Simultaneous and Visual Assay for Three 16Sr Groups of ‘Candidatus Phytoplasma’ Infecting Date Palm (Phoenix dactylifera L.) Targeting 16S rRNA Gene Sequences

Date palm is a staple food crop and crucial export agricultural commodity in Arab countries. Date palm diseases caused by phytoplasmas are devastating diseases that severely constrain the sustainable development of date palm cultivation and industry. The phytoplasmas associated with date palm diseases mainly consist of three taxa: ‘Candidatus Phytoplasma asteris’ of the 16SrI group, ‘Ca. Phytoplasma citri’ of the 16SrII group, and ‘Ca. Phytoplasma fraxini’ of the 16SrVII group. Based on the 16S rRNA gene sequences of the three groups of phytoplasmas, two sets of primer pairs (Da-1 and Da-5) enabling simultaneous visual detection of date palm phytoplasmas were designed and screened. Universal simultaneous visual detection methods were successfully established for different groups and candidate species of date palm pathogenic phytoplasmas. The whole detection procedure of this optimized method can be accomplished via isothermal amplification at 64 °C within 30–50 min, and the detection results can be directly and intuitively judged by the color variation of the reaction system. Sensitivity verification showed that for the three groups of phytoplasmas, the minimum detection limit of the Da-1 primer set for simultaneous visual detection ranged from 1 fg/μL to 10 fg/μL, while that of the Da-5 primer set ranged from 1 fg/μL to 100 pg/μL. The established LAMP assay demonstrated high sensitivity and specificity using synthetic DNA templates. This method provides a promising laboratory tool for the simultaneous visual detection of three major date palm phytoplasma groups. Further validation on naturally infected field samples is required before practical application in field diagnosis, epidemic monitoring, and port quarantine.