Phytochemicals and spore germination: At the root of AMF host preference?

Applied Soil Ecology 60 (2012) 98–104

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Phytochemicals and spore germination: At the root of AMF host preference? Walid Ellouze a,b,c,∗ , Chantal Hamel a , Andre Freire Cruz d , Takaaki Ishii d , Yantai Gan a , Sadok Bouzid c , Marc St-Arnaud b a

Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, P.O. Box 1030, 1 Airport Road, Swift Current, SK, Canada S9H 3X2 Institut de Recherche en Biologie Végétale, Université de Montréal and Jardin Botanique de Montréal, 4101, rue Sherbrooke est, Montréal, QC, Canada H1X 2B2 Département de Sciences Biologiques, Faculté des Sciences de Tunis, Université Tunis El Manar, Campus Universitaire, Tunis 1060, Tunisia d Lab of Pomology, Graduate School of Agriculture, Kyoto Prefectural University, 1-5 Shimogamohangi-cho, Sakyo-ku, Kyoto 606-8522, Japan b c

a r t i c l e

i n f o

Article history: Received 2 September 2011 Received in revised form 22 November 2011 Accepted 3 February 2012 Keywords: Chickpea (Cicer arietinum L.) Garbanzo bean Root extracts Biologically active compounds Plant genotype Arbuscular mycorrhizae Plant symbioses Plant breeding

a b s t r a c t Plants trigger various responses in the organisms living around them using a wide array of phytochemicals, which are components of their adaptation to a biological environment. The roots of five varieties of chickpea inoculated with Glomus intraradices were extracted, and extracts were fractionated, first based on solubility in methanol and further by HPLC. We found a relationship between chickpea genotype and root phytochemical composition. Several HPLC fractions repressed the germination of AM fungal spores in bioassays conducted in multi-well plates with extracts from the variety CDC Anna. This repression may be an expression of the control of the plant on the AM fungal symbionts. Glomus etunicatum and Gigaspora rosea spore germination responded differently to exposure to the HPLC fractions soluble in 25% methanol. A differential response of AM fungal species to plant phytochemicals could be involved in the so called “host preference” of AM fungi. Whereas extensin and other proteins were identified in a bioactive root extract fraction, other proteins undetected by LC–MS/MS analysis and non-peptidic compounds may be involved in AMF spore germination suppression. © 2012 Elsevier B.V. Open access under CC BY-NC-ND license.

1. Introduction The structure of rhizosphere microbial communities is affected by plants through the release of attractants and repellents from their roots (Estabrook and Yoder, 1998). Plants modify their biological environment using a wide range of phytochemicals. The production of volatile phytochemicals by plants was well studied (Arimura et al., 2009) and led to practical applications in the field of insect pests monitoring and control. Rhizospheric interactions are shorter range than phyllospheric interactions and phytochemicals with restricted mobility are also effective in the regulation of the biological environment in plant rooting soil. Strigolactones are important signals triggering branching in arbuscular mycorrhizal (AM) fungi, despite their spontaneous hydrolysis in soil water upon release by roots (Parniske, 2005, 2008), and plant peptides can act as attractant (Horii et al., 2009).

∗ Corresponding author at: Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Box 1030, 1 Airport Rd., Swift Current, SK, Canada S9H 3X2. Tel.: +1 306 778 7253; fax: +1 306 778 3188. E-mail address: [email protected] (W. Ellouze). 0929-1393 © 2012 Elsevier B.V. Open doi:10.1016/j.apsoil.2012.02.004

access under CC BY-NC-ND license.

AM fungi form symbiotic associations with the vast majority of land plants. They can provide their host plants with improved plant nutrient use efficiency and enhanced N2 -fixation in legumes, they reduce plant disease incidence, increase plant tolerance to stress and improve soil physico-chemical quality (Jeffries et al., 2003). Knowledge of the regulation of the AM symbiosis is accumulating. The initiation and the maintenance of the AM symbiosis appear to involve different regulatory mechanisms. Whereas the control of the AM fungi by the host plants in an established association involves hormonal regulation in processes similar to those involved in plant defence against pathogens (Vierheilig et al., 2008), the initiation of the symbiosis involves an exchange of signals between the plants and the fungi, and the recognition of these signals by the symbiotic partners (Parniske, 2008; López-Ráez et al., 2011). Chickpea is only a fair rotation crop, leading to lower yield in following wheat crops, as compared to medic, vetch or lentil (Ryan et al., 2010), although it imports atmospheric N into cropping systems through biological nitrogen fixation, like other legumes. The poor ‘rotation effects’ of a chickpea crop could come from its influence on the soil microbial community and, in particular, on the AM fungi. The inhibitory effects of chickpea roots on subsequent crops were shown to depend on the chickpea cultivar (Chaichi and Edalati-Fard, 2005). In this study, we hypothesize that certain

W. Ellouze et al. / Applied Soil Ecology 60 (2012) 98–104

biologically active compounds present in chickpea exudates influence AM fungal spore germination, and that different chickpea cultivars produce root exudates differing in composition. 2. Materials and methods 2.1. Experimental design and fungal isolates Compounds with bioactivity on AM fungal spore germination were sought within the mycorrhizal root extracts of different chickpea (Cicer arietinum L.) genotypes inoculated with Glomus intraradices (DAOM 181602). Five chickpea cultivars with contrasting phenotypes were selected for this study: Kabuli types CDC Frontier, CDC Xena and Amit, and Desi types CDC Anna and CDC Nika. Their roots were extracted in methanol, extracts were parsed based on solubility in methanol (MeOH), and materials soluble in 25% and 50% MeOH were further separated by HPLC as described below. HPLC fractions from CDC Anna were recovered and assayed on Glomus etunicatum (isolate NPI) and Gigaspora rosea (isolate DAOM 194757), for their effects on spore germination. G. intraradices (DAOM 181602) which was recently associated with the clade of Glomus irregulare (Stockinger et al., 2009), G. etunicatum (isolate NPI) and Gi. rosea (isolate DAOM 194757) spores were obtained from pot cultured leek (Allium porrum L.) and maize (Zea mays L.) plants, in calcined clay, in a greenhouse. 2.2. Plant materials and conditions Chickpea roots of each cultivar were produced in large flats (122 cm × 122 cm × 15 cm in depth) containing an autoclaved sand: calcined clay (Professional Gardener Co. Ltd., Calgary, AB) (1:1, v:v) mix and approximately 250 chickpea plants m−2 . To simulate field conditions, chickpea seeds were inoculated with the seed-coating inoculants Nitragin Nitrastick GC® (Nitragin Inc., Brookfield, WI) at the time of seeding, and with a root-soil inoculum of the AM fungus G. intraradices, which was mixed into the growth substrate prior to seeding. The AM inoculants was multiplied on maize (Z. mays L.) grown in calcined clay for 2 months in a greenhouse. Maize roots were chopped in segments of about 1 cm and returned to the calcined clay. Approximately 10 L of this AM fungal inoculant was mixed in the growth substrate of each flat, using a cement mixer. Plants were grown in the greenhouse under a 24/15 ◦ C (±2 ◦ C) day/night temperature regime. Natural day light was supplemented with high intensity discharge lamps (Alto 400 watt low pressure sodium, Philips, Somerset, NJ) providing photosynthetically active radiation for 16 h day−1 . Plants were examined daily and watered as needed. They received weekly increasing amounts of a modified Long Ashton nutrient solution (Hewitt, 1966) containing (in mg L−1 ) 554 KCl, 200 NaH2 PO4 ·H2 O, 244 MgSO4 , 520 CaCl2 ·H2 O, 1.7 MnSO4 , 0.25 CuSO4 ·5H2 O, 0.30 ZnSO4 ·7H2 O, 3.0 H3 O3 , 5.0 NaCl, 0.09 (NH4 )6 Mo7 O24 ·4H2 O and 32.9 NaFe-EDTA. Plant roots were harvested six weeks after emergence, washed in water, spun dry and frozen at −24 ◦ C until extraction a few days later. 2.3. Root extraction and extracts fractionation The scheme of the root extraction/fractionation procedure is summarized in Fig. 1. A 400 g sample of fresh roots from each cultivar was soaked three times in 80% MeOH solution for 24 h at room temperature. The extracts were concentrated in a rotary evaporator at 40 ◦ C and filtered before fractionation by flash chromatography on an octadecyl-silica 45 × 400 mm column through successive elution with 0%, 10%, 25%, 50%, 75% and 100% MeOH solutions. Fractions were concentrated in a rotary evaporator at

99

40 ◦ C. The 25% and 50% MeOH fractions were purified by autofocusing using a Rotofor (Bio-Rad) before separation by HPLC with a UV detector at 220 nm, and a C18 column. The mobile phase consisted of a 10% ethanol solution with a flow rate of 1.2 mL min−1 . The 21 peaks produced from HPLC separation of the 25% MeOH-soluble and 50% MeOH-soluble materials from the roots of chickpea cultivar CDC Anna (Fig. 2) were recovered. CDC Anna was selected based on its highest compatibility with the beneficial microorganisms in a field experiment (Ellouze, 2011). The 25% MeOH-soluble HPLC fractions were evaporated and dissolved in 5 mL of 25% MeOH, and the 50% MeOH-soluble fractions were evaporated and dissolved in 10 mL of 50% MeOH. 2.4. AM fungal spores germination bioassay One hundred spores of two AM fungal species were exposed to three concentrations of each HPLC fraction (10, 50, or 100 mg fresh root equivalent mL−1 ) of the 25% and 50% MeOH-soluble materials from CDC Anna root extract. Treatments were replicated four times. The germination assays were performed in sterile 8-well chambered coverglass (Lab-Tek) for G. etunicatum spores and in sterile 24-well culture plates for Gi. rosea spores. AM fungal spores were extracted from leek pot cultures by wet sieving (Gerdemann and Nicolson, 1963) followed by centrifugation in 60% sucrose (w:v) (Daniels and Skipper, 1982). Spores of G. etunicatum and Gi. rosea were surface sterilized for 12 and 15 min, respectively, in a solution made of 0.7 g chloramine-T, 5.6 mg streptomycin and 2.1 mg chloramphenicol in 100 mL distilled water, with a few drops of Tween 80 (Horii and Ishii, 2006; Yu et al., 2009). After sterilization, spores were rinsed seven times, re-suspended in sterilized distilled water and cold treated for four to six weeks at 4 ◦ C prior to use, to improve germination (Juge et al., 2002). Solutions were prepared by diluting volumes of the HPLC fractions to obtain the target concentrations of 10, 50 and 100 mg fresh root equivalent mL−1 , in distilled water. Control stock solutions were prepared using identical amounts of 25% or 50% MeOH solutions. One hundred surface sterilized AM fungal spores were placed in each well with 300 ␮L of a potentially bioactive or control solution (25% or 50%, MeOH). Spores of G. etunicatum were incubated in the dark at room temperature for 20 days, and spores of Gi. rosea, for 5 days. Spores were examined directly in the wells, using an inverted microscope (100–200×) and spores were considered to be germinated when germ tubes length was as long as the diameter of the spore or longer. The bioactivity of HPLC fractions was assessed through a series of 12 assays, each testing the effects of one of the three rates of the HPLC fractions soluble in either 25% or 50% MeOH, on either G. etunicatum or Gi. rosea. As a bioactive peptide was found in the 25% MeOH fraction of bahia grass, the proteins of the active fractions were analysed (Horii et al., 2009). 2.5. LC–MS/MS analysis Trypsic digestion and LC–MS/MS analysis were performed in a commercial laboratory (Institute for Research in Immunology and Cancer [IRIC]). The peak contents were digested with trypsin prior to LC–MS/MS analysis. A volume of 20 ␮L of the desalted compounds diluted to 50 ␮L was injected onto the column. The peptide mixture was separated on a in-house Jupiter C18 (3 ␮m, 150 ␮m × 100 mm) column using a Eksigent nanoLC-2D (Dublin, CA). The peptides were eluted at a flow of 0.6 ␮L min−1 using a linear gradient of acetonitrile with 0.2% formic acid from 5% to 56%, over 56 min. Eluted peptides were ionized at 1.7 kV and the ions analysed by a ThermoFisher LTQ-XL mass spectrometer (San Jose, CA). In the ion trap, the top 3 ions were selected for MS/MS by using a data dependent triple play scheme. Dynamic exclusion was set

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Fresh chickp ea roo ts (≈ 500 g fresh weight) Extracted with 80 % MeOH Filtered with Tovo No. 5 C paper Roo ts residue (discard) Evaporated to the aqu eous phase at 40°C Filtered with Tovo No. 5 C paper

Fractionated with a flash chromatograph equ ipp ed with a chromatorex ODS column (45mm in diameter and 400mm in length) Distilled water (4000 ml)

0 % MeOH fraction 10% MeOH (4000 ml) 10 % MeOH fraction 25% MeOH (4000 ml) 25% MeOH fraction 50% MeOH (4000 ml) 50 % MeOH fraction 75% MeOH (40 00 ml) 75 % MeOH fraction

100% MeOH (4000 ml) 100% MeOH fraction Concentrated by evaporation at 40°C Purification by a Rotofor (BIO-RAD)

Filtered with 22 μm filter HPLC (C18 ; 220 nm)

Peaks collect Fig. 1. Flow chart for separation of compounds present in the root tissues of chickpea cultivars. Modified from Ishii et al. (1997).

after one scan to facilitate identification of low abundance proteins. The acquired data were searched with Mascot 2.1 search algorithm. The compounds which gave a non-significant match with Mascot were researched using NCBI (nr) against the Viridiplantae database. 2.6. Statistical analysis The effect of treatments on AM fungal spore germination compared to the controls was assessed by analysis of variance (ANOVA) using JMP 3.2.6 (SAS Institute, Cary, USA). A P value of 0.05 was used as threshold to accept the significance of effects. The significance of difference between treatment mean values was assessed by the least significant difference (LSD), when significant treatment effects were found. The normality of residuals was tested using Shapiro-Wilk’s test and data transformation prior to analysis was done when required. A square-root transformation was applied to the G. etunicatum spore germination percentage at the two higher rates of 25% MeOH-soluble HPLC fractions, and cosines transformation was applied to the Gi. rosea germination percentage

at the lowest rate of the 50% MeOH-soluble fractions, to meet the requirement of normality before statistical analysis. Principal component analysis (PCA) was carried out to compare the biochemical composition of the different chickpea root extracts, as described by their profile of HPLC peak areas. The areas under the peaks were measured using the image-processing software ‘ImageJ’ version 1.42 (Abramoff et al., 2004) and the percentage of every peak in each extract was determined. The data were Hellingertransformed prior to PCA analysis (Legendre and Legendre, 1998; Legendre and Gallagher, 2001) using the R Project for Statistical Computing version 2.12.2 (R Development Core Team, 2011). 3. Results 3.1. HPLC analysis of root extracts Up to 14 different HPLC fractions of materials soluble in 25% MeOH and 13 HPLC fractions of material soluble in 50% MeOH were produced from the root extracts of the different chickpea

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Fig. 2. The HPLC chromatograms of the fractions of the root extracts of the Desi type chickpea cultivar CDC Anna. The arrows show the HPLC fractions collected (peaks) from the root extracts that are soluble in (a) 25% methanol and (b) 50% methanol.

genotypes (Fig. 3). The HPLC profiles varied with chickpea genotypes. The peaks identified as 5a, 5b, 10a and 10b in the 25% MeOH-soluble HPLC fractions (Fig. 4a) and those identified as 1a and 4a in the 50% MeOH-soluble HPLC fractions (Fig. 4b) were absent from the root extracts of the cultivar CDC Anna but present in the root extracts of at least one of the other cultivars. Similarity in the composition of root extracts from Desi and Kabuli cultivars was revealed by the PCA analyses (Fig. 4). The Desi chickpea CDC Anna and CDC Nika clustered separately from the three Kabuli cultivars, i.e., Amit, CDC Xena and CDC Frontier (Fig. 4), suggesting a relationship between chickpea root phytochemical production and genotype. 3.2. AM fungal spore germination assays The HPLC fractions affected spore germination differently in G. etunicatum and Gi. rosea. The 25% MeOH-soluble HPLC fractions of chickpea root extracts corresponding to peaks 3, 6, 7, 9 and 10 (Fig. 2) significantly inhibited G. etunicatum spore germination at the low concentration (Fig. 5a), and all peaks except peak 10 significantly inhibit G. etunicatum spores germination at the medium rate of application. At the highest concentration, all the 25% MeOHsoluble HPLC fractions significantly inhibited G. etunicatum spore germination as compared to the control. By contrast, only two fractions influenced the germination of Gi. rosea spores (Fig. 5b). Compounds contained in the 50% MeOH-soluble HPLC fractions of chickpea root extracts were more effective in repressing Gi. rosea spore germination. These fractions had a more similar effect on the germination of G. etunicatum and Gi. rosea spore than the fractions soluble in 25% MeOH, although three of them had a significant effect only on the spores of one or the other species (Fig. 6).

Fig. 3. The HPLC chromatograms of the fractions of the root extracts of five chickpea cultivars of type Desi or Kabuli that are soluble in 25% and 50% methanol.

3.3. Compound identification using LC–MS/MS A total of 21 HPLC fractions from the roots of chickpea cultivar CDC Anna were analysed by LC–MS/MS in this study. Of these, the proteins of 6 fractions were identified with some level of confidence (Table 1). However, no significant match was obtained in a database search and no proteins could be assigned to the peptides contained in the remaining HPLC fractions.

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Table 1 List of MS/MS identified chickpea root extract proteins. MeOH fraction (%)

Identification

gi no.

7 8 9

25 25 25

10 10 11

25 50 50

Extensin Extensin Putative retroelement polyprotein Putative retroelement pol polyprotein Unknown protein RPR1h ZCF37 Putative protein Receptor-like protein kinase Retrotransposon protein; putative; Ty1-copia sub-class P0004D12.20 ZCF37

1486263 1486263 9989054 4063762 15223263 4519938 18406390 7594561 166846 6273445 34906958 18406390

0.6

Peak no.

(a)

5

Nika

10b

Xena

2 6

4

5a 5b

-0.2

3

Frontier

-0.4

8

-0.4

-0.2

0.0

0.2

0.4

0.6

70

PC1 Spore germination (%)

60

0.2

6

0.0

Anna

4

9 2

Nika

8 11

Xena Frontier

7

-0.4

10 mg

50 mg

100 mg

50 40

*

30

*

*

* *

20

*

10

**

**

**

**

**

**

*

**

*

10 mg

50 mg

*

*

4a

70

Amit 1a 3

5

0.0

60

10

1

-0.2

(a)

0

0.2

0.4

0.6

PC1 Fig. 4. PCA biplots of the relationship between the compositions of root extract fractions soluble in (a) 25% and (b) 50% methanol, and chickpea cultivars. Numbers represent the peaks produced by HPLC analysis of the chickpea root extract, as shown in Fig. 2.

Spore germination (%)

0.4

(b)

-0.2

SPPPPPYK; SPPPPPIHK SPPPPPYK; SPPPPPIHK LLIAVAAAK LLLAVAAAK YNEIAKK + Deamidated (NQ)-N2 LSSLLTLNIK + Deamidated (NQ)-N8 QSIYSKK + Deamidated (NQ)-Q1 LVIAPPPPPLPSR LDLDNEK LAKFLLMR + Oxidation (M)-M7 SSWNSPYYDTSSYGAGSGGGGGGGR QSIYSKK + Deamidated (NQ)-Q1

The results presented here show that the roots of different chickpea genotypes produce different arrays of compounds, some of which can suppress the germination of AM fungal species. Interestingly the suppression of AM fungal spore germination by chickpea root compounds was selective. Specific colonization of different host plant species by different AM fungi was often reported (Vandenkoornhuyse et al., 2002; Gollotte et al., 2004; Scervino et al., 2005). The different response of G. etunicatum and G. rosea to exposure to certain compounds soluble in 25% and 50% MeOH, suggests that this so called ‘host preference’ is mediated at least in part through the production of phytochemicals. Seven 25% MeOHsoluble proteins and four 50% MeOH-soluble proteins (Table 1) were identified in HPLC fractions with negative impact on AM fungal spore germination. This concurs with reports of a methanolsoluble fraction exuded by pre-mycorrhizal infection myc− tomato mutant M161 inhibiting hyphal tip growth in Gi. gigantea and G.

0.4

Anna 0.0

PC2

0.2

10 7

110aAmit

PC2

Peptide sequences

58.43 49.36 44.51 44.51 42,7 41.97 40.2 44.54 44.06 44.44 42.08 41,34

4. Discussion

P9

-0.4

Score

(b)

100 mg

50 40 30

*

*

20 10 0

Fig. 5. Percentage of inhibition of spore germination caused by compounds contained in the HPLC fractions of chickpea root extracts that are soluble in 25% MeOH, in two isolates of AM fungi (a) G. etunicatum and (b) Gi. rosea. Stars indicate a statistically significant difference from the control (Student’s t test, P < 0.05).

W. Ellouze et al. / Applied Soil Ecology 60 (2012) 98–104 70

Spore germination (%)

60

(a)

10 mg

50 mg

50 40

30

*

20

*

10

* **

70 60

**

* **

0

Spore germination (%)

100 mg

(b)

10 mg

50 mg

100 mg

50 40

30 20

*

*

*

* *

*

**

non-protein, root produced compounds reported to inhibit AM fungal spore germination. Variations in the root extract profile of different chickpea genotypes observed here correspond with reports of variations in the presence (Stevenson et al., 1995) and abundance (Stevenson et al., 1994) of certain antifungal compounds in the exudates of wilt-susceptible and wilt-resistant chickpea genotypes. Similarities between the mechanisms operating in the regulation of the AM fungal symbiosis and those operating in plant defense against pathogens have shown that plants rely on the same mechanisms for the management of beneficial and pathogenic fungi (Vierheilig et al., 2008; Pozo et al., 2010). Genotypic differences in qualitative and quantitative composition of root exudates were reported in cultivated and wild plants differing in their level of tolerance to disease (Rengel, 2002) suggesting that root exudates can modify soil microbial community to the plant’s advantage. The larger similarity of root extract composition of cultivars within chickpea types (Desi and Kabuli) found here suggests a genetic basis to the production of phytochemicals by chickpea roots and the possibility to select chickpea genotypes for the production of phytochemicals improving the biological quality of chickpea roots environment.

*

10 0

103

**

Fig. 6. Percentage of inhibition of spore germination caused by compounds contained in the HPLC fractions of chickpea root extracts that are soluble in 50% MeOH, in two isolates of AM fungi (a) G. etunicatum and (b) Gi. rosea. Stars indicate a statistically significant difference from the control (Student’s t test, P < 0.05).

Acknowledgments The authors would like to gratefully acknowledge the excellent technical assistance of Keith Hanson and Mouna Fakhfakh. This project is supported by a grant from the Alberta Pulse Growers to Dr. Chantal Hamel and coll., a NSERC grant to Dr. Marc St-Arnaud and a scholarship to Mr. Walid Ellouze from the Islamic Development Bank Merit Scholarship Programme for High Technology. References

intraradices (Gadkar et al., 2003) and reducing Gi. gigantea and G. intraradices spore germination (David-Schwartz et al., 2001). Some of the proteins identified in bioactive fractions in this study are involved in plant defence response. The accumulation of hydroxyproline-rich glycoprotein (HRGP) extensin reported at the AM symbiotic interface (Bonfante-Fasolo et al., 1991) could be involved in the containment of AM fungal growth in host cells. Plant HRGP is involved in plant cell wall assembly. The size and repetitive co-polymeric block structure of HRGP give these molecules the capacity to self-assemble into wall scaffolds even outside of plant tissues (Wegenhart et al., 2006). Extensin may have structurally contained germ tube elongation by binding on spore wall. Among other proteins identified by LC–MS/MS, the receptorlike protein kinase (RLK) is a transmembrane protein controlling a wide range of processes including plant symbiont and pathogen recognition (Shiu and Bleecker, 2001; Morris and Walker, 2003). The retroelement pol polyprotein-like was found to be among the proteins up-regulated or down-regulated on the initial association with the AM fungus Gi. margarita (Deguchi et al., 2007). The retrotransposon protein, putative, Ty1-copia sub-class was found to be up-regulated in the shoot of the P deficiency line of rice NIL64 (Pariasca-Tanaka et al., 2009). How exposure to these proteins could inhibit the germination of AM fungal spores is unclear. Whereas extensin and other proteins were identified in a bioactive root extract fraction, other proteins undetected by LC–MS/MS analysis and non-peptidic compounds may also be involved in the suppression of AM fungal spore germination. The flavonoids biochanin A, genistein and hesperetin (Chabot et al., 1992), and isoflavone formononetin Phillips (Tsai and Phillips, 1991) were reported to inhibit the germination of spores of different AM fungal species. Glucosinolate degradation products (Roberts and Anderson, 2001) and isothiocyanate (Bainard et al., 2009) are other

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