First Report of <i>Diplodia seriata</i> and <i>Dothiorella viticola</i> Associated With Citrus Dieback Disease in Morocco

Between 2022 and 2024, diseased twigs and branches of Citrus clementina and Citrus sinensis trees exhibiting symptoms of dieback (Figure 1), cankers, bark cracking and gummosis were collected randomly from three major citrus-growing areas in Morocco: 200 samples from the Eastern region (Berkane), 150 samples from Beni-Mellal-Khenifera, and 150 samples from Rabat-Salé-Kénitra. Small tissue fragments (c. 3 × 3 mm) from leaves and transverse sections of twigs and branches were aseptically excised and surface-sterilized by immersion in 70% ethanol for 3 min, followed by 2% sodium hypochlorite (NaClO) for 3 min. Samples were then rinsed three times with sterile distilled water and air-dried on sterile filter paper. Subsequently, the disinfected tissues were placed onto potato dextrose agar (PDA) plates supplemented with streptomycin sulphate (150 µg/mL; Sigma-Aldrich, USA) and incubated at 25°C for 7 days. Pure cultures were obtained by transferring single hyphal tips to PDA amended with streptomycin antibiotic. Fungal colonies morphologically resembling members of the family Botryosphaeriaceae were recovered consistently from all wood fragments cultured on PDA. The initial colonies appeared white, exhibiting rapid growth, a dense, woolly texture, and abundant mycelium (Figure 2). As the colonies matured, the mycelium, initially white, gradually developed a grey-brown to black pigmentation. Conidia produced by these isolates were initially hyaline, becoming dark grey upon maturation. They were aseptate, ovoid in shape, with moderately thick walls, and measured 22.6–23.3 × 9.5–10.5 µm. Other isolates obtained were characterized by colonies bearing a prominent aerial, cottony mycelium and a centrally originating dark pigmentation (Figure 3). The conidiogenous cells observed in these colonies were cylindrical, hyaline and smooth-walled, measuring 8–14 × 3–7 µm. The conidia were brown, with subcylindrical, rounded apices, and exhibited a single septum at early developmental stages, measuring 16–26 × 7–12 µm. For species level identification, genomic DNA was extracted from pure cultures following the protocol of Doyle and Doyle (1990). Amplification of the complete internal transcribed spacer (ITS) region of the ribosomal DNA was performed using ITS1 and ITS4 primers (White et al. 1990). The products were sequenced and deposited in GenBank under Accession Nos. PV866973 (Dothiorella viticola) and PV866974 (Diplodia seriata). BLAST analysis revealed 99.6% identity with the type strain of Dothiorella viticola (CBS 117009; NR_111186) and 99.6% identity with the type strain of Diplodia seriata (CBS 112555; NR_111151), respectively. Pathogenicity assays were conducted on twelve one-year-old Citrus aurantium shoots for each fungal isolate. A wound approximately 1 cm in length was made on each shoot and inoculated beneath the epidermis with a mycelial plug excised from the actively growing margins of 15-day-old colonies cultured on PDA in the dark. Control plants (n = 12) received sterile PDA plugs as described by Jabiri et al. (2020). All inoculation sites were sealed with plastic film, and the plants were maintained in a controlled environment chamber at 25°C. Fifteen days post-inoculation, necrotic, brown-to-dark lesions developed at the inoculation points. Lesions on shoots inoculated with D. seriata and D. viticola had a mean length of 4.31 cm and 4.15 cm, respectively. No symptoms were observed in control plants. Re-isolation and re-identification of the pathogens from diseased tissue fulfilled Koch's postulates, confirming the ability of D. seriata and D. viticola to cause disease symptoms on citrus shoots. Notably, D. seriata has previously been implicated in causing branch canker and citrus dieback in China (Xiao et al. 2021). To our knowledge, this study is the first to establish a link between D. seriata and D. viticola and the symptoms observed on citrus in Morocco. This study was financially supported by the Phytopathology Unit of the Department of Plant Protection of the Ecole Nationale d'Agriculture de Meknès.