Molecular characterization of stem gall disease caused by Agrobacterium tumefaciens (= Agrobacterium biovar 1) on Citrus trees as a new host, in the Eastern Mediterranean region of Turkey

Citrus ( Citrus spp. L.), which has high nutritional and economic value, is one of the most commonly grown fruits in Turkey. In spring 2020, large aerial galls (tumors) were observed on trunks and main branches of mandarin ( Citrus reticulata cv. Satsuma) and lemon ( Citrus limon cv. Kutdiken) trees grafted to sour orange ( Citrus aurantium L.) in commercial citrus orchards in Hatay and Mersin provinces of Turkey. Disease incidence was 0.2–1.0% in the orchards inspected. Six bacterial isolates were obtained from young tumors on King's B (KB) medium. Several biochemical and morphological characteristics of all isolates were consistent with the description of the tumorigenic bacterium Agrobacterium tumefaciens (= Agrobacterium biovar 1). Identification of these isolates was confirmed by comparison with reference isolates of Agrobacterium tumefaciens (Syn. Rhizobium radiobacter ) using MALDI-TOF MS analysis. The pathogenicity of the isolates was confirmed on carrot slice and stems from 6-month-old Kalanchoe plants and 2-year-old mandarin (cv. Satsuma) saplings. For molecular identification, the 16S rRNA and virD2 genes were amplified with primer pairs 27F/1492R and VirD2A/VirD2C, respectively and the PCR products were sequenced for representative isolates (CAt21, CAt27). The partial sequences of 16S rRNA (accessions numbers MZ708735 and MZ708734) and VirD2A/VirD2C of the representative isolates (CAt21, CAt27) showed 100% nucleotide identity to the A. tumefaciens isolates available in GenBank. According to symptoms, biochemical characteristics, pathogenicity test and molecular analyses, the causative agent of the disease was identified as Agrobacterium tumefaciens (= Agrobacterium biovar 1). To our knowledge, this is the first report of Agrobacterium tumefaciens (= Agrobacterium biovar 1) on citrus in Turkey.