First Report of Fusarium oxysporum Causing Root Rot on Pleione bulbocodioides in China.

Abstract

The members of Pleione (Orchidaceae) are popular worldwide due to their beautiful flowers and medicinal values. In October 2021, we observed the typical symptoms of yellow or brown leaves, rotted root, and plant death on P. bulbocodioides (Sup. S1a). Nearly 30% of the plants showed disease symptom in the farm in Zhaotong city, Yunnan Province, China. Three fresh root samples with typical symptoms were collected from the plants of P. bulbocodioides in the field. The root sections (3mm × 3mm) from the border of the symptomatic tissue were cut and sterilized with 75% ethanol for 30 s, followed by 3% sodium hypochlorite (NaClO) for 2 min, and then rinsed three times with sterile water. The sterilized root tissues were inoculated on potato dextrose agar (PDA) at 28℃ in the incubator for 3 days. The colonies were obtained and sub-cultured from the hyphal tip to the new PDA to further purify. The colonies grew up on PDA at 28℃ for 1 week, the hyphal color from white turned to purple, the center of colony became brick red. The colonies produced abundant microconidia, macroconidia and chlamydospores, but no sporodochia was observed (Sup. S2). The microconidia were oval and irregularly oval, zero to one septate, and measured 2.0 × 5.2 to 4.1 × 12.2 µm (n = 20). The macroconidia were falcate, slender, with a distinct curve to the latter half of the apical cell, three to five septate, and measured 4.0 × 15.2 to 5.1 × 39.3 µm (n = 20). Morphological characterization showed that the three isolates were similar, and appeared to be Fusarium oxysporum (Leslie and Summerell, 2006). For molecular identification, total genomic DNA of two representative isolates DSL-Q and DSL-Y were extracted with CTAB method, and the PCR amplification were performed. The sequence of partial elongation factor (TEF1-α) gene was amplified using the primer pair EF-1/EF-2 (O'Donnell et al. 1998). The sequence of β-tublin gene (TUB2) was amplified using the primer pair T1/T22 (O'Donnell and Cigelnik, 1997). The sequences from the two isolates were obtained and sequenced. Clustal2.1 searches indicated that the sequences of the three loci of the two isolates revealed 97.8% to 100%similarity to F. oxysporum strains and were deposited in GenBank (accession nos. OP150481 and OP150485 for TEF1-α; OP150483 and OP186426 for TUB2). A pathogenicity test was performed to confirm Koch,s postulates. Inoculum was obtained from the two isolates by cultivating in 500 mL of potato dextrose broth on a shaker at 25℃. After 10 days, the hyphae grew into a cluster. The 6 individuals of P. bulbocodioides were divided into two groups. Three individuals grew in the bark substrate containing hyphae cluster, while another 3 individuals grew in the bark substrate containing sterile agar medium. The plants were kept in a greenhouse (constant temperature at 25℃, day and night for 12 h). After 20 days, the group inoculated with F. oxysporum isolates showed the same disease symptoms as observed on the plants in the field, while the control plants remained disease free. F. oxysporum was reisolated from the infected tissues (Sup. S1b, c). Phylogenetic dendrograms of Fusarium oxysporum were grouped by TEF1-α and TUB2 sequence (Sup. S3). The results confirmed that this fungus was identical to those identified by colony morphology, phylogenetic relationship and TEF1-α and TUB2 sequence. To our knowledge, this is the first report of F. oxysporum causing root rot on Pleione species in China. This is a pathogenic fungus in the production of Pleione species. Our study is helpful for the identification of root rot on Pleione species and the development of disease control strategy on cultivation.