First Report of Pestalotiopsis lushanensis Causing Black Leaf Spot on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum) is widely grown for forage in Southwest China. In December 2023, black leaf spots were observed on Italian ryegrass in a 10 hm² field in Hongya County, Sichuan Province, China (29°52'11''°N, 103°16'16''°E). The disease developed sporadically with an incidence rate of < 1%. Leaves showed typical symptoms of small, black spots. For isolation, six samples (about 0.5 cm × 1 cm) were cut with a sterilized blade at the junction of diseased and healthy tissues from two different plants. They were surface disinfected in 75% ethanol for 40s, rinsed thrice in sterilized distilled water, air-dried, plated on potato dextrose agar (PDA), and incubated in the dark at 25°C for 3-7 days. A Pestalotiopsis-like fungus with similar colony characteristics was consistently isolated from all samples. Pure cultures were obtained with the single-spore technique (Cai et al. 2009), and two strains (LJH17 and LJH18) were selected for further study. After 5 days in the dark at 25°C, colonies on PDA were cottony, whitish, 70-78 mm in diameter, with an entire edge. The reverse side of the colonies appeared white to light yellow. Conidia were fusoid, straight to slightly curved, 4-septate, with 2-4 (mostly 3) tubular apical appendages, measuring 17.3 to 25 × 6.1 to 9.9 μm (mean ± SD = 20.7 ± 2.3 × 7.9 ± 0.7 µm; n = 50; LJH17). For further identification, the internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA (ITS), partial β-tubulin (tub2) and translation elongation factor 1-α (tef1) genes from the two pure strains GMCC000083 (=LJH17; GMCC, Grassland Microbiome Culture Collection) and LJH18 were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson 1995; O'Donnell and Cigelnik 1997), and EF1-728F/EF2 (Carbone and Kohn 1999; O'Donnell et al. 1998). Sequences were deposited in GenBank (PP779763, PP781949, and PP781950 for LJH17; and PQ013725, PQ015095, and PQ015096 for LJH18). BLAST analysis showed 100% identity across all three segments with ex-type isolate LC4344 of Pestalotiopsis lushanensis (Liu et al. 2017). A randomized accelerated maximum likelihood (RAxML) phylogenetic tree based on concatenated ITS, tub2, and tef1 alignments showed that our two strains clustered with the previously reported P. lushanensis strains ((Liu et al. 2017; Jiang et al. 2022) (Fig. S1F). Eight-week-old healthy plants were grown from Italian ryegrass seeds in pots, with three plants per pot. Following our previous spray inoculation procedures (Xue et al. 2020): fresh 5-day-old mycelia from each strain were prepared as mycelial suspensions at about 2.5 × 10⁴ CFU/ml, and conidial suspensions were adjusted to about 2 × 10⁵ conidia/ml. For each strain (LJH17 and LJH18), five pots were separately spray-inoculated with conidial and mycelial suspensions (about 2 ml/pot). Five pots served as non-inoculated controls were sprayed with sterilized distilled water. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 23-29°C. After incubation for 15 days, both conidial and mycelial suspensions produced black spots on the plants (Fig. S1), resembling those previously observed in the field. No symptoms were observed in the control plants. Pathogenicity tests were conducted thrice using the same method. The same fungus was reisolated from the lesions and confirmed via the morphological and molecular methods described above, thus fulfilling Koch' s postulates. To our knowledge, this is the first report of P. lushanensis causing black leaf spot on Italian ryegrass in China or worldwide. This finding may aid in diagnosing, identifying the pathogen, and controlling the disease on Italian ryegrass.