First report of alfalfa leaf spot caused by Leptosphaerulina australis in China.

ABSTRACT

A disease was observed on alfalfa cultivar WL168 characterized by white to brown leaf spots of regular to round shapes, in Aluhorqin County, Inner Mongolia, China (120°13'23″ to 120°29'14″ E, 43°27'52″to 43°35'16″ N, 281.71m to360.13 m Altitude) during 2019 to 2020. The disease mainly presented in spring one month after re-greening and the incidence was 78.30% in this field. Twenty alfalfa plants with severe symptoms were used for pathogen isolation. The infected tissue was cut into 2 × 2 mm pieces, surface-sterilized (in 75% ethanol and 5% commercial bleach (NaClO) for 30 s and 2 min, respectively), rinsed five times with sterilized distilled water, and dried between sterile filter paper (Wang et al. 2019). The diseased tissue from each plant sample were cultured on potato dextrose agar (PDA) and incubated at 25 °C with 12 h light/day for ten days. A fungus was isolated from the diseased leaves at a 100% frequency. Fungal growth on PDA was round with a black surface, radial edge, and a dirty white center. The ascocarps were moved to a clean microscope slide to release asci and ascospores. Ascocarps were spheroidal, subglobose brown, 120 to 160 µm × 160 to 180 µm, which contain several ascus. The size of ascus were 31.0 to 41.6 µm × 75.0 to 87.5 µm and each asci having eight ascospores. Ascospores were ellipsoid to oblong with a gelatinous sheath, brown, 8.8 to 15.0 µm × 29.9 to 43.0 µm with 2 to 3 horizontal septums, and 0 to 2 vertical septums. A phylogenetic tree was constructed after DNA extraction, PCR with primers to amplify the ITS (VG9 5'- TTACGTCCCTGCCCTTTGTA-3' and ITS4 5'-TCCTCCGCTTATTGATATGC-3') and LSU (LR7 5'-TACTACCACCAAGATCT-3' and LROR 5'- GTACCCGCTGA ACTTAAGC -3') regions. The LSU (SUB8273071) and ITS (SUB8218291) amplicons showed 99% similarity with L. australis (EU754166.1) in the GenBank. To verify the pathogenicity, fungs plugs were inverted on three compound leaves of 20 alfalfa WL168 for two days. Agar plugs (PDA) were inverted on another 20 alfalfa WL168 three compound leaves which were control. All plants were maintained at 22 °C and 44% relative humidity in a growth chamber. Similar disease symptoms were observed on infected leaves ten days after inoculation, while control plants showed no symptoms. The same fungus was re-isolated from the lesions, and further morphological characterization and molecular assays, as described above. L. australis has been reported on various plants, including Prunusarmeniaca, Dolichos, Poa, Lolium, and Vitis in Australia (Graham and Luttrell., 1961), and also from Korean soil in 2018 (Weilan et al., 2018). Additionally, L. briosiana, which is common in the USA, China, and other countries, causes Leptosphaerulina leaf spot (Samacet al., 2015). L. trifolii is newly reported to occur in China (Liu et al., 2019). To the best of our knowledge, this is the first report of L. australis infecting alfalfa in China. Considering the large planting area in Inner Mongolia, this pathogen may losses to alfalfa cultivation. Hence, future studies should explore aspects of effective management of this disease.