First report of Bougainvillea spectabilis Willd bacterial leaf spot caused by Pantoea stewartii subspecies indologenes in China.

ABSTRACT

Bougainvillea spectabilis Willd. is an important ornamental flowering plant belonging to the family Nyctaginaceae. It is widely used in landscape designs in tropical and subtropical regions. In December 2020, severe disease-causing leaf spots were discovered on the leaves of B. spectabilis in the Modern Agricultural Park (110°19' E, 21°26' N) Zhanjiang City, Guangdong Province, China. Field surveys revealed that the disease was widespread, with an incidence of 60-80%. Early symptoms on the leaves appeared as tiny leaf spots that later developed into concentric circles surrounded by a yellowish halo (Fig. 1). Diseased leaves with typical symptoms were collected for pathogen isolation. The leading edges of the lesions were excised, sanitized in 75% ethanol for 30 s and in 3% sodium hypochlorite for 3 min, and rinsed three times with sterile distilled water (SDW). The diseased tissue was crushed in 1 mL SDW, soaked for 15 min, and then spread onto nutrient agar medium on a petri dish. Circular, bright yellow colonies with smooth margins were observed after 24 h of incubation at 28 °C. The isolate (SJM1) was a gram-negative bacillus with positive results for catalase, indole synthesis, maltose, and arbutin and negative results for sorbitol, lactose, salicin, and starch hydrolysis. The SJM1 genomic DNA was extracted using the TIANamp Bacterial DNA Kit, and partial 16S rDNA gene segments were amplified using the bacterial generic primers 27F and 1492R. The collated 16S rDNA gene sequences were submitted to the NCBI GenBank (MZ723935). BLAST analysis of the sequences revealed 99.38% identity with Pantoea stewartii (MG517424.1). Amplification using subspecies-specific primers galE (#562/564; Gehring et al. 2014), glmS (#356/341; Wensing et al. 2010), and pstC + pstS (#338/339; Wensing et al. 2010) revealed that the genes showed 99-100% identity with P. stewartii subsp. indologenes (galE = 100%, MZ754494.1; glmS = 99.79%, MZ75496.1; and pstC + pstS = 99.89%, MZ754495.1). Phylogenetic trees were constructed using the neighbor-joining method (MEGA X), with both the 16S rDNA sequence (Fig. 2 2A) and the concatenated 16S rDNA, galE, pstC + pstS, and glmS sequences (Fig.2 2B). The SJM1 isolate belonged to the same clade as P. stewartii subsp. indologenes and was 99% homologous to P. stewartii subsp. indologenes strain ZJ-FGZX1 (Fig. 2 2B; Ren et al. 2020). Pathogenicity tests were performed through prick wound inoculation. Sterile needles were used to create fresh wounds on healthy young leaves of one-year-old B. spectabilis plants. Wounds were inoculated with 20 µl bacterial suspension (1 × 108 CFU/ml) or SDW. Four leaves per plant and three plants per treatment were evaluated. The plants were incubated at 28 °C temperature and 80-90% relative humidity. After 4-7 days of inoculation, all plants inoculated with the bacterial suspension had spot symptoms with a halo, similar to those observed in the field. However, leaves inoculated with SDW alone did not show any symptoms. Furthermore, the colony morphology and 16S rDNA sequences of the strains isolated from the inoculated leaves were identical to those of the original isolates. These results verified Koch's postulates. Based on biochemical identification and sequencing analysis, the pathogen causing B. spectabilis leaf spot was identified as P. stewartii subsp. indologenes. Previous reports have shown that P. stewartii subsp. indologenes can cause diseases in Dracaena sanderiana, Cenchrus americanus, and Allium cepa (Zhang et al. 2020, Ashajyothi et al. 2021, Stumpf et al. 2018). To our knowledge, this is the first report of P. stewartii subsp. indologenes causing B. spectabilis leaf spot disease in China.