First Report of Nigrospora oryzae Causing Basal Rot on Siberian lily (Lilium spp.) Ornamental hybrids in China.

Abstract

Lilium 'Siberia', is extensively cultivated and has high economical value in China due to its popularity as an ornamental lily. In August 2023, lily basal rot was observed on about 60% of two-year-old plants at the Liaoning Academy of Agricultural Sciences Multiplex greenhouse ( in an area of about 2000 m² ) . Initial symptoms appeared on the base of the bulbs as brown lesions that expanded over time, and later the leaves turned yellow and about one month latter the whole plant died. To isolate the causal pathogen, tissues were collected from 5 symptomatic plants with basal rot. The infected bulbs were surface-sterilized with 75 % ethanol solution for 30 s after initial rinsing, followed by triple sterile-water washes, specimens were dried aseptically prior to pathogen isolation. Tissue sections 5 mm × 5 mm were transferred from plant lesion margins onto potato dextrose agar (PDA) medium after surface sterilization. Cultures were maintained at 25℃ for pathogen isolation. After 7 days, 3 consistent surface morphology strains(H1, H2 and H3) were prepared based on single spore isolates. After 10 to 15 days, mycelium had fully covered the plates. The hyphae were branched, septate, smooth, hyaline, Conidia were black, solitary, globose or subglobose, glossy aseptate of 8.3 - 11.4 μm × 12.4 - 14.9 μm (n=50) in size, and produced at the tips of hyaline and ampulliform conidiophores. The fungus showed similar morphological characteristics to Nigrospora oryzae (Wang et al. 2017). For molecular confirmation of the species identity, genomic DNA of three isolates (H1, H2 and H3) were extracted by the CTAB method (O'Donnell et al., 1998), and portions of three genes, the Intemal Transcribed Spacer (ITS) of the ribosomal RNA, the Translation Elongation Factor subunit 1-alpha (TEF1-ɑ) and the beta-tubulin (TUB2) genes were amplified by the PCR using the primers ITS1/ITS4,EF1-728F/EF-2 and Bt-2a/Bt-2b (Wang et al. 2017). A BLASTn analysis showed that the identity of the nucleotide sequences of isolates (H1, H2 and H3) were 99%(500/501bp; 488/488 bp; 495/499bp) for ITS, 99% (461/461 bp;466/467 bp;467/467 bp) for TEF1- ɑ; and 99% (331/333 bp; 376/384 bp; 381/388 bp) for the TUB2 sequences, with N. oryzae LC2702 in GenBank. The resulting sequences were submitted to GenBank under accession numbers PP203296, PP784618, PP784619 for ITS; PP417827, PP869279, PP886084, for TEF1-ɑ; PP448183, PP869277, PP869278 for TUB2 of isolates H1, H2 and H3 respectively. A neighbor-joining phylogenetic tree showed that the three isolates from Siberian lily clustered with the N.oryzae clade.Therefore, the isolates H1, H2 and H3 were identified as N. oryzae based on morphology and molecular evidence. One representative isolate(H1) was used for completing the Koch's postulates. A pathogenicity test was made on 1-year-old healthy Siberia lily bulbs, the bulbs were disinfected, using a sterile needle to prick the base of the bulbs, then they were planted in sterilized soil. A conidial suspension (1 × 10⁶ conidia/ml) was used to water the planted lily bulbs once every 5 days, and the controls were watered with sterile water. Each treatment contained 30 bulbs, and experiment was repeated three times. After 15 days of inoculation at 25℃, the bulbs inoculated with H1 began to turn black and to rot, and black perithecia were visible. The control plants did not show disease and no pathogen was isolated from them. We reisolated the pathogen from infected tissues, after observation of the morphological characteristics and ITS sequence ( PQ686265 ) the reisolated fungus was identified as Nigrospora oryzae. According to Koch's Postulates, Nigrospora oryza was the pathogen causing lily basal rot. To our knowledge, this is the first report of basal rot on Lilium'Siberia'(Lilium spp.) caused by Nigrospora oryzae in China.