First report of Sphingomonas yabuuchiae causing Bacterial Leaf blight Disease of rice in West Bengal, India

Rice (Oryza sativa) is one of the major cereal crops grown in West Bengal, India. During August 2024 bacterial leaf blight symptoms with white to straw-coloured streaks and wavy margins were observed over approximately 1.5 ha area at Lakshmipur (25.31539° N, 88.76996° E) with a disease incidence of 42.2% at the active tillering stage. Infected leaves samples were collected and cut into small fragments (5mm) from the margins between symptomatic and healthy tissues. The fragments were surface-sterilized with 70% ethanol for 1 min, rinsed thrice with sterile distilled water, and dried on sterilized filter paper. It was then transferred in sterilized test tubes containing 2 ml distilled water for 20 min followed by fourfold serial dilutions and streaking onto peptone sucrose agar (peptone 5 g, sucrose 20 g, K2HPO4 0.5 g, MgSO4·7H2O 0.25 g, and Bacto Agar 15 g per litre of distilled water). The isolated colonies were bright yellow, convex, opaque, mucoid, circular, and exhibited smooth margins after 48 h of incubation at 28±1°C. Purification was done taking single colonies in nutrient agar plates and stored at 4°C for future use. For pathogenicity testing, a bacterial suspension from a 48-hour-old nutrient broth culture was adjusted to 107 CFU/ml using 2000V Vis Spectrophotometer at OD 600 nm and inoculated onto 45-day-old rice cv. Samba Mahsuri (BPT 5204) using the clip inoculation method (Ke et al. 2017). Precautions were taken to avoid seed-borne infection by pre-treating seeds with 500 ppm streptocycline (90% streptomycin sulfate and 10% tetracycline hydrochloride, w/w) while control plants received sterile water. Initial symptom appeared within 6 days as brownish grey lesion progressing downward with wavy margins, and complete leaf blighting occurred with light brown necrosis within 20 d post-inoculation. No symptoms were observed in control plants. The pathogen was reisolated and molecular identification was performed by amplifying and sequencing the 16S rRNA (~1500 bp) and two housekeeping genes, atpD, using atpD-F and atpD-R primers (Tambong et al. 2021) and rpoB gene, using Univ_rpoB_F_deg and Univ_rpoB_R_deg primers (Ogier et al. 2019). The sequences obtained were deposited in GenBank under accession numbers PZ028620 (16S rRNA), PZ098258 (atpD) and PZ098259 (rpoB). Phylogenetic analysis conducted using the Neighbor-Joining method in MEGA 11 confirmed that the isolate closely clustered with Spingomonas yabuuchiae (NR028634), S. pseudosanguinis (PX502184) and S. pseudosanguinis (KP216807). To our knowledge, this is the first study to report S. yabuuchiae as a causative agent of bacterial leaf blight of rice in West Bengal, India. Previously, Sphingomonas has been reported in several countries, including Benin, Burkina Faso, Gambia, Ivory Coast, Mali, Nigeria, Tanzania, and Togo (Kini et al. 2017), as well as in southern Karnataka, India (Bijoy et al. 2023) but the species was not confirmed. The confirmation of S. yabuuchiae as the causal agent of bacterial leaf blight in rice will accelerate the development of effective prevention and control measures for this emerging disease.