First Report of Bacterial Dry Rot of Mango Caused by Sphingomonas sanguinis in China

Mango, Mangifera indica L., native to India and Southeast Asia, is an economically important crop in China. From 2016 to 2017, a new mango leaf and branch disease was observed on commercial mango orchards planted with cultivar Keitt in the Leizhou Peninsula region, Guangdong Province, China. Disease incidence reached 25% in severely infected fields. In some trees, more than 50% of the leaf and branch were infected. The early symptoms in leaf were browning, followed by general interveinal chlorosis and profuse brown necrotic spotting and scorching, eventually becoming dry. The initial symptoms on stems were dark green and irregularly shaped lesions, with xylem browning. At last, the fleshy stems became completely dry rotted. To identify the possible pathogens involved, 10 diseased tissue including buds, leaf petioles, and the stem from cultivar Keitt were disinfected with tap water and then 70% ethanol, followed by maceration in sterile distilled water, and 20 µl of the suspension was plated onto nutrient agar medium (NA) and incubated at 28°C for 48 h in the dark. A predominant type of colony was purified on NA, and a representative isolate designated NY01 was used for further study. The cultures formed a yellow lawn and were rod shaped, with a cell size around 0.7 to 1.4 μm, and a single polar flagellum with slow motility. In biochemical tests, NY01 was a gram negative, non-spore-forming, chemoheterotrophic, and strictly aerobic. Glucose is primarily utilized for growth, but a wide variety of other sugars such as lactose, fucose, mannose, melibiose, sucrose, trehalose, and xylose also are frequently assimilated. Genomic DNA was extracted from the isolate, with polymerase chain reaction amplification of the 16S ribosomal RNA region (primers 27F and 1541R), and the specific sequences of Sphingomonas spp. (primers Spf-429F and Sph-933R) were used to support morphological identification (Frank et al. 2008). The 16S rRNA gene sequence (GenBank accession no. MH061144) of NY01 isolate had 99% sequence identity with S. sanguinis accessions in GenBank (KR708909, KT766072, and KR708848). On the basis of morphological, physiological, and biochemical characteristics and sequencing of the 16S rRNA gene, the bacterium was identified as S. sanguinis. One-year-old mango seedlings planted in pots in a glasshouse were used for inoculation. Isolate NY01 was tested for pathogenicity by injecting a bacterial suspension (10 μl/inoculation site, 1 × 10⁸ CFU/ml) onto mango bud surfaces with sterile distilled water as a control. Plants were incubated in a growth chamber at 30 ± 1°C day and 26 ± 1°C night (12-h day/night cycle) at 80 ± 5% relative humidity. Five days after inoculation, typical bacterial apical necrosis symptoms were observed on all treated buds, and the controls were still healthy. The same bacterium was reisolated from the inoculated tissue. The isolated bacteria from the inoculated plants was reidentified as S. sanguinis, based on the morphological, biochemical, and sequencing of the 16S rRNA gene. Koch’s postulates were fulfilled. The strains of S. sanguinis are widely distributed in nature, having been isolated from many different land and water habitats. Because of their ability to survive in low concentrations of nutrients, they have been used for a wide range of biotechnological applications, from bioremediation of environmental contaminants to production sphingans used in the food and other industries (Yabuuchi and Kosako 2015). To our knowledge, this is the first report of S. sanguinis strain causing bacterial apical necrosis on mango in China. Meanwhile, this is the first report of S. sanguinis strain as a plant pathogen.