First Report of Burkholderia cepacia Causing Finger-Tip Rot on Banana Fruit in the Guangxi Province of China

Banana (Musa acuminata) is one of the most popular and widely consumed fruit crops in the world. During late October to early November 2020, a banana finger-tip rot disease was observed in the banana (cultivar 'Brazil', AAA group) orchard of about 12 hectares located in Zhongcun, Zhangmu Town, Fumian District, Yulin City, Guangxi province, China. The disease incidence was about 0.5% at the surveyed field. Infected fingers and their tips were usually normal in the appearances and then turned to brown to black discoloration in the central fruit pulp adjacent to the fingertips (Fig. 1A). In severe infection, diseased fingers showed brown to black discoloration in both the central and the periphery fruit pulp, and along the longitudinal axis throughout the fruit (Fig. 1B-C). The symptomatic banana fingers were surface-disinfected with 1% sodium hypochlorite for 30 sec, 75% ethanol for 30 sec then rinsed three times with sterile distilled water. The flesh tissues were ground in a sterile mortar and soaked in 1 ml of sterile distilled water for 30 min. A 50 μl of tissue suspensions was streaked onto Luria-Bertani (LB) medium. Single colonies were picked and re-streaked onto new LB medium. The cultures were incubated at 37°C for 24 h. Two representative strains, GX and GX2, were obtained from symptomatic pulps and used in the following studies. To molecularly identify the bacterial species, we performed a polymerase chain reaction (PCR) using 16S rRNA and recA primers (Turner et al. 1999; Lee and Chan 2007) and amplified 1,442 bp and 1,019 bp sequences, respectively. The amplified sequences were deposited in GenBank under the accession numbers MZ267253 and MZ961355 for the 16S rRNA and MZ287336 and MZ983484 for the recA genes. BLASTn searches shared more than 99% similarity with the reference sequences of B. cepacia strains (MK680073.1 and KC261418.1 for 16S rRNA; AY598028.1 and KF812859.1 for recA). Phylogenetic trees were constructed using the 16 rRNA and recA sequences and showed that the representative strains, GX and GX2, strongly clustered with B. cepacia type strains (Fig. 2). To further determine the genomovars of strain GX, we used specific PCR primers to the B. cepacia epidemic strain marker (BCESM), type III secretion gene cluster (bcscV) and cable pilin subunit gene (cblA) (Lee and Chan 2007; Ansari et al. 2019). The presence of bcscV and BCESM were confirmed by PCR, while cblA was not observed in the strains GX and GX2, suggesting that the isolated strains belong to B. cepacia genomovar III and are slightly different from the Iranian and Taiwan strains of B. cepacia (Lee and Chan 2007; Ansari et al. 2019). Pathogenicity test was conducted on banana fingers (cultivar 'Zhongjiao No.3') at the immature and full ripe stages. A final suspensions of 106 CFU/ml, was injected into the banana fingers (100 μl per finger) through the center of the stigma (Lee and Chan 2007; Ansari et al. 2019). The fingers inoculated with sterile water were used as negative control. To maintain humidity, the treated fingertips were wrapped with Parafilm. For each treatment, ten independent replicates were conducted. At 10 days post-inoculation (dpi), the pulp of immature bananas exhibited reddish brown decaying tissue, which symptoms were similar to those observed in the field (Fig. 1D). Moreover, the pulp tissues of ripe bananas showed a dark brown discoloration in the tip at 5 dpi, whereas the controls remained symptomless (Fig. 1E). The same bacterium was re-isolated from diseased tissues and its identification confirmed by 16S rRNA, thus fulfilling the Koch's postulates. This disease was first described in Honduras in Latin America, and then reported in Taiwan province of China, and Iran (Buddenhagen 1968; Lee et al. 2003; Ansari et al. 2019). To our knowledge, this is the first report of banana finger-tip rot caused by B. cepacia in the Guangxi province, China. It is necessary to determine the distribution of B. cepacia and to prevent its spread in Guangxi province of China.