Leaf Spots of Caused by s.s. and Newly Reported in China.

L. shows a great potential for restoration of contaminated water or soils and has a high ornamental value (Li et al. 2015). In mid-October 2021, a leaf spot disease, with an incidence of approximately 61%, occurred on leaves of 25-year-old on the campus of Nanjing Forestry University. On average, 65% of the leaves per tree were infected. Symptoms began as dark brown, irregular spots, and the centers were grayish white. The spots gradually enlarged with time. Fresh specimens were collected from 3 trees (10 leaves/tree). Small tissue pieces cut from lesion margins were surface-sterilized (Mao et al. 2021), plated on potato dextrose agar (PDA), and incubated at 25°C. Three representative isolates (NL1-7, NL1-10, and NL1-13) were obtained and deposited in The China Forestry Culture Collection Center. The colonies of 3 isolates were white, grayish white at the center. The conidia of 3 isolates were one-celled, straight, subcylindrical, hyaline, smooth, 14.6-18.6 × 4.3-6.7 µm, 13.8-16.7 × 4.7-6.0 µm and 12.1-16.9 × 5.4-7.5 µm (n = 50) for NL1-7, NL1-10, and NL1-13, respectively. The conidiophores of NL1-7 were hyaline to pale brown, septate, and branched, 18.9-48.0 µm (n = 50). Appressoria were one-celled, ellipsoidal, brown or dark brown, thick-walled. The conidiophores and appressoria of the other two isolates were almost identical to NL1-7. Based on morphological characteristics, the 3 isolates matched the species complex (Weir et al. 2012). DNA of the 3 isolates was extracted. The internal transcribed spacer region (ITS), actin (), calmodulin (), chitin synthase (), glyceraldehyde-3-phosphate dehydrogenase (), and β-tubulin 2 () loci were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-354R, GDF1/GDR1, and T1/Bt2b, respectively (Weir et al. 2012). The sequences were deposited in GenBank [Accession Nos. ON870951 and ON858477 to ON858481 for NL1-7; ON908707 and ON858482 to ON858486 for NL1-10; ON870949 and ON858487 to ON858491 for NL1-13]. BLAST result showed that ITS, , , , , and sequences of NL1-7 were identical to at a high level (>99%). The sequences of NL1-10 and NL1-13 were consistent with at a high level (>99%). A maximum likelihood and Bayesian Inference analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences (ITS, , , , , and ) placed NL1-7 in the clade of sensu stricto and NL1-10 and NL1-13 in the clade of . To confirm their pathogenicity, 9 healthy 3-yr-old seedlings, and 10 leaves/seedling were wounded with a sterile needle and inoculated with 10 µL of conidial suspension (10 conidia/mL) of the 3 isolates, respectively. Three control plants were treated with sterile water. Seedlings were covered with plastic bags after inoculation and kept in a greenhouse at 25 ± 2°C and RH 80%. Within 7 days, all inoculated leaves showed lesions similar to those in the field, and controls were asymptomatic. s.s. and were reisolated from the infected tissues. It was reported that species can cause many plant diseases, for example, causes twig canker (Swain et al. 2012), and causes willow anthracnose (Okorski et al. 2018), etc. However, some species are endophytic (Martin et al. 2021) and may only become pathogenic under the right conditions. This is the first report of s.s. and causing leaf spots on in the world. These data will help select appropriate strategies for managing this disease and further studies on the pathogen and the host.