First Report of Fire Blight Caused by <i>Erwinia amylovora</i> on Pear in Saudi Arabia

In the spring of 2020 and 2021, pear trees (Pyrus communis cv. Williams) in orchards (27°46'36.0"N 42°29'44.7"E, 30°00'00.2"N 40°15'11.8"E, and 28°44'52.9"N 36°18'47.8" E) in Hail, Al Jouf, and Tabuk regions exhibited fire blight symptoms. After removing bark, the affected trees showed shoot blight, brown-blighted shoot tips and blossom blight, dead flowers on the stems, and reddish-colored cankers. The disease incidence varied from 10% to 25%. Pathogen was isolated from 21 symptomatic samples including fruits, flowers, and shoots. Bacteria were isolated from washed tissues on King's B (KB) and semi-selective CCT media (Ishimaru and Klos, 1984). After 48 hours, colonies resembling Erwinia amylovora on KB media were 1.5-2 mm in diameter, white, circular, slightly convex, with a smooth surface, and exhibited no fluorescence under ultraviolet light. Colonies on CCT after 72 h were 3-4 mm in diameter, mucoid with shiny surfaces, semitransparent, speckled with craters, and slightly violet. All isolates were purified by subculturing on KB. All isolates were Gram-negative and rod-shaped, fermentative of glucose, positive for catalase and negative for oxidase, and potato rot. They induced a hypersensitive reaction when infiltrated in tobacco leaves (cv. Xanthi). Based on morphology and biochemical tests (EPPO, 2004), the strains were identified as Erwinia sp. Twenty-six strains from Saudi Arabia (SA) and the reference strain (NCPPB 683T) hydrolyzed gelatin and formed white, highly mucous colonies on the levan medium. These strains could not reduce nitrate to nitrite and tested negative for urease and indole production. All the isolates and the reference strain were confirmed to be E. amylovora based on a PCR 0.9-kb DNA fragment amplification with a species-specific primer set, A/B targets pEA29 (Bereswill et al. 1992). 16S-rDNA fragments from Saudi isolates were amplified with 27F and 1492R primers (Lane, 1991). Purified amplicons from PCR were sequenced (OR717505 and OR743536-OR743560), and a BLAST search of the GenBank database revealed 100% (927/927) homology with E. amylovora strain CP066796.1. The housekeeping gene rpoB was PCR amplified with primers CM7-F and CM31b-R (Rezzonico et al. 2012), and the products were sequenced (PP465516-PP465541). BLAST analysis showed 100% (944/944 nt) and 96.19% (908/944 nt) identities with the sequences of E. amylovora ATCC 15580 CP066796.1 and E. pyrifoliae CP201486 CP103445.1, respectively. To fulfill Koch's postulates, SA strains were inoculated on five healthy 3-month-old clones of apple (Malus domestica cv. Gala) per strain with a 10-μl bacterial suspension containing 107 colony-forming units per milliliter by injecting directly in the veins of the upper second leaf plus 5 healthy plants injected with sterile distilled water as control. Plants were incubated at 28°C for six days under a 12-hour light regime. Observed symptoms were similar to the ones observed in the field. The experiment was replicated twice. Bacterial colonies on CCT media were re-isolated from the inoculated apple rootstocks and confirmed by the A/B primer set. To our knowledge, this is the first peer-reviewed report of E. amylovora in SA since the fire blight-like symptoms were observed in SA in 2013 (Alhudaib, 2013). Further research will identify new host plants for the fire blight pathogen within SA which is important due to the importation of pome fruit seedlings (quince, apple, and pear) from neighboring Jordan where E. aylovora was reported (Tehabsim et al. 1992).