In vitro Propagation of Phytoplasma-Free Strawberry Plants and Molecular Characterization of the Pathogen

Abstract

Multiplication of strawberry plants in tissue culture has advantages as reduced exposure to many insects and several various diseases. During 2019 growing season, strawberry (Fragaria x ananassa Duchesne cv. Festival) plants showing phyllody fruit symptoms, in which petals and sepals become leaf-like, were detected in planting fields in Ismailia governorate, Egypt. Phytoplasma associated with phyllody of fruit in strawberry plants was detected using phytoplasma universal primers, accurately identified at the molecular level, and the DNA sequence has been deposited in GenBank under accession number MT757900 with Isolate name SAMA. DNA from all symptomatic plants yielded PCR products of 1278 bp, whereas DNA from asymptomatic plants collected in the field yielded no products. The phylogenetic analysis of the 16S rDNA sequence of SAMA-isolate indicated that the 16S rDNA gene shared 100% identity with StrawbPhF-isolate classified in a group 16SrI (AY102275) that belonged to new subgroup R. Meristems (0.1-0.3 mm in size) were excised from the terminal and axillary buds of the strawberry-runners and then cultured on hormone-free medium. The two sizes (0.2 mm and 0.3 mm) of meristem had pronounced effects on the phytoplasma elimination (100%), and survival rates (66.6 % and 90%, respectively), but the smaller size of meristem had a deleterious effect to survival rate (0%). After approximately seven months and a half, rooted plantlets raised from meristem culture were confirmed for phytoplasma-free by a nested PCR assay. Therefore, the production of strawberry plantlets that needs no examination for their phytoplasma infection could be expected by applying the meristem culture method.