First Report of Erwinia persicina Causing Leaf Blight of Geranium in Iran

Pelargonium spp., commonly known as geraniums, are among the economically important bedding and pot plants worldwide. In May 2020, irregular and water-soaked lesions were observed on the leaves of Pelargonium × hortorum (L.H. Bailey) in a commercial greenhouse (100 m2) in Rasht County, Gilan Province, with an incidence of 5%. Four diseased plants were sampled for pathogen isolation. Small pieces from the infection zone of symptomatic leaves were surface disinfected in 70% (v/v) ethanol for 2 min, blotted dry and macerated in 5 ml of sterile distilled water (SDW). Droplets of sample suspension were streaked on nutrient agar containing 1% sucrose (SNA) and the plates were incubated at 30°C for 72 h (Basavand et al. 2022). Four single colonies of the dominant bacterium with creamy-like, convex and round appearance on SNA were purified and characterized morphologically and biochemically (Schaad et al. 2001). The isolates were Gram-negative, facultatively anaerobic, catalase positive and positive in pectolytic activity and growth at 37°C. They were negative in production of fluorescent pigment on King's B medium, oxidase and arginine dihydrolase. The strains produced acid from glucose, sucrose, cellobiose, mannitol and raffinose and produced a water-soluble pink pigment on nutrient agar (NA) medium. The colony's color transitioned from white or creamy to pink as the culture aged due to production of carotenoid compounds. Two representative isolates (PL21 and PL22) were further identified by PCR amplification of the partial sequences of 16S rRNA, gyrB and atpD using FD1/RD1 (Weisburg et al. 1991), UP1/UP2r (Yamamoto and Harayama 1995), and 03-F/04-R (Brady et al. 2008) primers, respectively. The amplification products were sequenced and submitted to GenBank: 16S rRNA, PP599026 and PP599031; gyrB, PP619832 to PP619833, atpD, PV067134 to PV067135. BLASTn analyses of the obtained sequences revealed the following identities and query coverages with Erwinia persicina sequences of strains B64 and SR15 deposited in GenBank: 100.0% of 16S rRNA region (900/1200 bp), 99.84-100.0% of gyrB (747/800 bp) and 99.84-100.0% for atpD (629/640 bp). The phylogenetic tree based on the concatenated sequences of the gyrB and atpD genes using the neighbor-joining method with MEGA7 software showed that PL21 and PL22 isolates clustered solely with E. persicina. The cell suspensions of the isolates PL21 and PL22 in SDW were prepared from 24 h old culture grown on NA medium and adjusted turbidimetrically at 600 nm with spectrophotometer to contain 1 × 10⁷ CFU/ml. The suspensions were sprayed on the fully expanded leaves of Pelargonium plants using a refillable mini atomizer. After inoculation, plants were incubated in a greenhouse at 29 ± 2°C and 50 ± 2% relative air humidity. Experiments were repeated twice, each time with three leaf replicates per isolate. SDW was sprayed on negative control leaves. Symptoms were observed on inoculated plants 5 to 6 days post-inoculation, while no symptoms appeared on the plants sprayed with SDW. Bacteria were reisolated only from symptomatic leaves and again identified as E. persicina by 16S rRNA sequencing. To our knowledge, this is the first report of E. persicina causing leaf blight disease of geranium in Iran. This work will enhance awareness among geranium growers, emphasizing the importance of recognizing leaf blight disease caused by E. persicina and implementing effective control measures to minimize or prevent significant economic losses.