First Report of Alternaria alternata Causing Leaf Spot on Spinach (Spinacia oleracea) in Italy

Abstract

Spinach (Spinacia oleracea L.) is widely cultivated in many countries and is affected by a complex of foliar diseases (Correll et al. 1994). During March 2018, disease symptoms were observed on 15 to 30% of leaves of 90-day-old spinach cultivar Matador plants grown for the fresh market, at a density of 80 to 90 plants/m², in an open field of approximately 0.5 ha in Cuneo province in Piedmont, northern Italy. Symptoms first appeared on the basal and middle leaves as small circular spots (1 to 3 mm), developing as concentric rings, which were dark brown in color, and each spot was surrounded by a chlorotic halo. As the disease progressed, lesions expanded, sometimes covering the entire leaf surface. Small pieces (1 to 2 mm) of affected leaf tissues were surface sterilized in 1% sodium hypochlorite for 1 min, rinsed in sterile water, and plated on potato dextrose agar amended with 25 mg of streptomycin sulfate per liter. Alternaria sp. was consistently isolated from lesions on 15 plants after 4 days of incubation of the leaf sections at 22°C (Simmons 2007). Ten-day-old monoconidial cultures of AltSpin2-18, selected as representative of 10 morphologically similar isolates grown on potato carrot agar, produced chains of 10 to 13, multicellular, obclavate to obpyriform conidia measuring 14.7 to 41.0 µm (average 27.1 µm) in length and 7.2 to 12.1 µm (average 9.6 µm, n = 40) in width, with zero to three longitudinal and two to five transverse septa. When present, the beak measured 1.3 to 8.3 µm in length. Genomic DNA was extracted from the isolate AltSpin2-18, and the following genes were amplified: internal transcribed spacer (ITS) region of rDNA, endopolygalacturonase (endoPG) gene, β-tubulin (tub2) gene, and histone 3 (H3) (Woudenberg et al. 2015). Polymerase chain reaction products were sequenced at the BMR Genomics Centre (Padova, Italy). A BLASTn search of the sequences showed 100% identity to the ITS rDNA region of Alternaria alternata KX343153 and several other Alternaria spp. Moreover, the AltSpin2-18 isolate showed 100% sequence identity to A. alternata AB047682 (endoPG), MH521955 (tub2), and MF070866 (H3). The sequences of the AltSpin2-18 isolate were deposited to GenBank with accession numbers MK078634 (ITS rDNA), MK085978 (endoPG), MK085977 (tub2), and MK085979 (H3). The overall sequence analysis of these four loci assigned the species to A. alternata. Pathogenicity tests were performed by spraying leaves of 30-day-old healthy cultivar Matador plants grown in six pots (10 plants/pot) with a suspension (10⁶ conidia/ml) of AltSpin2-18. Plants sprayed with sterile water served as a control. Plants were covered with plastic bags for 5 days after inoculation at 19 to 22°C and maintained in a greenhouse. Lesions similar to those observed in the field first developed on leaves 6 days after inoculation, resulting in 50 to 60% disease severity 20 days after inoculation, whereas control plants remained healthy. Alternaria sp. morphologically identical to the original isolate was reisolated consistently (100% frequency) from the inoculated plants. Two independent replication runs of the pathogenicity tests provided the same results. Alternaria leaf spot caused by A. alternata on S. oleracea has been reported in the Kingdom of Saudi Arabia (Marraiki et al. 2012), Poland, India, and China (Farr and Rossman 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on S. oleracea in Italy. The disease could become a significant problem owing to the importance of the crop in Italy, covering approximately 5,540 ha in open field production and 43,141 ha under protection.