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Arbuscular mycorrhizal fungus causes increased condensed tannins concentrations in shoots but decreased in roots of Lotus japonicus L.
Author’s Accepted Manuscript Arbuscular mycorrhizal fungus causes increased condensed tannins concentrations in shoots but decreased in roots of Lotus japonicus L Zakaria M. Solaiman, Keishi Senoo www.elsevier.com
PII: DOI: Reference:
S2452-2198(17)30183-0 https://doi.org/10.1016/j.rhisph.2017.11.006 RHISPH91
To appear in: Rhizosphere Received date: 27 October 2017 Revised date: 28 November 2017 Accepted date: 28 November 2017 Cite this article as: Zakaria M. Solaiman and Keishi Senoo, Arbuscular mycorrhizal fungus causes increased condensed tannins concentrations in shoots but decreased in roots of Lotus japonicus L, Rhizosphere, https://doi.org/10.1016/j.rhisph.2017.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Arbuscular mycorrhizal fungus causes increased condensed tannins concentrations in shoots but decreased in roots of Lotus japonicus L.
Zakaria M. Solaimana* and Keishi Senoob a
SoilsWest, UWA School of Agriculture and Environment, and The UWA Institute of
Agriculture, Faculty of Science, The University of Western Australia, Crawley, WA 6009, Australia b
Laboratory of Soil Science, Department of Applied Biological Chemistry, Graduate
School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo113-8657, Japan *
Corresponding author: [email protected]
ABSTRACT Condensed tannins are a class of plant secondary metabolites which are formed by the condensation of flavanols, and play an important role in plant-soil-microbes interactions by influencing the colonisation of arbuscular mycorrhizal (AM) fungi. The aim of this study was to determine the influence of AM fungi on condensed tannins concentrations in roots, stems and leaves of birdsfoot trefoil (Lotus japonicus L.). The concentrations of condensed tannins in roots, stems and leaves of the Lotus japonicus plants were measured using histochemical staining and spectrophotometry. The extent of AM fungal colonisation in roots was also quantified. Soluble and insoluble condensed tannins concentrations in roots were lower in plants inoculated with AM fungi but higher in stems and leaves compared to plants not inoculated with AM fungi. The higher concentration of condensed tannins in the stems and leaves were associated with AM
1
fungi colonisation of roots. This had increased animal feed value and also could be influenced by the plant-soil-microbes feedback processes.
Keywords: Condensed tannins, Lotus japonicus, Flavonols, Fodder, Mycorrhizas
1. Introduction Condensed tannins, also known as proanthocyanidins, are a group of plant secondary metabolites and are part of a larger group of compounds, the polyphenols. Condensed tannins are present in most vascular plants and are thought to play various important roles associated with processes of plant defence against pests (Brillouet et al., 2013). The most common type of condensed tannins are produced from cyanidin which are metabolised by acid hydrolysis named as proanthocyanidins.
Condensed tannins are often found in great concentrations in tissues of coniferous trees and shrubs as well as in pasture plants (Ushio et al., 2013; Hartley and Jones, 1997; Jones et al. 1976) and formed by the polymerisation of catechins. The concentrations of condensed tannins can be up to 17 mg g-1 of the dry weight in birdsfoot trefoil sward tissues (Marshall et al., 2013). Condensed tannins have a significant role on the soil mineralisation processes (Ushio et al., 2013) and may also act in defence against pathogens (Brownlee et al., 1992; Min et al., 2003; Edwards, 1992), pests and herbivores (Swain 1979). A group of saprophytic fungi can utilise and grow on medium containing high concentrations of condensed tannins (Ushio et al., 2013). Furthermore, fungal inhibitory tannins concentration is relatively higher than the concentration that 2
inhibit bacteria (Scalbert, 1991). However, the biological activity of plant condensed tannins depends on their chemical structure and concentration (Dixon et al., 2005; Monagas et al., 2010; Dixon et al., 2013). Understanding the effect of AM fungi colonisation on condensed tannins and phenolic concentration in the roots, stems and leaves of birdsfoot trefoil (Lotus japonicus) are essential for improving feed quality (Frutos et al., 2004).
Birdsfoot trefoil is a fabaceae plant suitable for infertile, acidic soils and is highly persistent, productive, drought-tolerant and bloat-safe feed for livestock (Hancock et al., 2014). This plant has an extensive tap root system, therefore it can effectively mitigate dryland salinity through improving soil water use in recharge environments. Hence, birdsfoot trefoil has the potential for widespread use in many areas of Australia where there is a lack of an adapted pasture legumes (Moore et al., 2006). The model legume birdsfoot trefoil was used as a host plant for this study as this plant has been successfully used as AM hosts in previous studies (Senoo et al., 2000; Solaiman et al., 2000; Solaiman et al., 2007) where the investigators screened mycorrhizal mutant Ljsym72. Therefore, mycorrhizal mutant Ljsym72 was used in this study to mimic the control plant (Solaiman and Senoo, 2005). This mutant plant Ljsym72 formed a small quantity of AM colonisation characterised by the formation of branched appressoria on the root surfaces and the blocking of hyphal penetration at epidermis, however produced similar shoot weight of wild-type Gifu plant.
The formation and functioning of AM fungal symbiosis is assumed to involve signals between the host plant and the fungal symbiont, although the nature and mechanism of 3
the action of such signals is unclear (Takeda et al., 2015). Genre et al. (2013) proposed that short-chain chitin oligomers secreted by AM fungi are part of a molecular exchange with the host plant and that their presence in the epidermis leads to the activation of a SYM-dependent signalling pathway involved in the initial stages of fungal root colonisation. But the influence of flavonoid and isoflavonoid compounds on fungal growth, however, unclear whether they are essential signal molecules for the AM symbiosis. Previous studies reported phenolic compounds found in the cell walls of roots and have also been proposed as potential signal molecules (Harrison, 1997; Mandal et al., 2010). Comparisons of host and non-host plants have shown stimulatory and inhibitory compounds, but did not reveal a compound responsible for the formation of appressoria on host roots or for the lack of colonisation of non-host roots (Douds et al., 1996; Harrison, 1997). When screening for AM defective plant mutants, structures in trypan blue stained roots in addition to mycorrhizal structures were observed and subsequently identified as condensed tannins following vanillin-HCl histochemical staining procedures (Broadhurst and Jones, 1978). Here, the effect of AM fungal colonisation on condensed tannin synthesis in different parts of birdsfoot trefoil plant was investigated.
2. Materials and methods 2.1 Potting medium and mycorrhizal inoculum Washed sand was mixed with a low (10 µg/g) phosphorus-containing volcanic ash soil (v/v 1:1), sterilised by autoclaving (121 oC, 1 h) and amended with NH4NO3, KH2PO4 and KCl at a rate of 0.16 g, 0.008 g, and 0.032 g per pot (300 g sand and soil mix), respectively. AM fungal inoculum containing spores of single Glomus sp. R-10, species 4
name unidentified (Idemitsu Kosan Co, Ltd, Japan), was mixed thoroughly with the potting mixture (10%, v/v). Non-mycorrhizal treatment was amended with washing of inoculum (equivalent amount of inoculum was suspended in deionised water and filtered) to keep bacterial community similar.
2.2 Plant growth experiment The experiment was conducted following a two factorial randomised design including two plant genotypes ((wild-type Gifu B-129 (nod+fix+myc+) and a nod-fix-myc- mutant (Ljsym72)) × two mycorrhizal inoculum (inoculated and non-inoculated) with three replications. Mycorrhizal mutant Ljsym72 was used to mimic the control plant as this mutant formed <5% of deformed colonisation in the root epidermis. Seeds from wildtype Gifu B-129 and a mycorrhizal mutant (Ljsym72) were scarified using sand paper with the surface sterilised with 2% sodium hypochlorite solution containing 0.02% Tween 20 for ten minutes, rinsed in sterile distilled water and germinated on sterilised moist filter paper in Petri dishes at 25 oC under dark conditions. After germination, seedlings were transplanted into individual sections in nursery trays (six plants per tray) containing the sand-soil-inoculum mix substrate (300 g of mix per tray of plants). Three-week-old seedlings were then transplanted into pots (six plants per 500 ml pot) with three replicates (18 plants per treatment) and allowed to grow in a growth chamber. Additional seedlings of Gifu were grown in the sand-soil substrate without mycorrhizal inoculum added to provide a check for infectivity of the inoculum, as well as the extent and characteristics of colonisation in the parent plants. Plants were kept in the growth chamber (day: 20 h, 25 oC, 100 mmol m-2 s-1 photosynthetic photon flux density; night: 4 h, 22 oC) for nine weeks. Sampling was conducted at six and nine weeks after 5
transplanting of seedlings for the assessment of mycorrhizal colonisation, condensed tannins and flavanols.
2.3 Evaluation of mycorrhizal colonisation Root length colonised by the AM fungi was estimated at six and nine weeks after transplanting of seedlings. Root samples (0.5 g fresh weight) were cleared in 10% KOH and stained with trypan blue using a modification of the method by Phillips and Hayman (1970), in which lactoglycerol was used instead of lactophenol (Solaiman and Abbott 2008). Colonisation in the inoculated Gifu B-129 plants was determined as the percentage root length colonised under a dissecting microscope using the gridline intersect method (Tennant, 1975). Gridline intersects were scored for the presence of mycorrhizal structures at ×100 magnification.
The evaluation of mycorrhizal
colonisation was carried out in three replicates samples.
2.4 Histochemical staining procedure for condensed tannins Sections of roots, stems and leaves were stained with vanillin/HCl (one drop of 10% vanillin in absolute ethanol followed by one drop of 6 M HCl after 2 minutes). Tissue was mounted on slides and examined microscopically for tannins containing cells.
2.5 Chemical extraction of condensed tannins Each representative sample (roots, stems and leaves) was prepared from six plants comprising three representative fresh tissue samples per treatment of each mycorrhizal and non-mycorrhizal wild plant Gifu. Samples were frozen in liquid nitrogen at -80 oC before extraction. Mutant plant Ljsym72 was excluded from this chemical extraction 6
procedure due to the high similarity in the number of condensed tannins granules in roots of non-mycorrhizal wild plant Gifu (shown in histochemical staining). Fresh tissue was extracted with methanol followed by the method used by Stafford and Lester (1980), with minor modifications. Two hundred mg of fresh weight tissues from both harvests were homogenised in 2 × 1 ml aliquots methanol (80:20 v/v) with a mortar and pestle. The ground powder and washings were centrifuged (2000 × g, 5 minutes) and the supernatant liquid transferred to a new Eppendorf tube. After evaporation of methanol under vacuum between 30 and 35 oC, the aqueous fraction was extracted with petroleum ether to remove lipids. Petroleum ether was removed after evaporation, the remaining aqueous extract was adjusted to 5 ml with water, and aliquots analysed for soluble tannins and soluble flavanols. The residue after centrifugation was analysed for insoluble tannins.
2.6 Condensed tannins analysis Tannin analysis was based on the formation of cyanidin on acid hydrolysis. Condensed tannins concentration was determined with the improved acid butanol assay (Porter et al., 1986). Aliquots in a final volume of 0.1 ml were heated with 1 ml butanol-HCl (95:5, v/v) at approximately 95 oC for 30 minutes (Stafford and Cheng, 1980; Berard et al., 2011). The absorbance was determined at 550 nm, the peak absorption for cyanidin. Estimates of the condensed tannin as mg g-1 fresh weight of tissue were made at 550 nm (Stafford and Cheng, 1980) by using purified Quebracho tannin to standardise the measurements (Waterman and Mole, 1994).
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2.7 Flavanol analysis Flavanol analysis was based on the reaction with vanillin in acidic solution (Swain and Hills 1959). Half-a-millilitre aliquots of the extract was mixed with 4.5 ml 1% vanillin in 70% H2SO4 and the absorbance at 500 nm measured after 30 minutes. Flavonols had specificity to vanillin (Sarker and Howarth, 1976). Values are expressed as catechin equivalents. Vanillin-H2SO4 also reacts with oligomeric and polymeric flavanols and anthocyanins, but these were shown to be absent from the methanol-soluble extracts (Sarker and Howarth, 1976).
2.8 Statistical analysis Data were expressed as means. A normality test was applied before analysis of variance (ANOVA). The two-way ANOVA was used to compare the effects of treatments (genotypes × mycorrhiza inoculum), followed by Tukey’s post hoc test for multiple comparisons of means using GENSTAT 16th Edition (Lawes Agricultural Trust, 2014). Significant differences at p≤0.05 were considered.
3. Results Mycorrhizal plants L. japonicus produced higher biomass than the non-mycorrhizal plants at six and nine weeks of growth (Table 1). Inoculated non-mycorrhizal mutant plant Ljsym72 produced similar root dry weight as un-inoculated Gifu wild-type but had very similar dry weights of stem and leaf as the inoculated Gifu wild-type. Mycorrhizal root colonisation of wild-type Gifu was 54% at six weeks and 79% at nine weeks of growth after transplanting of seedlings (Table 1). In contrast, non-inoculated Gifu wild type did not show any mycorrhizal colonisation and inoculated mycorrhizal mutant
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plant Ljsym72 showed 4% of root colonisation at both six and nine weeks growth after transplanting of seedlings. Variation in mycorrhizal colonisation patterns in inoculated Gifu wide-type and mutant Ljsym72 were observed (Fig. 1A, B) as well as in the tannin granules (Fig. 1C).
Histochemical staining of condensed tannin showed that tannin granules were more present in non-mycorrhizal Gifu wild-type roots and mutant plants roots compared to mycorrhizal roots of Gifu wild-type (Fig. 2). In contrast, mycorrhizal plants shoots (stems and leaves) showed higher condensed tannin granules compared to shoots of non-mycorrhizal and mutant plants (Fig. 3). Soluble condensed tannin concentrations were reduced in mycorrhizal roots compared to non-mycorrhizal roots, however, was increased in mycorrhiza inoculated plant shoots (stems and leaves) compared to nonmycorrhizal plant shoots at six and nine weeks of growth (Table 2). Insoluble tannin concentration in roots was higher in non-inoculated plants only at nine weeks of growth whereas it was higher in leaves of inoculated plants both at six and nine weeks of growth (Table 2). Insoluble tannin concentration in stems was similar for both inoculated and non-inoculated plants at six and nine weeks of growth respectively. Flavanols concentration was increased in roots and stems of mycorrhizal inoculated plants compared to non-inoculated plants (Table 3). However, there was no mycorrhizal inoculation effect observed on flavanols concentration of plant shoots (stem and leaves) at both six and nine weeks of growth (Table 3).
4. Discussion
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Many signalling compounds produced by plants include both primary metabolites (carbohydrates, proteins, organic acids etc.) and secondary metabolites (flavonols, phenol, phytohormones etc.) (Singh et al., 2016). The signalling compounds play diverse roles in ensuring benefits to both parties of the plant–microbe interaction. Condensed tannins are a group of plant secondary metabolites and it’s concentration in roots, stems and leaves between AM fungi inoculated and non-inoculated birdsfoot trefoil plants differed significantly in the present study. Mutant plant Ljsym72 was excluded from the chemical extraction procedure of condensed tannins due to the high similarity in the number of condensed tannins granules in roots between mutant and non-inoculated control plants. However, future research should include this variable to clarify the confounding effect of growth differences between mycorrhizal inoculated and non-inoculated plants. The variation in tannins concentration of mycorrhizal and non-mycorrhizal wild-type Gifu plants strongly indicates the efficiency of AM fungi in maximising the production of primary and secondary metabolites in birdsfoot trefoil. This was observed in previous studies in plants such as stevia herb (Mandal et al., 2013), cebil seedlings (Pedone-Bonfim et al., 2013), medicinal herbs (Jugran et al., 2015; Lima et al., 2017) and in clover (Zhang et al., 2013). In some cases, AM fungi inoculation was more efficient in supporting the production of these compounds compared with the uptake of P (Nair et al., 1991). This increase is a direct effect of the AM fungal colonisation which should supply nutrients, especially P, to the plant by the mycorrhizal pathway (Smith and Read, 2008). However, in the present study, P analysis was not carried out to test the aforementioned hypothesis. The study also showed that mycorrhizal colonisation of plants relieved nutrient deficiency and enhanced the growth of the plants, and thus, reduced the tannins concentration of the roots and increased in 10
shoots (da Silva et al., 2014). The high tannins concentration of non-mycorrhizal roots may have been induced by deficiency of nutrients (N and P) in the plant tissue which is similar to non-fertilised plants (Jones et al., 1976; Ferwerda et al., 2005). Therefore, the mechanism(s) through which AM fungi affect tannins production in different parts of the plants may be related to availability of N and P to the plants and warrants further investigation. The low concentration of tannins in plant roots inoculated with AM fungi supports compatibility of the symbiotic partnership between birdsfoot trefoil and AM fungus (Campos-Soriano et al., 2012; Zakhia et al., 2003). Phytohormones, such as auxins and cytokinins act as signalling molecules and affect cell proliferation or modify root system architecture by overproduction of lateral roots and root hairs with a subsequent increase of nutrient and water uptake (Ortíz-Castro et al., 2009) and also play an important role in plant and AM fungi interactions (LudwigMüller and Güther, 2007). An assortment of secondary plant metabolites such as flavonoids and strigolactones, the latter of which is excreted by roots, also carry importance as signalling molecules in several symbiotic and pathogenic plant–microbe interactions (Steinkellner et al., 2007). For example, in response to the root exudation of signal molecules recognised as plant hormones strigolactones, hyphal branching of AM fungi induced and colonise plant roots (Akiyama et al., 2005; Haichar et al., 2014). In this study, AM fungus inoculation was efficient in increasing the concentration of flavonols and soluble tannins in the shoot (stems and leaves), thus showing the potential of AM fungi in increasing the production of plant secondary metabolites (Zhang et al., 2013). The increasing concentration of condensed tannins in the leaves of the forest plant such as Dacrydium than the Lithocarpus plant may be due to the influence of the microbial community composition and their functions in the rhizospheres which can 11
subsequently influence plant performance and induce plant-soil-microbes feedback processes (Ushio et al., 2013). The production of fabaceae plants with a high concentration of phenolic compounds adds value to the vegetative fodder biomass, making it a more attractive feed for animals. The phenolic-abundant plants also protect protein in the animal rumen and in silo, thereby reducing the rate of protein degradation and consequent nitrogenous losses to the environment (Waghorn and Shelton, 1992). The presence of condensed tannin can also reduce the incidence of pasture bloat (McMahon et al., 2000) and reduce ruminants’ methane emissions (Ramirez-Restrepo and Barry, 2005). Although for a long time tannins were thought to be detrimental to ruminants, their effect may be beneficial or harmful depending on the type of tannins consumed, its chemical structure and molecular weight, the amount ingested, and the animal species involved (Hagerman and Butler, 1991; Frutos et al., 2004). High concentration of tannins (generally > 50 g kg-1 of dry matter, DM) reduce voluntary feed intake and nutrient digestibility, whereas low to moderate concentrations (<50 g kg-1 of DM) may improve the digestive utilisation of feed mainly due to a decrease in protein degradation in the rumen and a subsequent increase in amino acid movement to the small intestine (Barry and Duncan, 1984). These effects on nutrition are shown in improved animal performance (Frutos et al., 2004). In this study, the concentration of tannins in stems and leaves are low which may increase animal performance through the aforementioned mechanism.
5. Conclusion In conclusion, increased condensed tannins concentrations were observed in shoots and decreased in roots of plants colonised with AM fungi. These results support the positive 12
signal in mutualistic AM symbiosis i.e. the recognition of AM fungal entry to the root epidermis as opposed to that of systemic resistance in plant-pathogen interaction. A better understanding of the factors controlling the biosynthesis, accumulation, and release of these secondary metabolites condensed tannins may represent a novel way to manipulate AM symbiotic systems.
Acknowledgements The authors thank Dr Motoshi Suzuki of Idemitsu Kosan Co., Ltd, Tokyo, Japan for supplying spores of Glomus sp. R-10. The author ZMS is grateful to the Japan Society for the Promotion of Science for providing postdoctoral fellowships.
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Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis. Springer, Berlin. Solaiman, Z.M., Abbott, L.K., 2008. Influence of arbuscular mycorrhizal fungi, inoculum level and phosphorus placement on growth and phosphorus uptake of Phyllanthus calycinus in jarrah forest soil. Biol. Fert. Soils. 44, 815-821. Solaiman, Z.M., Marschner, P., Wang, D., Rengel, Z.. 2007. Phosphorus uptake and rhizosphere properties of wheat and canola genotypes in alkaline oil under P-limiting conditions. Biol. Fert. Soils. 44, 143-153. Solaiman, Z.M., Senoo, K., 2005. Interactions between Lotus japonicus genotypes and arbuscular mycorrhizal fungi. J. Plant Interact. 1, 179-186. Solaiman, Z.M., Senoo, K., Kawaguchi, M., Imaizumi-Anraku, H., Akao, S., Tanaka, A., Obata, H., 2000. Characterization of mycorhizal colonization by Glomus sp. on root of hypernodulating mutants of Lotus japonicas. J. Plant Res. 113, 443-448. Stafford, H.A., Lester, H.H., 1980. Procyanidins (condensed tannin) in green cell suspension culture of douglas fir compared with those in strawberry and avocado leaves by means of C18-reversed-phase chromatography. Plant Physiol. 66, 1085-1090. Stafford, H.A., Cheng, T.Y., 1980. The procyanidins of Douglas-fir seedlings, callus and cell suspension cultures derived from cotyledons. Phytochem. 19, 131-135. Steinkellner, S., Lendzemo, V., Langer, I., Schweiger, P., Khaosaad, T., Toussaint, J.P., Vierheilig, H., 2007. Flavonoids and strigolactones in root exudates as signals in symbiotic and pathogenic plant-fungus interactions. Molecules 12, 1290–1306. Swain, T., 1979. Tannins and lignins, In: Rosenthal GA, Janzen DH (Eds.) Herbivores: their interactions with secondary plant metabolites, Academic Press, NY, pp 657-682. Swain, T., Hillis, W.E., 1959. The periodic constituents of Prunus domestica. I. The quantitative analysis of phenolic constituents. J. Agric. Food Chem. 10, 63-68. 18
Takeda, N., Handa, Y., Tsuzuki, S., Kojima, M., Sakakibara, H., Kawaguchi, M., 2015. Gibberellins interfere with symbiosis signalling and gene expression and alter colonization by arbuscular mycorrhizal fungi in Lotus japonicus. Plant Physiol. 167, 545-557. Tennant, D., 1975. A test of a modified line intersect method of estimating root length. J. Ecol. 63, 995-1001. Ushio, M., Balser, T.C., Kitayama, K., 2013. Effect of condensed tannins in conifer leaves on the composition and activity of the soil microbial community in a tropical mountain forest. Plant Soil 365, 157-170. Waghorn, G.C., Shelton, I.D., 1992. The nutritive value of Lotus for sheep. Proc. New Zealand Soc. Animal Prod. 57, 89-92. Waterman, P.G., Mole, S., 1994. Analysis of phenolic plant metabolites, Blackwell Science, Oxford. Zakhia, F., Ducousso, M., Mondolot, L., Cleyet-Marel, J.C., Andary, C. 2003. Spot inoculation of Medicago laciniata root by Sinorhizobium meliloti CI-4 or Glomus sp. S043 induces local accumulation of flavonoids. Acta Botanica Gallica 150, 275-283. Zhang, R.Q., Zhu, H.H., Zhao, H.Q., Yao, Q. 2013. Arbuscular mycorrhizal fungal inoculation increases phenolic synthesis in clover roots via hydrogen peroxide, salicylic acid and nitric oxide signaling pathways. J Plant Physiol. 170, 74-79.
Figures legends
Fig. 1. Mycorrhizal colonisation in roots of (A) wild-type Lotus japonicus L. var. Gifu; (B) mycorrhizal mutant Ljsym72 of Lotus japonicus L., and (C) non-inoculated wild19
type Lotus japonicus L. var. Gifu at 6 weeks of plant growth (×100). Arrows indicate external hyphae entry point in root surfaces (A and B) and condensed tannin granules (C). Representative images of three samples per treatment. Scale bar represents 50 µm.
Fig. 2. Visualisation of condensed tannins with vanillin stain in tissues of roots: (A) wild-type Lotus japonicus L. var. Gifu; (B) mycorrhizal mutant Ljsym72 of Lotus japonicus L., and (C) non-inoculated wild-type Lotus japonicus L. var. Gifu at 6 weeks of plant growth (×100). Arrows indicate condensed tannin granules. Representative images of three samples per treatment. Scale bar represents 50 µm.
Fig. 3 Visualisation of condensed tannins with vanillin stain in tissues of stems of wildtype Lotus japonicus L. var. Gifu: (A) mycorrhizal (+AMF) and (B) non-mycorrhizal (– AMF); and in leaf: (C) mycorrhizal (+AMF) and (D) non-mycorrhizal (–AMF) at 6 weeks of plant growth (×100). Arrows indicate condensed tannin granules. Representative images of three samples per treatment. Scale bar represents 50 µm.
Table 1. Biomass and root colonisation of mycorrhizal and non-mycorrhizal birdsfoot trefoil plants 6 and 9 weeks of sowing 20
Treatment Plant 6 weeks Gifu wild-type Ljsym 72 lsd p≤0.05 9 weeks Gifu wild-type
Root colonisation (%)
Mycorrhiza
Biomass DW (g plant-1) Roots Stems Leaves
Inoculation No inoculation
0.56a 0.27b
0.12a 0.05b
0.27a 0.10b
54a 0b
Myc- mutant
0.35b 0.12
0.09a 0.05
0.24a 0.11
4b 19
Inoculation No inoculation
0.84a 0.47b
0.26a 0.08b
0.57a 0.20b
79a 0b
Ljsym 72 Myc- mutant 0.56b 0.22a 0.44a 4b lsd p≤0.05 0.21 0.13 0.23 15 Data are the mean of three replicates (18 plants per treatment); Different letters indicate significant differences between treatments within sampling time
Table 2. Condensed tannins concentrations in mycorrhizal and non-mycorrhizal birdsfoot trefoil plants 6 and 9 weeks of sowing Soluble tannins (mg g-1 Insoluble tannins (mg g-1 Treatment DW) DW) Plant Mycorrhiza Roots Stems Leaves Roots Stems Leaves 6 weeks Gifu wild-type Inoculation 0.32b 2.92a 1.51a 1.18a 2.27a 2.01a No inoculation 0.76a 2.56b 0.98b 1.37a 2.20a 0.74b lsd p≤0.05 0.16 0.28 0.32 0.29 0.20 0.53 9 weeks Gifu wild-type Inoculation 0.74b 1.18a 1.02a 0.45b 2.81a 3.06a No inoculation 0.94a 0.88b 0.85b 0.87a 2.73a 2.55b lsd p≤0.05 0.12 0.16 0.11 0.26 0.18 0.40 Data are the mean of three replicates (18 plants per treatment); Different letters indicate significant differences between treatments within sampling time
21
Table 3. Flavanols concentrations in mycorrhizal and non-mycorrhizal birdsfoot trefoil plants 6 and 9 weeks of sowing Flavonols (catechin equivalents, mg g-1 Treatment DW) Plant Mycorrhiza Roots Stems Leaves 6 weeks Gifu wild-type Inoculation 2.50a 2.85a 2.80a No inoculation 0.69b 1.35b 2.57a lsd p≤0.05 0.53 0.27 0.25 9 weeks Gifu wild-type Inoculation 2.80a 1.76a 2.02a No inoculation 1.43b 1.07b 1.97a lsd p≤0.05 0.63 0.47 0.22 Data are the mean of three replicates (18 plants per treatment); Different letters indicate significant differences between treatments within sampling time
22
A
B
C
Fig. 1 23
A
B
C
Fig. 2
24
B
A
C
D
Fig. 3
25
PII: DOI: Reference:
S2452-2198(17)30183-0 https://doi.org/10.1016/j.rhisph.2017.11.006 RHISPH91
To appear in: Rhizosphere Received date: 27 October 2017 Revised date: 28 November 2017 Accepted date: 28 November 2017 Cite this article as: Zakaria M. Solaiman and Keishi Senoo, Arbuscular mycorrhizal fungus causes increased condensed tannins concentrations in shoots but decreased in roots of Lotus japonicus L, Rhizosphere, https://doi.org/10.1016/j.rhisph.2017.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Arbuscular mycorrhizal fungus causes increased condensed tannins concentrations in shoots but decreased in roots of Lotus japonicus L.
Zakaria M. Solaimana* and Keishi Senoob a
SoilsWest, UWA School of Agriculture and Environment, and The UWA Institute of
Agriculture, Faculty of Science, The University of Western Australia, Crawley, WA 6009, Australia b
Laboratory of Soil Science, Department of Applied Biological Chemistry, Graduate
School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo113-8657, Japan *
Corresponding author: [email protected]
ABSTRACT Condensed tannins are a class of plant secondary metabolites which are formed by the condensation of flavanols, and play an important role in plant-soil-microbes interactions by influencing the colonisation of arbuscular mycorrhizal (AM) fungi. The aim of this study was to determine the influence of AM fungi on condensed tannins concentrations in roots, stems and leaves of birdsfoot trefoil (Lotus japonicus L.). The concentrations of condensed tannins in roots, stems and leaves of the Lotus japonicus plants were measured using histochemical staining and spectrophotometry. The extent of AM fungal colonisation in roots was also quantified. Soluble and insoluble condensed tannins concentrations in roots were lower in plants inoculated with AM fungi but higher in stems and leaves compared to plants not inoculated with AM fungi. The higher concentration of condensed tannins in the stems and leaves were associated with AM
1
fungi colonisation of roots. This had increased animal feed value and also could be influenced by the plant-soil-microbes feedback processes.
Keywords: Condensed tannins, Lotus japonicus, Flavonols, Fodder, Mycorrhizas
1. Introduction Condensed tannins, also known as proanthocyanidins, are a group of plant secondary metabolites and are part of a larger group of compounds, the polyphenols. Condensed tannins are present in most vascular plants and are thought to play various important roles associated with processes of plant defence against pests (Brillouet et al., 2013). The most common type of condensed tannins are produced from cyanidin which are metabolised by acid hydrolysis named as proanthocyanidins.
Condensed tannins are often found in great concentrations in tissues of coniferous trees and shrubs as well as in pasture plants (Ushio et al., 2013; Hartley and Jones, 1997; Jones et al. 1976) and formed by the polymerisation of catechins. The concentrations of condensed tannins can be up to 17 mg g-1 of the dry weight in birdsfoot trefoil sward tissues (Marshall et al., 2013). Condensed tannins have a significant role on the soil mineralisation processes (Ushio et al., 2013) and may also act in defence against pathogens (Brownlee et al., 1992; Min et al., 2003; Edwards, 1992), pests and herbivores (Swain 1979). A group of saprophytic fungi can utilise and grow on medium containing high concentrations of condensed tannins (Ushio et al., 2013). Furthermore, fungal inhibitory tannins concentration is relatively higher than the concentration that 2
inhibit bacteria (Scalbert, 1991). However, the biological activity of plant condensed tannins depends on their chemical structure and concentration (Dixon et al., 2005; Monagas et al., 2010; Dixon et al., 2013). Understanding the effect of AM fungi colonisation on condensed tannins and phenolic concentration in the roots, stems and leaves of birdsfoot trefoil (Lotus japonicus) are essential for improving feed quality (Frutos et al., 2004).
Birdsfoot trefoil is a fabaceae plant suitable for infertile, acidic soils and is highly persistent, productive, drought-tolerant and bloat-safe feed for livestock (Hancock et al., 2014). This plant has an extensive tap root system, therefore it can effectively mitigate dryland salinity through improving soil water use in recharge environments. Hence, birdsfoot trefoil has the potential for widespread use in many areas of Australia where there is a lack of an adapted pasture legumes (Moore et al., 2006). The model legume birdsfoot trefoil was used as a host plant for this study as this plant has been successfully used as AM hosts in previous studies (Senoo et al., 2000; Solaiman et al., 2000; Solaiman et al., 2007) where the investigators screened mycorrhizal mutant Ljsym72. Therefore, mycorrhizal mutant Ljsym72 was used in this study to mimic the control plant (Solaiman and Senoo, 2005). This mutant plant Ljsym72 formed a small quantity of AM colonisation characterised by the formation of branched appressoria on the root surfaces and the blocking of hyphal penetration at epidermis, however produced similar shoot weight of wild-type Gifu plant.
The formation and functioning of AM fungal symbiosis is assumed to involve signals between the host plant and the fungal symbiont, although the nature and mechanism of 3
the action of such signals is unclear (Takeda et al., 2015). Genre et al. (2013) proposed that short-chain chitin oligomers secreted by AM fungi are part of a molecular exchange with the host plant and that their presence in the epidermis leads to the activation of a SYM-dependent signalling pathway involved in the initial stages of fungal root colonisation. But the influence of flavonoid and isoflavonoid compounds on fungal growth, however, unclear whether they are essential signal molecules for the AM symbiosis. Previous studies reported phenolic compounds found in the cell walls of roots and have also been proposed as potential signal molecules (Harrison, 1997; Mandal et al., 2010). Comparisons of host and non-host plants have shown stimulatory and inhibitory compounds, but did not reveal a compound responsible for the formation of appressoria on host roots or for the lack of colonisation of non-host roots (Douds et al., 1996; Harrison, 1997). When screening for AM defective plant mutants, structures in trypan blue stained roots in addition to mycorrhizal structures were observed and subsequently identified as condensed tannins following vanillin-HCl histochemical staining procedures (Broadhurst and Jones, 1978). Here, the effect of AM fungal colonisation on condensed tannin synthesis in different parts of birdsfoot trefoil plant was investigated.
2. Materials and methods 2.1 Potting medium and mycorrhizal inoculum Washed sand was mixed with a low (10 µg/g) phosphorus-containing volcanic ash soil (v/v 1:1), sterilised by autoclaving (121 oC, 1 h) and amended with NH4NO3, KH2PO4 and KCl at a rate of 0.16 g, 0.008 g, and 0.032 g per pot (300 g sand and soil mix), respectively. AM fungal inoculum containing spores of single Glomus sp. R-10, species 4
name unidentified (Idemitsu Kosan Co, Ltd, Japan), was mixed thoroughly with the potting mixture (10%, v/v). Non-mycorrhizal treatment was amended with washing of inoculum (equivalent amount of inoculum was suspended in deionised water and filtered) to keep bacterial community similar.
2.2 Plant growth experiment The experiment was conducted following a two factorial randomised design including two plant genotypes ((wild-type Gifu B-129 (nod+fix+myc+) and a nod-fix-myc- mutant (Ljsym72)) × two mycorrhizal inoculum (inoculated and non-inoculated) with three replications. Mycorrhizal mutant Ljsym72 was used to mimic the control plant as this mutant formed <5% of deformed colonisation in the root epidermis. Seeds from wildtype Gifu B-129 and a mycorrhizal mutant (Ljsym72) were scarified using sand paper with the surface sterilised with 2% sodium hypochlorite solution containing 0.02% Tween 20 for ten minutes, rinsed in sterile distilled water and germinated on sterilised moist filter paper in Petri dishes at 25 oC under dark conditions. After germination, seedlings were transplanted into individual sections in nursery trays (six plants per tray) containing the sand-soil-inoculum mix substrate (300 g of mix per tray of plants). Three-week-old seedlings were then transplanted into pots (six plants per 500 ml pot) with three replicates (18 plants per treatment) and allowed to grow in a growth chamber. Additional seedlings of Gifu were grown in the sand-soil substrate without mycorrhizal inoculum added to provide a check for infectivity of the inoculum, as well as the extent and characteristics of colonisation in the parent plants. Plants were kept in the growth chamber (day: 20 h, 25 oC, 100 mmol m-2 s-1 photosynthetic photon flux density; night: 4 h, 22 oC) for nine weeks. Sampling was conducted at six and nine weeks after 5
transplanting of seedlings for the assessment of mycorrhizal colonisation, condensed tannins and flavanols.
2.3 Evaluation of mycorrhizal colonisation Root length colonised by the AM fungi was estimated at six and nine weeks after transplanting of seedlings. Root samples (0.5 g fresh weight) were cleared in 10% KOH and stained with trypan blue using a modification of the method by Phillips and Hayman (1970), in which lactoglycerol was used instead of lactophenol (Solaiman and Abbott 2008). Colonisation in the inoculated Gifu B-129 plants was determined as the percentage root length colonised under a dissecting microscope using the gridline intersect method (Tennant, 1975). Gridline intersects were scored for the presence of mycorrhizal structures at ×100 magnification.
The evaluation of mycorrhizal
colonisation was carried out in three replicates samples.
2.4 Histochemical staining procedure for condensed tannins Sections of roots, stems and leaves were stained with vanillin/HCl (one drop of 10% vanillin in absolute ethanol followed by one drop of 6 M HCl after 2 minutes). Tissue was mounted on slides and examined microscopically for tannins containing cells.
2.5 Chemical extraction of condensed tannins Each representative sample (roots, stems and leaves) was prepared from six plants comprising three representative fresh tissue samples per treatment of each mycorrhizal and non-mycorrhizal wild plant Gifu. Samples were frozen in liquid nitrogen at -80 oC before extraction. Mutant plant Ljsym72 was excluded from this chemical extraction 6
procedure due to the high similarity in the number of condensed tannins granules in roots of non-mycorrhizal wild plant Gifu (shown in histochemical staining). Fresh tissue was extracted with methanol followed by the method used by Stafford and Lester (1980), with minor modifications. Two hundred mg of fresh weight tissues from both harvests were homogenised in 2 × 1 ml aliquots methanol (80:20 v/v) with a mortar and pestle. The ground powder and washings were centrifuged (2000 × g, 5 minutes) and the supernatant liquid transferred to a new Eppendorf tube. After evaporation of methanol under vacuum between 30 and 35 oC, the aqueous fraction was extracted with petroleum ether to remove lipids. Petroleum ether was removed after evaporation, the remaining aqueous extract was adjusted to 5 ml with water, and aliquots analysed for soluble tannins and soluble flavanols. The residue after centrifugation was analysed for insoluble tannins.
2.6 Condensed tannins analysis Tannin analysis was based on the formation of cyanidin on acid hydrolysis. Condensed tannins concentration was determined with the improved acid butanol assay (Porter et al., 1986). Aliquots in a final volume of 0.1 ml were heated with 1 ml butanol-HCl (95:5, v/v) at approximately 95 oC for 30 minutes (Stafford and Cheng, 1980; Berard et al., 2011). The absorbance was determined at 550 nm, the peak absorption for cyanidin. Estimates of the condensed tannin as mg g-1 fresh weight of tissue were made at 550 nm (Stafford and Cheng, 1980) by using purified Quebracho tannin to standardise the measurements (Waterman and Mole, 1994).
7
2.7 Flavanol analysis Flavanol analysis was based on the reaction with vanillin in acidic solution (Swain and Hills 1959). Half-a-millilitre aliquots of the extract was mixed with 4.5 ml 1% vanillin in 70% H2SO4 and the absorbance at 500 nm measured after 30 minutes. Flavonols had specificity to vanillin (Sarker and Howarth, 1976). Values are expressed as catechin equivalents. Vanillin-H2SO4 also reacts with oligomeric and polymeric flavanols and anthocyanins, but these were shown to be absent from the methanol-soluble extracts (Sarker and Howarth, 1976).
2.8 Statistical analysis Data were expressed as means. A normality test was applied before analysis of variance (ANOVA). The two-way ANOVA was used to compare the effects of treatments (genotypes × mycorrhiza inoculum), followed by Tukey’s post hoc test for multiple comparisons of means using GENSTAT 16th Edition (Lawes Agricultural Trust, 2014). Significant differences at p≤0.05 were considered.
3. Results Mycorrhizal plants L. japonicus produced higher biomass than the non-mycorrhizal plants at six and nine weeks of growth (Table 1). Inoculated non-mycorrhizal mutant plant Ljsym72 produced similar root dry weight as un-inoculated Gifu wild-type but had very similar dry weights of stem and leaf as the inoculated Gifu wild-type. Mycorrhizal root colonisation of wild-type Gifu was 54% at six weeks and 79% at nine weeks of growth after transplanting of seedlings (Table 1). In contrast, non-inoculated Gifu wild type did not show any mycorrhizal colonisation and inoculated mycorrhizal mutant
8
plant Ljsym72 showed 4% of root colonisation at both six and nine weeks growth after transplanting of seedlings. Variation in mycorrhizal colonisation patterns in inoculated Gifu wide-type and mutant Ljsym72 were observed (Fig. 1A, B) as well as in the tannin granules (Fig. 1C).
Histochemical staining of condensed tannin showed that tannin granules were more present in non-mycorrhizal Gifu wild-type roots and mutant plants roots compared to mycorrhizal roots of Gifu wild-type (Fig. 2). In contrast, mycorrhizal plants shoots (stems and leaves) showed higher condensed tannin granules compared to shoots of non-mycorrhizal and mutant plants (Fig. 3). Soluble condensed tannin concentrations were reduced in mycorrhizal roots compared to non-mycorrhizal roots, however, was increased in mycorrhiza inoculated plant shoots (stems and leaves) compared to nonmycorrhizal plant shoots at six and nine weeks of growth (Table 2). Insoluble tannin concentration in roots was higher in non-inoculated plants only at nine weeks of growth whereas it was higher in leaves of inoculated plants both at six and nine weeks of growth (Table 2). Insoluble tannin concentration in stems was similar for both inoculated and non-inoculated plants at six and nine weeks of growth respectively. Flavanols concentration was increased in roots and stems of mycorrhizal inoculated plants compared to non-inoculated plants (Table 3). However, there was no mycorrhizal inoculation effect observed on flavanols concentration of plant shoots (stem and leaves) at both six and nine weeks of growth (Table 3).
4. Discussion
9
Many signalling compounds produced by plants include both primary metabolites (carbohydrates, proteins, organic acids etc.) and secondary metabolites (flavonols, phenol, phytohormones etc.) (Singh et al., 2016). The signalling compounds play diverse roles in ensuring benefits to both parties of the plant–microbe interaction. Condensed tannins are a group of plant secondary metabolites and it’s concentration in roots, stems and leaves between AM fungi inoculated and non-inoculated birdsfoot trefoil plants differed significantly in the present study. Mutant plant Ljsym72 was excluded from the chemical extraction procedure of condensed tannins due to the high similarity in the number of condensed tannins granules in roots between mutant and non-inoculated control plants. However, future research should include this variable to clarify the confounding effect of growth differences between mycorrhizal inoculated and non-inoculated plants. The variation in tannins concentration of mycorrhizal and non-mycorrhizal wild-type Gifu plants strongly indicates the efficiency of AM fungi in maximising the production of primary and secondary metabolites in birdsfoot trefoil. This was observed in previous studies in plants such as stevia herb (Mandal et al., 2013), cebil seedlings (Pedone-Bonfim et al., 2013), medicinal herbs (Jugran et al., 2015; Lima et al., 2017) and in clover (Zhang et al., 2013). In some cases, AM fungi inoculation was more efficient in supporting the production of these compounds compared with the uptake of P (Nair et al., 1991). This increase is a direct effect of the AM fungal colonisation which should supply nutrients, especially P, to the plant by the mycorrhizal pathway (Smith and Read, 2008). However, in the present study, P analysis was not carried out to test the aforementioned hypothesis. The study also showed that mycorrhizal colonisation of plants relieved nutrient deficiency and enhanced the growth of the plants, and thus, reduced the tannins concentration of the roots and increased in 10
shoots (da Silva et al., 2014). The high tannins concentration of non-mycorrhizal roots may have been induced by deficiency of nutrients (N and P) in the plant tissue which is similar to non-fertilised plants (Jones et al., 1976; Ferwerda et al., 2005). Therefore, the mechanism(s) through which AM fungi affect tannins production in different parts of the plants may be related to availability of N and P to the plants and warrants further investigation. The low concentration of tannins in plant roots inoculated with AM fungi supports compatibility of the symbiotic partnership between birdsfoot trefoil and AM fungus (Campos-Soriano et al., 2012; Zakhia et al., 2003). Phytohormones, such as auxins and cytokinins act as signalling molecules and affect cell proliferation or modify root system architecture by overproduction of lateral roots and root hairs with a subsequent increase of nutrient and water uptake (Ortíz-Castro et al., 2009) and also play an important role in plant and AM fungi interactions (LudwigMüller and Güther, 2007). An assortment of secondary plant metabolites such as flavonoids and strigolactones, the latter of which is excreted by roots, also carry importance as signalling molecules in several symbiotic and pathogenic plant–microbe interactions (Steinkellner et al., 2007). For example, in response to the root exudation of signal molecules recognised as plant hormones strigolactones, hyphal branching of AM fungi induced and colonise plant roots (Akiyama et al., 2005; Haichar et al., 2014). In this study, AM fungus inoculation was efficient in increasing the concentration of flavonols and soluble tannins in the shoot (stems and leaves), thus showing the potential of AM fungi in increasing the production of plant secondary metabolites (Zhang et al., 2013). The increasing concentration of condensed tannins in the leaves of the forest plant such as Dacrydium than the Lithocarpus plant may be due to the influence of the microbial community composition and their functions in the rhizospheres which can 11
subsequently influence plant performance and induce plant-soil-microbes feedback processes (Ushio et al., 2013). The production of fabaceae plants with a high concentration of phenolic compounds adds value to the vegetative fodder biomass, making it a more attractive feed for animals. The phenolic-abundant plants also protect protein in the animal rumen and in silo, thereby reducing the rate of protein degradation and consequent nitrogenous losses to the environment (Waghorn and Shelton, 1992). The presence of condensed tannin can also reduce the incidence of pasture bloat (McMahon et al., 2000) and reduce ruminants’ methane emissions (Ramirez-Restrepo and Barry, 2005). Although for a long time tannins were thought to be detrimental to ruminants, their effect may be beneficial or harmful depending on the type of tannins consumed, its chemical structure and molecular weight, the amount ingested, and the animal species involved (Hagerman and Butler, 1991; Frutos et al., 2004). High concentration of tannins (generally > 50 g kg-1 of dry matter, DM) reduce voluntary feed intake and nutrient digestibility, whereas low to moderate concentrations (<50 g kg-1 of DM) may improve the digestive utilisation of feed mainly due to a decrease in protein degradation in the rumen and a subsequent increase in amino acid movement to the small intestine (Barry and Duncan, 1984). These effects on nutrition are shown in improved animal performance (Frutos et al., 2004). In this study, the concentration of tannins in stems and leaves are low which may increase animal performance through the aforementioned mechanism.
5. Conclusion In conclusion, increased condensed tannins concentrations were observed in shoots and decreased in roots of plants colonised with AM fungi. These results support the positive 12
signal in mutualistic AM symbiosis i.e. the recognition of AM fungal entry to the root epidermis as opposed to that of systemic resistance in plant-pathogen interaction. A better understanding of the factors controlling the biosynthesis, accumulation, and release of these secondary metabolites condensed tannins may represent a novel way to manipulate AM symbiotic systems.
Acknowledgements The authors thank Dr Motoshi Suzuki of Idemitsu Kosan Co., Ltd, Tokyo, Japan for supplying spores of Glomus sp. R-10. The author ZMS is grateful to the Japan Society for the Promotion of Science for providing postdoctoral fellowships.
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Figures legends
Fig. 1. Mycorrhizal colonisation in roots of (A) wild-type Lotus japonicus L. var. Gifu; (B) mycorrhizal mutant Ljsym72 of Lotus japonicus L., and (C) non-inoculated wild19
type Lotus japonicus L. var. Gifu at 6 weeks of plant growth (×100). Arrows indicate external hyphae entry point in root surfaces (A and B) and condensed tannin granules (C). Representative images of three samples per treatment. Scale bar represents 50 µm.
Fig. 2. Visualisation of condensed tannins with vanillin stain in tissues of roots: (A) wild-type Lotus japonicus L. var. Gifu; (B) mycorrhizal mutant Ljsym72 of Lotus japonicus L., and (C) non-inoculated wild-type Lotus japonicus L. var. Gifu at 6 weeks of plant growth (×100). Arrows indicate condensed tannin granules. Representative images of three samples per treatment. Scale bar represents 50 µm.
Fig. 3 Visualisation of condensed tannins with vanillin stain in tissues of stems of wildtype Lotus japonicus L. var. Gifu: (A) mycorrhizal (+AMF) and (B) non-mycorrhizal (– AMF); and in leaf: (C) mycorrhizal (+AMF) and (D) non-mycorrhizal (–AMF) at 6 weeks of plant growth (×100). Arrows indicate condensed tannin granules. Representative images of three samples per treatment. Scale bar represents 50 µm.
Table 1. Biomass and root colonisation of mycorrhizal and non-mycorrhizal birdsfoot trefoil plants 6 and 9 weeks of sowing 20
Treatment Plant 6 weeks Gifu wild-type Ljsym 72 lsd p≤0.05 9 weeks Gifu wild-type
Root colonisation (%)
Mycorrhiza
Biomass DW (g plant-1) Roots Stems Leaves
Inoculation No inoculation
0.56a 0.27b
0.12a 0.05b
0.27a 0.10b
54a 0b
Myc- mutant
0.35b 0.12
0.09a 0.05
0.24a 0.11
4b 19
Inoculation No inoculation
0.84a 0.47b
0.26a 0.08b
0.57a 0.20b
79a 0b
Ljsym 72 Myc- mutant 0.56b 0.22a 0.44a 4b lsd p≤0.05 0.21 0.13 0.23 15 Data are the mean of three replicates (18 plants per treatment); Different letters indicate significant differences between treatments within sampling time
Table 2. Condensed tannins concentrations in mycorrhizal and non-mycorrhizal birdsfoot trefoil plants 6 and 9 weeks of sowing Soluble tannins (mg g-1 Insoluble tannins (mg g-1 Treatment DW) DW) Plant Mycorrhiza Roots Stems Leaves Roots Stems Leaves 6 weeks Gifu wild-type Inoculation 0.32b 2.92a 1.51a 1.18a 2.27a 2.01a No inoculation 0.76a 2.56b 0.98b 1.37a 2.20a 0.74b lsd p≤0.05 0.16 0.28 0.32 0.29 0.20 0.53 9 weeks Gifu wild-type Inoculation 0.74b 1.18a 1.02a 0.45b 2.81a 3.06a No inoculation 0.94a 0.88b 0.85b 0.87a 2.73a 2.55b lsd p≤0.05 0.12 0.16 0.11 0.26 0.18 0.40 Data are the mean of three replicates (18 plants per treatment); Different letters indicate significant differences between treatments within sampling time
21
Table 3. Flavanols concentrations in mycorrhizal and non-mycorrhizal birdsfoot trefoil plants 6 and 9 weeks of sowing Flavonols (catechin equivalents, mg g-1 Treatment DW) Plant Mycorrhiza Roots Stems Leaves 6 weeks Gifu wild-type Inoculation 2.50a 2.85a 2.80a No inoculation 0.69b 1.35b 2.57a lsd p≤0.05 0.53 0.27 0.25 9 weeks Gifu wild-type Inoculation 2.80a 1.76a 2.02a No inoculation 1.43b 1.07b 1.97a lsd p≤0.05 0.63 0.47 0.22 Data are the mean of three replicates (18 plants per treatment); Different letters indicate significant differences between treatments within sampling time
22
A
B
C
Fig. 1 23
A
B
C
Fig. 2
24
B
A
C
D
Fig. 3
25