First report of Pyrenophora avenicola causing leaf spot disease on barley

In subtropical southern Brazil, the leaf spot complex, primarily consisting of brown spot and net-type net blotch, caused by Bipolaris sorokiniana and Pyrenophora teres f. teres, respectively, significantly affects barley production. A survey aimed at enhancing our understanding of the aetiology of the leaf spot complex using DNA barcoding techniques for pathogen identification was conducted in the state of Paraná, Brazil in 2021 and 2022. It revealed a consistent association between a Pyrenophora species distinct from P. teres f. teres and a light brown necrotic lesion with irregular margins surrounded by a yellow halo (Figure 1a), with an incidence ranging from 3% to 5% over the 18 hectares surveyed each year. Segments excised from the edges of diseased leaves were disinfected for 30 seconds in 0.5% sodium hypochlorite solution, washed with sterile water, and then transferred to Petri dishes containing V8-PDA medium (Jacques et al., 2021). The plates were maintained at a temperature of 22 ±2°C with a photoperiod of 12 hours for seven days for sporulation. Three single-spore cultures were used for DNA extraction and morphological analysis. The protocols of Marin-Felix et al. (2019) were used for DNA barcoding identification of Pyrenophora species by PCR. The sequences of the amplicons from the three isolates were identical. The examined loci were ITS (476 bp; GenBank Accession No. OP787066), LSU (833 bp; OP856948), GAPDH (468 bp; OP863215), and RPB2 (781 bp; OP874923). Phylogenetic analysis by Bayesian and Maximum Likelihood methods using the combined datasets was needed to complement identification by nucleotide BLAST searches of the individual datasets against the GenBank database. Phylogenetic analysis revealed that isolate UEM4601 grouped with ex-type P. avenicola CBS 307.84 (Marin-Felix et al., 2019) in a well-supported clade (Figure 2). Morphological characterisation of the three isolates revealed conidia cylindrical to obclavate, tapering towards the apex, pale brown to brown, 2–5 distoseptate, 27–73 × 10–14 μm, and conidiophores arising in groups, septate, straight or flexuous, geniculate at the upper part, up to 310 μm long, and 5–7 μm wide (Figure 3, 4). These characteristics match those of P. avenicola CBS 307.84. The pathogenicity of the three P. avenicola isolates was tested on barley plants by spraying a conidial suspension with 1 × 104 conidia per ml on the leaves of potted barley cv. Imperatriz plants at the stem elongation stage. Control plants were sprayed with sterile water. Four days after inoculation, leaves of the inoculated plants initially showed small, light brown, necrotic lesions with an irregular shape surrounded by a slight yellow halo. The necrotic lesions had a dark brown centre that expanded and became surrounded by an irregular yellow halo, seven days after inoculation (Figure. 1b-c). Pyrenophora avenicola was reisolated from the inoculated plants. Pyrenophora avenicola has been reported to infect oats in Sweden (Marin-Felix et al., 2019) and China (Chen et al., 2022) but this is the first report of the species causing leaf spot disease on barley worldwide. The results of this study will provide a better understanding of the aetiology of leaf spot diseases affecting barley in the humid subtropical climate of southern Brazil. This work was funded by the National Council for Scientific and Technological Development of Brazil (CNPq - Grant Number: 308948/2020-3) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES Finance Code 001).