First Report of Leaf Necrosis Caused by Alternaria alternata on Ceratostigma willmottianum in Italy

Abstract

Ceratostigma willmottianum Stapf, a member of Plumbaginaceae family, is a perennial shrub with a long summer flowering period, commonly grown in public and private gardens. During the autumn of 2018, leaf necrotic areas were observed primarily on lower leaves of 12-month-old plants of C. willmottianum growing either in pots or in the ground, in a private garden located in Biella province (northern Italy). Fifteen of 20 plants were affected (average disease severity 5%). Necrotic areas were light brown, irregularly polygonal, 1 to 4 mm in size, and surrounded by extensive chlorosis. Symptomatic leaves were washed for 1 min with sterile water and blotted dry, and small pieces of tissue were taken from the margins of necrotic areas and placed on potato dextrose agar for 7 days, at temperatures from 20 to 25°C. The developing dark green colored fungal colonies were transferred on the same medium, at the same temperatures, to obtain pure cultures. Successively, pure cultures were grown on sterilized leaves of C. willmottianum placed on potato carrot agar (Simmons 2007), under a light/dark regime of 10 h/14 h, from 20 to 23°C. On this medium, colonies produced branched conidiophores (up to two branches), supporting conidia borne in chains. Conidia were light gray-green, ovoid to obclavate, multicellular, and measured 13 to 43 × 4 to 12 µm (average 23 × 9 µm, n = 50). Conidia contained one to seven transverse and zero to three longitudinal septa. When present, beaks were 2 to 5 µm long. Based on these morphological characteristics, the fungus was identified as Alternaria sp. (Simmons 2007). DNA from one fungal isolate was extracted using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany), and a polymerase chain reaction was performed by amplification of the internal transcribed spacer (ITS) region of rDNA, the endoPG, the tub2, and the cmdA gene portions (Lawrence et al. 2013; Woudenberg et al. 2015). Four sequences with 529 (ITS), 476 (endoPG), 994 (tub2), and 671 bp (cmdA) (GenBank accession nos. MK204576, MK558219, MK558217, and MK558221, respectively) were obtained. A BLASTn search of these sequences showed 100% homology with Alternaria alternata (Fries) Keissler (accession nos. KU933199, MH728996, MH560610, and MG736308). A spore suspension of 0.8 × 10⁵ CFU/ml in sterile water was obtained from the pure cultures used for the observations described above. This suspension was sprayed (10 ml/plant) onto leaves of three healthy plants of C. willmottianum. Three plants treated with sterile water served as controls. All plants were placed in moistened plastic bags for 7 days and maintained in a greenhouse in which temperatures ranged from 20 to 22°C. The first leaf necrosis appeared 10 days after inoculation, causing an irregular necrotic area up to 4 mm in size. A. alternata was reisolated from affected leaves and identified by the amplification of the endoPG, tub2, and cmdA gene portions (GenBank accession nos. MK558220, MK558218, and MK558222). Control plants remained healthy, and the attempt to reisolate the pathogen failed. To our knowledge, this is the first report of A. alternata infecting C. willmottianum in Italy, as well as worldwide. This disease could become important owing to the reduction of the ornamental value of affected plants of C. willmottianum, the use of which is increasing in the landscape.