Colonization of tomato root by a nonpathogenic strain of Fusarium oxysporum

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1 Neu) Phytol. (\99~), 137, 481^94 Colonization of tomato root by a nonpathogenic strain of Fusarium oxysporum BY CHANTAL OLIVAIN AND CLAUDE ALABOUVETTE* Laboratoire de Recherches sur la Flore pathogene dans le sol INRA - CMSE, BV 1540, 17 rue Sully, DIJON Cedex, France {Received 21 February 1991; accepted 15 July 1997) SUMMARY A strain of non-pathogenic Fusarium oxysporum Schlecht. emend, Snyd. & Hans, has been selected for its capacity to reduce the incidence of Fusarium wilt of tomato. Among the possible modes of action of this strain, competition with the pathogen for the colonization of the root surface and tissues has been proposed, in order to study the pattern of root colonization, young Lycopersicon esculentum Miller (tomato) plants grown in a nutrient solution were inoculated by a suspension of F. oxysporum microconidia and processed at time-intervals for microscopic observations. The fungal strain was transformed with the Gus reporter gene to facilitate the observations. Within 24 h of inoculation the root surface was colonized by a dense network of hyphae, with the exception of the apex, which was colonized only after 48 h. A few hyphae were observed penetrating into the epidermis, leading to the internal colonization of the root cortex. This colonization was always discontinuous, since defence reactions of the plant limited the extension of the fungus. The barrier formed by thickenings and coilings of the cell walls and hypertrophied cells was most frequently observed in the external cortex and, sometimes, deeper in the internal cortex, close to the vessels which were never colonized. Typical defence reactions such as wall appositions, intercellular plugging and intracellular osmiophilic deposits, were frequently observed. This is the first report, based on microscopic observations, of the capacity of a non-pathogenic strain of F. oxysporum to colonize roots of tomato. Key words: Light microscopy, ultrastructural study, Gus gene marker, infection process, plant defence reactions. control fusarium wilts came from the study of soils INTRODUCTION ^ c 'i.. Tu naturally suppressive to fusarium wilts, 1 hese Strains of Fusarium oxvsporum are common inhabit- studies showed that most suppressive soils supported ants of soils and have a worldwide distribution. All large populations of non-pathogenic Fusarium spp. strains of F. oxysporum are successful as saprophytes (Smith & Snyder, 1971 ; Toussoun, 1975), and hut some are responsible for vascular diseases in prompted the hypothesis that these non-pathogenic many plants of economic importance. These patho- Fusarium spp. were responsible for soil suppressgenie strains show a high level of host specificity and iveness (Rouxel, Alahouvette & Louvet, 1979). Not are classified into more than 120 formae speciales all the T^wsarmm spp. isolated from suppressive soils and races, according to the plant species and cultivar were able to induce suppressiveness in disinfected they are able to infect (Armstrong & Armstrong, soil, but most of the strains of F. oxysporum studied 1981). Only inoculation of the host plant can be used showed some ability to limit the incidence of to determine the/orma ^peciwis, and all the strains fusarium wilts (Tamietti & Alabouvette, 1986; unable to induce symptoms on a given species can be Tamietti & Pramotton, 1990). Several teams have considered non-pathogenic towards this species. selected non-pathogenic strains of F. oxysporum to A strain belonging to a given/orma sp(!c?fl/is can be control fusarium wilts (Ogawa & Komada, 1984; used to protect an incompatible plant against its Mandeel & Baker, 1991; Alabouvette & Couteaudier, specific pathogen (Matta, 1989), and many attempts 1992; Postma & Rattink, 1992; Larkin, Hopkins & have been made to utilize strains of different formae Martin, 1996), and efficient control has been respeciales to control fusarium wilts through induced ported (Postma & Rattink, 1992; Alabouvette, resistance (Biles & Martyn, 1989). Lemanceau & Steinberg, 1993). The idea of using non-pathogenic F. oxysporum to The mechanisms by which these non-pathogenic strains control the disease are not totally understood. * To whom correspondence should be addressed..,,, r -i ^ ^i. aiabouvette@dijon.inra.fr At least three modes of action can contribute to the

2 482 C. Olivain and C. Alabouvette efficiency of these strains; competition for nutrients in soil and the rhizosphere, competition for infection sites and root colonization, and induced systemic resistance (Mandee! & Baker, 1991; Alabouvette et al., 1993). Competition for nutrients, especially for carbon, has been clearly demonstrated but competition for root colonization has been demonstrated only indirectly. It has been proved, using microbial isolation techniques, that most of the non-pathogenic strains are able to colonize to some extent the root surface and root tissues (Nagao, Couteaudier & Alabouvette, 1990; Mandeel & Baker, 1991). This indirect evidence does not, howe%'er, clearly indicate which root tissues are colonized. The objective of the present study was to utilize microscopic observations to describe the pattern of tomato root colonization by a strain of F. oxysporum (Fo5a4) previously used to demonstrate the existence of systemic induced-resistance (Tamietti & Matta, 1991; Olivain, Steinberg & Alabouvette, 1995). MATERIALS AND METHODS Fungal strain and inoculum preparation A non-pathogenic strain of Fusarium oxysporum Schlecht. emend. Snyd. & Hans. {Fo5a4) isolated from a suppressive soil (Tamietti & Alabouvette, 1986) has proved its capacity to reduce disease incidence when applied to roots or soil before the inoculation of tomato plant with a pathogenic strain oi F. oxysporum (Tamietti & Matta, 1991; Olivain et al., 1995). This strain was transformed with the Gus {E. coli /?-D-glucuronidase) gene according to the procedure described by Couteaudier et al. (1993). In the presence of 5-bromo~4-ch!oro-3-indolyl /f-d-glucuronide, the //-glucuronidase activit\' of the fungus enables its histochemical localization in tissues and plant cells. The Gus gene, being under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter, an enzyme catalysing the second step of glycolysis, the intensity of the turquoise staining resulting from the Gus activity is an indication of the metabolic activity of the transformed strain (Eparvier & Aiabouvette, 1994). After transformation, the biocontrol activity of the Gus-marked strain was compared in a biotest with that of the wild strain and found not to be significantly different. The fungus was stored at 80 C until use. The fungus was grown in 10 gt^ malt broth (Biokar Diagnostics, Beauvais, France) at 25 C on a rotary shaker (250 rpm). After 6 d culture, the mycelial nnats were removed by filtration through a 40 //m mesh. The microconidia in the filtrate were washed three times by centrifugation (11000 j^, 20 min) and resuspended in sterile distilled water. The density of the conidial suspension was determined with a haemocytometer. Conditions of plant growth and inoculation Tomato seeds {Lycopersicon esculentum Miller cv. Marmande Verte), provided by INRA (Station d'amelioration des Piantes, Montfavet), were surface-sterilized in l'25"o sodium hypochlorite for 20 min and rinsed three times in sterilized distilled water. Seeds were germinated on nutrient agar, prepared by adding 10 g T' agar in the nutrient solution used to grow tomatoes in rockwoo! (Hydrokani nutrient solution, Hydro-Azote, Neuilly, France). Five to 10 seeds were deposited on the surface of the nutrient agar in Petri dishes that were kept at an inclination of 60, and incubated in the dark for 3 d at 25 C. Seedlings showing a 1 5-cm-long radicle were gently removed from the agar surface to be inoculated. Inoculation was made by dipping the radicles in a conidial suspension (10* microconidia ml"^) for 1 h. The seedlings were then aseptically transferred into tubes containing a sterilized nutrient solution adapted to tomato crop in soil-less culture (Hydrokani nutrient solution). The plants were cultivated in a growth chamber at 25 C' with a day-night cycle of h. Uninoculated control plants were cultivated similarly. Radicles were sampled on days 1, 2, 4, 6, 8 and 10 after inoculation for observation. Six inoculated plants and three control plants were taken at each sampling time. All the plants were observed under a stereomicroscope, and three inoculated plants and one control were processed for microscopic observations of root sections. Light microscopy of whole roots The roots were transferred into a 1 mm solution of 5- bromo-4-chloro-3-indolyl /^-D-glucuronide (Sigma Paris, ref. B.665O) in 0\ M phosphate buffer ph 7-2 for 1 h at 25 C, and were observed under a stereomicroscope (Leitz) or a bright field light microscope (Leica). Microscopy of root sections For each radicle, three zones were characterized, where transverse sections were taken: (i) the apex and the elongation zone, (ii) the intermediate zone without secondary roots, (iii) the distal part of the root where secondary roots were present. These secondary roots were removed before processing the tap root. One-cm-long pieces were cut in these three zones and fixed with 1 % glutaraldehyde in phosphate buffer (0-1 M, ph 7-2) for 4 h at room temperature. After washing in phosphate buffer, the samples were postfixed with 1 % osmium tetroxide in the same buffer for 1 h at room temperature. Samples were dehydrated in a graded series of ethanol followed by propylene oxide and embedded

3 Root colonization by non-pathogenic Fusarium Figures 1-6. Light microscopy of the whole root, showing colonization of Lycopersicon esculentum roots by the Gus-marked Fusarium oxysporum. Figures h after inoculation: Figure 1. Intense colonization of the root hair zone. Figure 2. Discontinuous colonization of the elongation zone. Figure 3. Root tip without hyphae. Figures h after inoculation: Figure 4. Root tip with blue-stained hyphae. Figure 5. Intense colonization of the elongation zone. Figure 6. Secondary root showing localized patches of colonization (arrows). Scale bars = 40/^m.

4 484 C. Olivain and C. Alabouvette 11 I Figures 7-H. Light microscopy of whole Lycoper«co«escu/cn(uw roots, showing colonization by the blue Gusmarked Fusarium oxysporum.

5 Root colonization by non-pathogenic Fusarium 485 in a Epon-Araldite mixture (Glauert & Hall, 1991). Semi-thin {0-5 f,xn) and ultra-thin (0-1 //m) sections were cut using an ultramicrotome (Reichert Ultracut E). Semi-thin sections were stained with 1 % aqueous toluidine blue O and methylene blue O in 1 % sodium tetrahorate, then examined under a bright field light microscope (Leica), Ultra-thin sections were contrasted with a methanolic solution of uranyl acetate (2-5%) followed hy Reynold's lead citrate. Microscopic observations were made with a Hitachi 600 electron microscope operating at 75 kv. RESULTS Sections of uninoculated control plants showed the normal structure of tomato roots as described hy Charest, Ouellette & Pauze (1984), Secondary roots were generally less colonized than the tap root. At this stage, only the hyphae located at the emergence of the secondary roots showed blue staining (Fig. 7); other hyphae outside the root appeared unstained (Fig. 8), indicating that glucuronidase activity had ceased. The staining was still observed inside the root, showing limited patches of colonization in the cortical tissue (Fig. 8). In some cases, root apices showed restricted stained areas indicating the colonization of cap cells (Figs 9, 10) and subapical cells. A dense network of hyphae was still observed at the surface of the root 6-10 d after inoculation, but the blue staining had disappeared and chlamydospores had formed, indicating that the fungus was no longer actively growing but had entered a resting stage (Fig. 11). Colonization of the root surface Twenty-four h after inoculation of young plants cultivated in a nutrient solution, the surface of the radicle that was 3-4 cm long showed a dense network of hyphae of F. oxysporum, especially in the zone where root hairs were present. Not only hyphae outside the root hut also the root surface, some root hairs, and apparently some cortical cells, were stained hlue, indicating the colonization of the root hy metabolically active hyphae (Fig. 1). The elongation zone of the root, corresponding to tissues that had grown after inoculation of plants, was only slightly colonized, as indicated by small patches of blue staining randomly distributed, indicating a discontinuous colonization of the root (Fig, 2). In few places, the cortical cells appeared stained, although only rarely could hyphae be observed on the surface of the root. The root tip was apparently not colonized, since neither staining nor hyphae were detected at this stage (Fig. 3). Forty-eight h after inoculation, the pattern of root colonization was nearly identical to that observed previously. However, the apical zone of the root was surrounded by blue-stained hyphae (Fig. 4), and the elongation zone was intensively colonized (Fig. 5). The secondary roots that emerged in the upper part of the main root grew through the mycelial network and showed patches of colonization, especially in the subapical zone (Fig. 6). Four d after inoculation of plants, the tap root that was 6'5 cm long appeared covered by an intense network of hyphae. All the root zones were more or less colonized by the fungus, and hyphae were always observed at the surface of the root apex. Relationships between hyphae and the root surface Transverse sections made from surface-colonized parts of the young root showed three t\'pes of relationships between the fungus and the root. (i) In many places, many hyphae were observed outside the root but no direct contact between the fungus and epidermal cells or root hairs could be detected. Both semi-thin sections observed by light microscopy and ultra-thin sections observed by electron microscopy showed hyphae forming a ring of mycelium around the root (Fig. 12) but without any contact with the root surface (Fig, 15). (ii) In a few places, hyphae were observed in contact with epidermal cells. Hyphae were closely appressed to the cell surface and at the point of hyphai attachment the cell wall appeared swollen, but the cell was not invaded by the fungus (Fig, 17). (iii) In rare cases, it was possible to observe the penetration of the fungus into root cells (Fig. 13). Hyphae penetrating the cell wall were constricted at the point of penetration and showed an increased diameter inside the host cell (Fig. 16), There was apparently no specialized structure such as an appressorium; the penetration pegs were formed directly from hyphae, but never from germinating conidia. Intracellular penetration of the epidermis was observed 48 h after inoculation of plants, in the differentiated part of the root. Invasion of sloughed cells and penetration of intact cap cells were observed 4 d after inoculation of plants (Fig. 14), Intercellular penetration also occurred, since many hyphal tips were observed in the depression between two epidermal cells. Figures Four d after inoculation: Figure 7. Active hyphae at the site emergence of a secondary root. Figure 8. Intense network of unstained hyphae at the surface of the root (arrow) and colonization of cortical cells (arrowheads). Figures Root tips with blue-stained colonized cap cells (arrows). Figure 11. Ten d after inoculation, dense network of mostly metabolically inactive hyphae with chlamydospores (arrows) on the root surface. Scale bars = 40 /im.

6 486 C. Olivain and C. Alabouvette ^^i :«!,,;* sj-.ij.i-i^ v^t'toi'i^^^ie^e^s?^,.. j^v;., - -i ik^s\^,r^si3' «,.",:'>-.',,,-.,k, Figures Light micrographs of transverse semi-thin sections ai Lycopersicon esculentum roots colonized by the Gus-marked Fusarium oxysporum showing relationships between hyphae and the root (toluidine blue O and methylene blue O staining). Figure 12. Ring of hyphae around the root. Scale bar = 40 /tm. Figure 13. Penetration of the fungus into an epidermal cell (arrowhead) in the differentiated part of the root. Figure 14. Hyphae (arrowheads) in the cap cells, especially in the peripheral layer. Scale Bars = 10//m. CC, cortical cell.

7 Root colonization by non-pathogenic Fusarium 487 Figures Electron micrographs of transverse ultra-thin sections of Lycopersicon esculentum roots colonized by the Gus-marked Fusarium oxysporum showing relationships between hyphae and the root. Figure 15. A root hair surrounded by hyphae (arrows) but without contact between the fungus and the root. Scale bar = 2 fim. Figure 16. Penetration of the cell wall of an epidermal cell by a hypha. Constricted hypha at the penetration point. Figure 17. Attachment of hyphae to an epidermal cell, showing swelling of the eel! wall (arrow). Scale bars = 1 /.im. EC, epidermal cell; H, hypha; RH, root hair. Colonisation of root tissues At the apex of the root. At the apex, the fungus was in both cap cells (Fig. 18) and a few subapical cells. In the cap, the growth of the fungus was limited to the peripheral layer of cells by w-all thickenings (Figs 14, 18). Hyphae were embedded in an electron-dense material, and osmiophilic deposits were present in the \-acuole of adjacent cells (Fig. 23), The infected subapical cells often appeared dead, showing an aggregated cytoplasm (Fig. 22); the adjacent cejls were never invaded, indicating that the death of infected cells limited invasion by the fungus. In the adult root. Above the apical zone, where the cortex and the stele were differentiated, the fungus penetrated more or less deeply into the root tissue, but the colonization was always limited by a barrier formed by thickenings and coilings of the cell walls. This barrier was observed either immediately under the epidermis or deeper in the cortex (Figs 19-21). (i) When colonization was limited to the epidermis, which was most frequently observed, hyphae were scarce and appeared as cross sections, indicating that they were growing longitudinally rather than centripetaljy in the root. Epidermal ceils appeared misshapen and formed buckles entrapping the fungus (Figs 19, 24). (li) Less frequently, several layers of cortical cells were colonized, but the lesion was limited by a barrier formed by a layer of hypertrophied cells, showing thick walls (Fig. 20). In some places, the fungus was observed in the intercellular spaces, the cell walls showing dome-like or elongated protuberances w-ith a multi-textured structure. These wall appositions were surrounded by large bands of cytoplasm containing numerous mitochondria (Fig. 28). The infected cells outside the barrier appeared drastically altered, with a disrupted cytoplasm, and tended to be exfoliated (Figs 20, 25). (iii) Rarely, the barrier was observed in the vicinity of the stele, but the vessels were never

8 C. Olivain and C. Alabouvette Figures Light micrographs of transverse semi-thin sections showing colonization of Lycopersicon esculentum root tissues by a non-pathogenic Fusarium oxysporum (Fo5a4).

9 Root colonization by non-pathogenic Fusarium 489 infected (Fig. 21). All the cortical cell layers were invaded by many hyphae colonizing both the cells and the intercellular spaces. They appeared mainly as longitudinal sections, indicating centripetal growth of the fungus (Figs 21, 30). The physical barrier was fortned by collapsed and distorted cells in which the fungus was encased. Numerous electron-opaque flecks lined the thick cell-walls (Fig. 26). Striking modifications of the host cells were observed: the plasmalemnia appeared highly convoluted and retracted from the cell wall (Fig. 27), the intercellular spaces were filled with an electronopaque material and the wall showed multilayered thickenings (Figs 27, 30). In a few cases, occlusion papillae were observed in places where hyphae were perforating the cell wall (Fig. 29). When these reactions took place close to the stele, a band of electron-dense material coated the secondary wall of the vessels, where polymorphic bubbles accumulated (Fig. 31). The adjacent cells showed an intense cytoplasmic activity, and osmiophilic deposits were observed in the vacuoles (Fig. 31). This intense colonization of the cortex seenned to determine the degeneration of the root tip. The elongation of the root ceased, but some lateral roots elongated faster then and replaced the tap root. DISCUSSION This study showed that the non-pathogenic strain Fo5a4 of Fusarium oxysporum, effective in controlling fusariuni wilt of tomato (Olivain et al., 1995), is able to colonize the surface and all tissues of the tomato root except the vessels. This is the first time that microscopic observations have been used to demonstrate that non-pathogenic strains of F. oxysporum are able to colonize plant roots. Although the cultural conditions, in nutrient solution without oxygen supply were difterent from growth conditions in soil, these results are in agreement with previous observations acquired through indirect techniques on plants grown in soil (Mandeel & Baker, 1991; Eparvier & Alabouvette, 1994). Observations of the whole root showed that colonization occurs very quickly, since abundant hyphae were observed at the root surface 24 h after the inoculation of the plant. There is apparently no preferential zone for colonization by this strain of F. oxysporum, although after 24 h, colonization was tnainly observed in the zone with root hairs; after 48 h and later, hyphae were observed everywhere on the root surface, even the tip. However, most of these hyphae did not adhere closely to the root surface; attachment points were scarce. Secondary roots might become infected when they grow through the hyphae that surround the tap root. This pattern of root colonization might be related to the experimental design. Indeed, the young radicle was immersed for 1 h in a conidial suspension of the fungus, then allowed to grow in a nutrient solution without fungus. Therefore, all hyphae resulted from the germination of microconidia that were able to bind to the root surface within that hour. This could explain why 24 h after inoculation the elongation zone was less colonized than the root hairs zone, the latter corresponding to the part of the radicle that has been in contact with the conidial suspension; 48 h after inoculation, the root tip appeared surrounded by blue-stained hyphae. The colonization of the root tip results either from mycelium grow-ing from the upper part of the root, or from hyphae emerging from conidia bound to the cap cells. This second hypothesis is the more likely since very few hyphae are unlikely not to have been detected after 24 h incubation. The activity of the fungus revealed by the Gus reporter gene seemed to decrease rapidly. Indeed. the Gus gene, being under the control of the glyceraidehyde-3-phosphate dehydrogenase promoter, does not mark the wall of the fungus but reveals its metabolic activity; 4d after inoculation, blue-stained hyphae outside the root were only observed in discontinuous spots, especially at the apex and at the emergence of secondary roots, where exudation and slough are important, supporting the activity' of the fungus. Although the colonization of the root surface was intense, it was difficult to detect and observe penetration points because they were scarce and distributed randomly. The penetration of the fungus into the root took place during the 48 h following inoculation. Penetration always occurs from growing hyphae, never from germinating conidia, and specialized structures such as appressoria have never been observed. This penetration process of a non-pathogenic strain of Fusarium oxysporum did not differ from the process described for pathogenic strains of F. oxysporum such as F. oxysporum f. sp. lycopersici (Bishop & Cooper, 1983), F. oxysporum f. sp. asparagi (Smith & Peterson, 1983) and F. oxysporum f. sp. pini (Farquhar & Peterson, 1989). Figure 18. Colonization of cap cells (arrowheads). Figure 19. Colonization of epidermal cells limited by a barrier composed of thickenings and coilings of cell walls (between arrows). Scale bars 10 /(m. Figure 20. Colonization of several layers of cortical cells limited by a layer of hypertrophied cortical cells. Figure 21. Colonization of all the cortical cell layers limited by a barrier made of collapsed and distorted cells in close proximity to tbe stele. Scale bars = 40 /im. CC, cortical cells; DCC, dead cortical cells; HCC, hypertrophied cortical cells; ICC, infected cortical cells; MC, meristematic cells; XV, xylem vessel.

10 490 C. Olivain and C. Alabouvette Figures Electron micrographs of transverse ultra-thin sections showmgthe colonization of Lycopersicon esculentum root tissues by a non-pathogenic Fusarium oxysporum (Fo5a4). Figure 22. Hypha (arrow) in a dead subapical cell showing aggregated cytoplasm (arrowhead). Scale bars = 1 /iui. Figure 23. Colonization of cap cells (enlargement of Fig. 18), hyphae (arrows) embedded in osmiophilic rrjaterial and osmiophilic deposits (arrowheads) in the vacuoles of adjacent cells. Figure 24. Colonization of epidermal cells (enlargement of Fig. 19), hypha (arrow) in an intercellular space, surrounded by cell wall thickenings (arrowheads). Figure 25. Colonization of several layers of cortical cells (enlargement of Fig. 20),

11 Root colonization by non-pathogenic Fusarium 491 Penetration has never been observed later than 4 d after inoculation of the plant. One can assume that the process requires energy, explaining why only actively growing hyphae can penetrate into the root. The loss of activity after a few days, revealed by the absence of blue staining, might be caused by nutrient starvation. Indeed, the biomass at the root surface is determined by the available nutrients provided by root exudation and cell exfojiation, Therefore one may assume that, after a while, the adult part of the root no longer provided enough nutrients to support the growth and the penetration of the fungus. As soon as the hyphae were able to penetrate into the root either inside the cells or in the intercellular spaces, the cells showed defence reactions. At the apex, the defence reactions were rapid and intense, leading to cell death, and therefore the colonization was always limited to a very few cells, the meristematic zone never being colonized. In the differentiated zone of the root, wall appositions and thickenings, intercellular plugging, intracellular deposits and hypertrophied cells corresponding to defence reactions limited the extension of the fungus, both longitudinally and centripetally. This phenomenon, described as compartmentalization, has been reported in carnation infected by F. oxysporum f. sp. dianthi by Baayen, Ouellette & Rioux (1996). When the defence reactions occurred quickly, the penetration of the fungus was limited to the epidermis and the outer cortex. This superficial colonization was the most frequently obser\'ed and did not seem to modify root growth. In a few cases, an intense colonization of the cortex has been observed, the defence reactions taking place close to the stele. The root was so damaged that it was difficult to establish clearly which layer of cells formed the barrier that was observed. In these cases, the growth of the root seemed impaired and the root tip appeared possibly no longer functional. This \ ery intense colonization might result from the high inoculum pressure to which the experimental procedure submitted the root. In contrast to pathogenic strains, this nonpathogenic strain of F. oxysporum has never been observed in the vessels. The defence reactions observed in tomato colonized by this non-pathogenic strains of F. oxysporum did not differ from those described in tomato and other plant species infected by compatible or incompatible formae speciales of F. oxysporum (Charest et al., 1984; Brammall & Higgins. 1988; Teissier, Mueller & Morgham, 1990). The chemical nature of the substances that accumulated in the cells and the intercellular spaces of infected plants has been determined by histochemistry and immunocytochemistry. Callose and phenolic compounds are the most frequently cited molecules. They appear in reaction to plant infection in both susceptible and resistant varieties, but usually they are formed more rapidly, and accumulate in greater amounts, in resistant than in susceptible varieties (Jordan, Endo & Jordan, 1988; Benhamou f^/a/., 1991 ; Shi, Mueller & Beckman, 1992). These defence reactions always limit the extension of the fungus in the resistant cvs and generally do not allow the invasion of the vessels. Therefore, the defence reactions observed in a susceptible variety of tomato infected by a nonpathogenic strain of i^. oxysporum could be compared to those observed in a resistant variety colonized by a pathogenic strain of F. oxysporum. Regarding the role of the colonization of the root by the non-pathogen in biological control, one may assume that the intense colonization of the root surface that appears I'ery quickl)' after inoculation, constitutes a physical barrier that prevents direct contact of the pathogen with the root surface. This colonization also presents intense competition for root exudates; therefore, the pathogen might not find the nutrients required for propagule germination. However, the colonization of the root surface, although intense, is never complete, and some noncolonized areas might allow the pathogen to reach the root. Moreover, the Gus activity decreased quickly and, 4 d after inoculation of the plant most of the hyphae did not show any Gus activity' at the root surface. The fungus might enter a resting stage resulting from nutrient starvation. Later on, these hyphae might undergo lysis and provide nutrients for other micro-organisms, including the pathogen if it is in contact W'ith the root. Therefore, competition for colonization of the rhizoptane between the pathogen and the non-pathogen might be limited in space and time to areas where the non-pathogenic strain is growing actively. Competition might also occur inside the plant tissues, as suggested by Postma & Luttikholt (1996). Indeed, the non-pathogenic strain was able to colonize the epidermis and the cortex of tomato roots, although this internal colonization was always sparse, leaving large healthy spots available for colonization by another strain of F. oxysporum. The presence of the fungus in the root also triggered defence mechanisms of the plant. Indeed, the same types of plant defence reactions, which constitute a barrier to the extension of the fungus, have been observed in tomato after application of chitosan, hyphae (arrows) in dead cortical cells. Figure 26. Colonization of all the cortical cell layers (enlargement of Fig. 21); hyphae (arrows) in collapsed and distorted cells; electron-opaqueflecksline the cell walls (arrowheads). Scale bars = 2 fim. CW, cell wall; DCC, dead cortical cells; EC, epiderma) ceil; HCC. hypertrophied cortical cell.

12 492 C. Olivain and C. Alabouvette Figures Electron micrographs of transverse ultra-thin sections showing the defence reactions of Lycopersicon esculentum. against a nonpathogenic Fusarium oxysporum (Fo5a4). Figure 27. Infected cell showing a highly convoluted plasmalemma (thin arrows), wall thickenings with a multilayered structure, intercellular plugging (thick arrows) and intracellular osmiophilic deposits (arrowheads). Figure 28. A dome-like protuberance papilla (arrow) formed in a cell adjacent to an invaded intercellular space. Figure 29. An occlusion papilla (arrow) formed around a hypha penetrating through a cell wall. Figure 30. Plugged intercellular spaces on each side of a hypha penetrating the wall between two cortica) cells. Scale bars = 1 //m. Figure 31. Defence reactions in the stele; coating material (thick arrows) on the secondary wall and bubbles in the vessels (arrowheads) and deposits in the vacuoles of adjacent cells (thin arrows). Scale bar = 5 fim. H, hypha; IS, intercellular space; WT, wall thickening; XV, xylem vessel. effective in controlling F. oxysporum f. sp. radicis lycopersici by systemic-induced resistance (Benhamou, Lafontaine & Nicole, 1994; Lafontaine & Benhamou, 1996). Previous results have shown that strain Fo5a4, utilized in this study, was able to decrease disease incidence when it was inoculated on

13 Root colonization by non-pathogenic Fusarium 493 one side of a split-root system of tomato (Olivain et al, 1995). This study demonstrates clearly that the nonpathogenic strains of F. oxysporum are able to colonize both the rhizoplane and root tissues of tomato. Therefore, competition for infection sites between the pathogen and the non-pathogen is one of the modes of action of the non-pathogen. However this mode of action can not explain alj the biocontro] activity of the non-pathogen F. oxysporum^ which probably results from the complementarity of several modes oi action. ACKNOWLEDGEMENTS This study has been partly supported by a grant from Conseil Regional de Bourgogne. We thank Paul Bremeersch for technical help, Josette Reiot for photographic work (Centre for Microscopy applied to Biology, Universite de Bourgogne), Jeannine Lherminier for technical advice and Patrice Richard for typing the manuscript. 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14 494 C, Olivain and C. Alabouvette Teissier BJ, Mueller WC, Morgham AT Histopathology Toussoun TA Fusarium-suppressive soils. In: Bruehl and ultrastructure of vascular responses in peas resistant or GW, eci. Biology and Control of Soil-borne Plant Pathogens. St susceptible to Fusarium oxysporum i. sp. pisi. Phytopatkologv Paul, MN, USA: The American Phytopathological Society, 80:

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