Scopoletin production and degradation in relation to resistance of Hevea brasiliensis to Corynespora cassiicola

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-Scopoletin production and degradation in relation to resistance of Hevea brasiliensis to Corynespora cassiicola FREDERIC BRETON, CHRISTINE SANIER,

and JEAN

D'AuZAC

Laboratoire de Biotechnologie et Physiologie Vegetale Appliquees, Universite Montpellier II, Case 002, Place Eugene Bataillon, F-34095 Montpellier cedex 5; France Received February 12, 1997· Accepted March 26, 1997

Summary

Inoculation of leaves of resistant and susceptible Hevea brasiliensis clones with Corynespora cassiicola induced foliar necrosis and biosynthesis of scopoletin (Scp), considered as a Hevea phytoalexin. Foliar symptoms appeared, as soon as 24 h after infection, and precede Scp accumulation in inoculum droplets (peak: 48 h after inoculation). Scp concentration was 5-fold higher in the susceptible than in the resistant clone 48 h after infection. Nevertheless, a fungitoxic effect of Scp on spore germination and on mycelium growth was shown in bioassays, but the efficient concentrations were greater than those found with another foliar pathogenic fungus of rubber tree such as Microcyclus ulei and Colletotrichum gloeosporioides. Bioassay showed that the low Scp fungitoxicity to C cassiicola could also be related to the ability of fungus to detoxify Scpo Isoelectric focusing analysis of peroxidase activity in Hevea infected leaflets has shown an increase in acidic and basic isoperoxidases that are able to use Scp as substrate. The rapid increase of Scpoxidase activity (as soon as 16 h after inoculation) was higher in the resistant than in the susceptible clone. In vitro testing of Scp peroxidation products has shown a toxic effect on C cassiicola (conidia germination and mycelium growth), but not significantly higher than ScpoIt appears that the level of Scp accumulation was a balance between its synthesis and its degradation by the pathogen orland by foliar Scp-peroxidases. In conclusion, these results lead to the fact that Scp cannot be considered as a major defense mechanism of Hevea towards C cassiicola disease.

Key words: Hevea brasiliensis, Euphorbiaceae, Corynespora cassiicola, Scopoletin, Phytoalexin, Peroxidases. Introduction

Hevea brasiliensis, a perennial tropical plant, is the major source of natural rubber in the world. Since 1975, a newly reported disease, the bird's eye spot, has been detected in Asia (Anonymous, 1975) and later in Africa (Chee, 1988). The pathogenic agent, Corynespora cassiicola, infects the leaves, leading to brown necrotic lesions along the veins and finally to massive and repeated defoliation. This results in serious loss in Hevea growth and rubber yield. Since the first descriptions of the disease, only field observations reported the existence of some resistant Hevea clones to C cassiicola (Liyanage. 1987). while the resistance mechanisms remain unknown. High disease resistance is often correlated with an increase in phenolic content (Ampomah and Friend. 1988; Assabgui et aI .• 1993). Among the range of enhanced phenols. some of

J Plant Physiol Vtli. 151. pp. 595-602 (1997)

them are newly induced antimicrobial compounds. the phytoalexins. In many infected host plants, a higher rate of phytoalexin production is considered as one of the most important mechanisms for localized resistance (Darvill and Albersheim. 1984; Smith. 1996). The efficiency of phytoalexins depends not only on their fungitoxicity but also on the rapidity and the intensity of their accumulation in host tissues and on the parasites ability to detoxify them (Tal and Robeson, 1986 b; Van Etten et al., 1989). Moreover, Alh Goy et al. (1993) suggested that the toxicity of phytoalexin could be enhanced in situ by peroxidation reactions. giving rise to new products with higher toxicity. In rubber tree, the first evidence of phytoalexin plant-response was described in 1975 by Tan and Low. Later, this new induced compouod, detected in infection of Hevea leaves with Microcyclus ulei, was identified as a hydroxycoumarin

596

FREDERIC BRETON, CHRISTINE SANIER, and JEAN D'AuZAC

called scopoletin (Scp) (Giesemann et al., 1986). More recently, Scp accumulation in Hevea infected leaves was directly associated with resistance to Microcyclus ulei and Colletotrichum gloeosporioides (Garcia et al., 1995 b; Breton et al., 1994). Resistance to fungi is also frequently related to the increase in peroxidase activity (Shimoni et al., 1991; Candela et al., 1994; Dalisay and Kuc, 1995). Garcia (1994) demonstrated in HevealMicrocylus ulei interaction a positive correlation between peroxidase activity and an intensive lignification closely linked to disease resistance. This peroxidase activity could also be involved in a wide range of phenol oxidations leading either to the appearance of necrotic lesions or to a strong antimicrobial activity (Alam et al., 1991; Moerschbacher, 1992). In accordance to previous studies on Hevea response to M ulei and C gloeosporioides, leading to the description of resistance indicators (Garcia et al., 1995 a-b; Breton et al., 1994), the aim of this present work was to study the accumulation of Scp and the variation of peroxidase activity level during the establishment of H. brasiliensislC cassiicola interaction. The Scp accumulation kinetics in Hevea infected leaves are described for the first time in relation to clonal foliar symptoms of resistance to the disease. The Scp efficiency against C cassiicola and the involvement of some foliar peroxidases as defense response are discussed in relation to Scp accumulation.

ter. The extract was filtered through a 0.22 f.lm Millipore membrane and vacuum-dried. The sample was resuspended in methanol (100 f.lL) and analysed by two dimensional TLC on a cellulose plate with 12 % acetic acid and benzene/acetic acid/water (6: 7: 3). The chromatogram was observed under UV light (254 and 366 nm) and a single blue fluorescent compound was characterized by its Rf(0.41 and 0.43 for each solvent, respectively). Identification and quantification were also performed by HPLC with a reverse phase column (Spherisorb CIS' 250x4mm) equipped with a precolumn (Deltapak, Guard-Pak Waters). Elution at 1 mL min-I was programmed as a linear gradient (4 to 96 % acetonitrile in water, pH 2.6) for 20 min. HPLC analysis was coupled with a fluorescent detector (Waters 420-AC) equipped with an aflatoxin lamp, a 337 nm interference filter and a 425 nm long pass filter. Standard Scp (Extrasynthese) was used as control. This Scp identification agrees with the previous determination by Tan and Low (1975), Giesesemann et al. (1986) and Garcia et al. (1995 b). Results were expressed in flM of Scp accumulated in droplets.

Extraction ofScp-peroxidases from leaf tissues Leaf tissues were ground in liquid nitrogen and suspended (1 : 5 w/v) for 15 min at 4 'C in 0.5 M Na-acetate buffer (pH 5.2) containing insoluble polyvinylpolypyrolidone (40 % w/w). The homogenate was centrifuged at 40,000 gn for 20 min at 4 'c. Afrer two successive extractions, the supernatant was filtered through a 0.45 f.lm Durapore membrane and stored at -80'C prior to use. Protein concentration was estimated by the method of Bradford (1976) using bovine serum albumin as a standard.

Materials and Methods

Plant material Two Hevea brasiliensis clones (PB260 and GTl) that differ in their degree of susceptibility to C cassiicola were grown in a tropical greenhouse in order to obtain 6 to 8-day-old leaflets (BrC stage, as described by Halle and Martin, 1968).

Assays ofScp-peroxidase activity Scp-oxidase activity was estimated in 0.1 mollL acetate buffer (pH 5.5) with 0.1 mmollL Scp (dissolved in ethanol, 1 % final) and 4 mmollL H 2 0 2 • Activity was measured by the decrease in absorbance at 345 nm. Scopoletin oxidation is characterized by the appearance of a new blue substance that is rapidly converted to a yellow product (Andreae and Andreae, 1949).

Fungal culture and in vitro leaflet inoculation A monospore strain of C cassiicola, obtained from infected leaves of Hevea in the Philippines, was grown in the dark at 25 'C on PDA (POtato Dextrose Agar) medium with 0.05 % chloramphenicol. Sporulation was induced on a 7-day-old thallus after 3 days at 28 'C in continuous light (Philips fluorescence tube, daylight). The conidial suspension was titrated at 2.3 X 104 spores mL -I in sterile distilled water. Inoculation was performed by positioning conidial suspension droplets (10flL) on the abaxial side ofieafIets (3 dropletscm- 2). Controls were treated with sterile distilled water droplets. Leaflets were incubated in Petri dishes in moist conditions at 28 'C in continuous light (Philips fluorescence tube, daylight).

Isoelectric focusing (IEF) and peroxidase staining Native IEF was performed using the method of Robertson et al. (1987) on vertical minigels containing 2 % of ampholines (SERVA). Isoperoxidases were separated on a pH gradient ranging from pJ:I 2-11. Protein bands with guaiacol-oxidase activity were detected directly by soaking the gel in 0.1 M phosphate buffer, pH 6, containing 0.32 % guaiacol and 20 mM H 20 2 • Scp-oxidase isozymes were revealed as blue colour bands after incubating the gel in 0.1 mollL acetate buffer, pH 5.5, supplemented with 1 mM Scp (dissolved in ethanol, 3 % final) and 5 mmollL H 20 2 •

Scanning electron microscopy Necrotic wnes from infected leaves, previously fIXed, were dehydrated in a gradual series of alcohols (70 %, 80 %, and 90 %) and three times in absolute alcohol. Specimens were dried in a criticalpoint drier under CO 2, coated with gold-palladium and observed under a Jeol JSM 6300F scanning electron microscope.

Identification and analysis ofScp accumulation Inoculum droplets were recovered at different times after inoculation and inoculated wnes were rinsed twice with 10 flL distilled wa-

Obtention ofScp peroxidation products A solution of 5 mmollL standard Scp, dissolved in ethanol (7% final), was oxidized with 20 mmollL H 20 2 and 10 U mL -I of horseradish peroxidase (Sigma) in 0.1 mollL acetate buffer (pH 5.5) at 37·C. Afrer 48 h, the Scp oxidation products were filtered on a 0.45 f.lm Durapore membrane and freeze dried. The powder was resuspended in distilled water and stored at -80'C prior to use. The standard was performed under the same conditions but without ~cp. Complete Scp oxidation, followed by HPLC analysis, was obtamed after 48 h under these conditions.

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Fig. 1-3: 1 Scanning electron microscopy observations: (1 A) Germ tube enveloped (GT) by a fibrillar sheath (FS) showing a strong attachment to the leaf surface (L). (1 Band 1 C) Direct mycelium penetration through cuticular surface leaflets (L) 12h after inoculation. (2) Appearance of the first necrosis (arrows) in resistant (2A) and in susceptible clones (2 B), only 24h after infection. (3) Development offoliar symptoms 48 h after inoculation in resistant (3A) and susceptible clones (3 B).

Bioassays The toxic effects of Scp and Scp oxidation products were tested on spore germination (2.3 X 104 conidia mL -I in sterile distilled water) and on mycelium growth. Four concentrations (1 to 4 mmollL) of scopoletin (dissolved in ethanol) and of Scp oxidation products were tested. For Scp bioassays, the final ethanol concentration (3%) was the same in tests as in the control. Germination tests were performed at 25 'c in the dark on glass slides under moist conditions.

The inhibition rate of conidia germination was determined after 24h by microscope observations (6 samples of 100 conidia) in comparison with the control. Growth tests were realized in Petri dishes by subculturing a mycelium plug (5 mm diameter) on PDA medium containing Scp or Scp oxidation products. Fungal cultures were incubated for 7 days in the dark at 25 'c. Mycelia growth was expressed as the surface area of the mycelium development comparatively to the control.

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Scp, previously dissolved in ethanol, was filter sterilized on a 0.2 J,1m membrane and incorporated into Czapek liquid medium (Borjesson et al., 1990) at a final concentration of 2 mM and 3 % ethanol. A thallus fragment (5 mm in diameter) was transferred into the liquid medium and incubated at 25 ·C in the dark. Scp degradation was quantified in the culture filtrate every 2 days by the HPLCspectrofluorometry method as previously described. Controls were performed without fungus.

clone, necrotic spots do not extend out of inoculated zones (Fig. 3 A). Just the opposite was found in the susceptible clone: yellowish brown necrosis appeared and enlarged into circular or irregular pale grey lesions (Fig. 2 B). Fourty eight hours after inoculation, most of the necrotic lesions were widely spread and associated with brown discolouration of the veins around them, giving a typical «fish-bone» appearance (Fig. 3 B).

Scp accumulation Results

Infection process and symptomatology The germ tubes of C. cassiicola developed an extracellular fibrillar sheath apparently involved in the mycelial adhesion to the leaf (Fig. 1A) and also in fungus penetration (Figs. 1 B and 1 C). No differences were observed in conidia germination and in fungus sheath aspects between infected leaves of susceptible (PB260) and resistant (GTI) Hevea clones. Twelve hours after inoculation, germ tubes invaded the first cell layers of abaxial epidermis by direct penetration through the plant surface rather than by the rare stomata penetrations (Figs. 1 B and 1 C). The fungal progression in host tissues was accompanied by the appearance of necrotic lesions as soon as 24 h after infection. Necrosis of the two Hevea clones differed in their aspect and in their size (Figs. 2A-2 B). In inoculated wnes of the resistant clone, necrosis appeared as diffuse spots resembling a «hypersensibility reaction» (Fig. 2A). Mycelium development was rapidly stopped in foliar tissues and was thus restricted to a few cells around the penetration site. Thus, in the resistant

Twenty four hours after inoculation, direct observation of inoculum droplets (under UV light, 254 and 366nm) on the two clone's leaves revealed a blue fluoresence, which rapidly increased and reached a peak at 48 h. Fluorescence of inoculum droplets was higher for susceptible (Fig. 4 A) than for resistant (Fig. 4 B) clones and was never observed in distilled water droplets on controls. This fluorescence was due to a single compound identified as, Scp by HPLC and TLC (see Materials and Methods). Scp, abundantly secreted into inoculum droplets, was quantified in droplets 24, 48, 72 and 96 h after inoculation (Fig. 5). For the susceptible clone Scp concentration reached a higher level (8.49 ± 1.86 ~mollL) than for the resistant clone (1.79 ± 0.93 ~mollL) 48 h after inoculation. During the time course of the experiment, no Scp accumulation was detected in control droplets.

LeafScp-peroxidase activity Peroxidases capable of using Scp as substrate were identified in a crude extract of susceptible and resistant Hevea infected leaves. This Scp-oxidase activity increased significantly

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growth than on spore germination, and was positively correlated with their concentration (Table 1). The efficiency ofScp oxidation products to inhibit spore germination was lower than Scp under a concentration of 3 mmol/L and slighty greater (20 %) for 4 mmol/L. The oxidation products at 1mmol/L were also less efficient in liming mycelium development in comparison with Scp (Table 1) . The cultivation of Corynespora on a Czapek liquid medium containing 2 mmol/L of Scp (which inhibits 50 % of fungal growth) shows that Scp gradually disappeared (Fig. 8). In spite of Scp fungitoxicity, the decrease observed suggests that C cassiicola is able to degrade this phytoalexin. The mechanism of fungus Scp degradation is unknown, but no peroxi-

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after inoculation, to become 2-fold higher in the resistant than in the susceptible clone after 48h (Fig. 6). Isoelectric focusing analysis of the Scp-oxidase activity revealed two identical acidic and basic isoperoxidase groups present in the leaves, whatever the treatment and the clone tested, excluding the appearance of new induced isoenzymes (Fig. 7 A). The same observations can be made for the guaiacol-isoperoxidase pattern (Fig. 7B). However, all guaiacol-oxidase isoenzymes were not able to use Scp as substrate (Figs. 7A and 7B). The higher activity of basic and some acidic Scp-isoperoxidases in the resistant clone fits well with the difference previously observed with kinetics of total Scp-oxidase activity between the two clones (Fig. 6).

Fungitoxicity ofScp and ofits peroxidation products The fungitoxicity of Scp and Scp oxidation products, tested in vitro on C cassiicola, was higher on mycelium

Fig. 7: Isolectric focusing patterns of Scp-isoperoxides (7 A) and guaiacol-isoperoxidases (7B) from healthy (C) and C cassiicola-inoculated Hevea leaflets (I). Staining peroxidase activities was performed for resistant (GT 1) and susceptible (PB260) clones, 24 h after inoculation. Soluble proteins of foliar extracts, equivalent to 20 J.Lg, were deposited per tract. Markers of pI are given at the margin of the gel.

Table 1: Fungitoxic effect of increasing concentration of Scp and Scp oxidase products on the germination of C cassiicola conidia (24-h incubation) and on mycelium growth (7 day incubation). Inhibition effects were expressed in percent of the control. The concentrations that inhibit spore germination and mycelium growth by 50 % (150) were 2.2 mmollL and 1.4 mmollL for Scp, and 2.7 mmollL and 1.6 mmollL for Scp oxidation products, respectively. Concentrations (mM) 1

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FREOERIC BRETON, CHRISTINE SANIER, and JEAN O'AUZAC

centration was 5-fold higher in the susceptible (PB260) than in the resistant clone (GTl). Comparable discordant results were obtained in resistant or susceptible tobacco cultivars to Phytophthora parasitica var. nicotianae or Pseudomonas solanacearum (Sequeiera and Kelman, 1962; Snook et al., 1991). These data illustrate the controversy concerning the ability of Scp to confer, strictly by itself, the resistance to plant disease. Our results confirm that the fungitoxic effect of phytoalexin could be related not only to its concentration around the penetration site, but also to the rapidity of its accumulation after infection (Bailey, 1982; Tal and Robeson, 1986b). Comparative to Microcyclus ulei, Colletotrichum gloeosporioides and Ceratocystis fimbriata leaf diseases, in Hevea/C. cassiicola inter0,0 +---.---,.---r---,r----...--"'T"'""'-.---r--r---,r----~ action, the onset of Scp accumulation appears too late to 12 o 2 4 6 8 10 limit the very fast C. cassiicola invasion in host tissues (Garcia Incubation time (days) et al., 1995 b; Breton et al., 1994; El Modafar et al., 1995). Bioassays with C. cassiicola showed a fungitoxic effect of Fig. 8: lime course of scopoletin degradation by C cassiicola grown Scp on mycelium growth (Iso = 1.4 mM) and on conidia geron Czapek liquid medium containing Scp (initial concentration: mination (Iso = 2.2 mM). The Scp concentration inhibiting 2mmoVL). spore germination was higher for C. cassiicola (Iso = 2.2 mM) than for other fungi such as Microcyclus ulei (Iso = 0.5 mM), dase activity was measured, neither in mycelium, nor in Cza- Colletotrichum gloeosporioides (Iso = 1.1 mM) and Ceratocystis fimbriata (150 =0.4 mM) (Garcia et al., 1995 b; El Modafar et peck liquid medium (data not shown). al., 1995); C. cassiicola could be relatively resistant to the toxic effect of Scpo However, in vitro, Scp is efficient in the millimolar range, whereas it is present in the inoculum dropDiscussion lets in a micromolar range concentration. The same observations were reported with many pathogens such as Cercospora Time course ofScp accumulation in relation to disease nicotianae, Phytophthora parasitica var. nicotianae, Pseudomoresistance nas syringae pvs tabaci and syringae, TMV (Ahl Goy et al., Inoculation of Hevea leaflets with C. cassiicola leads, as 1993). It seems obvious that Scp concentration in the host soon as 24 h after infection, to well-developed necrotic le- pathogen interface must be higher than those estimated in tosions, which are much more important in the susceptible tal foliar extract or in inoculum droplets. Ahl Goy et al. (PB260) than in the resisrant clone (GT1). The necrosis ap- (1993) suggested that local concentration at the sites of pearance precedes, for the rwo clones, the accumulation of fungal penetration could be 10 to SO-fold higher than those Scp, previously characterized as a Hevea phytoalexin (Giese- in global leaf or in inoculum droplets. This hypothesis, allowmann, 1986). Recently, Scp concentration in inoculum drop- ing to reach a Scp concentration in millimolar range at the lets of Hevea infected leaflets has been positively correlated first contact point of the leaf, has been recently confirmed by with the degree of clonal resistance to Microcyclus ulei and Garcia (1994). This author estimated that the Scp concentraColletotrichum gloeosporioides (Garcia et al., 1995 b; Breton et tion in droplets must be multiplied by about 60-fold to obal., 1994). The Scp concentration in inoculum droplets was tain the apoplastic Scp concentration of the rwo first layers of comparable (micromolar range) berween these three patho- epidermial cells in contact with the fungus. This uneven resystems and also in plane tree inoculated with Ceratocystis partition has also been shown in infected cotton cotyledons fimbriata £sp. platani (El Modafar et al., 1995). In this latter and sorghum leaves (Pierce and Essenberg, 1987; Snyder and interaction the concentration of Scp in inoculum droplets Nicholson, 1990). Thus, the uneven repartition of Scp in inand in foliar extracts was nearly identical (El Modafar et al., fected tissues could explain its efficiency observed for higher 1995). Consecutively, it was' assumed that the differences in concentrations according to the in vitro data. Scp concentration in C. cassiicola inoculum droplets berween PB260 and GTI clones also reflects the inoculated leaf conScp degradation tent. The presence of Scp has often been detected in response to attack by viruses (Andrea and Andrea, 1949; Clarke and Phytoalexin instability is an important factor that may imBaines, 1976), bacteria (Sequeira, 1969) and fungi (El Moda- pair its in situ efficiency (Van Etten et al., 1982). It is well esfar et al., 1993; Tal and Robeson, 1986 a; Zeringue, 1984). tablished that some phytopathogenic fungi are tolerant to Resistant cultivars of tobacco, rubber tree and plane tree pro- their hosts' phytoalexins in relation to the fungus' capacity to duced more Scp than the susceptible ones in response to metabolize them (Desjardins et al., 1989; Tal and Robeson, infection with TMV, Microcyclus ulei, and Ceratocystis fimb- 1986 b; Sbaghi et al., 1996). In our pathosystem, rwo factors riata, respectively (Ahl Goy et al., 1993; El Modafar et al., can directly affect the level of in situ Scp accumulation. The 1995; Garcia et al., 1995 b). The authors suggested that this first is the ability of C. cassiicola to degrade in vitro Scp, such phytoalexin could be an important factor in disease resist- as previously demonstrated for Alternaria helianthi, Ceratoance. In contrast, in Hevea/C. cassiicola interaction, Scp con- cystis fimbriata and Gibberella pulicaris (Tal and Robeson, 2,0-cr-----------------.

Scopoletin and Corynespora-Hevea leaf disease

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1986 b; EI Modafar et al., 1993; Gardner et al., 1994). The BORJESSON, T., U. STOLLMANN, and T. SCHNURER: Volatile metabolites and other indicators of Penicillium aurantiogriseum growth second may be the activity of foliar peroxidase capable of on different substrates. Appl. Environ. Microbiol. 56, 3705using Scp as substrate. Reigh et al. (1973) reported that Scp 3710 (1990). could be an endogenous substrate for a specific peroxidase BRADFORD, M.: A rapid and sensitive method for the quantification isoenzyme isolated from callus tobacco. In the Hevea resistant of microgram quantities of protein utilizing the principle of proclone, Scp-oxidase activity was rapidly induced and was tein-dye binding. Anal. Biochem. 72,248-259 (1976). higher than in the susceptible one. Among all of the guaiacol- BRETON, E, C SANIER, and J. D'AuZAC: Biochemical characterizaperoxidase isoenzymes separated by isoelectric focusing, two tion of Hevea brasiliensislColletorriehum gloeosporioides interacgroups, an acidic and basic one, are also capable of oxidizing tion. In: Proc. Soc. Fr. Physiol., Plant Science, Saint-Malo, Scpo Their role in plant pathogen interaction remains unclear. France, pp. 325 (1994). Alternatively, two hypotheses may be advanced. The first one CANDELA, M. E., R. MUNOZ, M. D. ALcAzAR, and A EsPIN: Isoperoxidase involvement in resistance of Capsicum annuum to inimplies the detoxification of phytoalexin to protect unaffection by cucumber mosaic virus. J. Plant Physiol. 143,213-217 fected host tissues from the phytotoxicity of the plants' own (1994). phytoalexins (Moerschbacher, 1992). Then, peroxidation of phytoalexins could indirectly help to increase the virulence of CHEE, K. H.: Studies on sporulation, pathogenicity and epidemiology of Corynespora eassiieola on Hevea rubber. J. Nat. Rubb. Res. pathogens (Van Etten et al., 1989). According to the second 3,21-29 (1988). hypothesis, phytoalexin peroxidation products could be more CLARKE, D. D. and P. S. BAINES: Host control of scopoletin acfungitoxic than phytoalexin itself (Moerschbacher, 1992). cumulation in infected potato tissue. Physiol. Plant Pathol. 9, Bioassays have shown that Scp oxidation products also dis199-203 (1976). playa toxic effect on C cassiicola (spore germination and my- DALISAY, R. E and J. A. Kuc: Persistence of reduced penetration by celium growth), but not significantly higher than Scpo Hevea Colletotriehum lagenarium into cucumber leaves with induced sysfoliar Scp-peroxidase activity does not seem to be involved in temic resistance and its relation to enhanced peroxidase and acincreasing the fungitoxic effect of Scpo tivities. Physiol. Mol. Plant Pathol. 47, 329-338 (1995). In conclusion, the Scp accumulation in Hevea leaves may DARVILL, A. G. and P. ALBERSHEIM: Phytoalexins and their elicitorsA defense against microbial infection in plants. Ann. Rev. Plant result from the balance between its synthesis after infection Physiol. 35, 243-275 (1984). and its degradation by the fungus and/or by the host's Scpperoxidase activity. Morever, unlike in Microcyclus ulei and DESJARDINS, A E., H. W. GARDNER, and R. D. PLATTNER: Detoxification of the potato phytoalexin lubimin by Gibberella puliearis. Colletotrichum gloeosporioides Hevea leaf diseases, Scp acPhytochem. 28, 431-437 (1989). cumulation during HevealCorynespora cassiicola interaction EL MODAFAR, C, A. CLERIVET, A. FLEURIET, and J. J. MACHEIX: does not seem to strongly contribute to the disease resistance Inoculation of Platanus aeerifolia with Ceratocystis fimbriata inand thus could not be used as a resistance marker. The reladuces scopoletin and umbelliferone accumulation. Phytochem. tively low fungitoxicity of Scp for C cassiicola, the ability of 34, 1271-1276 (1993). the host and the pathogen to degrade this phytoalexin and its EL MODAFAR, C, A. CLERIVET, A. VIGOUROUX, and J. J. MACHEIX: delayed accumulation compared with the rapidity of the CoAccumulation of phytoalexins in leaves of plane tree (Platanus rynespora invasion could probably, at least partially, explain spp.) expressing susceptibility or resistance to Ceratocystis fimthe inefficiency of Scp characterized elsewhere as an efficient briata f.sp. platani. Eur. J. Plant Pathol. 101,503-509 (1995). phytoalexin for the rubber tree. GARCIA, D.: Contribution it l'etude de la resistance totale et partielle dans l'interaction hote-parasite Hevea spp. - Microcyclus ulei, References AHL GOY, P., H. SIGNER, R. REIST, R. AICHHOLZ, W. BLUM, E. SCHMIDT, and H. KEsSMANN: Accumulation of scopoletin is associated with the high disease resistance of hybrid Nicotiana glutinosa X Nicotiana debneyi. Planta 191,200-206 (1993). AuM, M., A SATTAR, and K. K. JANARDHANAN: Changes in phenol and peroxidase in leaves of Java citronella infected with Curvularia andropogonis. BioI. Plantarum 33, 212-215 (1991). AMpOMAH, Y. A and J. FRIEND: Insoluble phenolic compounds and resistance of tuber disc to Phytophthora and Phoma. Phytochem.

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