Characterization and molecular differentiation of 16SrI-E and 16SrIX-E phytoplasmas associated with blueberry stunt disease in New Jersey

Abstract

A nested PCR assay was employed to detect the presence of phytoplasmas in 127 blueberry plants exhibiting typical or a portion of blueberry stunt (BBS) syndrome collected in 2010 and 2011, from 11 commercial farms predominantly located in two counties in New Jersey, USA. Ninety plants exhibiting typical stunt syndrome tested positive for phytoplasma infection. Restriction fragment length polymorphism (RFLP) analysis indicated that two distinct phytoplasmas were associated with BBS-diseased plants. About 95% of phytoplasmas detected were very closely related to BBS phytoplasma strains BBS3-AR (subgroup 16SrI-E) and BBS1-MI (unidentified) identified previously, and 4.4% of phytoplasmas detected belonged to the pigeon pea witches'-broom phytoplasma group (16SrIX). Sequence and phylogenetic analysis of cloned 16S rDNA further indicated the subgroup 16SrI-E related phytoplasmas represented a variant of 16SrI-E reference strain BBS3-AR, while the 16SrIX related phytoplasmas were closely related to juniper witches'-broom (JunWB) phytoplasma (16SrIX-E), representing a 16SrIX-E variant. Ribosomal protein (rp) and secY gene-based phylogenies revealed that BBS3-AR and BBS-NJ 16SrI-E strains belonged to a closely related lineage, while BBS-NJ 16SrIX-E strains and JunWB strains represented two distinct lineages. Single nucleotide polymorphisms (SNPs) analyses of rp and secY gene sequences further revealed that no specific rp gene SNPs and only two specific secY gene SNPS were present between BBS-NJ 16SrI-E strains and BBS3-AR. In contrast, BBS-NJ 16SrIX-E strains/clones had 15 consensus rp SNPs and 28 consensus secY SNPs that separated them from JunWB strains/clones. For the first time, two distinct phytoplasmas that cause BBS-disease in the U.S. was revealed.