First Report of Bacterial Blight of Anthurium Caused by Xanthomonas campestris in Iran

Abstract

In May 2023, symptoms of bacterial blight were observed on anthurium (Anthurium andraeanum Linden ex André) cultivars ‘Pistache’ and ‘Midori’ in four commercial greenhouses in Pakdasht County, Tehran Province, Iran. The initial symptoms were small water-soaked lesions that turned dark brown and necrotic, irregular in shape and 2 to 4 cm in diameter. The disease incidence was around 15% in a total greenhouse area of 4 ha. Leaf pieces (5 × 5 mm) excised from the margins of symptomatic tissues were surface-disinfested, macerated in sterile distilled water (SDW), and the resulting suspension was streaked onto yeast dextrose calcium carbonate agar (YDC) and sucrose nutrient agar (SNA). Bright yellow, circular, convex, and mucoid bacterial colonies were consistently recovered. Six representative isolates, originally obtained from six symptomatic plants, were randomly selected for further characterization. Based on their phenotypic features (Schaad et al. 2001) and genus-specific PCR amplification using primers X1/X2 (Maes 1993), the isolates were identified as belonging to the genus Xanthomonas. Phenotypically, they were Gram-negative, aerobic, catalase-positive, oxidase-negative, and arginine dihydrolase-negative. Furthermore, the isolates hydrolyzed aesculin, gelatin, starch, and Tween-80. To accurately identify the isolates, partial sequences of the housekeeping genes, gyrB, dnaK (Young et al. 2008) and gltA (Almeida et al. 2010), were amplified and sequenced. The resulting amplicon sequences were deposited in GenBank under the following accession numbers: PZ019873–PZ019875 and PP817750–PP817752 for gyrB; PZ019867–PZ019869 and PP817753–PP817755 for dnaK; and PZ019870–PZ019872 and PV258277–PV258279 for gltA. Nucleotide BLAST analysis of these three genes (gyrB: 600/700 bp, dnaK: 890/910 bp, and gltA: 498/501 bp) revealed 98–99% identity (100% query coverage) with the ATCC 33913 (AE008922), the type strain for X. campestris pv. campestris (Xcc). A multilocus phylogenetic analysis based on the concatenated sequences of these genes revealed that the isolates clustered within the clade composed of X. campestris, supported by a bootstrap value of 99%. Koch’s postulates were fulfilled in two independent experiments using four anthurium cultivars (‘Pistache’, ‘Midori’, ‘Sunglow’, and ‘Angel’). For each isolate, a bacterial cell suspension containing approximately 1 × 106 CFU/mL was spray applied on the plant leaves. Another set of plants of the same cultivars was inoculated by injecting 100 μL of the inoculum into the leaves using a sterile disposable syringe fitted with a hypodermic needle. Control plants were treated either by spray application of SDW or with a cell suspension of reference strain Xcc ICMP 6541, originally isolated from cauliflower (Young et al. 2008). Three leaf replicates were used per isolate. After 5 to 6 days, all inoculated anthurium cultivars developed the same leaf spot symptoms, specifically dark brown and necrotic lesions, that visually resembled those observed in the greenhouse, whereas all control plants remained asymptomatic. Bacteria reisolated from the symptomatic leaves of inoculated plants exhibited identical colony morphologies on YDC and SNA plates to those originally described, and their identity was reconfirmed as X. campestris based on phenotypic traits and gyrB sequencing. This study is the first report of X. campestris naturally infecting anthurium in Iran. Given Pakdasht County's status as a leading center for floricultural production, the implementation of urgent phytosanitary measures and the development of resistance breeding programs are strongly recommended.