Occurrence of Xanthomonas arboricola pv. pruni Causing Bacterial Leaf Spot and Shot-Hole on Peach in Montenegro

Abstract

During May and June 2019, bacterial leaf spot was observed on peach (Prunus persica L. Batsch) in a young plantation (2017, 2018) on four different cultivars (Sugar Time, Britney Lane, Royal Bell, and Royal Time) in the Podgorica region of Montenegro. Diseased trees appeared sporadically, and incidence of symptomatic leaves per infected tree was 10 to 15%. Angular water-soaked spots surrounded by chlorotic tissue were observed along leaf midribs or margins, turning light brown in later stages. Necrotic lesions sometimes dropped out, giving the leaves a “shot-hole” appearance. When spots coalesced, large areas of necrotic foliar tissue formed. Eventually leaves turned yellow, resulting in premature defoliation. Margins taken from healthy tissue and necrotic lesions were homogenized in sterile distilled water (SDW) and spread onto YDC agar (Schaad et al. 2001), on which mostly pale yellow, translucent, circular, raised, and mucoid Xanthomonas-like colonies were observed after 3 days incubation at 26°C. Twelve representative isolates coded as Xp1 to 12 (Sugar Time, Xp1 to 3; Britney Lane, Xp4 to 6; Royal Bell, Xp7 to 9; and Royal Time, Xp10 to 12) were aerobic, gram negative, catalase positive, and oxidase and arginine dihydrolase negative; hydrolyzed esculin and gelatin but not starch; produced H2S but not indole; and did not reduce nitrate (Schaad et al. 2001). All reactions corresponded with the reactions of the reference strain NCPPB 3156 of Xanthomonas arboricola pv. pruni (Xap), causal agent of leaf spot, shot-hole, and canker of stone fruit. The identity of the isolates was confirmed by PCR using the Xap-specific primer pair XapY17-F/XapY17-R for the ABC transporter ATP-binding protein (Pothier et al. 2011). A single unique target band of 943 bp was amplified for all isolates tested. Furthermore, sequencing of the housekeeping genes fyuA, rpoD, and gyrB (Young et al. 2008) showed 100% identity of tested isolates with genomic sequences of Xap strains deposited in the NCBI database (CFBP 411, 3921, 5580, 6653, 7099, and 7100) based on BLAST analysis and a neighbor-joining phylogenetic tree. The sequences of four isolates representing each cultivar were deposited in GenBank under accession numbers MN520623 to 26 for fyuA, MN520627 to 30 for rpoD, and MN520631 to 34 for gyrB gene for Xp1, Xp4, Xp7, and Xp10, respectively. Pathogenicity was confirmed on (i) young, fully expanded, healthy, detached peach leaves using a hypodermic syringe without a needle, pressed against the abaxial side of the leaf (bacterial suspension at 107 CFU/ml from a 72-h YDC culture) and (ii) immature fruits stabbed and pressing the bacterial suspension (107 CFU/ml) until overflow. Xap reference strain NCPPB 3156 served as a positive and SDW as a negative control. Inoculated leaves were kept under controlled conditions at 25°C, 16/8-h day/night photoperiod, and 60 to 80% relative humidity. All tested bacterial isolates and the reference strain developed water-soaked spots on leaves 3 days and on fruits 4 days after inoculation. Ten days after inoculation the spots became dark brown and necrotic. SDW-treated controls were negative. Xap was successfully reisolated and confirmed to be Xap using PCR (XapY17-F/XapY17-R). This study demonstrates Xap on peach as a new host in Montenegro. Xap was previously reported from other (15 of 44) European countries (https://gd.eppo.int/taxon/XANTPR/distribution), mainly on peach and plum, and from Montenegro on almond (Panić et al. 1998). As the causal agent of a major disease of stone fruits, Xap is a quarantine organism in Europe (EPPO A2 List of Pests recommended for regulation for EU). Therefore, it is necessary to implement containment and/or eradication measures to prevent further spread to new areas and/or new hosts. Furthermore, a nation-wide survey on the (re)occurrence of Xap in other hosts such as almond (reported by Panić et al. 1998), apricot, and plum advised.