First Report of Achromobacter Xylosoxidans Causing Stem Soft Rot on Amorphophallus Konjac in China

Abstract

Amorphophallus konjac is one of the important commercial crops cultivated in south China and has long been used as a food source and a traditional medicine, because it is the only species with glucomannan and other trace elements (Ban et al. 2009; Melinda et al. 2010). In June of 2021, an outbreak of stem soft rot disease was observed on A. konjac plants in more than 2,000 square meters of agricultural planting fields in the Fuyuan country (25°34'50″N, 104°04'21″E), Qujing City, Yunnan Province, China. The disease incidence ranged from 30 to 35% in severely infested fields. The diseased plants displayed the first symptoms were damp brown spots. As the brown spots expanded, dark brown water stains appeared at the basal part of the stem and the bulbs were rotting with a foul smell, gradually extending to the underground parts. Progressively, the whole plants wilted and collapsed, and even the plants ultimately died. To identify the pathogen, symptomatic stems were cut into pieces, surface sterilized with 75% (v/v) ethanol, and placed on LB (tryptone/yeast extract/NaCl) medium for 24 to 48 hours at 28 ± 2°C. Six single-colony isolates were obtained from the diseased stems. The colonies on LB present a raised milky white opaque colonies moisture on the surface, round and convex in shape, with neat edges. Scanning electron microscopy showed that the cells were short rods (0.3∼0.5) × (1.9∼2.1)μm in size without any flagellum and were often arranged in pairs or clusters at certain angles. The 16S rDNA sequence of the randomly selected strain MY-G1 with primers 27F/1492R (Ying et al. 2012) and the housekeeping genes nusA, eno, lepA and nuoL (Spilker et al. 2012) were amplified and sequenced. The 16S rDNA sequence of the 1326 bp product was deposited in GenBank (accession no. ON786717) and showed a 99.77% similarity to A. xylosoxidans strain E2 (accession no. MK849863.1). The nusA (OP680477), eno (OP680479), lepA (OP680481) and nuoL (OP680482) sequences showed 94.71%, 97.24%, 94.64% and 95.95% similarity to A. xylosoxidans strain DN002 (accession no. CP045222.1), respectively. The phylogenetic trees built based on 16S rRNA and the nusA-eno-lepA-nuoL multilocus analysis showed the isolate MY-G1 to cluster with A. xylosoxidans. Based on morphological and molecular analysis, the isolated MY-G1 was identified as A. xylosoxidans, which indicates that MY-G1 is a new strain of A. xylosoxidans. Pathogenicity tests were confirmed on the stem and petiole of one-year-old A. konjac. The wounds were made by puncturing with a MY-G1 bacteria suspension containing 108 CFU/ml (15ul/inoculation site). As a negative control, control seedlings were injected with the same amount of sterilized distilled water. Control and inoculated seedlings (each six) were kept in greenhouses and watered as needed in controlled conditions: 28°C, 75% relative humidity. Inoculated seedlings presented similar symptoms of stem soft rot, inner medulla disintegration, and wilt of leaves on developed within 3 to 9 days. The bacterial pathogen was re-isolated from inoculated seedlings and identified by morphological and molecular methods to fulfill Koch's postulates test. According to previous research, A. xylosoxidans can cross-kingdom infect animals and plants (Aisenberg et al.,2004; Ye et al.,2018). To the best of our knowledge, this is the first report of A. xylosoxidans causing stem soft rot of A. konjac in China, expanding the known pathogen for the soft rot of A. konjac, and also the host range of A. xylosoxidans.