First Report of Spot Blotch of Barley Caused by Cochliobolus sativus in Morocco

Barley (Hordeum vulgare L.) is grown on ∼2 million hectares in Morocco (av. 1.2 t/ha), with ∼70% under semiarid conditions (FAO 2017). Historically, net form net blotch (NFNB) (Pyrenophora teres f. teres [Ptt]) and spot form net blotch (SFNB) (P. teres f. maculata [Ptm]) have predominated in Morocco, causing up to 29% yield losses (El Yousfi and Ezzahiri 2002). Often, barley spot blotch (SB) caused by Cochliobolus sativus (anamorph: Bipolaris sorokiniana [Sacc.] Shoem.) is misdiagnosed as SFNB. Disease surveys were conducted in six regions (Abda, Rommani, Choiya, Dokhala, Gharb, and Rahamna) in 2015 (54 fields) and 2018 (49 fields) at heading stage and covered three cultivars (Annoucer, Rabat 071, and Arig 08) representing 34, 41, and 25% of fields, respectively. Leaf samples (∼10) from three plants per field were collected. From each site, a representative sample with SFNB symptoms (20 leaf segments, 1-cm2 pieces) was used for isolation. Samples were surface sterilized with 50% ethanol for 30 s and 5% bleach for 60 s, followed by rinsing in sterile deionized water twice for 5 min. Samples were dried between two layers of sterile filter paper, followed by incubation on PDA under 12 h light/dark at 22 ± 1°C. After 4 to 5 days, dark brown elliptical conidia of 60 to 120 × 12 to 20 µm with 5 to 9 septa per cell were observed. Cultures sporulated profusely with light to dark gray or brown cottony mass with wavy colony margins (Kumar et al. 2002). Based on morphology, isolates were identified as C. sativus. Of 103 samples, C. sativus was recovered from 11 (11%), and 92 samples were Ptt (40%) and Ptm (50%). To test pathogenicity of the SB isolates, two SB differential genotypes (NDB5883 and Bowman) were used as checks, along with three cultivars (Rabat 071, Annoucer, and Oussama). Seeds (∼4 to 5 per genotype per replicate) were sown in three replications in peat moss with 14-14-14 NPK; seedlings were raised in a growth chamber with 16 h light/8 h dark at 20 ± 1°C. To produce inoculum, lyophilized agar plugs of monoconidial SB isolates were incubated on V8PDA in the dark for 4 to 5 days at 22 ± 1°C, followed by 22 ± 1°C with 12 h light/dark for 7 to 8 days. V8PDA plates were flooded with 5 to 10 ml of sterile distilled water, and the conidia were harvested followed by filtration with a double layer of cheese cloth. Spore density was adjusted to 5,000 conidia/ml supplemented with surfactant (0.01% Tween 20). Seedlings at the two-leaf stage (∼14 days old) were inoculated with the two isolates independently until runoff and incubated at 100% RH at 20 ± 1°C in the dark for 24 h. Seedlings were transferred to a greenhouse with 16 h light/8 h dark at 20 ± 1°C. After 10 days, the seedlings were scored on a 0 to 9 scale (Fetch and Steffenson 1999). All infected seedlings produced lesions as described by Kumar et al. (2002) and Fetch and Steffenson (1999). The experiment was carried out twice to confirm the repeatability of SB symptoms. To satisfy Koch’s postulates, leaf tissue from infected seedlings of all genotypes for both SB isolates was surface sterilized and incubated on PDA as described above. Monoconidial cultures were induced for sporulation on V8PDA; upon inoculation, similar symptoms were observed, confirming SB. Both SB isolates were grown in Fries medium, and genomic DNA was isolated (Benslimane et al. 2013). PCR with ITS1-F/ITS2-R produced a single band of ∼500 bp and was subjected to DNA sequencing from both ends. Homology-based BLASTN searches using sequences from both SB isolates produced 100% identity (E-value 0) to 98 GenBank accessions of SB partial sequence of small and large subunit rRNA, complete sequence of ITS1, 5.8S rRNA gene, and ITS2 (MK676001, MK905507, MH864698). Conidia and colony morphology, symptomology, Koch’s postulates, and DNA sequencing corroborate C. sativus (perfect stage) causing SB in barley. The queried sequences were deposited in GenBank as MN444781, 82, 84, and 85. This is the first report of SB in Morocco. This has implications for future barley breeding and cultivation under dry areas, as SB so far has been a disease of hot and humid conditions of South Asia.