First Report of Pantoea ananatis Causing Bacterial Leaf and Panicle Blight of Rice in Louisiana, USA.

Abstract

In July 2023, panicle and leaf blight-like symptoms were observed from the rice (Oryza sativa) variety, PVL03, in research field plots in Louisiana (Rayne, LA 70578, USA; 30.21330⁰ N, 92.37309⁰ W). We observed that chlorosis started on the tips and spread throughout the whole leaf. We also observed panicle blight-like symptoms (grain discoloration of rice hulls) on infected rice plants at the heading stage, which ranged from dark brown to black. Disease incidences were approximately 70% in the observed plots. Five plants were sampled from five plots (one plant per plot). Leaf tissues including both symptomatic and healthy parts (~ 1 cm²) were surfaced sterilized and placed on CCNT medium, a semi-selective medium for the rice bacterial panicle blight pathogen Burkholderia glumae (Kawaradani et al., 2000), and on Luria-Bertani (LB) agar supplemented with cycloheximide (50 µg/ml) for non-selective isolation of bacteria. For the panicle samples, the surfaced sterilized rice kernels were dehulled and ground in sterile distilled H₂O using a sterile mortar and pestle. The homogenate was spread on the CCNT media and LB agar supplemented with nitrofurantoin (50 µg/ml) and cycloheximide (50 µg/ml). After ~72 h at 41⁰C, round, smooth and yellow-colored bacterial colonies were observed on both media. The two isolates, one from an infected rice leaf (LPL-1) and the other from the infected rice panicles (LPP-1), were initially identified as Pantoea ananatis based on PCR-amplified 16S rDNA sequences (Weisburg et al., 1991). The taxonomic identity of these two isolates was further confirmed through whole genome sequencing (NCBI Accession ID: PRJNA1092676), which was performed through a hybrid sequencing approach using Oxford Nanopore sequencer & Illumina NextSeq2000 system (Plasmidsaurus Inc., South San Francisco, CA, USA). To satisfy Koch's postulates, we inoculated six one-month-old rice plants (cv. Kitaake) with LPL-1 and LPP-1, respectively, by pricking the stems with a sterile toothpick touched to the overnight grown bacterial culture on LB agar (~ 5-8 x 10⁵ bacterial cells). This virulence assay was conducted twice, and rice plants inoculated with sterile distilled water were included as the negative control. By 27 days post inoculation (DPI), the inoculated plants developed symptoms on leaves and panicles, which were like the symptoms originally observed in the field, and additional symptoms such as shot holes and stem necrosis were also observed. From leaf and panicle samples of the artificially inoculated plants, we reisolated bacteria and confirmed the identity of the bacteria using the P. ananatis specific PCR primers PANA_1080 61F and PANA_1080 1009R (Asselin et al., 2016). To the best of our knowledge, this is the first report of bacterial panicle and leaf blight of rice caused by P. ananatis in Louisiana. Since P. ananatis is a recently emerging pathogen of rice worldwide, including the United States (Yu et. al., 2022; Lu et al., 2022; Luna et al., 2023), it is crucial to monitor the occurrence of this pathogen in other rice-growing areas as a potential threat to US rice production.