First report of Pseudomonas syringae pv. atrofaciens Causing Leaf Spot to barley (Hordeum vulgare) in the Republic of Korea.

Abstract

Barley (Hordeum vulgare L.) is one of the staple food in the Republic of Korea, with 68,000 metric tons produced on 24,000 ha in 2022 (Ministry of Agriculture, Food and Rural Affairs 2023). In April 2018, leaf spots with brownish black edges were observed on barley leaves at the early tillering stage in the field of 1,100 m² at Iksan-si (35°56'21.4"N 126°55'56.5"E), with an incidence of 5%. Early symptoms on the leaf appeared as small, dark brown to black spots, then enlarged to gray lesions with brownish-black edges. Leaf cuttings (5 x 5 mm) from three symptomatic plants were surface-sterilized with 70% ethanol for 1 min, followed by 1% hypochlorite for 1 min, then macerated in sterile distilled water. The macerates were streaked on Nutrient Agar (Difco, MD, USA) media and incubated at 28 °C in the dark for 2 days. After single colony isolation, three isolates from three different plants were designated as isolates Ik 510-1, Ik511-1, and Ik513-1, then deposited in the Korean Agricultural Culture Collection (https://genebank.rda.go.kr) under the accession numbers KACC 23555, 23556, and 23557, respectively. Bacterial colonies were circular, convex, and cream-colored on NA media and produced fluorescent blue-green pigment on King Agar B media (Millipore, Merck, Darmstadt, Germany). The morphology of the three isolates was identical and showed the same characteristics as Pseudomonas syringae (von Kietzell and Rudolph 1997). Biolog Identification System (Biolog Inc., Calif., USA) and BLAST similarity of the 16S rRNA gene sequence (GenBank accession no. MK140998, MK140999, and MK141000, respectively) with the primer set of F27/R1492 (Heuer et al. 1997) showed that the three isolates belonged to the Pseudomonas syringae species complex, and MLSA (multi locus sequence analysis) with partial sequences of gltA, gapA, gyrB, and rpoD genes (Hwang et al. 2005) showed that the three isolates were on the same clade with Pseudomonas syringae pv. atrofaciens LMG5095^PT. For confirmation, PCR with the primer set of Psat-F/Psat-R (Animal and Plant Quarantine Agency 2023) amplified the 477 bp amplicons of the target region, thus identifying them as Pseudomonas syringae pv. atrofaciens. To determine the pathogenicity to barley, bacterial suspensions (O.D.₆₀₀=0.1 in PBS, phosphate buffered saline) of the three isolates was infiltrated at approx. 0.05 ml into leaves of three 2-week-old plants 'Keunalbori1ho' grown in pots using a needless syringe, while PBS was infiltrated as negative control, respectively. Infiltrated plants were grown in a growth chamber at 25℃ with a 16-h/8-h light/dark cycle. 5 days after inoculation, the infiltrated areas showed water-soaked symptoms, then dried to gray with characteristic brown-black edges only on leaves infiltrated with bacterial suspension and no symptoms on leaves infiltrated with PBS. The pathogenicity test was repeated three times, and each time the pathogen was re-isolated and identified by 16S rRNA gene sequences, thus fulfilling Koch's postulates. In the Republic of Korea, the average temperature and rainfall in the spring season has increased by 0.24℃ and by 1.8 mm per decade in the last 106 years (Kim et al. 2018), resulting in an increased risk of the disease due to cool and humid conditions (Toben et al. 1991). To the best of our knowledge, this is the first report of Pseudomonas syringae pv. atrofaciens causing leaf spot to barley in the Republic of Korea.