First report of black root rot of lemon (<i>Citrus limon</i>) caused by <i>Sordaria sclerogenia</i> in Egypt

In November 2023, lemon trees (Citrus limon) showing black root rot, defoliation and mortality were observed in Wadi El-Natroun, Al-Behera governorate, Egypt (30° 59′ 16.5″N, 30°29′11.7″E), with a disease incidence of 25% (Fig. 1). Yellowing and dieback of the trees started from one side then developed to cover the whole tree. The diseased trees showed gummosis with a large quantity of exudates oozing out along the tree branches and trunk before death of the trees. Black, distorted and partially decayed roots were observed. Diseased roots were cut into small sections (1 cm), surface sterilised and transferred onto potato dextrose agar (PDA) plates supplemented with chloramphenicol (50 mgl−1). After incubation for three days at 20°C, the fungal colonies were purified using the hyphal tip technique onto new PDA plates. Colonies appeared similar to each other. The mycelium of one colony (SY25) had a cottony appearance with some scattered dense tangles, turning black with age, radiating, and covering the full plate after three days at 25°C in the dark (Fig. 2). Sclerotia-like structures formed on the aerial hyphae after two days. These were subglobose to irregular, dark brown turning to black at maturity, and measured 78.0-205.2 × 60.8-194.1 µm. Black ostiolate protoperithecia formed on the mycelia when cultured on modified corn meal dextrose agar medium (Fields & Grear, 1966). These measured 350–415 × 280–300 µm (Fig. 3). The phenotypic description of isolate SY25 was consistent with Sordaria sclerogenia (Fields & Grear, 1966). The ITS (White et al., 1990), and rpb2 (Liu et al., 1999) and tef1-α (Carbone & Kohn, 1999) genes were sequenced (GenBank Accession Nos. PQ869508, PQ879694 and PQ879693, respectively). The tef1-α sequence showed 100% identity with S. sclerogenia FGSC 2741 (FR774390). To identify the phylogenetic relationship, the tef1-α sequence of this fungus and that of closely related species from the GenBank database were aligned using MEGA software (ver. 11) using the maximum likelihood method. The fungus clustered with S. sclerogenia, confirming the identification of the fungal isolate (SY25) as S. sclerogenia (Fig. 4). No ITS or rpb2 sequences of S. sclerogenia were found in GenBank. Five pots (20 cm in diameter) filled with sterilised field soil and planted with one-year-old lemon trees (cv. Balady), grafted on Volkamer lemon rootstock, were used in a pathogenicity test under greenhouse conditions. Inoculum of S. sclerogenia isolate SY25 was prepared by culturing the fungus on sorghum/soil medium (3:1 v/v) and incubating for seven days at 20°C in the dark. Soil inoculation was performed by mixing 3% of the fungal inoculum with the surface layer of soil in each pot. Two weeks after inoculation, all lemon trees in the inoculated pots showed leaf yellowing, rolling, drying, defoliation and black root rotting (Fig. 5). Lemon trees that did not receive the fungal inoculum did not develop disease symptoms. Sordaria sclerogenia was isolated from the black rotted roots fulfilling Koch's postulates. Sordaria sclerogenia was isolated from soil and identified for the first time in 1966 by Fields & Grear (1966). Other species of Sordaria have been reported as pathogens (Petrović et al., 2024; Song et al., 2024). This is however the first report of S. sclerogenia as a pathogen causing black root rot of lemon trees. The authors are grateful to Prof. Ahmed M. Moharram from Assiut University Mycological Centre, Assiut, Egypt for his kind help in morphological identification of the fungus.