Lasiodiplodia citricola, a new causal agent of Acacia spp. dieback

Acacia species are woody perennial trees belonging to the family Mimosaceae and native to Australia. In summer 2021, unusual necrotic sunken lesions and wood discolouration were observed at the stem level both in the rootstock and in the scion, as well as the graft union on young A. dealbata plants grafted on A. retinodes in a nursery in Milazzo (north-western Sicily, Italy). In addition, gumming from cracking of the bark and shoot blight were observed (Figure 1). The disease incidence was estimated around 20% on approximately 2,500 plants. Symptoms were different from those previously observed on A. retinodes in Sicily (caused by Cylindrocladium pauciramosum) (Polizzi & Catara, 2001). Diseased vascular stem tissue segments were cut and transferred onto potato dextrose agar medium (PDA) and incubated at 25°C in the dark for seven days. Colonies resembling Botryosphaeriaceae was consistently isolated (Figure 2). Pycnidia grown on pine needles agar (PNA) were observed using a stereoscopic microscope and harvested. Conidia were hyaline, non-septate, and ellipsoid. The length × width of 50 conidia were 22.52 × 12.54 μm (Figure 3). The internal transcriber spacer region (ITS) was amplified with primers ITS5/ITS4 (White et al., 1990), and primers Bt2a/Bt2b were used for the partial β-tubulin (tub2) (Glass & Donaldson, 1995). Resultant sequences were deposited in GenBank with Accession Nos. OM891495-OM891502 for the ITS and OM918761-OM918768 for tub2. For comparison, 66 additional sequences were selected and aligned according to recent literature on the Botryosphaeriaceae (Zhang et al., 2021). Maximum parsimony analysis of tub2 region was performed in PAUP v.4.0a (Swofford, 2002). Clade support was assessed by 1,000 bootstrap replicates. Neodeightonia phoenicum served as an outgroup. Our isolates clustered within the group of Lasiodiplodia vaccinii (65% bootstrap value) (ex-type CGMCC 3.19022), described by Zhao et al. (2019), and now synonymised with L. citricola (Zhang et al., 2021). Based on both morphological and molecular results, the organism was identified as L. citricola (Figure 4). Pathogenicity tests were conducted on three one-year-old A. retinodes rootstocks and three one-year-old A. dealbata grafted pot plants. The inoculum consisted of a mycelial plug from a seven-day-old culture inserted at three different inoculation points on each plant wounded with a cork borer (15 cm from each other) along the stem. Controls were inoculated with sterile PDA. After one week, symptoms of wood discolouration and dieback were observed and then 70% of inoculated plants died (Figure 5). Once symptoms appeared, re-isolations were conducted as described above showing colonies resembling Lasiodiplodia, therefore Koch's postulates were fulfilled. To our knowledge, this is the first report of L. citricola causing shoot blight and canker on A. retinodes and A. dealbata worldwide. High humidity is an important factor for spore dispersion, therefore sprinkler irrigation should be avoided in greenhouses. Moreover, grafting is a crucial step, through which infections can easily occur, especially in the case of L. citricola, which has previously been associated with graft failure (Chen et al. 2013). Disease management starts with sanitation of the tools and of the propagation material to avoid infection with Botryosphaeriaceae. Funding was provided by the Programma Ricerca di Ateneo MEDIT-ECO UNICT 2020–2022 Linea 2-University of Catania (Italy); Starting Grant 2020, University of Catania (Italy); Fondi di Ateneo 2020–2022, University of Catania (Italy), Linea Open Access. Research Project 201–62018, Univ: 5A722192134.