Occurrence of Crown Gall Disease on Japanese Spindle (Euonymus japonicas var. Green Rocket) Caused by Agrobacterium rosae in Iran

Crown gall disease caused by diverse Agrobacterium species is one of the main biotic constraints in the ornamental plants industry in Iran (Mafakheri et al. 2017). In August 2019, Japanese spindle (Euonymus japonicus var. Green Rocket) plants showing crown gall symptoms were observed in a commercial greenhouse in Tehran, Iran. Infected plants were characterized by a visible overgrowth on their stems and crown. Bacterial isolation from the gall tissues was performed on nutrient agar (NA) and 1A media as described by Moore et al. (2001). The six resulting bacterial strains (A.E1 to A.E6) were evaluated using PCR primer pair F8360/F8361 amplifying a 453-bp DNA fragment in the recA gene and confirmed as Agrobacterium sp. (Shams et al. 2013). Pathogenicity of the strains was evaluated in two independent assays on Japanese spindle plantlets as well as 10- to 15-day-old tomato (Solanum lycopersicum cv. Sunseed 6189) and sunflower (Helianthus annuus cv. Armavirski) plants in greenhouse conditions using the needle prick method as described previously (Mafakheri et al. 2019). The reference strain A. radiobacter ICMP 5856 and sterile distilled water were used as positive and negative controls, respectively. Crown gall symptoms appeared 20 to 25 days post inoculation on the Japanese spindle plantlets as well as tomato and sunflower plants inoculated with the strains isolated in this study, while the negative control plants remained asymptomatic. Koch’s postulates were accomplished by reisolating on NA medium and PCR-based identification of the inoculated strains from the symptomatic plants. The representative strain A.E1 was subjected to multilocus sequence analysis (MLSA) using the sequences of four housekeeping genes (i.e., atpD, gyrB, recA, and rpoB) as described previously (Mafakheri et al. 2019). MLSA results revealed that the strain A.E1 is phylogenetically closely related to A. rosae. The sequences were deposited into GenBank under the accession numbers MT007962 to MT007965 for atpD, gyrB, recA, and rpoB, respectively. Further, the strain A.E1 was subjected to whole genome sequencing using the Illumina HiSeq X platform. DNA extraction was performed using NucleoSpin Microbial DNA kit (Macherey-Nagel, Düren, Germany), DNA libraries were obtained with Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, U.S.A.), and de novo sequence assembly was performed using SPAdes genome assembler. The resulting whole genome sequence was deposited into the GenBank database under the accession number JAFJZW000000000. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were calculated among all the type strains of Agrobacterium species/genomospecies using standard criteria as detailed previously (Chen et al. 2021; Osdaghi et al. 2020). The strain A.E1 had 97% ANI and 72% dDDH values with A. rosae strain NCPPB 1650, suggesting that the bacterial strains isolated from Japanese spindle in Iran belong to A. rosae. This is the first report of A. rosae causing crown gall disease on Japanese spindle in Iran. The new crown gall disease could negatively affect the ornamental shrub production industry in central Iran unless strict sanitary measures are taken into the account in the nurseries in these areas. Further nationwide surveys and samplings are warranted to elucidate the economic impact of the pathogen on ornamental plant industry in the country.