First report of Neofusicoccum parvum causing canker and dieback on Alnus glutinosa in Portugal

In March 2022, sunken cankers were observed on the stem and branches of young and mature Alnus glutinosa trees in central Portugal (Canelas, 40°42’17’’N; 8°33’59’’W). Cankers were characterised by necrotic lesions on the outer and inner bark that progressively girdled the circumference of stem and branches causing extensive canopy dieback (Figure 1). The incidence of the disease was estimated at 70% (linear plot of 50 m long). To isolate the causal agent, 20 samples including twigs, branches and stems were collected from seven diseased trees chosen at random along the transect. Isolations were performed from ten chips (5 mm2) of inner bark and xylem tissues cut aseptically from the margin of the necrotic lesions and transferred onto Petri dishes containing potato dextrose agar (PDA). Isolations yielded twenty fungal colonies showing a greyish-white aerial mycelium typical of Botryosphaeriaceae species. Based on colony appearance and morphology of conidia (hyaline, fusiform, aseptate and thin-walled) produced after 15 days on sterile Fraxinus ornus twigs on PDA at 25°C, isolates were identified as Neofusicoccum parvum (Figure 2). The identity was confirmed by sequencing the internal transcribed spacer region of two representative isolates, using the universal primers ITS1 and ITS4. DNA extraction, PCR amplification and sequencing were performed according to Linaldeddu et al. (2020). A BLASTn search showed 100% identity with sequence AY236943 of the ex-type cultures of N. parvum (CMW9081). The ITS sequence of a representative isolate (CBP109) was deposited in GenBank (Accession No. OQ547268). Pathogenicity tests were performed by inoculating an agar mycelium plug (3 mm diameter) cut from the margin of an active colony (isolate CBP109) growing on PDA onto the wounded stem of two-year-old common alder seedlings using the methodology reported by Linaldeddu et al. (2020). Ten seedlings were inoculated with the fungus and ten, used as controls, with a sterile PDA plug. Seedlings were kept in controlled conditions at 21°C and watered regularly for 30 days. At the end of the experimental period, all seedlings showed necrotic lesions similar to those observed in the field; six out of ten seedlings also showed wilting symptoms (Figure 3). Average lesion size was 38 ±14 mm (mean ±SD). Control plants remained asymptomatic. Neofusicoccum parvum was successfully re-isolated from all inoculated seedlings, thus fulfilling Koch's postulates. This study represents the first report of N. parvum affecting Alnus glutinosa in Portugal and in Europe. Neofusicoccum parvum is a plurivorous pathogen reported on about 300 plant hosts (Phillips et al., 2013; Batista et al., 2021). In Portugal N. parvum has been reported in both forest and urban environments and its pathogenicity confirmed on different hosts such as pine and oak trees (Lopes et al., 2016; Batista et al., 2020). The occurrence of this invasive species on declining Alnus glutinosa trees and the recent discovery of several Phytophthora species in the same sites (Bregant et al., 2023), could explain the rapid devastation of extensive alder ecosystems observed recently in Portugal. This research has been financially supported by the Land Environment Resources and Health (L.E.R.H.) doctoral course (University of Padova) and project F4F—Forest For Future (CENTRO-08-5864-FSE-000031) from Programa Operacional Regional do Centro (Centro 2020), Portugal 2020 and Fundo Social Europeu. Thanks are due to the Portuguese Foundation for Science and Technology (FCT/MCTES) for the financial support to CESAM (UIDP/50017/2020 + UIDB/50017/2020 + LA/P/0094/2020) and the PhD grants of Sandra Hilário (SFRH/BD/137394/2018) and Eduardo Batista (PD/BD/135535/2018).