Fungal Isolation from Plant Tissues

The tissue separation method was adopted for culturable fungal isolation (Attitalla et al., 2010 ). Isolation was done either on potato dextrose agar (PDA) or alkyl ester agar (AEA) according to the method of Chen X. et al. (2021 (link)). Diseased and healthy tissues (5 × 5 mm pieces) were directly inoculated on both PDA and AEA plates. The remaining tissues were placed in the dark at 28°C for 5 days, and the hyphae were then ready to be transferred to a fresh plate. Pigment, growth rate, and morphological traits were the distinct criteria for fungal isolation. Purified cultures were stored at 4°C on PDA slants. Molecular identification was conducted according to the method of Huang et al. (2021 (link)). The PCR products were electrophoresed on a 1% agarose gel and the purification of the ITS 500 bp fragment was exercised using Gene JET (Thermo Fisher Scientific, Waltham, MA, United States). Subsequently, the fragments were then sequenced and used as a query in a BLASTN search of the NCBI nr database1. For the purpose of identification of each isolate, matches with identity values >98% were used.