An Emerging Bacterial Leaf Disease in Rice Caused by Pantoea ananatis and Pantoea eucalypti in Northeast China

Abstract

Rice production faces new challenges from emerging diseases due to intensive cultivation practices and climate warming in China. A new rice leaf bacterial disease has recently occurred in Northeast China. The symptoms of the disease are similar to those of bacterial leaf blight. Disease lesions spread along leaf edges and are later dried up due to water loss. In this study, 17 bacterial isolates were identified as the causal agents of the new disease following Koch's postulates. These strains are categorized into two groups based on colony morphology and molecular characterization. Phylogenetic analysis using the five housekeeping genes leuS, gyrB, fusA, pyrG, and rplB reveals that the two groups of the isolates belong to Pantoea ananatis and P. eucalypti, respectively. The new rice disease is caused by P. ananatis, P. eucalypti, or a combination of both bacterial species. A complete genome map has also been assembled for P. eucalypti. Meanwhile, some important virulence factors have been predicted based on gene annotation and determination of extracellular enzymes. Collectively, this study represents the first report of a new rice leaf disease caused by P. eucalypti and the first high-quality genome assembly of P. eucalypti that infects rice leaves.

Keywords: Pantoea; genome; pathogen identification; rice.

Figure 1

Symptoms of a new rice disease occurred in paddy fields in Northeast China. (A) Plant wilting caused by a new disease in paddy fields. (B) Disease symptoms on rice leaves at the early infection stage. (C) Disease symptoms on rice leaves at the late infection stage.

Figure 2

Colony and cell morphologies of the JMS78-1 and GY78-10 bacterial isolates. (A,B) Colony morphology of JMS78-1 (A) and GY78-10 on LA medium plates after culturing at 28 °C for 2 days (B). (C,D) Representative transmission electron microscope (TEM) images showing the morphology of JMS78-1 (C) and GY78-10 (D) cells (Scale bar: 1.0 μm).

Figure 3

Pathogenicity assays on rice leaves for the bacterial isolates JMS78-1 and GY78-10. (A) Disease symptoms on the inoculated leaves by the indicated bacterial isolates. The leaves were inoculated with LB medium as a negative control. (B) The average length of disease lesions on the inoculated leaves caused by different bacterial isolates. Disease lesion lengths were measured at 14 dpi. The representative data from three independent experiments are shown as mean ± standard deviation (SD, n =  10). Lowercase letters (a–c) represent significant differences in disease lesion length caused by different bacterial isolates (p < 0.05).

Figure 4

A phylogenetic tree was constructed based on the concatenated sequences of leuS, gyrB, fusA, pyrG, and rplB genes. The phylogenetic trees constructed through BI and ML methods were consistent. The numbers displayed at the nodes represent the posterior probabilities from the Bayesian analysis (MrBayes) and the bootstrap values based on the 1000 replicates of the ML analysis (IQ-TREE), respectively. Red stars indicate the bacterial isolate identified in this study. The scale bar represents units of substitutions per site.

Figure 5

The genome and plasmid maps and functional annotation of P. eucalypti GY78-10. The circles from outside to center: Genomic size representation; COG (+), COG functional categories of CDSs on the positive strand; rRNA, tRNA on the positive strand; CDSs, rRNA, tRNA on the negative strand; COG (−), COG functional categories of CDSs on the negative strand; GC content. Detailed information and corresponding gene numbers in distinct functional categories in COGs indicated by different uppercase letters are provided in Supplementary Table S4. rRNA: ribosomal RNA; tRNA: transfer RNA.

Figure 6

The ability to secrete different extracellular enzymes in P. eucalypti GY78-10, P. ananatis JMS-78-1, and X. oryzae pv. oryzae PXO99A. (A–C) Detection of extracellular proteases (Prt), cellulases (Cel), and pectate lyases (Pel) in GY78-10, JMS-78-1, and PXO99A strains on culturing plates. Upper panels, Prt images were captured at 24 h after culturing, while Pel and Cel images were captured at 14 h after culturing. Lower panels, the radius of the clear area caused by extracellular enzymes. Data are shown as mean  ± SD (n = 3). Bacterial cultures (20 μL, OD₆₀₀ = 1.0) were loaded onto assay plates. All the plates were incubated at 28 °C and stained for visibility. Radius of the inhibition zone was measured.