The first report of Alternaria cantlous causing leaf spot on lentil (Lens culinaris)

Abstract

During a survey in May 2019, lentil (Lens culinaris cv. Syria 229) plants cultivated in an open field in Tiaret, Algeria showed chlorotic leaf spots with an incidence of about 10%. The symptoms began as small, irregular lesions and expanded with time from the edge to the centre of the leaves. To identify the causal agent, diseased leaves were disinfected in 2% NaOCl for three minutes, rinsed thrice with sterile water, plated onto potato dextrose agar and incubated at 25±1°C for seven days. Fungal colonies were dark grey with dense growth. Conidiophores were brown, erect, simple or branched, 2.5-3.75 × 21–42.5 μm. Conidia were mostly ovoid or ellipsoidal, light to dark brown with 1 to 3 transverse septa and 0 to 2 longitudinal septa and ranged in size from 8.75-17.5 × 7.5-15 μm (n = 25) (Fig. 1). Based on morphological characters, isolates were identified as Alternaria sp. (Simmons, 2007). To confirm the identity of the fungus, a PCR was done to amplify ITS and tef1 regions of a representative isolate, ST2 (White et al., 1990; Carbone & Kohn, 1999). The obtained sequences (GenBank Accession Nos. OQ256241, OQ269665) were analysed with BLASTn and had 99.58 and 99.52% identity, 479/481(99%) and 209/210 (99%) pairs matching, with the ex-type sequences of Alternaria cantlous (CBS 123007) for ITS (KC584245) and tef1 (KC584739), respectively. Phylogenetic analysis of concatenated sequences of ITS and tef1 using the maximum likelihood method, clustered isolate ST2 consistently with the ex-type strain of Alternaria cantlous (Yong Wang bis & X.G. Zhang) (Woudenberg et al., 2013), with bootstrap support of 96% (Fig. 2). Based on the BLAST search and phylogenetic analysis, isolate ST2 was identified as Alternaria cantlous. A pathogenicity test was performed under greenhouse conditions by spraying a conidial suspension of 105 conidia/ml onto mature leaves of three lentil (cv. Syria 229) plants. Control plants were sprayed with sterile water. All plants were covered with a transparent plastic bag for 72 hours. After 20 days, the inoculated plants showed similar symptoms to those observed in the field (Fig. 3), while no symptoms were observed on control plants. The fungus was reisolated from all inoculated plants to fulfill Koch's postulates. To the best of our knowledge this is the first report of A. cantlous causing leaf spot on lentil in Algeria and worldwide. The authors wish to express their sincerest gratitude to Professor Madani Benouycef from University Mustapha Stambouli of Mascara, Department of Earth Sciences for providing the necessary facilities and equipment for the research work.