the Genomics Core Facility of Penn State University. The nucleotide sequences were analyzed using Geneious software (Biomatters, Auckland, NZ) for sequence alignments and using iPhyClassifier (http://www.ba.ars.usda.gov/data/mppl/iPhyClassifier.html) for group-subgroup and ‘Candidatus Phytoplasma’ species assignments. The classification into 16Sr groups and subgroups was established by virtual RFLP analysis of PCR products compared with reference strains. Through combined analysis of nucleotide sequence and RFLP pattern, the phytoplasma strain detected in all three apple trees (GenBank KX219759) was found to belong to subgroup 16SrIII-A (X-disease subgroup). The 16S rDNA sequences were identical to each other and to ‘Candidatus Phytoplasma pruni’ reference strain (JQ044393). The virtual RFLP pattern of 16S rDNA F2nR2 fragment was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group III, subgroup A (L33733). The nucleotide sequence of ribosomal protein genes from the apple tree phytoplasma (KX219761) had 100% identity (1,252/1,252) to reference strain ‘Ca. P. pruni’ strain PX11CT1 (JQ360960.1), while the secY gene (KX219760) had one nucleotide difference 99.92% (1,252/1,253) from ‘Ca. P. pruni’ strain PX11CT1 (JQ268254.1). Representative samples from phytoplasma-infected apple trees were sent to USDA-ARS and CPHST Beltsville Lab for confirmatory testing and were determined positive for ‘Ca. P. pruni’ by nucleotide sequence analysis of 16S rRNA gene sequences amplified in end point PCR. While Adams County has a history of X-disease on peaches (Wheeler and Stimmel 1981), apple has not been known to host X-disease group phytoplasmas (Davis et al. 2013). Both positive farms had peach blocks less than a mile from positive apple trees. X-disease on peach trees was detected on the same farm as the positive apple. The nucleotide sequence of 16S rDNA from the peach tree phytoplasma was identical to the one from apple. To our knowledge, this report is the first to indicate that M. domestica can serve as a host of ‘Ca. P. pruni.’ With a set of only three positive trees, the extent of disease spread on apples remains undetermined. This report could have important epidemiological and plant health quarantine consequences for the fruit tree industry.
All Science Journal Classification (ASJC) codes
- Agronomy and Crop Science
- Plant Science