First report of Alternaria alternata complex causing leaf spot on Vitis vinifera in Italy.

Grapevine is one of the main cultivated crops in the Northern Italy. During the summer 2023, high incidence (80% out of 350 plants) of brown, irregularly necrotic spots, measuring up to 2-3 cm, were observed on experimental potted two-years-old Vitis vinifera (cv. "Sauvignon Blanc" grafted onto K5BB (n = 180)) at Susegana (TV) (GPS coordinates: 45°51'07.7"N 12°15'19.9"E). The same symptoms were also observed on ungrafted potted two-years-old Vitis rootstocks (Kober 5BB, SO4, 110 Richter, 420A, Fercal, Rupestris Du Lot), located in the same area (n = 170).Symptomatic leaves were collected, underwent superficial sterilization in EtOH 70% and then washed twice in sterile water. Small leaf tissue fragments (± 3 mm) were cut from the margins of necrotic spots and plated on Czapek Yeast Autolysate agar (CYA) amended with Rose Bengal (Frisvad and Samson 2004; Ottow 1972). The plates were incubated at 28°C for one week in dark condition and, the fungal isolate, was sub-cultured on CYA. Fungal isolate, initially appearing greyish-white, later transitioned to black or darkish olive-green with a lighter border. Under a microscope at 40x magnification, conidia were light brown and obclavate to elliptical, divided by transverse and vertical septa. Their dimensions were 31 to 87 µm (avg. ± 56 µm) length and 11 to 32 µm (avg. ± 24.8 µm) width (n = 50). Morphological characteristics resembled those of the small-spored group of the Alternaria genus (Simmons 2007). Fungal genomic DNA was isolated from lyophilized mycelium using the phenol-chloroform method (Nerva et al. 2019). The ITS, ATPase, GAPDH, CAL and ACT sequences were amplified using primer pairs ITS1/ITS4, ATPDF1/ATPDR1, GPD-1/GPD-2, CALDF1/CALDR1 and ACTDF1/ACTDR1 respectively (Lawrence et al. 2014; White et al. 1990; Lawrence et al. 2017). Subsequently, the PCR products were Sanger-sequenced and uploaded to GenBank under the accession numbers: OR976478 (ITS), PP024642 (ATPase), PP456742 (GAPDH), PP456740 (CAL) and PP456741 (ACT). A maximum likelihood phylogenetic analysis, based on the concatenated gene sequences, was performed using W-IQ-TREE (Trifinopoulos et al. 2016). The analysis showed that the isolate was closely related to A. tenuissima and A. alternata, belonging to the A. alternata complex (Woudenberg et al. 2015). Both in vitro and in vivo pathogenicity tests have been performed. A detached leaf assay was conducted in vitro: asymptomatic grape leaves cv. "Sauvignon Blanc", previously water-washed, were placed in Petri dishes containing sterile water - 1% agar. A conidial suspension was prepared by scraping one-week old fungal cultures grown at 28°C on CYA medium with sterile water, followed by filtration. A final concentration of 1 x 105 conidia/mL was sprayed onto the leaves. Three leaves were sprayed with sterile water as a control group. All Petri dishes were enclosed in plastic bags to maintain high humidity and were incubated at 32 °C under dark conditions. After one week, leaf spots similar to the above-described symptoms appeared on the inoculated leaves, whereas no symptoms were observed in the control group. The in vivo pathogenicity test was carried out twice spraying the same inoculum concentration on leaves of two years old healthy potted grape plants (cv. "Sauvignon Blanc", n = 10), grown under greenhouse conditions (26 ± 2°C). All plants were kept in a plastic bag for seven days; three control plant were sprayed with sterile water. Irregularly brown leaf spot, similar to those observed initially, appeared on inoculated plants 15 days after inoculation. The same fungus was reisolated from lesions, confirming the Koch's postulates. To our knowledge this is the first report of A. alternata as the causal agent of leaf spot disease on Vitis vinifera in Italy. Considering the significant role of grapevine cultivation, particularly in Veneto region, it is essential to allocate greater focus on this new pathogen.