First Report of Leaf Spot Caused by Alternaria alternata on Phlox maculata in Italy

Abstract

Phlox maculata (common name spotted phlox), Polemoniaceae family, is an herbaceous perennial plant with showy panicle inflorescences, commonly grown in mixed borders. During summer 2019, about twenty 5-year-old plants of P. maculata growing in a garden located in the Biella province (northern Italy) showed symptoms of a leaf spot, starting from the basal part of the foliage. Symptoms consisted in brown spots with not well-defined margins, irregularly circular to polygonal in shape, up to 2 to 3 mm in diameter, sometimes confluent. The internal part of spots dried. Chlorosis and yellowing surrounded the spots, involving the most part of affected leaf surfaces, due to the spread of the disease. The ornamental value of affected plants was significantly reduced. Several symptomatic leaves were washed in sterile water for 1 min. Isolations were carried out, taking several small fragments of tissues from the margins of the spots and placing them on potato dextrose agar. Fungal colonies that developed were dark green at the end of development. Successively, single-spore colonies were obtained, and pure cultures were transferred on potato carrot agar (PCA, Simmons 2007) under a light/dark regime of 11 h/13 h. On this medium, colonies formed branched conidiophores with brown, multicellular conidia, ovoid to obclavate in shape, containing one to four (average 2.4, n = 50) transverse septa and zero to one (average 0.1, n = 50) longitudinal septa. Conidia measured 10.8 to 29.3 × 5.3 to 11.2 µm (average 18.7 × 7.6 µm, n = 50). If present, the beak was 2.1 to 11.0 µm (average 5.5 µm) long. These morphological characteristics permitted identifying the fungus isolated from P. maculata as Alternaria section alternata (Woudenberg et al. 2015). The fungal DNA from the 19/33 isolate was extracted using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany), and PCR was performed using the ITS (White et al. 1990), the rpb2, and the endoPG primers (Woudenberg et al. 2015). Three sequences with 529 bp (ITS), 889 bp (rpb2), and 452 bp (endoPG) (GenBank accession nos. MN871825, MN871979, and MN871978, respectively) were produced. A BLASTn analysis of these sequences exhibited 100% identity with the reference strain CBS 117.44 of A. alternata (Fries) Keissler in rpb2 and endoPG portions (accession nos. KP124772 and KP124001, respectively) and 99.6% identity in ITS region (accession no. KP124303). One P. maculata fungal isolate (19/33) was grown on PCA. A spore suspension obtained from 15-day-old cultures was sprayed at the concentration of 10⁵ CFU/ml onto leaves of three healthy plants of P. maculata. Three control plants were treated with sterile water. All plants were closed in moist plastic bags for 7 days and maintained at temperatures ranging from 15 to 28°C. The first symptoms of leaf spot appeared 10 days after the inoculation, and A. alternata was reisolated from affected leaves. Control plants remained symptomless. The pathogenicity test was repeated once using the isolate 19/40, obtaining the same results. To our knowledge, this is the first report of A. alternata on P. maculata in Italy as well as worldwide. The spread of A. alternata on P. maculata and the difficulty to manage this disease, particularly in public gardens, may discourage the use of this ornamental in Italy.