Investigation of Fungi Causing and Associated with Narcissus Leaf Spot Disease in Southern Khorasan Province

Narcissus (Narcissus tazetta) is one of the perennial herbaceous and ornamental bulbous crops that is used as a cut flower garden plant and potted plant. Fungal diseases are one of the harmful factors of this ornamental plant in different parts of the world. In 2018, symptoms of Narcissus leaf spot (NLS) were observed in some planting areas of this plant in South Khorasan province. This study was carried out to identify the casual and associated fungi with NLS in Southern Khorasan province.

Samples with leaf spot symptoms were collected from two regions, Khoosf and Tabas, from 2018 to 2019. Plant tissues were transferred to the laboratory of the Department of Plant Protection in separate paper bags. The tissues were cultured on a Potato Dextrose Agar (PDA) medium and purification was performed on a 2% water-agar medium using the Hyphal tip method. Fungal agents were isolated and identified based on morphological and molecular characteristics.  The DNA of representative fungal isolates was extracted according to the CTAB method. To molecularly confirm the species, part of the rDNA gene was amplified using ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'- TCCTCCGCTTATTGATATGC-3') primers. Sequencing of PCR products was done by Pishgam Biotechnology Company and deposited in the GenBank with Accession number: MN829259. Sequences were edited with Chromas software and the edited sequences were saved in FASTA format. The ITS-based phylogenetic tree was constructed using the Maximum Likelihood Method with MEGA ver6.0 software. For the pathogenicity test, the Narcissus plant was inoculated with mycelia plugs. In order to maintain moisture and establish the fungus, the inoculation area was covered with parafilm.  The plants were covered with polythene bags for 24 hours. The bags were removed after 24 hours. In the control plants, the potato-dextrose-agar culture medium without fungal mycelium was used. All plants were examined after 3 to 14 days and the pathogen was re-isolated from the inoculated plant that showed leaf spot.

Symptoms of narcissus leaf spot (NLS) disease in the sampled areas included elongated, elliptical, and red to brown circles on the leaf. A total of 50 samples of different leaf spot symptoms were collected. In this study, 20 fungal strains were isolated and identified based on morphological and molecular characteristics. The growth rate of this isolate on the PDA medium was relatively fast and the average growth rate after 7 days was 6.6 cm. Pycnidia were formed abundantly on the PDA medium after 7 days. The pycnidiospores were transparent, mostly single-celled (3-4×5-7), sometimes two-celled (11.5-15 ×5.8), round to oval and curved. Chlamydospores (8-15) were dark brown, unicellular and multicellular, intercellular and rarely terminal. Based on morphological characteristics, the strains were finally identified as Phoma narcissi.The NCBI blast search revealed that the ITS region Phoma narcissi in our study had similarities to Didymella curtisii, Phoma narcissi, and Phoma sp. 100%, 100%, and 99% respectively. Phylogenetic analysis confirms that the examined strain belongs in a clade with strains of Ph. narcissi. In Pathogenicity tests, symptoms of the disease were observed on all plants 7 days after inoculation. No symptoms developed on non- inoculated plants. The original pathogen was re-isolated from the leaf spots of inoculated plants. Phoma s.l. is one of the most ubiquitous fungal genera, characterized by its great ecological diversity and difficult identification. According to our knowledge, the main cause of NLS disease in the east of the country is the Ph. Narcissi, Fusarium sp. and Cladosporium sp. were associated agents with this disease. Since the identification of the disease agent is effective in choosing effective and efficient strategies for disease control, the results can help to adopt effective methods in disease management.