23/07/2020
APS Publications. DISEASE NOTES

First Report of Leaf Spot Caused by Alternaria alternata on Anemone japonica in Italy

Anemone japonica L. (Japanese windflower) is a late summer flowering perennial ornamental belonging to the Ranunculaceae. In August 2019, symptoms of a leaf spot were observed on 10-year-old plants, growing in a private garden located near Biella in northern Italy (45°36′00″N, 8°03′00″E). Initial infections appeared as brown to black, circular to oval spots, 5 to 30 mm in diameter, present on both leaf surfaces. Leaf spots gradually developed concentric rings, which then coalesced into large infected areas, resulting in defoliation. At temperatures ranging between 15 and 28°C (average monthly temperature in August of 24°C), 25 to 30% of about 100 plants were affected. Isolations of the causal agent were made by excising infected leaf tissue pieces, previously immersed in a solution containing 1% sodium hypochlorite for 1 min, rinsing in sterile tap water, and placing them on potato dextrose agar (PDA) amended with 25 mg/liter streptomycin sulfate. PDA plates were incubated for 5 days at 20 to 22°C with 12 h of daylight, after which olivaceous to black fungal colonies developed on 90% of the plates. Monoconidial cultures of two fungal isolates were designated 19-51-31 and 19-51-36. On potato carrot agar (Simmons 2007), the two isolates incubated for 10 days at 22°C with 8 h of daylight developed multicellular, obclavate to obpyriform brown conidia that were produced on branched conidiophores, and measured 49 to 115 μm long × 2.4 to 4.7 μm wide, presenting a similar sporulation pattern as for group 4 (Simmons and Roberts 1993). Conidia were formed mainly in chains of 10 to 14 and measured 11.5 to 42.0 µm (average 27.7 µm, n = 40) in length and 3.2 to 13.9 µm (average 9.9 µm, n = 40) in width. Conidia contained zero to three longitudinal and two to five transverse septa, and, when present, beaks measured 0.9 to 10.5 µm. The fungus was identified as Alternaria sp. based on morphological characteristics (Simmons 2007). DNA extraction for molecular identification of the two fungal isolates was carried out using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). PCR assays were done using primers for ITS (White et al. 1990), rpb2, Alt a 1, endoPG, and OPA10-2 (Woudenberg et al. 2015). Sequences with 528 to 530 bp (ITS), 933 to 939 bp (rpb2), 439 to 444 bp (Alt a 1), 459 to 460 bp (endoPG), and 615 to 619 bp (OPA10-2) were obtained (GenBank accession nos. MT044149, MT043353, MT043357, MT185590, and MT043355 for fungal isolate 19-51-31; and MT044150, MT043354, MT043358, MT185591, and MT043356 for isolate 19-51-36, respectively). The sequences of both isolates using a BLASTn analysis showed 100% identity with the ex-type CBS 916.96 of Alternaria alternata (Fries) Keissler in ITS, rpb2, and Alt a 1 portions (accession nos. AF347031, KC584375, and AY563301, respectively) and 99% identity in endoPG and OPA10-2 portions (accession nos. JQ811978 and KP124632, respectively). Phylogenetic analysis of endoPG sequences grouped the 19-51-31 and 19-51-36 isolates with the reference isolates of A. alternata and not with the isolates of other Alternaria species. Pathogenicity tests were performed by spraying leaves of 1-year-old healthy potted A. japonica plants with spore suspensions of both fungal isolates that contained 105 conidia/ml. Plants sprayed with tap water only served as negative controls. Five plants per treatment were used in tests carried out twice. Plants were covered with plastic bags for 5 days after inoculation and maintained in a glasshouse at temperatures ranging between 22 and 26°C. Leaf spots, similar to those observed on the garden plants, developed 7 days after inoculation. Control plants remained with no symptoms. To our knowledge, this is the first report of A. alternata on A. japonica in Italy, as well as worldwide. A. alternata previously has been reported on Anemone occidentalis in the United States (Woudenberg et al. 2015). The disease has appeared in several gardens on A. japonica, influencing floral characteristics and reducing market value.

The author(s) declare no conflict of interest.

Funding: This work was funded by the Ministry of Education, Universities and Research (MIUR), Local research (ex 60%).