First report of <i>Klebsiella variicola</i> Causing Maize Bacterial Top Rot in Yunnan Province, China

Maize (Zea mays L.) is one of the most important staple crops in China. In July 2024, most conventional maize varieties suffered from maize bacterial top rot (MBTR) with loss rates ranging from 20-80% in Ning'er County (23.0628°N, 101.0449°E), Yunnan province, China. Typical symptoms, including leaf chlorosis, marginal necrosis, leaf twisting and curling, stem rot, tip collapse accompanied by a foul odor, and severe dwarfing, were consistent with those of MBTR caused by Klebsiella pneumoniae (Huang et al. 2016) and Phytobacter ursingii (Xiang et al. 2026), yet more severe, with an incidence rate reaching as high as 80%. The boundary of the lesion of leaves and stems (n=83) were surface sterilized using 75% ethanol for 3 min, then rinsed 3 times with sterile distilled water (SDW), and subsequently cut into small pieces and immersed in 1 mL of SDW for 5 min. The suspension was evenly spread onto Luria-Bertani agar plates which were incubated at 32 ℃ for 48 h. Preliminary pathogenicity tests were performed with 3 colonies of each unique colonial morphology. Kv1, the most virulent strain with circular, convex, and smooth colonies, was selected for further study. Biochemical tests showed Kv1 was positive for D-glucose, D-sorbitol, raffinose, and urea hydrolysis, but negative for adonitol and indole, matching the profile of K. variicola Kv4 (Zhang et al. 2024). Partial gene sequences of 16S rRNA (Frank et al. 2008), RNA polymerase subunit beta (rpoB) and DNA gyrase subunit B (gyrB) (Huang et al. 2016) were amplified, sequenced and deposited in GenBank (PZ230834, PZ238913, PZ238912), respectively. The sequence identity of 16S rRNA, rpoB, and gyrB of Kv1 with the type strain K. variicola F2R9T (NR_025635.1, AY367356.2, CP072130.1) were 98.83% (815/833 bp) , 98.25% (959/976 bp), and 98.18% (1130/1151 bp) respectively. The phylogenetic tree of 16S rRNA-rpoB-gyrB tandem sequences was constructed using maximum likelihood analysis by MEGA 7.0.21. The gene sequences of Kv1 clustered with K. variicola F2R9T. Kv1 was identified as K. variicola through comprehensive analysis of biochemical and phylogenetic characteristics. Healthy maize seedlings (cv. Mitiannuo 2) with two fully expanded leaves were inoculated with bacterial suspension of Kv1 (108 cfu/mL) by stem injection (30 µl/plant) and leaf surface infiltration (20 µl/plant), respectively, then cultured in the plant growth chamber (light intensity 13,000 lux, 10 h, 28℃/ dark, 14 h, 23 ℃). SDW was served as a control. Each treatment consisted of 6 plants with 3 replicates, and total 3 independent experiments were conducted. Typical symptoms of MBTR, including leaf twisting and curling, marginal necrosis, and stunting, appeared at 4 days post-inoculation. No symptoms were observed in the control group. The reisolated strains were identified as K. variicola based on 16S rRNA, rpoB and gyrB sequenceing, fulfilling Koch's postulates. It has been confirmed that K. variicola can infect banana (Fan et al. 2023), tomato(Zhang et al. 2024), sorghum(Malik et al. 2023), carrot (Chandrashekar et al. 2018), and onion(Ashrafi et al. 2024). To our knowledge, this is the first report of MBTR caused by K. variicola in China. It emphasized that under the changing climatic and environmental conditions as well as the evolving farming systems, the host range of pathogenic microorganisms has been continuously expanding, and highlighted the importance of ongoing monitoring of the occurrence and development of major diseases.