First report of Ficus carica leaf spot caused by Bipolaris sorokiniana

Abstract

Iran is the third largest producer of figs globally, with more than 76.41 tonnes of fruit produced per annum. The following pathogens have been recorded on fig (Ficus carica, family Moraceae): Phomopsis cinerascens causing cankers (Banihashemi & Javadi, 2009); Neoscytalidium dimidiatum causing dieback (Ray et al., 2010); Neofusicoccum parvum causing stem cankers and twig blight (Aiello et al., 2020); Lasiodiplodia theobromae causing stem cankers; Alternaria alternata causing fruit rot (Alam et al., 2021); and Cerotelium fici causing rust disease. During a routine survey in May 2022, leaf samples from a fig tree with leaf spot symptoms were collected from an orchard in Bardaskan county, Khorasan Razavi province, Iran. The leaf spot was brown, irregular, and medium to large (5–12 mm) with a thin yellowish margin (Figure 1). The fungus was isolated from leaves using the method of Refaei et al. (2011). The fungal isolate was purified on potato dextrose agar (PDA) medium using the hyphal tip method. On PDA the colony was velvety, grey-black, with an irregular margin, sporulating abundantly, and conidiophores were single, 100–300 × 6–8 µm (n = 30), straight to flexuous, and septate. Conidia produced terminally, curved or straight, obclavate, cylindrical, fusiform or broadly ellipsoidal, 30–75 × 15–21 µm (n = 50), mostly tapering toward the ends, dark olivaceous brown-golden brown, 3–12 distoseptate. Sexual morph was not observed (Figure 2). These morphological characteristics were consistent with Bipolaris sorokiniana, as described by Manamgoda et al. (2014). For confirmation genomic DNA was extracted from seven-day old mycelium using the method provided by Zhong and Steffenson (2001). The complete internal transcribed spacer (ITS) region of the rDNA gene was amplified using ITS1 and ITS4 primers (White et al., 1990. After sequencing, isolate FIC1 was deposited in GenBank (Accession No. ON819428). A BLAST analysis revealed a 97% identity with isolates of B. sorokiniana (MH864697.1, MH864698.1). Phylogenetic analysis was performed in MEGA X software and revealed that the isolate clustered with another isolate of Bipolaris sorokiniana with high bootstrap support of 89% (Figure 3). To confirm pathogenicity, 15 surface-sterilised, detached healthy Ficus leaves were inoculated with a conidial suspension (106 conidia/ml) of isolate FIC1, and incubated at 25°C with a 12-hr photoperiod. Sterile distilled water was used for controls. Brown spots developed on inoculated leaves after seven days, whereas control leaves remained symptomless. The same pathogen was re-isolated, fulfilling Koch's postulates. Based on these results, this is the first report of B. sorokiniana as the causal agent of leaf spot disease on F. carica in Iran and globally. We are thankful to the University of Tehran for supporting this research.