First Report of Xanthomonas arboricola Causing Bacterial Blight on Pecan Trees in Portugal

Abstract

Pecan (Carya illinoinensis) is an economically important nut-producing tree in North America and widely cultivated in many countries (Thompson and Conner 2012), with a smaller commercial relevance in Portugal. During field surveys performed in June 2014 and July 2016, necrotic lesions were observed on leaves of pecan trees in an orchard in Alcobaca, Portugal. The lesions were similar to those on walnut (Juglans regia) caused by Xanthomonas arboricola pv. juglandis. Symptoms were characterized by small brown spots that coalesced to form necrotic areas surrounded by chlorotic halos. No damage was observed on fruits under these field conditions. Lesions from leaves collected during the two surveys were plated on yeast extract dextrose calcium carbonate medium and incubated for 4 days at 26°C. Yellow, mucoid colonies typical of Xanthomonas spp. were then streaked on a fresh nutrient agar medium to obtain pure colonies. Isolates were able to induce a hypersensitivity response on Nicotiana benthamiana leaves 72 h after inoculation (Lelliot and Stead 1987). A portion of the gyrB gene from two isolates (CPBF 765, GenBank accession no. MG897454; CPBF 1494, no. MG897455) was sequenced with primers XgyrB1F and XgyrB1R (Young et al. 2008). Both isolates were assigned to X. arboricola, showing 98 to 99% identity with sequences from X. arboricola pathovars. Pathogenicity tests were performed on C. illinoinensis and J. regia ‘Hartley’, both members of the family Juglandaceae. For each of the two isolates, three plantlets from each species, with at least four fully expanded leaves, were inoculated by spraying with bacterial suspensions of 10⁸ CFU/ml, optical density of 0.1. Plants inoculated with X. arboricola pv. juglandis LMG 747ᵀ were used as positive controls and plants sprayed with sterile distilled water as negative controls. All plantlets were kept under a 16-h photoperiod (24°C day and 18°C night) and observed daily for 4 weeks. Spots on walnut and pecan leaves were first observed 1 week after inoculation with CPBF 765 and CPBF 1494. Disease developed progressively, forming small necrotic areas surrounded by chlorotic halos. Similar symptoms were produced on the positive controls but absent on the negative controls. Bacterial colonies showing characteristics similar to the original isolates were reisolated from both plant species tested. In addition, multiplex polymerase chain reaction for detection of X. arboricola pv. juglandis (Fernandes et al. 2017) was negative for both strains. The genetic relationship of CPBF 765 and CPBF 1494 was further compared with representative strains of X. arboricola by multilocus sequence analysis using partial sequences of seven genes (Fischer-Le Saux et al. 2015): atpD, GenBank accession nos. MG897444 to MG897445; dnaK, MG897446 to MG897447; efp, MG897448 to MG897449; fyuA, MG897450 to MG897451; glnA, MG897452 to MG897453; gyrB, MG897454 to MG897455; and rpoD, MG897456 to MG897457. X. arboricola CPBF 765 and CPBF 1494 were genetically distant from monophyletic clusters of Xanthomonas pathovars pruni, corylina, juglandis, and populi. The isolates did not cluster together, making allocation to pathovar level unlikely. CPBF 1494 was more closely related to X. arboricola CFBP 1022 isolated from J. regia, whereas CPBF 765 was more closely related to X. arboricola CFBP 4021 and X. arboricola pv. celebensis CFBP 7150 isolated from Magnolia spp. and Musa spp., respectively (Fischer-Le Saux et al. 2015). This first report of X. arboricola causing leaf blight symptoms on pecan trees is a warning to countries with important pecan-growing areas.