First Report of <i>Ilyonectria ilicicola</i> Causing Root Rot on <i>Panax notoginseng</i> in Maguan, China

Panax notoginseng, an important Chinese medicinal plant, is susceptible to various pathogens (Li et al. 2024). In June 2023, root rot symptoms were observed on P. notoginseng grown in China fir (Cunninghamia lanceolata) forest in Maguan County, Yunnan Province, China (23.01°N, 104.52°E). However, root rot symptoms were not observed on Chinese fir. Disease incidence ranged from 5% to 10%, affecting approximately 54 ha of production in five plantations. Fifteen P. notoginseng roots with typical symptoms were used to isolate the pathogen. The diseased roots were cut into 5 × 5-mm pieces, then surface-sterilized (75% ethanol for 30 S, 2% sodium hypochlorite for 1 min) and rinsed with sterilized water three times. After dried with sterile filter paper, three to five root pieces were plated onto a potato dextrose agar (PDA) plate with streptomycin sulphate (300 mg/liter) and incubated at 25 ± 1°C in the dark. Sixteen strains were obtained from four samples. Each strain was obtained from a single spore and examined (morphology, molecular analyses, pathogenicity). Multi-locus sequencing analyses and morphological characterization shows that the sixteen strains are identical to Ilyonectria ilicicola. The symptom developments on P. notoginseng of the sixteen strains were highly consistent across three independent pathogeny trials and all inoculated strains were reisolated. Therefore, only strain LGF34-4-6 is discussed. On PDA, macroconidia were 1-, or 2- septate, rarely 3-septate, straight, cylindrical, with obtusely rounded ends, measuring 16.3 to 37.5 × 4.5 to 8.8 μm (n = 100). Microconidia were aseptate to 1-septate, ellipsoidal to fusiform (occasionally oval), measuring 6.5 to 16.1 × 3.6 to 6.5 μm (n = 100), with laterally displaced hila. Aseptate microconidia were formed on simple conidiophores. Chlamydospores were globose to subglobose, mostly lateral (rarely intercalary), solitary, measuring 11.0 to 18.8 × 9.8 to 19.6 μm (n = 100). These morphological features matched descriptions of Ilyonectria ilicicola (Mora-Sala et al. 2018). Total DNA of strain LGF34-4-6 was extracted using E.Z.N.A.® HP Fungal DNA Kit (OMEGA Bio-tek, Inc., USA). Partial sequences of beta-tubulin (tub2), histone H3 (his3), internal transcribed spacer (ITS), and translation elongation factor 1-α (TEF1α) were amplified and sequenced using the primers T1/T2 (O’Donnell &amp; Cigelnik 1997), CylH3f /CylH3r (Crouse et al. 2004), ITS1F/ITS4 (White et al. 1990), and EF1/EF2 (O’Donnell et al. 1998), respectively. Phylogenetic tree was constructed using Neighbor-joining method in MEGA 11 based on concatenation of the four sequences (Tamura et al. 2021). The obtained tub2 (PV461865), his3 (PV461847), ITS (PV453038), TEF-1α (PV461700) sequences were 100% (604/604), 99.8%(496/439), 100% (521/543), and 100% ( 807/763) identical to the corresponding DNA sequences of I. ilicicola strains (GenBank: tub2, KY676877; his3, KY676866; ITS, OK090965; TEF-1α, KY676871). Phylogenetic analysis confirmed clustering with I. ilicicola. Pathogenicity was tested using the sixteen isolates on one-year-old P. notoginseng plants, with five plants per isolate. The P. notoginseng roots were washed with sterilized water, and then surface-sterilized with 75% ethanol. Before inoculation the roots were wounded with a sterile needle. Root of each plant was inoculated by brushing 1 ml of a conidial suspension (1 × 106 spores/ml) of the corresponding strain; five control plants were received sterile water. Plants were grown in 25 cm plastic pots (five plants per pot) filled with sterilized sand-vermiculite substrate (1:1, v/v) and maintained in the greenhouse at 20 to 26°C and 80% RH. After 15 days, inoculated plants developed root rot symptoms similar to those observed in the field, whereas controls remained symptomless. The experiment was repeated twice with similar results. The pathogen was reisolated and confirmed as I. ilicicola via TEF-1α sequencing. Ilyonectria ilicicola has previously been reported to cause root diseases on Ilex sp. (Mora-Sala et al. 2018). This is the first report of I. ilicicola causing root rot on P. notoginseng in Maguan county, China. In view of the economic importance of P. notoginseng in China, this finding highlights the need for targeted management strategies against this emerging pathogen.