First report of Enterobacter cloacae causing leaf spot of chilli pepper (Capsicum annuum) in India

Abstract

India produces 5.7 million tonnes of chilli (Capsicum annuum L.) from 100,000 hectares of cultivated plants annually (Directorate of Economics and Statistics, 2020). During June-October, 2022 and 2023, chilli cv. Pusa Jwala cultivated at the Indian Agricultural Research Institute, New Delhi developed irregular, necrotic lesions surrounded by a chlorotic halo and brown necrosis at the leaf tip followed by defoliation (Figure 1). Disease incidence was 52% over an area of c. 800 m2. Bacterial colonies isolated from ten infected leaf samples, collected randomly across three fields, were circular, 2–3 mm, convex and greyish to white after 48 hours at 28±1°C on nutrient agar. They were positive in 3% KOH and oxidase tests (Schaad et al., 2001). They produced a hypersensitive reaction in tobacco cv. BX61 two days after injection with a suspension containing 109 CFU/ml. One isolate and a positive control strain (Enterobacter cloacae subsp. dissolvens NAIMCC-B-01345 from NAIMCC, India) were positive for ONPG, urease, nitrate reduction, Voges-Proskauer, aesculin hydrolysis, utilisation of ornithine, malonate, citrate, arabinose, xylose, trehalose, and glucose, and negative for phenylalanine deamination, H2S production, methyl red, indole, utilisation of lysine, adonitol, and lactose using a KB003 Hi25-Enterobacteriaceae identification kit (Hi Media, India). The isolate did not utilise the carbohydrates rhamnose, cellobiose, melibiose, saccharose, and raffinose in contrast to the positive control strain. The pathogenicity of the ten isolates was tested by syringe injection of a bacterial suspension (107 CFU/ml) into leaves of 30-day old chilli plants (cv. Pusa Jwala); sterile water was used as a negative control. Dark brown necrotic patches developed after two days in the injected region (Figure 2), followed by leaf decay and defoliation. No symptoms were observed on the negative control. DNA was extracted from bacteria re-isolated from inoculated plants using a modified CTAB protocol (Wang et al.,2010). PCR was done using primer pairs 27F/1492R and Hsp60-F/Hsp60-R which amplify the 16S rRNA and Hsp60 genes, respectively (Poussier et al., 2000; Hoffmann & Roggenkamp, 2003). Amplicons of the expected size were Sanger sequenced by Barcode Biosciences Pvt. Ltd (Bengaluru, India) and aligned manually. Sequences were deposited in Genbank with Accession Nos. OP897636 (16S rRNA) and OP918670 (Hsp60). An isolate (DLC-1) was also deposited in the Indian Type Culture Collection, New Delhi with accession number ITCCBY0001. A phylogenetic tree based on the concatenated sequences of both genes revealed that isolate DLC-1 clustered with two E. cloacae strains, SBP-8 and GGT036 from India and Republic of Korea, respectively (Figure 3). Enterobacter cloacae is a ubiquitous Gram-negative, facultatively anaerobic human pathogen, that has been reported to infect plants such as Allium cepa, Capsicum annuum and Manihot esculenta (García-González et al., 2018). Enterobacter cloacae strains SD4L and FQY013 were reported infecting rice and tomato plants recently (Cao et al., 2020; Jin et al., 2023), indicating the bacterium has a wide host range. To the best of our knowledge, this is the first report of E. cloacae causing chilli leaf spot, marginal necrosis and defoliation symptoms in the field in India and globally. Further research is needed to investigate the presence and impact of E. cloacae strains on plants in India.