First Report of Pantoea agglomerans and Pseudomonas sp. Causing Bacterial Wilt of Strawberry in Iran

Cultivated strawberry (Fragaria × ananassa) is a major soft fruit crop worldwide (Sretenović et al. 2024). In July 2024, we surveyed two 2-ha greenhouses in Varamin County (Tehran Province, Iran) to investigate bacterial wilt observed on cultivar Sabrina. Affected plants showed rapidly expanding, water-soaked, black to brown necrosis on leaves and attached stems, sometimes with bacterial ooze; diseased plants failed to fruit. The average disease incidence was 10% in each of two greenhouses sampled using a five-point method (100 plants point) (Jiang et al. 2023). Ten symptomatic leaves and stems from different plants were surface sterilized and macerated in sterile distilled water (SDW). Aliquots were streaked on sucrose nutrient agar and incubated at 28°C. After 48h, yellow and creamy colonies, respectively, were consistently recovered; no fungi were observed. Standard bacteriological tests (Schaad et al. 2001) indicated Gram-negative, indole- and arginine dihydrolase-negative, catalase-positive bacteria. Two yellow colonies (EBC1 and EBC4) and one creamy colony (EBC8) were selected. Genomic DNA was extracted, and a gyrB fragment was amplified using universal primers (Yamamoto and Harayama 1995). BLASTn analysis of the gyrB sequences showed that EBC1 and EBC4 (GenBank accession nos. PQ807161 and PQ807162) shared 99.9% sequence identity with 100% query coverage (;940/1,000 bp) to Pantoea agglomerans reference strains (e.g., SUH1, CFBP8774, and DAPP-PG734), whereas EBC8 (PQ807163) showed 98.40% identity to Pseudomonas sp. strain B21-021. To refine the identification, atpD (for Pantoea) and rpoD (for Pseudomonas) were amplified following Brady et al. (2008) and Mulet et al. (2009), respectively. The atpD sequences of EBC1 and EBC4 (PQ807164 and PQ807165) showed 99 to 100% identity with 100% query coverage to P. agglomerans reference sequences (e.g., ASB05 and CFBP8774). The rpoD sequence of EBC8 (PQ807166) showed ;95.05% identity (100% coverage) to two Pseudomonas sp. (B16-257 and 3-2). Phylogenetic analysis (neighbor-joining, MEGA7) of concatenated targets clustered the EBC1/EBC4 with P. agglomerans and EBC8 with Pseudomonas sp. strains, with a high bootstrap value (99%). EBC1/EBC4 also yielded the expected amplicon with P. agglomerans-specific primers PagR-210F/626R (Shin et al. 2022). Isolates have been deposited at the Agricultural Biotechnology Research Institute of Iran (ABRII) as ABRIICC 21003 (EBC1), 21004 (EBC4), and 21005 (EBC8). Pathogenicity was assessed by spraying a cell suspension (1 × 106 CFU/ml) of isolates EBC1, EBC4, and EBC8, as well as a combination of EBC1 and EBC8, onto the leaves of 4-week-old potted strawberry plants (cvs. ‘Sabrina’ and ‘Camarosa’). When inoculation was carried out with a mixture of isolates, their ratio in the suspension was equal. Control plants were sprayed with SDW and cell suspension of P. agglomerans type strain ATCC 27155. Plants were placed in a plastic chamber (28 ± 2°C ;90% RH) for 24 h and then maintained in a greenhouse (27 ± 2°C). Experiments were performed twice, each with three plant replicates per isolate. Seven to 8 days post-inoculation, all plants inoculated individually with EBC1, EBC4, or EBC8, as well as those inoculated with the combination of EBC1 and EBC8, developed symptoms resembling those seen in the greenhouses. Control plants grew normally and remained asymptomatic. The bacteria were reisolated and identified as above, fulfilling Koch’s postulates. These results document P. agglomerans and a Pseudomonas sp. associated with strawberry bacterial wilt in Iran. To our knowledge, this is the first report of these bacteria causing wilt of strawberry in the country. The sequence accessions provide a baseline for diagnostics and surveillance.