First Report of Brown Stripe Disease Caused by on Rice in Guangxi (China).

Rice, being the most important food crop in China, is also the world's largest producer and consumer, accounting for 30% of global production and consumption. In 2023, brown stripes were observed on the leaf sheaths of diseased rice seedlings in a Jingxi rice field (22°51'N, 105°56'E) in Guangxi, China. The investigated area of rice fields, which was about 1.5 hectares, had an estimated disease incidence of 15 ± 5%. Leaf samples exhibiting symptoms of suspected brown stripe disease was collected and stored at 4 °C for bacterial isolation. The junction margins of lesions were surface-sterilized with 75% ethanol-saturated cotton swabs, and sterilized scissors were used to excise leaf sections (5 mm × 5 mm) from the diseased-healthy tissue interfaces. Within a sterile environment, these leaf fragments were soaked in 75% ethanol for three minutes, then ground and cultured on KB medium at a temperature of 28 °C for two days. A single colony (strain JX258) was then inoculated into sterile KB medium and incubated at 28 °C with a rotation speed of 220 rpm for 15 hours to obtain a bacterial suspension. Universal primers (27F/1492R) (Kinoshita et al., 2021) were used to amplify the 16S rRNA gene from strain JX258. A 1,394 bp fragment (GenBank PQ465223) amplified from strain JX258 was obtained and showed 97.80% sequence identity with Paracidovorax oryzae ATCC 19882 (Li, X et al. 2017). Specific primers (Aaaf3/ Aaar2, Aaaf3/ Aaar2) (Osdaghi E. 2023) successfully confirmed that this particular strain is indeed responsible for causing brown stripe disease in rice plants. Rice seeds were soaked in a bacterial suspension (1 × 10^6 CFU·mL⁻¹) for two days before undergoing germination followed by sowing in perlite under moist conditions (Supplemental Figures 1A-D). Brown lesions developed on leaves by 7 days post-inoculation (dpi), whereas mock-inoculated controls (10 mM MgCl₂) remained symptom-free. The seedlings soaked in a suspension of strain JX258 exhibited significantly reduced viability, germination, height, and growth rate, ultimately leading to seed mortality. Moreover, healthy rice leaves that were treated with a bacterial suspension (1 × 10^4 CFU·mL⁻¹) via spray inoculation, resulted in the manifestation of brown rice stripe disease symptoms within 7 days post-inoculation, P. oryzae was reisolated from the infected rice leaves, similar to those observed in the field, fulfilling Koch's postulates (Supplemental Figures 1E-F). The whole genome of JX258 was sequenced using the Sanger method, assembled, and analyzed to identify the strain at the species level. Whole-genome sequencing of JX258 (GenBank JBHJCP000000000.1) revealed 91.20% digital DNA-DNA hybridization (dDDH) and 98.14% average nucleotide identity (ANI) with P. oryzae ATCC 19882 (Meier-Kolthoff et al., 2013; Yoon et al., 2017). In the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), JX258 showed a similarity with a match probability of 73% to Paracidovorax avenae. The utilization of the unique carbon source on BIOLOG GEN III reagent strip by JX258 is shown in Supplemental Table 1. The pathogen was identified as Paracidovorax oryzae based on technological means of pathogenicity assessment, biochemical analysis, phylogenetic analysis (Supplemental Figure 1G), and molecular characterization. To our knowledge, this is the first report of Paracidovorax oryzae causing brown stripe disease on rice in Guangxi, China. Therefore, phytosanitary measures have to be implemented to prevent the introduction and spread of the pathogen in other areas.