First report of Needle Blight on Pinus sylvestris var. mongolica caused by Sydowia polyspora in China

Pinus sylvestris var. mongolica characterized by its windbreak, sand-fixation, drought resistance, and tolerance to infertile soils (Yu et al. 2023.). In July 2023, a severe needle blight disease has affected the P. sylvestris var. mongolica trees in Honghua’erji area, Inner Mongolia, China, which seriously restricted the local ecological construction. Symptoms were observed on 60% of 10 ha area in Toudaoqiao Forest Farm (48.275750°N, 120.000670°E). In the early stages of the disease, symptoms on needles yellow - to tan - coloured bands or spots, while those had been dead appeared greyish-brown or dark grey, eventually leading to extensive wilting. Thirty diseased needles on ten trees were collected in a zigzag manner at five locations within the sample area (20 × 20 m). Each needle pieces (3 mm 2 ) from the margin of lesions were surface sterilized with 0.2% NaClO solution for 60 s, 75% ethanol for 30 s, rinsed 3 times in sterile ddH 2 O, then transferred to potato dextrose agar (PDA) for incubation at 25℃ for 7 d in the dark. Using the single hyphal-tip isolation method, eighteen similar fungal colonies were successfully obtained. Colonies on PDA of 18 similar obtained isolates were all greyish-green initially with white margins, reverses were same. After 10 days of incubation, the pure culture became dark brown to black, and the fungal colonies are covered with creamy spore masses, non-septate, ellipsoid, hyaline and multiguttulate spores were produced. Conidia were measured to be on average 7.6 - 11.5 (6.3 ± 0.34) × 3.9-5.1 (3.3 ± 0.22) μm (n = 50). These morphological characteristics were consistent with those of Sydowia polyspora (Tinivella et al. 2014.). The genomic DNA of the two representative isolates (NEFU-ZJY-1 and NEFU-ZJY-3) were extracted using a Fungal DNA Kit (D3390-01, OMEGA, USA). The internal transcribed spacer (ITS) and the large subunit region (LSU) region were amplified using primers ITS1/ITS4 and NL1/NL4 respectively (Liang et al. 2024.). BLAST search results showed a maximum homology of 100% with Sydowia polyspora (MK907720.1, 549/550), (OQ240195.1, 573/574). The sequences of ITS and LSU were deposited in GenBank (NEFU-ZJY-1: PP809391, PP869089; NEFU-ZJY-3: PP869159, PP869101). The voucher specimens were deposited in the Heilongjiang Province Key Laboratory of Forest Protection. Moreover, a phylogenetic tree based on the two genes revealed that all two isolates were closest to S. polyspora. Based on these results, the two isolates were identified as S. polyspora which has been found to infect Abies nordmanniana, Pinus yunnanensis and Pinus halepensis (Talgo et al. 2010; Pan et al. 2018; Cakar et al. 2022). To confirm pathogenicity, the spore suspensions (1 × 10 7 spores/ml) of NEFU-ZJY-1 were sprayed on the 2-year-old healthy P. sylvestris var. mongolica seedlings (n = 15), equal volume of sterile water was sprayed for the control (n = 5). The seedlings were then placed in a greenhouse at 25°C. After 30 days, the chlorotic pine needles exhibited yellowing and browning symptoms which were those previously observed in the forest, while the control plants showed no symptoms. The re-isolated fungus, was identical to S. polyspora based on morphological and molecular analysis, thus fulfilling Koch’s postulate. To our knowledge, this is the first report in China of needle blight on P. sylvestris var. mongolica caused by S. polyspora. This study provides valuable information for developing effective methods to manage this disease.