First Report of Stem Rot Caused by Fusarium annulatum on Anoectochilus roxburghii (Wall.) Lindl. in China

Anoectochilus roxburghii (Wall.) Lindl., a rare and highly valued medicinal species within the genus Anoectochilus of the Orchidaceae family, is predominantly found in subtropical regions where it favors shaded and humid habitats. Its cultivation is of increasing economic importance in Guangdong Province, South China, where it is grown for use in traditional medicine. In April 2025, an outbreak of stem rot affecting A. roxburghii was identified in Boluo County, Guangdong Province (23.21234°N, 114.04051°E), with an observed incidence rate of 30% in one approximately 300 m² field plot. The disease symptoms manifested as water-soaked lesions located at the basal or mid-stem regions, which subsequently developed into yellowed, desiccated, and constricted brown necrotic streaks resembling thread-like structures (Figure S1A). To identify the causal agent responsible for stem rot, stem segments measuring 1 cm were excised from the junction of diseased and healthy tissues and subjected to surface sterilization by immersion in 75% ethanol for 45 seconds, followed by treatment with 3% sodium hypochlorite for one minute. Subsequently, the segments were rinsed with sterile distilled water, dried on sterilized tissue paper, and inoculated onto PDA plates. The Petri dishes were incubated at 28°C for a period of three days. Subsequent single-spore isolation yielded five pure cultures. Among these, one predominant isolate, designated as isolate Z2, was obtained from 80% (32/40) of the plated diseased tissue segments. The remaining four isolates, identified through preliminary sequencing, were recovered at significantly lower frequencies and did not consistently align with the observed disease symptoms; therefore, they were not evaluated further. After seven days of growth on PDA, the colony exhibited a circular morphology with woolly aerial mycelium and an off-white coloration (Figure S1B). Microscopic examination revealed hyphae with distinct septation. Microconidia were abundant, mono- or poly-phialidic, variable in shape, mostly oval, ellipsoidal to clavate, 0- to 1-septate, smooth-walled, and measured 3.3–15.7 × 1.6–4.1 μm (mean 8.2 × 2.8 μm, n=30). Larger conidia were less frequent, fusiform to cylindrical, 1- to 3-septate, measuring 17.1–45.3 × 3.5–6.0 μm (mean 25.8 × 4.8 μm, n=30). Typical multi-septate (3–5 septa) falcate macroconidia were observed but rare under the tested culture conditions (Figure S1C-D). Genomic DNA of isolate Z2 was extracted from the mycelium employing the CTAB method. PCR amplification was conducted utilizing the primer pairs EF1/EF2, Fa/G2R, and 5f2/7cr, which specifically targeted the TEF1-α, RPB1, and RPB2 gene regions, respectively (O'Donnell et al., 2022). The sequences were deposited in the GenBank database. BLASTn analysis revealed that the TEF1-α (PX531326.1), RPB1 (PX569646.1), and RPB2 (PX583276.1) sequences from isolate Z2 exhibited high sequence similarities (each 99%) with the corresponding sequences of Fusarium annulatum strain CBS 140150. Phylogenetic analysis was performed based on the concatenated nucleotide sequences of TEF1-α, RPB1 and RPB2, employing the IQ-TREE algorithm implemented within the PhyloSuite software (Fig S2) (Zhang et al., 2020). The phylogenetic analysis revealed that isolate Z2 grouped within the same evolutionary clade as F. annulatum. Based on both morphological traits and sequence data, the isolated isolate Z2 was identified as F. annulatum, consistent with the taxonomic framework proposed by Yilmaz et al. (2021). To evaluate the pathogenic potential of isolate Z2, a 10 μL droplet of a spore suspension (10⁶ spores/mL) was placed on a shallow wound made on the stem with a sterile needle. For the negative control, an equal volume of sterile distilled water was inoculated onto healthy stems using the same method. The inoculated specimens were maintained under controlled conditions of 28°C, 80% relative humidity, and a 12-hour photoperiod. Ten days following inoculation, water-soaked rot symptoms appeared on the stems, progressing to desiccation and ultimately causing plant lodging (Fig. S1F). In contrast, the negative control plants remained healthy and asymptomatic (Fig. S1E). Subsequently, F. annulatum isolate Z2 was successfully re-isolated from the symptomatic stem tissues, and its identity was confirmed by sequencing the TEF1-α, RPB1, and RPB2 genes. To our knowledge, this study constitutes the first report of F. annulatum inducing stem rot in A. roxburghii within Guangdong Province, China. These results provide critical insights that may inform the development of effective disease management strategies.