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The existence of a complex relationship between soil microbes and ginsenoside contents in their host plants has been reported by previous studies. Here, we analyzed the interaction between the root pathogens and the ginsenoside content in the roots of American ginseng. Two fungal pathogen species were isolated from diseased American ginseng roots, and identified as Fusarium oxysporum (isolate C1) and Fusarium solani (isolate F19) by molecular sequencing analysis. To determine the effect of Fusarium-mediated infection of American ginseng roots, the contents of three ginsenosides, ginsenosides Rb 1 , ginsenosides Re and ginsenosides Rg 1 , were monitored over a time course of 120 h post infection using high performance liquid chromatography (HPLC). We found that the level of Rb 1 was rapidly upregulated upon fungal infection, whereas the contents of Re or Rg 1 were not altered significantly. Furthermore, the presence of Rb 1 , but not Re or Rg 1 , significantly inhibited conidium germination of both Fusarium species. Thus, Rb 1 is likely the disease-resistance compound produced by American ginseng roots in response to the pathogenic fungi. Our study is the first to report the three ginsenosides with different chemical structure respond differently to soilborne pathogen infection.

Asian ginseng (Panax ginseng) is an economically important perennial herb, mainly cultivated in Jilin Province, China. In September 2013, Asian ginseng plants in Jilin showed rusty root symptoms. Typical symptoms included rusty superficial lesions of irregular shapes and margins. Ten symptomatic roots were collected from each of five fields for investigation. To isolate the pathogen, root epidermal tissues with typical lesions were excised, surface-sterilized, and placed on potato dextrose agar (PDA) amended with 50 μg/ml tetracycline. After incubation at 20 ± 1°C in the dark for a week, 18 single-spore isolates out of 50 samples were obtained and identified as Ilyonectria robusta (A.A. Hildebr.) A. Cabral & Crous based on morphological characters and DNA sequence analysis (1). After incubating 7 days on PDA in the dark at 20°C, colonies were cottony to felty in texture and orange white to brownish grey in color with average diameters of 60 ± 3 mm. Isolates were cultured on synthetic nutrient-poor agar for conidial measurements. Macroconidia formed on simple conidiophores predominately, with mostly one and occasionally up to three septa, and were cylindrical with both ends broadly rounded. Macroconidia varied in size depending on the number of cells as follows: one-septate, 7.0 ± 0.6 × 27.7 ± 2.7 μm; two-septate, 7.3 ± 0.7 × 33.3 ± 2.1 μm; three-septate, 7.4 ± 0.6 × 33.4 ± 2.2 μm. Microconidia that formed on complex conidiophores were ellipsoid to ovoid and ranged in size from aseptate 3.7 ± 0.5 × 8.7 ± 1.1 μm to one-septate 5.0 ± 0.6 × 13.1 ± 1.6 μm. Brown chlamydospores were abundantly produced on PDA, globose to subglobose in shape, and in size of 10.9 ± 1.3 × 11.8 ± 1.5 μm (n ≥ 30 observations per structure for each measurement). The isolates were further classified by amplifying and sequencing the ITS1-5.8S rRNA-ITS2 region and histone H3 gene with primer pairs ITS5 and ITS4 (4), and H3-1a and H3-1b (3), respectively. Sequences of the two loci (GenBank Accession Nos. KM015300 and KM015299) showed 100% identity among the three examined isolates and the published I. robusta isolates (JF735268 and JF735517). To confirm the pathogenicity, bare roots of 3-year-old Asian ginseng were inoculated with mycelial plugs of three isolates of I. robusta selected randomly. Four roots were inoculated as replicates for each isolate with pathogen-free agar plugs as a control. One week post-inoculation in the dark at 20 ± 1°C, all the inoculated ginseng roots showed light-brown to dark-brown lesions. I. robusta was recovered from symptomatic roots and confirmed by analyzing the DNA sequence of the histone H3 gene. The inoculation experiment was repeated, and both trials showed the same results. The ginseng tissue under the control agar plugs remained symptomless, and no fungi were isolated. To our knowledge, this is the first report of I. robusta causing rusty root of P. ginseng in China (1,2,5). References: (1) A. Cabral et al. Mycol. Prog. 11:655, 2012. (2) I. Erper et al. Eur. J. Plant Pathol. 136:291, 2013. (3) N. L. Glass et al. Appl. Environ. Microbiol. 61:1323, 1995. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (5) X. Lu et al. Plant Dis. 98:1580, 2014.

In northeastern China, Asian ginseng (Panax ginseng) roots exhibited reddish brown lesions of various sizes, irregular shapes, and diffuse margins, typical of rusty root disease. The lesions remain superficial, smooth, and limited to the epidermal and peridermal tissues. In September 2013, 10 symptomatic roots were collected from each of three fields in Jilin and Heilongjiang provinces. One piece of symptomatic skin tissue from each root was excised, surface-disinfested in 1% NaClO for 3 min, rinsed three times with sterile water, and then placed on tetracycline-amended (50 μg/ml) potato dextrose agar. After incubation at 22 ± 1°C in the dark for a week, small olivaceous black colonies developed from the symptomatic tissue from five of the 30 samples. No spores were observed. A single hyphal tip of each colony was transferred to a fresh V8 agar plate to purify the culture. Two-week-old colonies on V8 agar were olivaceous gray, and 42 to 46 mm in diameter with an outer white margin (3 to 5 mm wide). Conidia produced in V8 broth after 3 weeks with a 12-h photoperiod were straight and hyaline, cylindrical or subcylindrical with no or one septum. Mature conidia were 12.8 to 21.8 × 2.2 to 4.5 μm (mean 18.2 × 3.0 μm, n = 100 conidia for each of three isolates). Three isolates selected randomly were further identified by analyzing the partial sequences of the ITS region of rDNA with primers ITS4 and ITS5 (5), and partial sequences of β-tubulin with the primers tub2F and tub2R (1). Sequences of the three isolates (GenBank Accession Nos. KJ149287, KJ149288, and KJ149290 to 93) showed 99% to 100% homology with previously identified and deposited Rhexocercosporidium panacis isolates (DQ2499992 and DQ457119) for both loci (3). Therefore, the three isolates were identified as R. panacis and deposited in China General Microbiological Culture Collection Center (CGMCC3.17259 to 61). Pathogenicity of R. panacis in Asian ginseng was investigated using these three isolates as described previously with slight modifications (4). Bare roots of 3-year-old Asian ginseng were surface-disinfested as described above, and inoculated with mycelial plugs (4 mm diameter) cut from the margin of actively growing colonies of the isolates on V8 agar. Three mycelial plugs were placed on each root at 3-cm intervals and four roots (replicates) were inoculated for each isolate. Four additional roots were inoculated with non-colonized agar plugs as control. The treated roots were placed on moist filter paper in an enamel tray. The plates were sealed with plastic wrap to prevent desiccation and incubated in the dark at 18 ± 1°C. Four weeks post inoculation, all the inoculated ginseng roots showed red-brown lesions, which turned to dark red or black over time. R. panacis was recovered from symptomatic roots for all isolates and confirmed by ITS sequence analysis. The mock-inoculated control roots remained symptomless and no R. panacis was isolated. The inoculation experiment was repeated and showed the same results. R. panacis was reported in 2006 to infect roots of Panax quinquefolius (2,3,4). To our knowledge, this is the first report of R. panacis causing rusty root of P. ginseng. References: (1) P. R. Hirsch et al. Mycol. Res. 104:435, 2000. (2) Z. K. Punja et al. Can. J. Plant Pathol. 35:503, 2013. (3) R. D. Reeleder. Mycologia. 99:91, 2007. (4) R. D. Reeleder et al. Phytopathology 96:1243, 2006. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

American ginseng (Panax quinquefolius) is an important medicinal plant cultivated in China since the 1980s. Its dried roots are used for food, health care products, and medicine in China (Yuan et al. 2010). Root rot caused by Fusarium spp. was a major disease, with 33 to 41% incidence surveyed in main production areas of Wendeng County (121.80 °E, 37.09 °N) in Shandong Province, China in 2016 to 2019. Symptoms included soft, water-soaked, dark brown to black lesions on the roots. Lesions progressed and the inner parts gradually disintegrated. One-year-old diseased roots were collected in September 2016. Symptomatic tissues were surface-sterilized in 75% ethanol for 30 s and 0.8% NaOCl for 3 min, rinsed in sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in darkness. Single colonies were then obtained and transferred to carnation leaf agar (CLA) (Burgess et al. 1993) for growth at 25°C with a 12-h photoperiod. Colonies cultured on PDA for 7 days were white to light pink, turning to apricot pigmentation in color. After 30 days on CLA, the colonies produced elongate, falcate macroconidia having 3 to 5 septa, with a long, tapering and curved apical cell, and having the size ranging from 31.1 to 45.6 μm long x 4 to 4.6 μm wide. Microconidia were zero to 1septate, ellipsoid to ovoid and varied in size from 9.5 to 16.8 μm long x 3 to 3.2 μm wide. Chlamydospores formed abundantly, in chains or clusters. This fungus was identified as F. armeniacum (Burgess et al. 1993). Identification was confirmed by sequencing three DNA regions including the internal spacer ribosomal DNA (ITS), elongation factor 1α and β-tubulin genes (Lu et al. 2019). The three DNA regions (MN417271, MG457199, and MN427653) had 100% homology to the sequences of F. armeniacum (KJ737378, HM744664 and HQ141640) (Wang et al. 2015, Yli-Mattila et al. 2011). Pathogenicity tests were conducted on 1- to 2-year-old bare roots and 2-year-old whole plants. For root inoculation, 14 healthy roots were inoculated with two mycelial PDA plugs/root. After 3 to 10 days at 25°C, all the inoculated roots showed water-soaked and root rot symptoms while no lesions were observed in the control roots. For plant inoculation, eight seedlings planted in pots filled with sterilized soil were inoculated by pouring a conidial suspension of 1×105 conidia/ml at 30 ml/pot. Eight seedlings inoculated with sterilized water served as the controls. After 90 days, only 37.5% of the roots survived with typical root rot symptoms whereas the control plants remained symptomless. F. armeniacum was re-isolated from symptomatic roots but not from the control roots. Besides F. armeniacum, F. solani and F. oxysporum that have been reported to be associated with American ginseng root rot in China and Canada (Reeleder et al. 2002; Punja et al. 2008) were also obtained from the diseased root samples in this study. However, the development of root rot caused by F. armeniacum was much more rapid and its symptoms were more severe. Moreover, F. armeniacum could directly infect American ginseng with no wound requirement. F. armeniacum was previously reported on Glycine max (Leguminosae) (Ellis et al. 2012), Platycodon grandiflorus (Campanulaceae) (Wang et al. 2015) and natural grasses (Poaceae) (Nichea et al. 2015). This is the first report of F. armeniacum causing root rot on American ginseng in China. As this species is more virulent to American ginseng, more research is needed to work on this disease.

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