First report of rice bacterial leaf blight and grain discolouration caused by Pantoea agglomerans in India

Abstract

A disease survey was carried out in Tamil Nadu, India during 2021 to assess the prevalence of diseases in major rice-growing areas and characterise the pathogens associated with infected plants. During the survey, rice plants at the seedling to grain-filling stage showing water-soaked, elongated, light brown lesions in the leaf margins and tips (Figure 1) were collected along with grains in a polythene bag, labelled and sealed. Leaves showing yellowing and drying from tips and margins were cut into small pieces, surface-sterilised in 1% sodium hypochlorite for one minute, rinsed three times with sterile distilled water and then allowed to air dry. After drying, the tissues were soaked for 30 minutes at room temperature in 0.5 ml of sterile distilled water and then a loop full of suspension was streaked onto nutrient agar (NA) medium. For the isolation of pathogen from infected seed, discoloured grain samples were surface sterilised in 3% sodium hypochlorite for one minute, rinsed thrice with sterile distilled water and allowed to air dry. Surface-sterilised grains were placed in a Petri plate containing NA medium. These plates were incubated at 28°C for one to two days. Upon incubation, straw to yellow-coloured colonies emerged. The colonies were further purified through single colony isolation on NA. The purified colonies were yellow in colour, translucent and undulate. (Figure 2.) The colonies exhibited umbonate surfaces with slightly concave centres (Al-Kidsawey et al., 2020; Delétoile et al., 2009). The pathogenicity of the isolates was demonstrated by inoculation of isolates on rice seedlings (cv. Co 39). For this, bacterial isolates were incubated in nutrient broth for 48 hours at 27°C. The bacterial suspension was adjusted to a concentration of 109 cfu/ml by adding sterile water. The bacterium was inoculated by cutting the tip (about 3 to 4 cm) of the fully expanded uppermost leaf with scissors and dipping in the bacterial suspension. Controls used sterile distilled water instead of the bacterial suspension. The inoculated plants were covered with polythene sheet overnight and incubated under laboratory conditions. The inoculated leaves developed progressive necrosis with colour shifts from pale green to brown within 3 to 14 days post inoculation, whereas the control plants exhibited no symptoms. Koch's postulates were fulfilled by isolating the bacterium on NA which produced yellow, flat to convex translucent colonies after 48 hours incubation. The bacterium was further subjected to biochemical tests using the KB002 HiAssorted Biochemical Test Kit (Himedia, Germany). The bacterium showed a positive reaction for citrate utilisation, phenylalanine deamination, nitrate reduction, glucose and arabinose utilization. It showed a negative reaction to lysine utilisation, ornithine utilization, urease, H2S production, adonitol, lactose and sorbitol utilization. Based on the morphological characters and biochemical tests, the bacterium was identified as a Pantoea sp. To further characterise the pathogen, DNA extraction was done, and a PCR assay was performed using universal primers 27F and 1492R targeting the 16S rRNA gene. Sequencing of the amplicon revealed that the bacterial isolates had 93.7% identity with Pantoea agglomerans type strain SGAir0210 (GenBank Accession No. CP028033.1). The partial 16S rRNA gene sequences of two strains of P. agglomerans were deposited in GenBank (OP784578 and OP784743). Pantoea agglomerans causing bacterial leaf blight and glume discolouration has been reported from Brazil, South Korea, Turkey and Venezuela (Aksoy & Boluk, 2019; Carrer Filho et al., 2018; González et al., 2015; Lee et al., 2010). Pantoea agglomerans also causes infection in humans (Al-Kidsawey et al., 2020; Delétoile et al., 2009)). Bacterial leaf blight in rice caused by P. stewartii subsp. indologenes has been reported from Tamil Nadu, India (Vinodhini et al., 2017). However, to our knowledge, this is the first report of P. agglomerans causing rice leaf blight and grain discolouration in India. Further studies are needed to ascertain the prevalence of P. agglomerans in commercial rice cultivars in other parts of India. We gratefully acknowledge the facilities provided by DST-FIST Lab, Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.