First report of Xanthomonas euvesicatoria pv. euvesicatoria causing bacterial leaf spot in chilli pepper (Capsicum sp.) in Indonesia

Chilli pepper (Capsicum spp.) is an important crop with increasing global consumption; the top three chilli pepper-producing countries are China, Mexico and Indonesia (FAOSTAT, 2023). One of the main bacterial diseases of chilli pepper is bacterial leaf spot which causes yield loss due to damaged and unsaleable fruits (Utami et al., 2022). The disease has been reported in Indonesia but there is no information about the causal agent. In April and May 2022, bacterial leaf spots were observed in a commercial plantation in Bantul, Yogyakarta, Indonesia (8°00'5.5" S and 110°18'46.9" E). Small black lesions were observed on the leaves (Figure 1a). These lesions were surrounded by yellow circles or irregular, dark brown or black greasy spots (Figure 1b). A total of 100 diseased leaves were collected and surface-sterilised with 70% ethanol then wiped with absorbent paper. Squares containing individual lesions were excised from the samples using a sterile scalpel and bisected. One half of each lesion was placed on a drop of sterile water on a microscope slide and overlaid with a coverslip to inspect for oozing (×100 magnification), the other half was placed in an another drop of water for culturing and as a crude PCR template. Bacterial oozing was observed in almost all lesions sectioned. For six ooze-positive samples, bacteriological streaks on nutrient agar plates supplemented with 2% starch were made from the second set of samples. Plates were sealed with plastic film and incubated for 48 hours at 28°C. Following isolation and successive subculturing (×3) from single colonies, one isolate was obtained (BY1) and an amylase test (Schaad et al., 2001) was conducted to differentiate X. euvesicatoria pv. euvesicatoria from X. vesicatoria and X. euvesicatoria pv. perforans which also cause bacterial leaf spot. After incubation at 28°C for 48 hours, three drops of 10% iodine solution were added onto the edge of the colonies and were observed as amylase-negative owing to the lack of clearing in the medium. The bacterium was further identified by molecular characterisation. Six primer pairs Xeu2.4/Xeu2.5 (Moretti et al., 2009), gyrB-F/gyrB-R (Kyeon et al., 2016), and Bs-XeF/Bs-XeR, Bs-XvF/Bs-XvR, Bs-XgF/Bs-XgR and Bs-XpF/Bs-XpR (Koenraadt et al., 2009) were used to screen ooze samples as well as aqueous suspensions of pure cultures of the isolated bacterium. Aqueous suspensions were made by picking a single colony with a sterile pipette tip and swirling in 100 μl distilled water. PCR was performed using 12.5 μl 2X Phire reaction buffer (Thermo Fisher Scientific, USA), 0.5 μl of each primer (10 μM), 10.5 μl Milli-Q water, and 1 μl template with the following thermocycle: 95°C for 5 minutes followed by 40 cycles of 95°C for 20 secs, 64°C for 30 secs, and 72°C for 25 secs, followed by 72°C for seven minutes. The PCR products amplified with gyrB-F/gyrB-R and Xeu2.4/Xeu2.5 were sequenced and the sequences deposited in GenBank (Accession Nos. OQ943232 and OQ943233). Trimmed sequences were aligned with reference sequences of Xanthomonas spp. and the gyrB gene region showed 99.85% identity to X. euvesicatoria pv. euvesicatoria (KY658944.1 and EU015388.1) (Figure 2). The region amplified using the Xeu2.4/Xeu2.5 primers was identical to X. campestris pv. vesicatoria AM039952.1, a strain now classified as X. euvesicatoria pv. euvesicatoria (Figure 3). A pathogenicity trial was conducted in a greenhouse using two chilli pepper species (C. frutescens cv. Trisula Hijau and C. annuum var. annuum). The isolate was grown on nutrient agar and incubated for 48 hours at 28°C. Single colonies obtained from the pure cultures were suspended in distilled water and adjusted to 1 × 108 CFU/ml. Three five-week-old chilli pepper plants were inoculated with the bacterial suspension by spraying 1 ml bacterial suspension per leaf. Control plants were treated with sterile distilled water. Black spot symptoms, consistent with those observed in the field, were seen after four days in both chilli pepper species but no symptoms were seen on control plants. Symptoms on inoculated plants were ooze-positive, and the ooze was confirmed positive for X. euvesicatoria pv. euvesicatoria using the Xeu2.4/Xeu2.5 and gyrB-F/gyrB-R primers. Based on the biochemical, molecular and pathogenicity tests, the strain isolated from chilli pepper in Indonesia was identified as X. euvesicatoria pv. euvesicatoria. Bacterial leaf spot on chilli pepper has probably existed for some time in Indonesia but this is the first report of the aetiology of the disease. This research was supported by Australia Awards Scholarship- John Allwright Fellowship 2019–2023 and ACIAR SLAM/2018/145.