Development of a real-time PCR assay using locked nucleic acid probe for the detection of octopine, nopaline, and vitopine strains of tumorigenic Allorhizobium vitis in grapevines

TURGUT A., BASIM H.

CROP PROTECTION, cilt.145, 2021 (SCI-Expanded) identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 145
  • Basım Tarihi: 2021
  • Doi Numarası: 10.1016/j.cropro.2021.105620
  • Dergi Adı: CROP PROTECTION
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Environment Index, Veterinary Science Database
  • Anahtar Kelimeler: Real-time PCR, Allorhizobium vitis, Crown gall disease, Detection, Vitis vinifera, AGROBACTERIUM-TUMEFACIENS BIOVAR-3, CROWN-GALL, SENSITIVE DETECTION, GENETIC DIVERSITY, SEQUENCE-ANALYSIS, IDENTIFICATION, PRIMERS, EFFICIENT, TUMORS, SPP.
  • Akdeniz Üniversitesi Adresli: Evet

Özet

Crown gall disease caused by Allorhizobium vitis is one of the most destructive diseases in grapevine cultivation from an economical perspective. The bacterial pathogen exists systemically in grapevines and spreads through cuttings used for propagation material obtained from grapevines, living vines, and mother blocks, which poses a high risk for certified grapevine nursery material cultivation, nursery industry, and growers. Therefore, in this study, real-time PCR-based methods were developed for effectively and accurately determining the presence and the density of the bacterial pathogen in grapevine nursery material and the data used for indexing programs. Octopine-, nopaline-, and vitopine-catabolizing A. vitis strain-specific primer and locked nucleic acid (LNA) probe sets were determined depending on the ocs, nos, vis, vitopine iaaM, and vitopine virD2 genes. Primer-probe sets were then used to amplify the octopine synthase gene (62 bp) from octopine strains, the nopaline synthase gene (78 bp) from nopaline strains, and the vitopine synthase gene (60 bp) from vitopine strains, as well as the vitopine iaaM gene (60 bp) and the vitopine virD2 gene (66 bp) of A. vitis. The detection of A. vitis strains from bacterial suspension or extracted plant sap could be achieved within a short time (i.e., 20?25 min) with real-time PCR-based assays using the designed primer and probe sets. For the real-time PCR-based methods, the bacterial cell sensitivity threshold was defined as sensitivity threshold for one bacterial cell and at the DNA level, which was 1.4?1.9 fg. In real-time PCR-based methods, the corresponding PCR efficiencies for octopine and nopaline strains were 97.12% and 97.87%, respectively and for vitopine strains were 94.39%, 96.76%, and 99.01%. In this study, a new real-time PCR-based method was developed that uses the LNA probe, and different opine strains of A. vitis were detected and quantified from vegetative materials of infected grapevine.