First Report of Powdery Mildew Caused by Erysiphe pisi on Medicago ruthenica in Inner Mongolia, China.

Medicago ruthenica, a perennial forage species in the Leguminosae, is widely distributed in Northern China, Siberia and Mongolia. It is considered a high-quality protein source for livestock, and a valuable gene resource for alfalfa genetic improvement (Yin et al., 2021). However, M. ruthenica is susceptible to powdery mildew. In September 2021, an investigation of 500 m2 plant nursery at Inner Mongolia Agriculture University (111.73°E, 40.83°N), Hohhot, China, found that 90% leaves of M. ruthenica cv. Zhilixing were infected. Initially, small, white, and diffuse spots appeared on the adaxial leaf surface, then expanded to white mildew layer. Severely, orange or brown spots formed on the top of the layer, and abaxial leaf surface, stems and pods were also affected. The pathogen structures are as follows: conidia (n = 50) were produced singly, smooth, and elliptical or cylindrical, 19 to 38 × 10 to 18 µm, with a length/width ratio of 1.6 to 2.6; chasmothecia (n = 50) were black-brown, spherical, or subspherical, with a diameter of 75 to 130 μm. Approximately 17 colorless appendages may be contained in chasmothecia, whose average length was 50 μm. Chasmothecia contains 4 to 8 asci with 58 to 75 × 30 to 44 µm. The ascus contains 3 to 4 monospora and oval ascospores, 18 to 28 × 10 to 15 µm. Based on these morphological characteristics, the fungus was tentatively identified as an Erysiphe pisi (Ainsworth and Bisby, 1995; Hawksworth et al., 1995). For further confirmation, the internal transcribed spacer (ITS) and 28S large subunit (LSU) regions of one isolate (MNPM-MruZB1) were amplified using ITS4/ITS5 and NL1/TW14 primers (Innis et al., 1990; Takamatsu and Kano, 2001; Mori et al., 2000). Sequences of about 750 bp size were obtained and submitted to GenBank (accession number: PQ299565 and PV017896). They showed 99.70% (664/666) identity for ITS (KY661137.1) and 99.01% (808/816) for LSU (ON314806.1) with the previously reported sequences of E. pisi, respectively. Furthermore, the amplified sequences were used to construct a phylogenetic tree in MEGA7.0, which revealed the MNPM-MruZB1 isolate to be closely related to E. pisi. Pathogenicity tests were repeated three times on two-months old seedlings of three M. ruthenica varieties (Zhiling, Mengnong No.1 and Mengnong No.2). Three pots of healthy seedlings, from each variety, were inoculated by gently pressing diseased leaves, and the other three pots served as control inoculated with sterile distilled water. The seedlings were incubated at 21℃ and 70% relative humidity with a 16 h/8 h light/dark photoperiod. Seven days after inoculation, the three M. ruthenica varieties showed powdery mildew symptoms, while the control plants remained asymptomatic. Likewise, the results of morphology and sequence characteristics were also consistent with those of previous studies. E. pisi was identified as the causal species of powdery mildew of M. sativa and M. truncatula previously, whereas the pathogen of M. ruthenica powdery mildew is still unknown globally (Edmunds et al., 1998; Gupta et al., 2020). To the best of our knowledge, this is the first report of powdery mildew caused by E. pisi on M. ruthenica. It could serve as a basis for the identification, diagnosis, and prevention of M. ruthenica powdery mildew.