Morphological and molecular characterization of Podosphaera lini on Linum usitatissimum in IRAN

INTRODUCTION

Origin of flax (Linum usitatissimum L.), is the Near East and Mediterranean region, somewhere in Turkey, Iran and Syria (Cullis, 2007). Linseed cultivars have been planted to get fibers and oils. Powdery mildew diseases are widely distributed all around the world. They are economically important diseases on wild as well as cultivated plants. Powdery mildew disease of flax has been described from most flax-growing areas of the world (Reddy et al. 2013). Up to 38%, substantial yield loss has been reported as a result of its incidence (Pandey & Misra, 1992). In order to identify the powdery mildew collection on flax from Iran, morphological studies have been carried out and supplemented by phylogenetic analysis using the nuclear ribosomal DNA internal transcribed spacer (ITS) sequences.

MATERIALS AND METHODS

In the winter of 2019, germplasms of flax cultivars were planted for the regeneration of seeds on research fields of Seed and Plant Improvement Institute, Karaj, Alborz province, Iran. During spring and summer of 2020, the flax plants have been observed with powdery mildew symptoms on all aerial parts of plants, including stem, leaves, pods and fruits (Fig. 1).

The fungal structures were studied by means of a standard light microscope. Total DNA was extracted from fungal specimens by the Chelex method (Hirata & Takamatsu 1996). The ITS region of the nuclear rDNA (ITS1-5.8 S-ITS2) was amplified by polymerase chain reaction (PCR) with the powdery mildew specific primers PMITS1 (5'-TCGGACTGGCC(T/C)AGGGAGA-3') (Cunnington et al. 2003) and PM11 (5'-TACCGCTTC ACTCG CCGTTA-3') (Bradshaw & Tobin, 2020) for the first, and PM10 (5'-GGCCGGAAAGTTGTCCAA AC-3') (Bradshaw & Tobin 2020) and PM11 for the second PCR (semi-nested PCR). The nucleotide sequence was obtained using direct sequencing in a 3500 Genetic Analyzer (Applied Biosystems, USA) in Codon Genetic Group (Iran, Tehran). Sequences were analyzed and edited using MEGA 7.0 (Kumar et al. 2016). The obtained sequence was submitted to NCBI to find closely related sequences using a BLASTN search method. Several sequences with high query coverage and identity (97-100%) were selected for comparison and phylogenetic analyses. The phylogenetic tree was obtained using the Minimum Evolution method in MEGA 7.0 (Kumar et al. 2016). Sawadaea koelreuteriae (MT008194) was selected as the outgroup. The ITS sequence generated in this study was deposited in the GenBank under accession number MZ540779.

RESULTS AND DISCUSSION

Flax plants typically showed disease symptoms with different severity. In the early stages, the upper side of leaves covered with effuse, thin, white mycelium. The mycelium then developed to both sides, which often results in covering the entire surface of the leaves, stems and pods, along with quick covering the whole areal, especially the upper part of the plants. There were two types of mycelia. Primary mycelium was tender, hyaline, and thin-walled, while secondary mycelium was persistent, slightly thick-walled, white to yellowish, straight to sometimes flexuous, septate and branched. Hyphal appressoria were rare, solitary and nipple-shaped, sometimes with the crenulate margin occasionally indistinct. Conidiophores were solitary, straight, rarely curved, arising from top and lateral end of the hyphal mother cells with 70–150 (–220) μm length and 7.5–11 (–14) μm width. Foot cells were cylindrical or subcylindrical, mostly straight, occasionally curved, 22–53 (–130) μm long, sometimes widening at the base or apical part, followed by 1–3 (−4) shorter cells. Conidia were colorless, one-celled, thin-walled, not very swollen, bearing 3–6 in chain (Euoidium-type), usually ellipsoid to cylindrical, 25–30 (–36) × 8–16 (−18) μm, with a length/width ratio (L/W) 1.7-4.5. Conidia were typically containing fibrosin bodies. Conidial germination was observed almost perihilar, and sometimes lateral (Figs. 2). Chasmothecium was not observed. The specimen was deposited in Fungarium of University of Guilan (accession number GUM 1785).