First report of Cryphonectria parasitica causing dieback on Sweet Chestnut (Castanea sativa Mill.) in Algeria.

Abstract

In July 2023, chestnut tree mortalities were reported in the parc châtaigneraie, situated in the Akfadou mountain range, in Tizi Ouzou, Algeria. Dead sweet chestnut (Castanea sativa) trees exhibited symptoms including crown dieback, withered and dried leaves clinging to the branches, trunk and branch cracks and cankers, with a proliferation of epicormic shoots under the cankered areas (Fig 1). Examination of the cankers with a hand magnifying lens, exposed orange masses, erupting from under the bark. Cankers were found on the lower part of the trunk, with few occurrences on the branches. Flat mycelial fans were observed throughout the bark. Samples of infected wood were collected and subjected to further analysis in the laboratory. Close observation of fragments of infected wood under the stereomicroscope showed orange to dark orange fruiting structures (pycnidia), originating in the cambium, which showed a discoloration in cross section. After incubation (24 hours) in a humid chamber, twisted yellowish tendrils oozing from the pycnidia were observed (Fig 2). Pycnidia were isolated from the bark and transferred onto full strength potato dextrose agar (PDA). Fast growing whitish then turning to yellow-orange colonies developed within 5 days, with a growth rate of 7 mm/day at 30°C. Conidiospores were hyaline, aseptate, oblong to cylindrical, 2-4 x 1.5-2 µm in size. Total genomic DNA was extracted and the Elongation factor 1a (EF-1a) (Carbone & Kohn, 1999) and Beta-tubulin (βT1) (Glass & Donaldson, 1995) gene regions were amplified using the Phire Direct PCR kit (ThermoFisher) and PCR amplicons were subsequently sequenced. The EF-1a (PQ416673) and βT1 (PQ416670) sequence identity was determined using the Genbank database with 99.69% and 100% identity to Cryphonectria parasitica (OM112254.1 and MK578073.1) respectively. Based on disease symptoms, cultural and morphological characters, and DNA sequence similarity, the fungal pathogen was identified as Cryphonectria parasitica. Koch's postulates were performed to confirm the pathogenicity of C. parasitica by using chestnut tree detached twigs as described in Hunter et al. (2013). Twelve Chestnut twigs (20 cm long and 2 cm in diameter) were surface sterilized then put to dry in a laminar hood under a constant air flow for 10 minutes. Seven day old mycelial plugs from cultures of C. parasitica were used to inoculate two branches with three replicates. The six controls were inoculated with PDA plugs. After 5 weeks incubation at 20°;C (9/15 hours light/dark regime, respectively), canker zones developed around the inoculation point, from which the pathogen was reisolated. The recovered C. parasitica isolate was confirmed using methods described above. The resulting consensus sequences of the recovered pathogen matched C. parasitica with EF-1a 99.71-100% identity (PQ416674, PQ416675) and βT1 100% identity (PQ416671, PQ416672). This represents the first report of C. parasitica in Algeria, and the second report in Africa (EPPO, 1994). According to forestry archives, the chestnut groves of Tizi-Ouzou infected with C. parasitica, come from the Cévennes, in France (Rabhi & Messaoudene, 2018). Further field surveys need to be conducted to detect the presence of this disease in other chestnut orchards across Algeria, and phytosanitary regulation measures must be undertaken in order to counter the spread of this disease in Algeria and the North African region.