Whole-genome analysis of Pantoea alfalfae HF1 isolated from shot hole lesions in peach leaves and symptom reproduction by wound inoculation

Abstract

Bacterial leaf shot-hole disease has become an increasingly serious threat to peach production and related industries. Here, we isolated a bacterial strain, designated HF1, from symptomatic peach leaves infected with shot holes in Hefei City, Anhui Province, China. 16S rRNA gene sequencing revealed that the isolate belongs to the genus Pantoea. Furthermore, we used Illumina and MinION Nanopore sequencing technologies to analyze the whole-genome sequence of this strain. The complete genome comprises four distinct genomic elements: one circular chromosome measuring 4,008,864 bp (G + C content of 55.37%) and three circular plasmids measuring 527,106, 161,006, and 104,931 bp long, with G + C contents of 53.13%, 52.34%, and 53.92%, respectively. Multilocus sequence analysis of five housekeeping genes revealed that strain HF1 belongs to a clade containing Pantoea alfalfae CQ10, which was isolated from alfalfa. Average nucleotide identity and DNA-DNA hybridization analyses supported the phylogenetic proximity of HF1 to P. alfalfae CQ10. Based on these results, strain HF1 was reclassified as P. alfalfae HF1. In addition, HF1 was compared with three genetically related representative strains: P. alfalfae CQ10, P. ananatis DZ-12, and P. vagans C9-1. A comparative analysis was conducted using a bacterial pan-genome pipeline, and virulence factors were identified using a virulence factor database. The findings revealed that HF1 contains a number of genes associated with potential virulence traits involved in bacterium-host interactions, including motility, biofilm formation, the type VI secretion system (T6SS), and nutrient acquisition. Compared with its closest pathogenic relative, P. alfalfae CQ10, HF1 possesses three distinct T6SS clusters, including several copies of genes encoding predicted effector proteins such as Hcp, VgrG, and PAAR. Interestingly, incomplete AHL-mediated quorum-sensing system was identified in both P. alfalfae strains, and HF1 was unable to synthesize AHL; in vitro experiments corroborated these findings. Under wound inoculation conditions, strain HF1 reproduced symptoms resembling shot hole disease in peach leaves. Collectively, these findings indicate that strain HF1 is a Pantoea species associated with leaf shot hole symptoms in peach trees. This strain harbors diverse genetic features potentially related to virulence, including motility, biofilm formation, and the T6SS, which may contribute to its ability to induce symptoms under the tested conditions.

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