First Report of <i>Phytobacter ursingii</i> Causing Maize Bacterial Top Rot in Yunnan Province, China

Maize (Zea mays L.) is the most important grain and feed crop in China. In August 2023, a destructive maize bacterial top rot (MBTR) occurred with 60% incidence rate in the affected fields (more than 13.3 ha) of Ning’er County (23.0628° N, 101.0449° E), Yunnan Province. The symptoms included water soaking and chlorosis of leaves, marginal leaf necrosis, leaf curling, stem rot, and tip collapse accompanied by a foul odor, similar to the symptoms caused by Klebsiella pneumoniae (Huang et al. 2016). The lesion margin of leaves and stems (n=107) were surface disinfected with 75% ethanol for 3 min and then rinsed with sterile distilled water (SDW) 3 times, then cut aseptically into small sections and immersed in 1 mL SDW for 5 min. A loop of suspension was streaked on the surface of Luria-Bertani (LB) agar plate (LBP) which was then incubated at 32°C for 24 h. Three colonies of each unique morphological type (n=15) were selected randomly to perform preliminary pathogenicity tests. Then two distinct morphological types of bacteria with higher virulence were initially identified as Phytobacter ursingii and K. pneumoniae by 16S rRNA gene sequencing. The dominant, milky-white isolate Ne8, with smooth circular colonies and a central raised profile on LBP, was used for further study. Biochemical assays showed Ne8 was positive for indole production, L-sorbose and D-glucose fermentation and Voges-Proskauer test, but negative for adonitol fermentation and urease production, consistent with those of P. ursingii ATCC 27989T (type strain) described by Pillonetto(2018). Partial sequences of the 16S rRNA(Frank et al. 2008), gyrB (Huang et al. 2021), rpoB (Mollet et al. 1997), atpD, and infB genes were amplified, sequenced, and deposited in GenBank (PQ897941, PV297767, PV216706, PV948963, PV948964). These sequences of Ne8 showed 99.41% (1355/1363 bp), 98.48% (1164/1181 bp), 98.89% (982/993 bp), 99.08% (647/653 bp) and 99.24% (912/919 bp) identity to the sequences of P. ursingii ATCC 27989T (FJ611881, CABDWT010000003, CABDWT010000004) respectively in BLASTn analysis. Phylogenetic trees of 16S rRNA and combined gyrB-rpoB-atpD-infB sequences were constructed respectively using MEGA 11.0. The gene sequences of Ne8 clustered with P. ursingii ATCC 27989T. Ne8 was identified as P. ursingii by a comprehensive analysis of phenotypic and phylogenetic characteristics. Healthy maize plants (cv. Jingdian 8) with two fully expanded leaves were inoculated with bacterial suspension of Ne8 (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, 13000 lux, 10 h, 28˚C/dark, 14 h, 23˚C, 80-90% relative humidity) (Huang et al. 2020). SDW served as control. There were 9 plants and 3 replicates per treatment, and 3 independent experimental runs. Typical symptoms including leaf chlorosis, margin necrosis, and curling appeared on the inoculated plants at 4 days post-inoculation, while controls remained symptomless. atpD and infB gene-sequencing were used to confirmed the reisolated pathogen as P. ursingii fulfilling Koch’s postulates. Previously, P. ursingii was reported as a plant-associated nitrogen fixing bacterium (Pillonetto et al. 2018), and a human pathogen causing sepsis (Choice et al. 2024, Smits et al. 2022). Herein, we firstly revealed that P. ursingii is the causal agent of MBTR, highlighting the growing threat posed by emerging pathogens to agricultural production and human health.