First Report of <i>Burkholderia gladioli</i> Infecting <i>Dendrobium officinale</i> in China

Dendrobium officinale is a highly valued traditional Chinese medicinal herb with a cultivation area in China exceeding 8,000 ha (Wang et al, 2019). In May 2024, a new D. officinale disease was discovered in Gutian Town, Shanghang County, Longyan City, Fujian Province, with an incidence rate of approximately 10% observed in the 0.8 ha greenhouse. Symptoms initially appeared as stunted growth of new leaves, followed by leaf yellowing and brown leaf sheaths. Upon removing the leaf sheaths, water-soaked, brown necrotic spots were observed between the fleshy stem internodes. As the disease progressed, the leaves dried up and fell off, ultimately leading to plant death. Diseased tissues were surface-sterilized (75 % ethanol, 20 s), rinsed three times with sterile water, homogenized, plated on nutrient agar medium (NA), and incubated at 28℃ for 2 days. Colonies on NA were yellowish, round, fluid, convex, with smooth edges. Seven isolates (FJSH1-FJSH7) from five symptomatic plants were selected for further analysis. All isolates and Burkholderia gladioli type strain LMG6882 were Gram-negative, grew at 41°C, and were positive for potato tuber rot and nitrate reductase, but negative for phenylalanine deaminase, and utilized trehalose, mannitol, sorbitol, sodium tartrate, and xylose (Zafdri et al, 2022). Genomic DNA of FJSH6 and FJSH7 was extracted with a Tiangen DNA Extraction Kit and served as template for PCR amplification of 16S rRNA, gyrB, and rpoD genes with primers 27F/1492R (Lane, 1991), gyrB F/ R and rpoD F/ R (Maeda et al, 2006), respectively. The 16S rRNA, gyrB and rpoD sequences of strain FJSH6 and FJSH7 were deposited in GenBank (16S rRNA:PQ892059–PQ892060) and CNCB (https://www.cncb.ac.cn, gyrB: PRJCA049130; rpoD:PRJCA049174). They share 99.93-100%, 99.42-99.43%, and 99.63-99.65% nucleotide identity, respectively, to B. gladioli strain MAFF 302385 (AP024338.1). A phylogenetic tree was constructed based on 16S rRNA, gyrB, and rpoD genes using the maximum-likelihood approach. Sequences of FJSH6, FJSH7, and B. gladioli strains LMG 11626 and MAFF 302385 formed a distinct, highly supported branch (bootstrap support = 100%). Pathogenicity was tested by stab inoculation; sterile needles dipped in bacterial suspension (OD600=1.0) were gently stabbed into healthy, current-year succulent stems of D. officinale. Each bacterial strain was tested on three separate plants, and the experiment was replicated three times. Inoculated plants were incubated at 28℃, 85% relative humidity, under a 16 h light /8 h dark cycle. Water-soaked lesions emerged at the inoculation site in 4 days post-inoculation (dpi). Then, the lesions turned brown and spread upwards and downwards along the stem. The upper leaves of the inoculated plants turned yellow at 7dpi, and then the plants withered and died, while the control inoculated with sterile water had no symptoms. Bacteria re-isolated from symptomatic plants were confirmed as B. gladioli by colony morphology and 16S rRNA sequencing, thus fulfilling Koch’s postulates. Keith et al (2005) documented B. gladioli damaging Dendrobium in Hawaii, and You et al (2016) reported B. gladioli infecting Phalaenopsis in China. To our knowledge, this is the first report of D. officinale bacterial leaf blight caused by B. gladioli in China. Ignoring the disease could cause major economic losses in D. officinale cultivation. Effective prevention strategies remain an urgent research priority.