First Report of <i>Plectosphaerella cucumerina</i> Causing Wilt on <i>Leymus chinensis</i> in China

Leymus chinensis is one of the dominant forage grasses in the typical pastures of Inner Mongolia, recognized for its high productivity, protein content, and palatability (Wang et al. 2024). Since 2023, L. chinensis wilt has been observed in pastures of Subuerga Town, Ejin Horo Banner, Inner Mongolia (E109°26′19.48″, N39°30′4.51″), with an average disease incidence ranging from 24.6% to 41.7%. Wilt symptoms were noted on older leaves, severely infected plants showed symptoms of complete wilting. The rhizomes of the affected seedlings exhibited color changes, necrosis, and rot. Symptomatic roots and stem bases collected from 25 plants were surface-sterilized with 70% ethanol for 30 s, dried, and subsequently placed on water agar (WA) at 25 °C for 5 days. Hyphal tips growing from the edges of the plant tissue were then transferred to potato dextrose agar (PDA) and incubated at 25 °C for 14 days. The 14-day-old colonies on PDA were buff with a slimy surface. Aerial hyphae were sparse with simple or branched conidiophores. Conidia were hyaline, smooth, ovoid, sometimes one-septate, measuring 4.36 to 8.32 × 1.97 to 3.87 µm (average 6.05 × 2.98 µm) (n = 50). Seventeen isolated strains showed similar morphology, and two isolates (QY-1 and QY-2) were subjected to further analysis. The genomic DNA was extracted using the CTAB method (Guo et al. 1998). Partial sequences of the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-alpha gene (TEF), and β-tubulin gene (TUB2) were amplified and sequenced using primers PcCF1/PcCR1 (Atkins et al. 2003), EF1-668F/EF1-1251R (Alves et al. 2008), and β-tubulin-T1/β-tubulin-Bt1b (Ariska et al. 2019), respectively. BLAST homology analysis revealed that the ITS (GenBank accession no. PQ625907.1 and PQ625908.1), TEF (PQ807359.1 and PQ807360.1), TUB2 (PQ807355.1 and PQ807356.1) sequences of isolate QY-1 and QY-2 exhibited 98.67 to 99.63% identity with the Plectosphaerella cucumerina (PV240436.1, KY964491.1 and MZ209393.1). Based on morphological and molecular analyses, the isolates were identified as P. cucumerina (Cui et al. 2024). Pathogenicity was determined using the soil-pathogen mix inoculation method. L. chinensis seeds were sown in soil mixed with 1 × 107 spores per gram of soil, and soil mixed with sterile water was used as a control. All plants were placed in the greenhouse with a temperature set at 25 ℃ and an illumination period of 12 hours. After 45 days, the growth rate of L. chinensis plants inoculated with QY-1 and QY-2 was significantly slower than that of the control, and the leaves began to turn yellow and wither. After 80 days, the inoculated plants died, while the controls remained healthy. We re-isolated strains that were morphologically and molecularly identical to the QY-1 and QY-2 from the inoculated plants, thus fulfilling Koch’s postulates. Based on molecular, morphological, and pathogenic properties, P. cucumerina is the causal fungal pathogen of Leymus chinensis wilt disease. Previously, P. cucumerina has been reported as the causal agent of wilt disease in strawberry and coptis chinensis in China (Yang et al. 2023; Cui et al. 2024). This first report of P. cucumerina causing wilt disease on Leymus chinensis in China highlights a significant new threat to pastoral ecosystems and necessitates further research to understand its potential spread and develop effective management strategies.