Pythium irregulare subsp. cultures Buisman

2.3. In vitro exposure of P. irregulare to purified ginsenoside extract and pure ginsenosides The effects of a purified total ginsenoside extract and selected pure protopanaxadiol (Rb1 (1), F2 (4 )) and protopanaxatriol ginsenosides (Re, 6) on the growth of P. irregulare in vitro allowed us to gain a more complete understanding of the effect a point source of ginsenosides had on the growth pattern of the pathogen. Differential growth of mycelia from P. irregulare was monitored as it progressed toward ginsenoside-treated and non-treated disks on minimal Czapek-Dox Mineral Agar (Fig. 5). Mycelial growth appeared to be unaffected by the presence of the control disks (evaporated MeOH) as well as disks infused with 0.5 mg of the 20(S)-protopanaxatriol ginsenoside Re (6, Fig. 5B). Mycelia grew directly through these disks, showing no visible alteration in growth habit up to five days after initiation of growth. Exposure of P. irregulare to purified total ginsenoside extract (GSF; 1 mg) resulted in enhanced aerial mycelial growth and a distinct accumulation of mycelia around treated disks, albeit a short distance away from the filter disk (Fig. 5A). This accumulation persisted for several days, forming a visible ring around the filter disk. Mycelia continued to project out from this ring and accumulate directly around the assay filter disk until the end of the monitoring period of five days. Similarly, exposure of P. irregulare to 20(S)-protopanaxadiol ginsenoside Rb1 (1) (0.5 mg; Rb1 (1) represents ca. half of the total GSF) (Fig. 5C) resulted in an accumulation of mycelia around the treated disk. After five days, the ring of mycelia around the filter disk was complete and mycelial growth projected beyond it. That ginsenoside Re (6) had no effect, positive or negative, on the growth of P. irregulare in vitro, while both GSF and Rb1 (1) alone did, suggests that the protopanaxadiol ginsenosides are also the more bioactive towards this pathogen. These are also the only ginsenosides shown to be enzymatically altered by P. irregulare by extracellular glycosidases (Yousef and Bernards, 2006; Neculai et al., 2009; Ivanov and Bernards, 2012). The ring of enhanced mycelial growth around assay disks infused with GSF or ginsenoside Rb1 1 presumably represents the point of primary contact between the pathogen and the ginsenosides because otherwise P. irregulare mycelia grew thinly and in all directions until within close proximity to the treated disks. While there was extensive mycelial growth and hyphal branching observed around the GSF and Rb1 (1) treated disks, upon close inspection, it was evident that hyphal growth was not inhibited, and spread directly through the medium around and under the treated filter disks the same way it did through the control disks. In contrast, the growth of mycelia was clearly inhibited around the assay disk infused with ginsenoside F2 (4) (Fig. 5D; 0.5 mg). Within two days of growth, inhibition of mycelial growth towards the F2 (4) infused assay disk was visually evident. While the inhibitory effect of F2 (4) persisted throughout the five day time course, it did appear to diminish, as mycelia began to grow around the filter disk, almost completely surrounding it by the end of the experiment. However, mycelia were never seen to grow through the filter; neither was any enhanced aerial growth observed at any time during the time course. Alternatively, the presence of relatively large doses of ginsenosides in the rhizosphere may have simply altered the growth pattern of the pathogen in the soil, as seen when P. irregulare is grown in medium supplemented with ginsenosides (Nicol et al., 2002, 2003; see also below). Moreover, P. irregulare has been shown to directly interact with ginsenosides in vitro, by deglycosylating them into ginsenoside F2 (4) (Yousef and Bernards, 2006; Neculai et al., 2009; Ivanov and Bernards, 2012). It is possible, therefore, that this interaction results in altered patterns of growth in the soil, which may have the secondary effect of delaying infection. Unfortunately, our attempts to directly assess the extent of P. irregulare growth in the pots, using ITS region nuclear ribosomal DNA markers (Okubara et al., 2005; Schroeder et al., 2006; Kernaghan et al., 2007; Kernaghan et al., 2008) were unsuccessful. The effects of all ginsenoside treatments on mycelial growth, except ginsenoside Re (6), were dose dependent (see Supporting Information Figs. S1–S 4). That is, enhanced mycelial growth increased with ginsenoside concentration around filters treated with GSF and ginsenoside Rb1 (1), while the zone of inhibition around disks infused with ginsenoside F2 (4) increased with concentration. It is not clear from these in vitro data whether the enhanced growth of P. irregulare in the presence of protopanaxadiols was due to ginsenosides acting as chemoattractants or growth factors. Furthermore, while ginsenoside Rb1 (1) appeared to stimulate pathogen growth on its own, the effect of the ginsenoside mixture was greater and correlated to the increased concentration of ginsenosides applied. Ginsenoside Rb1 (1) applied in the same concentration as the ginsenoside mixture showed an equivalent level of growth stimulation (Supporting Information Figs. S4.1 – S4.4). Therefore, while ginsenoside Rb1 (1) appears to be involved in the growth stimulation of P. irregulare, it is not the only ginsenoside that has this effect. Based on these data, it remains unclear why P. irregulare invests the energy to convert the common ginsenosides (e.g., Rb1 (1), Rd (2), gypenoside XIIV (3 )) into ginsenoside F2 (4) via extracellular glycosidases (Yousef and Bernards, 2006), when in fact the growth of this organism is visibly inhibited by the pure F2 (4) compound. It is possible that the presence of common ginsenosides stimulates a change in P. irregulare metabolism, leading to the expression of genes encoding extracellular glycosidases, as well as genes associated with the utilization of F2 (4) (e.g., as a growth stimulant). By contrast, direct exposure to pure F2 (4) may circumvent the metabolic shift needed to utilize this compound and avoid the potential deleterious effects it can have on growth. Regardless, the impact of GSF and Rb1 on P. irregulare growth habit in the in vitro assays may inform our observations in the in vivo assays. That is, when hyphae of P. irregulare encounter ginsenosides in the soil surrounding roots of ginseng, it is likely that forward progress is temporarily halted while the organism metabolizes these compounds, building up hyphal mass in the process. Such a change in growth habit would explain the apparent delay in infection of ginsenoside-treated roots, which would have a higher concentration of ginsenosides in the rhizosphere.

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