First Report of Bacterial Leaf Spot Disease on Ginger Caused by Enterobacter quasiroggenkampii in China.

Abstract

In autumn 2023, an unknown leaf spot disease has occurred on ginger (Zingiber officinale Roscoe) in two fields of approximately 1800 m2 in Yongning District (22°49'N; 108°48'E), Nanning, China, with a incidence of 20-30%. The symptoms began as yellow spots on the leaves, expanding into elliptical to irregular lesions with yellow edges, the middle of the lesion turning grey-white in dry weather. Finally, multiple spots caused necrosis of the whole leaf. Twelve diseased leaves from six plants of two fields were collected, surface disinfected and ground. The ground samples were diluted and plated on nutrient agar (NA) medium at 28 °C for 48-72 h. The purified colonies appeared milky white and round, with smooth edges. Three isolates (GL1, GL2 and GL3) were selected for identification and pathogenic determination. They were gram negative, could utilize sorbitol, mannitol, inositol, raffinose, melibiose, disaccharides, and citrate; negative for methyl red, phenylalanine decarboxylase, hydrogen sulfide, urease; positive for voges-proskauer test and ornithine decarboxylase. These characteristics were consistent with Enterobacter genus (Wu et al., 2020). Genomic DNA was extracted from three isolates. The 16S rDNA region was amplified using 27F/1492R primers (Weisburg et al. 1991) and sequenced (accession no. PP837703-PP837705). Blastn analysis revealed that 16S rDNA sequences for GL1 was 99% identical (1373/1387 nt), GL2 96% (1364/1422 nt) and GL3 95% (1365/1435 nt) to Enterobacter quasiroggenkampii WCHECL1060 (NR_179166). To determine the species, the sequences of gyrB, rpoB and atpD genes were amplified using primers gyrB 01-F/gyrB 02-R, rpoB CM7/rpoB CM31b, and atpD 01-F/atpD 02-R, respectively (Lin et al. 2015; Zhu at al. 2010; Zhang et al. 2013). The GenBank accession numbers for the sequences were PP857680-PP857688. A multilocus phylogenetic tree was constructed with the concatenated sequence of 16S rDNA-gyrB-rpoB-atpD by using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA6 software. The three isolates clustered with E. quasiroggenkampii. Fifteen Darou ginger variety plants at the 4-5 leaf stage were tested for pathogenicity. Two to three leaves of each ginger plant were pricked with a syringe needle of 0.36mm in diameter or not and inoculated by spraying the bacterial suspension (108 CFU/mL), sterile water was used as a control. Five plants were inoculated with each isolate and the test was repeated three times. After 3-4 days of inoculation, all wounded leaves and about 10% of the unwounded leaves showed symptoms similar to those observed in the field. Control plants did not develop symptoms. Enterobacter quasiroggenkampii isolates were re-isolated from the inoculated leaves with symptoms, and their identity was confirmed by gyrB sequencing and colony morphology, completing Koch's postulates. Enterobacter quasiroggenkampii is a pathogen of humans that can cause nosocomial infections (Wu et al., 2020). In Guangxi, E. quasiroggenkampii was identified as one of the pathogens causing mulberry wilt (Jiao, 2022). To our knowledge, this is the first report of E. quasiroggenkampii causing bacterial leaf spot disease of ginger. The results of this study not only have practical significance for the control of ginger leaf spot, but also can provide excellent materials for the study of the differentiation and pathogenic mechanism of the genus Enterobacter, which has important academic value.