First Report of Bacterial Spot Caused by Xanthomonas arboricola pv. pruni on Apricot in Hungary

In June 2018, leaf spot symptoms suggesting Xanthomonas arboricola pv. pruni (Xap) infection were detected on almond (Prunus dulcis) cultivar ‘Fournat de Brezenaud’ and in October also on almond cultivars ‘Tetenyi bőtermő’, ‘Tetenyi rekord’, and ‘Tetenyi kedvenc’ in a 1.5-ha almond orchard within a 44-ha stone fruit plantation in Pest county, Hungary. Disease incidence was low (<5%) in June and high (50 to 90%) in October. Initial symptoms appeared mostly on the abaxial surface of leaves as 1 to 2 mm, angular, water-soaked spots. Subsequently, 3 to 5 mm circular, light yellow spots, the center of which underwent necrosis, were observed on the adaxial surface of leaves as well. Occasionally the spots coalesced, and necrotic areas sometimes fell out. Infected fruits exhibited water-soaked or dark-brown sunken lesions on the almond epicarp and endocarp that sometimes coalesced, with gummy exudates. Isolations from diseased leaf and fruit samples were carried out on a modified Tween-80 medium (Schaad et al. 2001). Thirty isolates forming yellow, mucoid colonies on yeast dextrose calcium carbonate agar medium were selected that induced a hypersensitive response in Nicotiana tabacum L. ‘Samsun’ (10⁸ CFU/ml). All isolates proved to be Gram-negative rods, oxidase negative, strictly aerobic, with typical biochemical characteristics of the genus Xanthomonas (Schaad et al. 2001). Sixteen isolates were selected for further analysis and identified as Xap on the basis of quinate metabolism, starch hydrolysis, Bio-PCR (Ballard et al. 2011), and duplex-PCR (Pothier et al. 2011). PCR products of isolates XapHU3 (isolated from cv. Tetenyi bőtermő) and XapHU4 (isolated from cv. Tetenyi rekord) obtained with gyrB- (Parkinson et al. 2007) and ftsX-specific (Pagani 2004) primers were sequenced. Obtained sequences of gyrB and ftsX were 100% identical in XapHU4 and XapHU3 (sequences of XapHU3 were submitted to GenBank, MK561379 and MK561380, respectively). Database searches in NCBI GenBank by nucleotide BLAST indicated that in both strains (XapHU4 and XapHU3) obtained sequences of gyrB and ftsX have 100% identity with the corresponding reference genes of the Xap strain CFBP 3894 (NZ_LOMI01000009.1, NZ_LOMI01000020.1) isolated in New Zealand and the Spanish Xap strain IVIA 2626.1 (NZ_LJGN01000040.1, NZ_LJGN01000033.1) based on the reference genomic sequence database. Furthermore, gyrB and ftsX sequences of XapHU4 and XapHU3 are also 100% identical to corresponding genes of several other Xap strains including the Hungarian Xap strains isolated earlier from apricot (XapHU1, KX950802.1, KY039173.1) and from peach (XapHU2, MG049922.1, MG049921.1), respectively. Pathogenicity tests carried out for the 16 isolates mentioned above were conducted on symptomless almond leaves (cv. Tetenyi bőtermő) by a detached leaf bioassay (Randhawa and Civerolo 1985) by injecting a bacterial suspension (10⁷ CFU/ml) into two leaves per strain. Three days after the treatment, water-soaked, angular spots appeared on all inoculated leaves except the water-treated controls. Leaf spots became necrotized 6 days after inoculation. The pathogen was reisolated from lesions of inoculated leaves and its identity confirmed by duplex-PCR (Pothier et al. 2011). Xap was first identified in Hungary on Japanese plum by a local plant protection service. The recent detection of Xap on apricot and peach causing severe fruit and leaf spot symptoms (Schwarczinger et al. 2017, 2018) certainly indicates the disease’s spread. Although almond production in Hungary is on a small scale, Hungarian almond growers from now on should expect recurrent infections by Xap, because during our survey in June 2019, we found more severe symptoms in the same almond orchard as in 2018. This is the first report of Xap on almond in Hungary.