Fusarium crown rot (FCR) is an important disease on wheat (Triticum aestivum L.) all over the world. Fusarium pseudograminearum is reported the main causal agent of FCR in China (Deng et al. 2020). In 2020, FCR occurred in wheat in Langfang, Hebei Province (116.31°E, 38.82°N) with observed incidence of 37.2% (48 out of 129 plants in total). The diseased wheat showed brown lesions at the crown and then stem necrosis. Samples with diseased symptom were collected from fields in late May 2020 (at the premature stage, 36 weeks after planting) (e-Xtra 1A). To perform fungal isolation, 0.3 cm2 samples excised at the symptomatic crown were surface disinfested with 75% ethanol for 10 s, and 0.1% HgCl2 for 40 s, then washed three times with sterile ddH2O. When cultured on potato dextrose agar (PDA), the colonies of two isolates out of Langfang (11.8% frequency) initially arewhite becoming violet with age, with violet pigments produced on PDA (e-Xtra 1B). Single-spored isolates were acquired, macroconidia were slender, thin walled, with 3- to 5-septate, measurements of 15.7-31.4 µm × 2.7-6.3 µm (n=50) (e-Xtra 1C). The pure culture were named as HWA94 and HWA97, respectively. DNA was extracted from the single-spored mycelium of HWA97 using the CTAB method (Leslie and Summerell, 2006). Internal transcribed spacer (ITS) region, partial sequences of actin (ACT), translation elongation factor 1-α (EF-1α) gene, 28S ribosomal RNA (LSU), and DNA-dependent RNA polymerase II largest subunit rpb1 (RPB1) were amplified using primers ITS1/ITS4, EF-1F/EF-1R, ACT512F/ACT728R, LR/LROR and RPB1B-F/ RPB1B-R and sequenced. The ITS, EF-1α, ACT, LSU, and RPB1 sequences were deposited in GenBank under accession numbers OM459813 to OM459817. These sequences showed 99.64%, 100%, 100%, 100%, and 100% similarity with the reference strain F. nygamai CBS749.97, respectively, resulting in HWA97 being identified as F. nygamai. To confirm the pathogenicity, inoculum was prepared by inoculating fully colonized F. nygamai (HWA97) PDA plugs on sterile wheat grain medium, cultured 7 days at 25℃ till massive mycelium formed, and hand shaken every two days to mix the wheat grains and the F. nygamai mycelium completely. Ten wheat seeds (cv. Jimai22, susceptible to FCR) for each 10-cm pot were inoculated with 10 g of inoculum when planting, then covered with soil. Mock inoculated wheat seeds with sterile grain without inoculum were used as control. The experiment was conducted in greenhouse, and repeated three times. Symptoms (brown necrosis at the crown) appeared 35 days after inoculation (dai) (e-Xtra 1D), with 91.5% incidence and 49.5±2.6 disease index. Mock-inoculated plants remained symptomless (e-Xtra 1E). Fusarium nygamai was re-isolated from the symptomatic stem and identified by morphological and molecular analysis, fulfilling Koch's postulates. Fusarium nygamai has been previously reported and recovered from wheat root and stalk (Fard et al. 2017) and causes root rot on wheat in Iraq (Minati, 2020), rice in Sardinia (Balmas et al. 2000), sugar beet in China (Cao et al. 2018), as well as lentil (Lens culinaris Medikus) in Pakistan (Rauf et al. 2016). To our knowledge, this is the first report of F. nygamai causing FCR of wheat in China. This study contributes useful information for epidemiologic studies for FCR. Additional studies will be needed to determine the distribution, aggressiveness, and impact on yield of F. nygamai compared with the dominant causal agent F. pseudograminearum.
Keywords: Causal Agent; Crop Type; Field crops; Fungi; Pathogen detection; Subject Areas; cereals and grains.