Endophytic fungi from the roots of horseradish (Armoracia rusticana) and their interactions with the defensive metabolites of the glucosinolate - myrosinase - isothiocyanate system

Abstract

BackgroundThe health of plants is heavily influenced by the intensively researched plant microbiome. The microbiome has to cope with the plant’s defensive secondary metabolites to survive and develop, but studies that describe this interaction are rare. In the current study, we describe interactions of endophytic fungi with a widely researched chemical defense system, the glucosinolate - myrosinase - isothiocyanate system. The antifungal isothiocyanates are also of special interest because of their beneficial effects on human consumers.ResultsSeven endophytic fungi were isolated from horseradish roots (Armoracia rusticana), from the genera Fusarium, Macrophomina, Setophoma, Paraphoma and Oidiodendron. LC-ESI-MS analysis of the horseradish extract incubated with these fungi showed that six of seven strains could decompose different classes of glucosinolates. Aliphatic, aromatic, thiomethylalkyl and indolic glucosinolates were decomposed by different strains at different rates. SPME-GC-MS measurements showed that two strains released significant amounts of allyl isothiocyanate into the surrounding air, but allyl nitrile was not detected. The LC-ESI-MS analysis of many strains’ media showed the presence of allyl isothiocyanate - glutathione conjugate during the decomposition of sinigrin. Four endophytic strains also accepted sinigrin as the sole carbon source. Isothiocyanates inhibited the growth of fungi at various concentrations, phenylethyl isothiocyanate was more potent than allyl isothiocyanate (mean IC50 was 2.30-fold lower).As a control group, ten soil fungi from the same soil were used. They decomposed glucosinolates with lower overall efficiency: six of ten strains had insignificant or weak activities and only three could use sinigrin as a carbon source. The soil fungi also showed lower AITC tolerance in the growth inhibition assay: the median IC50 values were 0.1925 mM for endophytes and 0.0899 mM for soil fungi.ConclusionsThe host’s glucosinolates can be used by the tested endophytic fungi as nutrients or to gain competitive advantage over less tolerant species. These activities were much less apparent among the soil fungi. This suggests that the endophytes show adaptation to the host plant’s secondary metabolites and that host metabolite specific activities are enriched in the root microbiome. The results present background mechanisms enabling an understanding of how plants shape their microbiome.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1295-4) contains supplementary material, which is available to authorized users.

A Simple Method for On-Gel Detection of Myrosinase Activity

“…Successful detection of glucosinolate-decomposing enzymes was also achieved from two of the endophytic fungi of horseradish, Fusarium oxysporum and Macrophomina phaseolina , which were recently shown to possess decomposing activity towards plant glucosinolates [ 8 ] ( Figure S6 ). As allyl-isothiocyanate-GSH conjugate was detected from the spent medium, but desulfoglucosinolates were not [ 8 ], these organisms also likely possess myrosinase activity. The crude extract of the fungi (prepared with the same methodology as the crude plant extract) were shown to be orders of magnitude less active than that of the vegetables.…”

“…Selected horseradish endophytes from our previous study [ 8 ] ( Fusarium oxysporum , Macrophomina phaseolina ) were grown in inactivated horseradish extract until reaching stationary phase of growth in which decomposition of sinigrin usually initiates (5 and 7 days, respectively). The inactivated horseradish extract was prepared using boiling methanol, therefore it does not contain any active enzymes.…”

“…It was standardized to contain 12 mM (5 mg mL −1 ) sinigrin. The preparation of the inactivated horseradish extract and the inoculation parameters were the same as in our recent work [ 8 ].…”

“…The default products are isothiocyanates, or, in vivo in the presence of so called specifier proteins, other less toxic volatiles can be formed. The activity itself is shown to be present in various Brassicaceae plants [ 3 , 4 ], microorganisms [ 5 , 6 , 7 ], and organisms associated with such plants, like endophytes from horseradish roots [ 8 ] or some insects feeding on host plants with such metabolites [ 9 , 10 , 11 ]. A simple plant can contain various myrosinase isoenzymes, as shown in Arabidopsis thaliana [ 12 ] and other Brassicaceae plants [ 13 , 14 , 15 , 16 ].…”

Effects of Endophytic Fungus MF23 onDendrobium nobileLindl. in an artificial primary environment

“…Guo et al [9–10] obtained a number of fungi from the Dendrobium, which could effectively promote the germination of Dendrobium seeds. Fungi also affected the growth of the plant [11], through the way of invading the root cells, and forming mycorrhizal symbioses with the root. It could supply the water and nutrients such as nitrogen and phosphorus to the host plant in a further thought [12].…”