First report of seed decay and seedling damping‐off of wheat (Triticum aestivum) caused by seed‐transmitted Alternaria rosae in Iraq

During the 2022 to 2023 growing season, a survey was conducted of the 13 wheat varieties (Adna, Baghdad, Chad, French, Ibaa 99, Jihan, Maxipak, Rashed, Research 22, Sham, Tigris1, Tigris 2, and Wafeia) most commonly grown in Karbala Governorate, Iraq. A persistent problem of seed germination failure rates, as high as 30% during the period of seedling emergence, had been noted in the Al-Sahrawea and Ain Altemr regions. This study was done to determine the reasons for this failure in seed germination, including the presence of seed-transmitted pathogens. One hundred seeds of each of the 13 varieties were collected from wheat fields in the Al-Khayrat, Al-Hurr, Al-Sahrawea, Ain Altemr and Al-Jadual Al Garby regions in Karbala. The seeds were disinfected using a 2% sodium hypochlorite solution for three minutes, then washed three times in sterile distilled water and dried on sterile filter paper. The disinfected seeds were then sterilised using 70% ethanol for 30 seconds and left to dry for 10 minutes. The sterilised seeds were placed on sterile potato dextrose agar medium supplemented with streptomycin sulphate (100 mg/l), incubated in the dark at 25°C and monitored daily. After one week, fungal colonies growing around the seeds were purified using the colony tip method. Fifty-one purified fungal isolates were obtained, of which eleven appeared similar. Colonies of these eleven isolates were primarily greyish-white before turning dark olive green, and had plentiful cotton-like aerial hypha (Figure 1). The microscopic characteristics of conidia were obclavate with 1–3 transverse septa and 0–3 longitudinal brown septa (Figure 2). The septa (n = 50) measured 13–28.5 × 6–14 µm. These morphological features were consistent with previous descriptions of Alternaria species (Simmons, 2007). Pure cultures of three representative isolates were sent to Macrogen, Inc. (South Korea) for DNA extraction and multilocus sequence typing. Four gene markers were amplified and sequenced partially using a specific primer set for each of these markers. The rDNA-ITS region was amplified with primers ITS5 / ITS4 (White et al., 1990), the transcription elongation factor 1-a with EF1-983F/EF1-2218R primers (Rehner & Buckley, 2005), the RNA polymerase II second largest subunit (RPB2) with RPB2-5F2/fRPB2-7cR primers (Sung et al., 2007), and actin (actA) using ACT-512F/ACT-783R primers (Lahuf et al., 2020). BLASTn analysis showed 100% identity with isolates of Alternaria rosae E.G. Simmons & C.F. Hill (KU375630.1, ON601003.1, XM_046168987.1, and XM_046175284.1). The sequences of one of the isolates were deposited in GenBank under Accession Nos. PP732704.1, PP750863.1, PP750862 and PP750861.1. The pathogenicity of A. rosae isolate Karbala-1 was evaluated using a Petri plate method (Lahuf et al., 2019). A 5-mm-diameter disk, taken from the edge of a one-week-old pure colony of A. rosae, was placed in the centre of a water-agar plate and incubated in darkness at 25°C for three days. Two-hundred certified seeds of wheat cv. Ibaa 99 were disinfected as described previously. The seeds were then divided equally into two groups. The first group was placed around A. rosae colonies (10 seeds/plate). The second group was placed in a non-inoculated water-agar plate as a control. All plates were incubated at 25°C. Seed germination and inhibition percentages were measured a week after planting. None of the inoculated seeds germinated whereas all the control seeds germinated. Half of the germinated control seeds were then planted in peat moss (10 seedlings/pot) and inoculated with 10 mL of 1×105 conidia of A. rosae isolate Karbala-1. The remaining control seedlings were inoculated with 10 mL of sterile distilled water. After a week, all the inoculated seedlings had weak growth with shoot wilting and yellowing and light to dark brown zones on the roots. To fulfill Koch's postulates, the pathogen was re-isolated and sequenced to confirm the presence of A. rosae. These results clearly show the importance of ensuring the quality of wheat seeds, which must be taken into consideration when growing this essential crop. To our knowledge, this report is the first of A. rosae in Iraq as a seed-transmitted pathogen of wheat. The authors would like to express their appreciation to the Plant Protection Department, Agriculture College, University of Kerbala, and the Iraqi Ministry of Higher Education and Scientific Research for their support.