International audienceThe specific activities of cinnamyl alcohol dehydrogenase (EC 1.1.1.-) and cell wall-bound peroxidases (EC 1.11.1.-) were enhanced in roots of Hevea brasiliensis 2 weeks after inoculation with Glomus mosseae, simultaneously with an increased lignin content. However, no scopoletin accumulation was observed by this time. These defence-like reactions were caused by the first penetration of the AM fungus but they were transient

COUDRET, Alain

Ducher, Mireille,

Laffont-Schwob, Isabelle

Sallanon, Huguette

Ducher, Mireille, M.

Coudret, Alain

HAL Clermont Université

An Arbuscular Mycorrhizal Fungus (Glomus mosseae) Induces a Defence-like Response in Rubber Tree (Hevea brasiliensis) Roots

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An Arbuscular Mycorrhizal Fungus (Glomus mosseae)
Induces a Defence-like Response in Rubber Tree (Hevea
brasiliensis) Roots
Isabelle Laffont-Schwob, Huguette Sallanon, Mireille Ducher, Alain Coudret
To cite this version:
Isabelle Laffont-Schwob, Huguette Sallanon, Mireille Ducher, Alain Coudret. An Arbuscular Mycor-
rhizal Fungus (Glomus mosseae) Induces a Defence-like Response in Rubber Tree (Hevea brasiliensis)
Roots. Journal of Plant Physiology, Elsevier, 2000, 156, pp.284 - 287. ￿hal-02095618￿
J Plant Physiol. Vol. 156. pp. 284-287 (2000) 
http://www.urbanfischer.de/journals/jpp 
Short Communication 
(I JOURNAL OF 
PLANT PHYSIOLOGY 
© 2000 URBAN & FISCHER Verlag 
An Arbuscular Mycorrhizal Fungus (Glomus mosseae) 
Induces a Defence-like Response in Rubber Tree (Hevea 
brasiliensis) Roots 
ISABELLE SCHWOB 1*, MIREILLE DUCHER
2
, and ALAIN COUDRET2 
1 Laboratoire Dynamique et Ressources du Vegetal, Universite de Provence case 17, 3 place Victor Hugo, F-13331 
Marseille cedex 3, France 
2 Laboratoire de Physiologie et Biotechnologies Vegetales, Universite Blaise Pascal, 24 avenue des Landais, 63177 Aubiere 
cedex, France 
Received March 1, 1999 . Accepted (Revised) September 10, 1999 
Summary 
The specific activities of cinnamyl alcohol dehydrogenase (EC 1.1.1.-) and cell wall-bound peroxidases 
(EC 1.11.1.-) were enhanced in roots of Hevea brasiliensis 2 weeks after inoculation with Glomus mosseae, 
simultaneously with an increased lignin content. However, no scopoletin accumulation was observed by 
this time. These defence-like reactions were caused by the first penetration of the AM fungus but they 
were transient. 
Key words: Glomus mosseae, Hevea brasiliensis (rubber tree), lignification, mycorrhization, scopoletin. 
Abbreviations: AM == arbuscular mycorrhizal; CAD == cinnamyl alcohol NADPH dehydrogenase; PO == 
cell wall-bound peroxidases. 
Introduction 
The formation of a functional symbiosis involves a com-
plex sequence of interactions between fungal hyphae and 
plant root cells (Harrison and Dixon, 1993) Unlike patho-
genic biotrophs, which generally elicit typical defence respon-
ses in host plants, AM fungi do not induce a lasting response 
(Codignola et al., 1989; Volpin et al., 1994), acting only tran-
siently during the early stage of colonization (Spanu and Bon-
fante-Fasolo, 1988; Morandi et al., 1984). 
An analysis of data collected on H. brasiliensis infected with 
Rigidoporus lignosus and Phellinus noxius suggests that lignifica-
tion is important in tree defence (Nicole et al., 1986, 1992). 
The enzymes specifically involved in the lignin process include 
cinnamyl alcohol NADPH dehydrogenase (CAD). However, 
the role of this enzyme in the first steps of mycorrhization is 
* To whom correspondence should be addressed. 
not well documented. Cell wall-bound peroxidases (PO) cat-
alyse phenol oxidation in the cell walls to polymerize lignin 
precursors. 
Isoflavonoid phytoalexins are antimicrobial phenylpropa-
noid derivatives that often accumulate rapidly in plant tissues 
resistant to pathogen attack (see Dixon et aI., 1995; Smith, 
1996). In the rubber tree, scopoletin is a coumarin induced 
by pathogenic fungi and identified as a phytoalexin (Giese-
mann et al., 1986; Garcia et al., 1995). 
Glomus mosseae forms mycorrhizae with Hevea brasiliensis 
roots under controlled conditions (Schwob et al., 1998). No 
study has apparently been conducted on the first enzymatic 
phenomena occurring in the early stages of AM colonization 
in rubber tree roots. Our purpose was to ascertain whether a 
transient host defence response occurs in H brasiliensis roots 
during G. mosseae installation. CAD and PO activities were 
monitored, and scopoletin and lignins were quantified during 
the early stages of colonization. 
0176-1617/2000/1561284 $ 12.00/0 
An AM Fungus Induces a Defence-like Response in Hevea Roots 285 
Materials and Methods 
Plant culture and fongus inoculation 
Surface-sterilized seeds of Hevea brasiliensis Miill. Arg. were pre-
germinated 2 weeks in sand soil and transplanted into individual 
250-mL pots, each containing twice-sterilized sand and peat soil (111, 
v/v), pH 6.2. Plants were grown in a phytotron (16H/8 day/n~ht 
cycle and 27± 1/24 ± 1°C day/night temperature at 250 J.Lmol m- s-I 
daylight [MazdafluorJ). Leek (Allium porrum L.) was used as a source 
of inoculum of Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe. 
Each pot with a 2-week-old plant was inoculated by adding 10 g of 
soil containing propagules of mycorrhized leek. Control plants were 
inoculated with 10 g of twice-sterilized soil containing propagules. 
Each pot initially received 50 mL of Long Ashton solution (Hewitt, 
1966) with 130 J.LmollL phosphorus. Plants were allowed to grow for 
a maximum of 5 weeks. The root samples were collected each week 
after inoculation for 5 weeks. Part of the roots were frozen in liquid 
nitrogen and stored at -80°C until required for the enzyme and 
scopoletin assays. The rest were dried for the lignin assay. 
Visualisation of colonisation 
The course of infection was visualized employing 0.05 % trypan 
blue in lactophenol (Merck) as described by Phillips and Hayman 
(1970) after clearing for 10 min in 10 % KOH at 90°C. 
Enzyme extraction and assay 
All of the enzyme assays were performed on crude extracts. 
CAD (EC 1.1.1.-) activity was assayed as previously described 
(Sarni et al., 1984). Oxidation of coniferyl alcohol to coniferyl alde-
hyde was measured by increasing absorbance at A 400 nm at 30°C. 
Enzyme activity was expressed in nkat mg -I protein. PO (EC 1.11.1.-) 
activity was assayed as previously described (De Jaegher, 1986) by 
measuring the variation of absorbance at A 530 nm after 4 min at room 
temperature. Enzyme activity was expressed in ,1.(OD) mg-I min-I 
protein. 
Three replications of 4 measurements were done for each sample. 
Protein was determined by the Bradford method (1976) using BSA 
as standard. 
Lignin contents 
Lignin contents were determined on fresh roots, on the cell wall 
residue obtained after successive extractions of the air-dried and 
ground wood. The method of De Jaegher et al. (1985), used to pre-
pare cell wall material, was modified by replacing, for safety, ben-
zene by toluene as recommended by Monties (1989). The final prep-
aration was dried at 80°C. Klason lignin content was estimated as 
described by Kirk and Obst (1988). Lignins were expressed as a per-
centage of the original sample. 
Scopoletin assay 
Scopoletin was extracted from rubber tree roots by two-dimen-
sional chromatography as described by Mabry et al. (1970) modified 
by Garcia et al. (1995). Fresh ground roots were extracted at room 
temperature for 3 days in methanol (75 mL). Afrer the plant mate-
rial was removed by filtration, the extract was evaporated to dryness 
using a water pump vacuum in a rotary evaporator (Rotavapor, Fla-
wi!, Schweiz). The residue was dissolved in 1 mL of methanol. A 20-
J.LL aliquot of the solution was spotted on cellulose plates without 
fluorescent indicator (Merck). A first migration was done in a ter-
tiary butanol: glacial acetic acid: water (4/115, v/v/v) solution. Afrer 
migration, the chromatogram was dried. A second migration was 
done in 2 % glacial acetic acid solution. The chromatogram was 
viewed in UV light (254 nm) with the help of a trans-illuminator 
(Polylabo, Strasbourg, France). Spots of scopoletin were isolated and 
immediately eluted in 2 mL methanol at room temperature. The re-
maining gel was removed by centrifuging at 2,000 Kn for 5 min. The 
supernatant was analysed by spectrophotometry (spectrophotometer 
SECOMAN 70) at A 365 nm using pure commercial scopoletin 
(Merck) as standard (Giesemann et al., 1986). Scopoletin content 
was calculated for each sample compared with standard and ex-
pressed as a percentage of the original sample. 
Statistical analysis 
To test the main and interactive effects of the sampling time (1 to 
5 weeks) and the inoculation (control or inoculated), data were pro-
cessed by two-way analysis of variance (AN OVA) (SYSTAT, Delta-
sofr), and means were compared with Scheffe's test. 
Results and Discussion 
Fungal colonization 
One week after inoculation, no external mycelium was vis-
ible in the root system of the inoculated plants. Mter 2 
weeks, the first external mycelia were observed, and thereafter 
external and internal mycelia were present in roots of the inoc-
ulated plants. After 5 weeks, few hyphae were visible with 
some arbuscules, but no vesicules were present, demonstra-
ting that mycorrhization was not fully expanded but develop-
ing. The control plants remained uncolonized throughout the 
5 weeks of experimentation. 
Enzyme assay 
CAD activity was constant for 3 weeks, and then fell in 
the following weeks for the control plants (Fig. 1 A). When 
plants were inoculated with Glomus mosseae, higher CAD ac-
tivity was observed 2 weeks after inoculation. Values then de-
creased during weeks 3, 4 and 5 to reach control values. 
PO activity was constant during the first 3 weeks in the con-
trol plants. It then increased during the following weeks. The 
PO activity of inoculated plants was higher after 2 weeks, de 
creased at week 3 and then increased again (Fig. 1 B). 
Two weeks after inoculation, the first contact of the fungus 
with the root cortex occurred. CAD and cell wall-bound per-
oxidase activity peaks were observed in rubber seedling roots 
by this time. Spanu and Bonfante-Fasolo (1988) observed a 
similar increased peroxidase activity on Allium porrum inocu-
lated with Glomus versiforme. Observed peaks of enzyme ac-
tivity were related to first penetrations of mycorrhizal fungus 
in our experiment. 
Lignin contents 
Lignin percentage in inoculated plants was higher than in 
control plants 2 weeks after inoculation (Fig. 1 C). No signifi-
cant difference was observed between inoculated and control 
plants for the other time measurements. 
Lignin deposition and peroxidase activities increased with 
time while CAD activity decreased. 
286 ISABELLE SCHWOB, MIREILLE DUCHER, and ALAIN COUDRET 
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Fig. I: Enzyme activities, and lignin and scopoletin contents in inoculated and control root tissues of rubber trees during the first 5 \,eeks 
after inoculation. A: CAD activity; B: PO activity; C: lignin percentage; D: scopoletin percentage. Values are the mean of 3 replications of 4 
measurements. Means with the same letter are not different by Scheffe's test (p<O.05). 
Scopoletin quantification 
A blue fluorescence was localized for all of the samples at 
the same spot of the commercial scopoletin, under UV view-
ing. The Rr value in butanol: acetic acid: water was 0.90 and 
the Rfvalue in acetic acid was 0.22, as previously described in 
rubber tree leaves by Garcia et al, (1995). The extract was 
identified by spectral absorption from 240 to 400 nm com-
pared with commercial scopoletin. No significant difference 
was observed between inoculated and control plants (Fig. 
1 D). Hence, scopoletin was constitutively present in rubber 
seedling roots. However, no scopoletin accumulation was ob-
served during the first penetration of the AM fungus. Scharff 
et al. (1997) reported no detectable increase in the phytoalexin 
levels of mycorrhizal soybean roots. 
Statistical analysis 
ANOVA showed an inoculation effect on all of the meas-
urements of PO activity and lignin percentage (Table I), Sam-
Table 1: Main factors and interactive effects of inoculation (control 
and inoculated) and sampling time (I to 5 weeks) of CAD ani PO 
activities, and lignin and scopoletin contents (ANOVA results). 
CAD activity PO activity Lignin 
content 
Scopoletin 
content 
Inoculation (0 NS Fl.2o = 4.4* Fl.2o = 8.6** NS 
Sampling time (Tl F4.2o = 15.9** Foo = 9.0** F4.20 = 64.0** NS 
IxT F4.2o =3.1* NS F4.20 = 3.2* NS 
NS: not significant; *: p ~0.05; **: p ~O.OOI. 
An AM Fungus Induces a Defence-like Response in Hevea Roots 287 
pIing time greatly influenced the variations between the vari-
ous parameters except for scopoletin contents (P<O.05). CAD 
activity and lignin percentage showed conjugate effects ofinoc-
ulation and sampling time. 
Hence, as reviewed by Gianinazzi-Pearson (1996) and Azcon-
Aguilar and Barea (1996) on other plant-AM fungus models, 
when mycorrhizal symbiosis initiates between H brasiliensis 
and C. mosseae, a weak, transient uncoordinated activation of 
inducible defence responses occurs that differs from the re-
sponse elicited in plant-pathogen interactions. 
Acknowledgements 
The authors thank Christian Plenchette for the Glomus mosseae 
strain, Jacqueline Grima-Pettenati for helpful discussion on CAD 
assay, and the Michel' n Tire Company for Hevea brasiliensis seeds. 
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An Arbuscular Mycorrhizal Fungus (Glomus mosseae) Induces a Defence-like Response in Rubber Tree (Hevea brasiliensis) Roots

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