Mycol. Res.

103 (9) : 12101216 (1999)

Printed in the United Kingdom

1210

Eects of nutritional sources on growth of one non-pathogenic strain and four strains of Fusarium oxysporum pathogenic on tomato

C. S T E I N B E RG1*, J. M. W H I P PS2, D. A. W O O D2, J. F E N L ON3 A N D C. A L A B O U V E T TE1

" INRA-CMSE, Laboratoire de Recherches sur la Flore PathogeZ ne du Sol, 17 rue Sully-BV1540, F. 21034 Dijon Cedex, France # Department of Plant Pathology and Microbiology $ Biometrics Department, Horticulture Research International, Wellesbourne, CV35 9EF, Warwick. U.K.

As part of an investigation into the factors inuencing the colonization of the rhizosphere and the root tissues of host plant, by pathogenic and non-pathogenic strains of Fusarium oxysporum, the eect of a range of carbon (C) sources on the growth habits of ve strains of F. oxysporum were compared. The strains used were two F. oxysporum f. sp. lycopersici (Fol strains), two F. oxysporum f. sp. radicis-lycopersici (Forl strains) pathogenic on tomato and strain Fo47, a non-pathogenic strain of F. oxysporum, currently used as a biocontrol agent to reduce severity of fusarium diseases on several crops. Radial extension rates on solid media were measured using soluble saccharides (glucose, xylose and D galacturonic acid), polysaccharides (carboxymethyl cellulose, xylan and pectin) and soluble and insoluble extracts of tomato roots as C sources. Growth parameters were estimated by tting a logistic equation to biomass data recorded from liquid culture using the same C sources. The strains were characterized by means of the pattern of radial extension rates on the various C sources, and the two Forl strains were discriminated further from the other strains. The growth parameters were unique features of each F. oxysporum strains, whatever the C source. The pathogenic strains did not exhibit particular abilities in degrading the cell wall components. It was concluded that growth habits related to carbohydrate utilization are unique to each strain of F. oxysporum and that these traits are not related to pathogenicity.

The ability of Fusarium oxysporum Schtdl. : Fr. isolates to grow in the rhizosphere and at the root surface may be an important prelude to infection of roots of host plants by either pathogenic or non-pathogenic isolates. F. oxysporum includes both plant pathogenic and non-pathogenic strains, with the former showing a high degree of specicity toward host plant species, grouped into formae speciales (f. spp.) according to the host (Armstrong & Armstrong, 1981). Tomato pathogenic strains responsible for wilt disease belong to f. sp lycopersici (Sacc.) W. C. Snyder & H. N. Hansen while other pathogenic strains, responsible for root rot, belong to f. sp. radicislycopersici Jarvis & Shoemaker. Both pathogenic and nonpathogenic F. oxysporum strains are aggressive colonisers of the rhizosphere and root tissues. Consequently, non-pathogenic strains have been selected on the basis of their ability to reduce disease severity (Paulitz, Park & Baker, 1987 ; Postma & Rattink, 1992 ; Alabouvette, Lemanceau & Steinberg, 1993 ; Minuto, Migheli & Garibaldi, 1995). It has not, however, been clearly established whether pathogenic or non-pathogenic strains have particular advantages either in soil, in rhizosphere soil or at the root surface of the host plant. Several studies have addressed aspects of this problem. For example (Couteaudier & Alabouvette, (1990) demonstrated that sapro* Corresponding author. Present address : Consultant in Microbial Technology, 6 Dunstanville Terrace, Falmouth, TR11 2SW, Cornwall, U.K.

trophic competence of F. oxysporum populations in soil was related to their competitive ability to use carbon sources, but not to their pathogenicity. Hyphal development of both pathogenic and non-pathogenic F. oxysporum was strongly stimulated in the vicinity of tomato roots by molecules diusing from the roots (Steinberg et al., 1999). Sensitivity to tomatin and rishitin, two phytoalexins produced by infected tomato, was tested on a range of pathogenic and nonpathogenic F. oxysporum isolates (Suleman et al., 1996). A high level of variation in sensitivity was found among all isolates, although non-pathogens were generally more susceptible to tomatin than pathogens. The occurrence of a binding process between F. oxysporum conidia and the surface of tomato roots was recently demonstrated (Recorbet & Alabouvette, 1997) but no dierence was observed in terms of binding anity among several strains of F. oxysporum diering either in pathogenicity or in host range. Similarly, Eparvier & Alabouvette (1994), using serological methods showed that the ability to colonize the root depended on the strain but was not related to pathogenicity. Olivain & Alabouvette (1997) observed that non-pathogenic strains were able to build up a dense mycelial network around the root but with only few attachment points. Moreover, they showed that while pathogenic F. oxysporum colonized all root tissues, nonpathogenic strains colonised the root surface and the root tissues but not the vessels. Soilborne fungi are able to utilize a variety of carbohydrates

C. Steinberg and others but their growth rates vary, depending on the range and quantitative amounts of hydrolytic enzymes each fungal strain produces. Glucose is universally assimilated by fungi and most hexoses provide a readily usable C source for growth and development. Xylose and most pentoses are utilized more slowly than hexoses (Anderson et al., 1975) and organic acids can also sustain fungal growth although some may favour sporulation instead of vegetative growth (Anderson & Solomons, 1984). In contrast, utilization of polysaccharides as C sources can reduce the growth rate of fungi since depolymerization can be energetically expensive. For instance, the maximum growth rate of F. graminearum decreased when cultivated on glucose, maltose and maltotriose respectively as C sources, (Anderson & Solomons, 1984). It may, therefore, be expected that pathogenic and non-pathogenic strains of Fusarium could be discriminated between on material such as polysaccharides derived from roots. Cellulose, hemicellulose (mostly xylan), pectin and lignin would be useful polymers to use for this assessment as they constitute the main cell wall ' squez, Heluane & Figueroa, 1995). components of plants (Va Consequently, the aim of this study was to determine whether discrimination between a non-pathogenic strain currently used as a biocontrol agent and strains belonging to f. sp lycopersici (Fol strains) or f. sp. radicis-lycopersici (Forl strains) could be achieved on the basis of their growth habits (radial extension rate and biomass production), evaluated on polysaccharides representative of root cell wall components on solid medium and in liquid culture, respectively. MATERIALS AND METHODS Fungi Five strains of F. oxysporum were used from the FPFS collection (INRA Dijon, France). Strain Fo47 is non-pathogenic, originating from the suppressive soil of Chateaurenard, in France (Rouxel, Alabouvette & Louvet, 1979). It is commonly used as a biocontrol agent against pathogenic F. oxysporum in greenhouses (Alabouvette, Lemanceau & Steinberg, 1996). Strains Fol8 and Fol15 are responsible for wilt disease on tomato and are f. sp. lycopersici, while Forl19 and Forl22 are responsible for crown and root rot on tomato and are f. sp. radicis-lycopersici. They were stored in liquid nitrogen and were subcultured on potato dextrose agar (PDA, Oxoid) at 25 mC. Liquid subcultures were performed at 25m in synthetic mineral medium (MM)jglucose (5 g l"). MM composition consisted of (l" distilled water) : Na NO 2 g, KH PO 1 g, # $ # % MgSO ;7H O 2n5 g, KCl 0n5 g, trace element solution 2 ml. % # Trace element solution composition was, l" distilled water : citric acid 5 g, ZnSO ;7H O 5 g, FeSO ;7H O 4n75 g, % # % # Fe(NH ) (SO ) ;6H O 1 g, CuSO ;5H O 0n25 g, MnSO ; %# %# # % # % H O 0n05 g, H BO 0n05 g, NaMoO ;2H O 0n05 g. # $ $ % # Hyphal extension rate measurements The strains were rst subcultured in Petri dishes on MMA (MMjagar, 15 g l") for 6 d at 25m. Plugs of 3 mm diam. were aseptically transferred from MMA on to the dierent media. Four replicates were done (Petri dish, 90 mm diam.). The dierent media used were : MMA, PDA, MMAjglucose

1211 5 g l", MMAjcarboxymethyl cellulose (CMC, low viscosity, degree of substitution 0n4, BDH) 4n82 g l", MMAjD galacturonic acid (DGA) 5n4 g l", MMAjpectin (from citrus fruits, Sigma) 5n4 g l", MMAjxylose 5 g l", MMAjxylan (from birchwood, Sigma) 5 g l", MMAjmannose, 5 g l", MMAjmannan (from Saccharomyces cerevisiae, Sigma) 5 g l", MMAj80 % ethanol soluble extracts, and MMAj80 % ethanol insoluble extracts. When DGA was used, 1n5 g KOH was added l" of medium to raise the pH to 5n5 before autoclaving to allow the agar to solidify. Pectin, CMC, and xylan were solubilized in the MM in a boiling waterbath before autoclaving. The concentration of carbon sources were determined to supply an equal amount of 2 g C l" of medium. All the media were autoclaved for 15 min at 121m and 0n1 MPa. Ethanol soluble and insoluble extracts were obtained from root tips (34 cm long) of 7-to-8-day old tomato seedlings as described by Steinberg et al. (1999). Soluble extracts were lter sterilised (0n2 m). One hundred l of the 80 % ethanol soluble extracts, corresponding to approximately 15 root tips were put into each Petri dish before pouring molten MMA into the plate. Insoluble extracts were crushed in liquid nitrogen using a pestle and a mortar. Two mg of crushed insoluble extracts, also corresponding to 15 root tips, were put into each Petri dish before pouring molten MMA into the plate. In both cases, Petri dishes were gently shaken to ensure uniform incorporation of the extracts in the medium before solidication. Once inoculated, plates were incubated at 25m. The colony diameters were recorded twice a day for 6 d. The hyphal extension rate was calculated by linear regression of the colony radius as a function of time for each replicate. Growth kinetics in liquid medium Liquid subcultures of the F. oxysporum strains grown for 24 h in MMjglucose (5 g l") were ltered on sterile sintered glass funnel (max. pore size 40100 m). The concentration of conidia in the ltrate was determined by direct microscopic counts and the suspension was adjusted to 10' conidia ml" sterile distilled water. One ml of this suspension was used to inoculate thirty 250 ml conical asks containing 100 ml of a liquid medium. The media used were : MMjglucose 5 g l", MMjCMC 4n82 g l", MMjDGA 5n4 g l", MMjpectin 5n4 g l", MMjxylose 5 g l", MMjxylan 5 g l". Pectin, CMC, and xylan and were solubilized in the MM as above. When DGA was used, the medium was buered at pH 5n5 with 17n6 g MES l" medium. (MES l 2-(N-morpholino) ethanesulphonic acid). This concentration was sucient to buer the medium but did not allow F. oxysporum to use MES as a carbon source (Lemanceau et al., 1993). The concentration of carbon sources were determined to supply an equal amount of 2 g C l" of medium. All media were autoclaved for 15 min at 121m and 0n1 MPa. Once inoculated, the asks were incubated in a controlled environment incubator shaker (New Brunswick Scientic Co.) at 25m, 150 rpm. At regular intervals, three asks were selected at random for an estimation of the dry weight content of the culture. Each sample was vacuum ltered on a pre-weighted glass microbre lter (Whatman GF\A, 90 mm diam.). The biomass mat was rinsed with

Nutrition and growth of Fusarium oxysporum approximately 50 ml of sterile water. The lters were then placed in a drying oven (80m) for 3648 h and weighed again for the determination of dry biomass content. Three uninoculated asks for each of the media were treated in the same way as the samples, at least once, to ensure that the C sources (especially CMC, xylan and pectin) remained solubilized and did not interfere with the biomass measurements. Statistical analysis Hyphal extension rates were compared using analysis of variance (ANOVA). For growth kinetics in liquid media, where strains clearly grew, the data for biomass were tted with the logistic curve. This model was chosen to describe the evolution of fungal populations over time as the experimental curves show a typical sigmoid pattern. The model is given by the equation : xl K , 1je(Pt)/R
Table 1. Mean radial extension rates (4 reps ; m h") of Fusarium oxysporum strains on solid media Fo47 MM PDA Glucose CMC Xylose Xylan DGA Pectin Mannose Mannan EtOH sol EtOH insol Mean 26 31 26 27 25 25 27 31 26 27 28 28 27 0n5 0n8 Fol8 26 32 28 27 19 22 27 29 28 27 25 27 26 Fol15 25 27 24 25 25 24 24 28 24 26 25 26 25 Forl19 28 33 27 29 27 27 30 31 28 30 28 27 29 Forl22 29 34 27 28 26 26 28 31 27 29 30 28 29

1212

Mean 27 31 26 27 24 25 27 30 26 28 27 27

... (for strains) ... (for media)

where x is the dry matter biomass, K is the upper asymptote for biomass (i.e. the carrying capacity of the medium for the particular isolate), P is the time taken to reach K\2, 1\R is the growth rate of the strain and t is time. The expression 2iloge(19)iR represents the time for the biomass to increase from 5 % to 95 % of its estimated capacity and this has also been presented. The equations were tted using the nonlinear regression module of Genstat (Payne et al., 1993). The shape of the responses were not consistent across dierent media so it was not possible to t reduced models to each isolate. Individual parameters of the non-glucose C sources have, however, been expressed relative to that of glucose as a percentage (K ), excess time (P), or a multiplication factor (R). RESULTS Extension rate on solid media with root cell wall components as C source Radial extension rate was assumed to be the slope of the linear regression of colony radius as a function of time for each replicate. The mean radial extension rates of each strain on each of the 12 C sources are shown in Table 1. ANOVA revealed highly signicant (P 0n01) dierences among strains, among media and a highly signicant (P 0n01) interaction, which must be attributable to the high level of reproducibility among the replicate assays. In fact, there is little real dierence between the various combinations : with the exception of Fol8 on xylose and xylan (19 and 22 m h") the growth rates varied between 24 m h" (several on Fol15) and 34 m h" (Forl22 on PDA). A simple analysis of variance based on the means (and using the interaction variation as error) is instructive : it reveals that the two Forl strains gave signicantly (P 0n05) higher growth rates than the other three strains. Among the C sources, PDA and pectin produced signicantly faster (P 0n05) growth rates than other sources. Retaining the two anomalous values cited above, xylose and

xylan appeared signicantly (P 0n05) slower growing than the remainder ; otherwise all but PDA and pectin were not signicantly dierent in their growth rates. It is worthwhile noting that radial extension rate did not reect the apparent growth of the strains. Mycelia were dense, aerial and uy when growing on PDA, glucose and, to a lesser extent, on xylose and mannose while they were very thin and sparse on MMA, CMC, xylan and mannan. They were thin and dense on pectin and DGA. Although there were slight dierences in mycelial morphology among strains, however, such dierences were not taken into account because they could not be accurately quantied. Growth kinetics in liquid medium In all cases, the C source contained in the liquid media was well solubilised and therefore did not account for biomass measurements. Apart from CMC, plots of biomass against time produced sigmoid-shaped curves which could be welldescribed by a logistic equation. In the case of CMC, for all strains, the growth was almost linear for more than 90 h, but very slow : the biomass achieved by F. oxysporum was some 20 mg at the end of the experiment, whereas it was approximately 10 times greater on glucose, for example. Fig. 1 shows the growth patterns (and tted curves) for Fol15 as an example. Growth parameters for all ve strains on glucose, xylose, xylan, pectin and DGA were estimated (Table 2). Maximum values of biomass (given by K, the carrying capacity) occurred with glucose on four out of the ve cases or with xylose ; K was smallest for xylan in all ve strains. The logistic curve has rotational symmetry about the value t l P, so that P gives a measure of the half-life of the production process. Taking account of the approximate standard error (..) of P it can be seen from Table 2 that glucose induced the fastest (or equal fastest) time to maximum growth on four of the strains ; only on Forl19 was it outstripped by the other growth media. P does not give a measure of the lag phase of the growth process, but combined with R, the expression P-loge(19)*R gives an estimate of the 5th percentile of the growth curve, i.e. the time at which

C. Steinberg and others

025
, , , glucose xylose xylan , , , CMC pectin DGA

1213

020

Dry weight (g)

015

010

005

000 0 10 20 30 40 50 Time (h) 60 70 80 90 100

Fig. 1. Data and tted curve for Fol15. Determination of dry biomass content of the culture (100 ml, incubator shaker : 150 rpm, 25m) was achieved by vacuum ltering the culture (3 replicates) on a pre-weighed glass microbre lter. A logistic equation was tted to the data to estimate growth curve and parameters.
Table 2. Growth parameters of Fusarium oxysporum estimated by tting a logistic curve to the biomass data (K l biomass at plateau ; P l time to K\2 ; 1\R l growth rate). A single .. (pooled from individual ..s) is given for each parameter to indicate approximate precision Variance accounted for (%) 99n2 99n3 98n9 99n2 96n4 99n1 98n8 94n2 98n6 97n8 99n1 99n0 93n2 98n5 97n3 97n1 97n1 98n5 98n2 99n0 96n6 98n2 97n6 lagb P-a (h) 34n3 35n7 37n1 22n6 57n5 35n6 62n2 30n5 30n3 30n2 28n2 33n3 41n4 24n8 43n1 38n1 34n1 27n1 38n2 39n2 30n9 31n9 22n0 3n17 0n72 7n94 lin. phaseb 2a (h) 13n9 27n6 29n2 34n5 42n1 14n6 34n3 72n5 33n6 41n0 19n4 22n7 20n6 40n0 38n7 30n1 36n5 37n3 19n2 29n9 33n5 51n0 70n8 2n26 0n65 4n05

Strain Fo47

Medium Glucose Xylose Xylan Pectin DGA Glucose Xylose Xylan Pectin DGA Glucose Xylose Xylan Pectin DGAa Glucose Xylose Xylan Pectin DGAa Glucose Xylose Xylan Pectin DGA Approx .. Min. Max.

K (mg ..) 215 191 111 164 127 215 234 134 170 152 228 228 112 192 230 195 113 144 226 220 106 202 236 38n9 14n3 97n3

P (h) 41n2 49n5 51n7 39n9 78n5 42n9 79n3 66n8 47n1 50n7 37n9 44n6 51n7 44n8 62n5 53n1 52n3 45n7 47n8 54n1 47n7 57n4 57n4 2n47 0n323 7n00

1\R (h") 0n423 0n214 0n202 0n171 0n140 0n404 0n172 0n081 0n175 0n144 0n303 0n260 0n286 0n147 0n152 0n196 0n161 0n158 0n307 0n197 0n176 0n115 0n083 0n0233 0n00229 0n0760

Fol8

Fol15

Forl19

Forl22

a For Fol15 the tted curve for DGA did not converge to a logistic the response was still in the exponential phase when sampling stopped ; for Forl19 a sample was not done. b Simple functions of the parameters describing the lag phase (time to 5 % response) and the 595 % or linear phase ; a l log (19)iR. e

Nutrition and growth of Fusarium oxysporum

Table 3. Variation of the three growth parameters (K, P and R) of all ve Fusarium oxysporum strains on C sources other than glucose expressed relative to the corresponding growth parameter estimated for glucose. Fo47 K[as % of Kgluc] Xylose Xylan Pectin DGA P[h Pgluc] Xylose Xylan Pectin DGA R[as Rx\Rgluc] Xylose Xylan Pectin DGA Fol8 Fol15 Forl19 Forl22

1214 oxysporum to use some carbohydrates which can be assimilated to cell wall components as C sources and to use these data to determine whether it is possible to discriminate these strains according to their pathogenicity. It was clearly established that the ve strains were characterized by the pattern of radial extension rates on the various C sources tested, and that the two Forl strains could be then discriminated from the other strains. Subsequently, it was shown that growth parameters were unique feature of each of the F. oxysporum strains, whatever the C source, and that pathogenic strains did not exhibit particular abilities in degrading root cell wall polysaccharides. Several sources of carbohydrates, from glucose to complex polysaccharides, were used to try to discriminate between pathogenic and non-pathogenic strains as well as among pathogenic strains. In this study, CMC was used in preference to cellulose as it could be solubilized and the polysaccharides (CMC, pectin, and xylan) were used alone as well as a combination of polysaccharides in the form of 80 % ethanol insoluble extracts of tomato roots. All strains established particular extension rates on the dierent media providing a pattern of radial extension rate for each strain on the specic set of media used in this study. The values found in this study were consistent with other reports (Allan & Prosser, 1985 ; Farina, Tonetti & Perotti, 1997). It has been suggested that the colony radial extension rate is specic for an organism and the culture conditions (Trinci, 1974 ; Farina et al., 1997). Our results indicate that the patterns of radial extension rate obtained using dierent substrates would be more useful to characterize closely related strains of fungi, rather than using a single C source, since similar values can be achieved by two or more strains on a single given C source. Comparing the strains on the basis of their patterns of radial extension rates, the two Forl strains consistently had the greatest radial extension rates. They behaved similarly on polysaccharides (CMC, pectin, xylan, 80 % ethanol insoluble extracts) but also on MMA. MMA was not supplied with an extra C source but it could constitute a C source to fungi since Fusarium spp. have been reported to be able to scavenge any substrate to obtain C (Tribe & Mabadeje, 1972). It is likely, however, that a very little C is available from agar. The radial extension rates observed on MMA, therefore, and on the polysaccharides in this study, may reect diculties for the Forl strains in utilising these carbohydrates. This sparse mycelial morphology and the hyphal extension rates achieved by the Forl strains on these four polysaccharides indicates that both strains were prospecting for a readily usable C source rather than exploiting the actual C source. Such behaviour has been proposed as a strategy for fungi in soil to colonize heterogeneous environments (Dix & Webster, 1995). Although these two Forl strains were responsible for root rot, they did not seem to be able to use root cell wall components as single C source for growth and development. Nevertheless, the Forl strains behaved similarly with regard to C utilization and growth whereas the Fol strains did not. Moreover, the pattern of radial growth in response to C sources of the nonpathogenic Fo47 was in between that of the Forl and Fol strains. These results indicate, therefore, that pathogenic strains do not have specic abilities compared to non-

89 52 76 59 8n3 10n5 k1n3 37n3 1n98 2n10 2n47 3n03

109 62 79 71 36n4 23n9 4n2 7n8 2n35 4n99 2n31 2n81

100 49 84 6n7 13n8 6n9 1n17 1n06 2n06

85 49 63 k9n4 k10n2 k16n8 0n78 0n94 0n96

97 47 89 104 6n3 k0n1 9n6 9n6 1n56 1n74 2n67 3n70

* For Fo115 the tted curve for DGA did not converge to a logistic ; For Forl19, a sample was not done

biomass has reached 5 % of its maximum value. This measure can be variable (for glucose it varies between 28 and 43 h) but gives an estimate of approximately when the process goes into its fast growth phase. The parameter 1\R is the growth rate and was consistently highest on glucose except for Forl19. The growth rate exceeded 0n4 h" for Fo47 and Fol8 on glucose and 0n3 h" for Forl22 and in all these cases gave a slope much greater than on other sources. For Fol15 growth rates were greater than 0n25 h" on glucose, xylose and xylan, but Forl19 grew faster on all other media than glucose. It can be seen from Fig. 1 that the growth parameter estimates are dierent among C sources for the same isolate ; equally, the estimates are dierent among strains on a common C source. To evaluate the ability of either Fol, Forl or the non-pathogenic strain to metabolize the two polysaccharides (xylan and pectin) and their respective monomers (xylose and DGA), glucose was chosen as the reference C source. Table 3 shows how the model parameters for the various C sources vary relative to glucose. For K, the carrying capacity, it can be seen that xylose matched glucose in almost all cases, but that xylan achieved between 47 and 62 % of the capacity given by glucose, with pectin being intermediate. Only three out of ve values were available for DGA, and while these were similar to xylan and pectin for Fo47 and Fol8, for Forl22 DGA gave a higher carrying capacity than glucose. The value of P was some 10 h less for xylose and xylan than glucose on Forl19 (17 h less for pectin), but for all the other strains glucose reached K\2 faster than the other C sources. The value of R gives a measure of the growth phase of the process, and Table 3 shows that except for Forl19 (faster) and Fol15 (marginally slower for xylan and xylose) the growth phase of other strains took generally twice as long on other sources as it did on glucose. DISCUSSION The aim of this study was to compare the ability of two Fol strains, two Forl strains and a non-pathogenic strain of F.

C. Steinberg and others pathogenic strains in utilizing cell wall components as C sources to extend at the root surface. These results are in agreement with previous ones indicating that both hyphal extension and branching of F. oxysporum were stimulated in the root vicinity of the tomato but no dierence was found either between Fol and Forl strains or between pathogenic and non-pathogenic strains of F. oxysporum (Steinberg et al., 1999). A positive relationship may exist between the linear increase of radial length and the exponential increase of dry weight on solid medium when strains are provided with rich medium (Farina et al., 1997). Because such a relationship was not likely to exist on polysaccharides such as those used in this study, biomass measurements were carried out in liquid medium using three solubilized polysaccharides as well as their respective monomers. Unexpectedly, the ve F. oxysporum strains never grew well on CMC and, therefore, no growth parameter could be estimated on that substrate even though carboxymethylcellulase and -glucosidase have already been described in F. oxysporum strains (Christakopoulos, Macris and Kekos, 1990 ; Mehta et al., 1992). Ocamb & Kommedahl (1994) evaluating several Fusarium species for rhizosphere competence on corn by measuring mycelial dry weight produced in liquid culture after 12 d found, surprisingly, that four times more fungal biomass was produced on microcrystalline cellulose than on glucose. Possible non-hydrolysed cellulose remaining in the medium might have been retained on the lter used to estimate fungal biomass. Such an eventuality was not considered by the authors. In the case of the ve other C sources, biomass measurements provided for each strain and each substrate a data set which allowed subsequent mathematical modelling of fungal growth. Because of its simplicity, the logistic model has generally been accepted as the standard model for singlespecies population growth (Rose, 1987). Fitting the logistic equation to each set of data allowed the characterization of the growth curves by three meaningful parameters : These growth parameters are valuable tools for strain characterization (Gilbert, Cother & Nicol, 1995 ; Steinberg et al., 1997). As expected, glucose was generally the best carbohydrate for all of the strains to produce rapidly a large amount of biomass while the organic acid DGA was the worst. The values of growth rates found in this study were consistent with other values reported in the literature. For instance Anderson & Solomons (1984) reported growth rates of 0n28 and 0n22 h" for a strain of F. graminearum cultivated on glucose and maltose respectively. Wiebe et al. (1995) found a growth rate of 0n26 h" for another F. graminearum strain, while growth rates of 60 isolates of F. oxysporum on glucose ranged from 0n058 to 0n353 h" (Steinberg et al., 1997). Because Forl strains induce root rot and root tissue maceration, they might be able to hydrolyse pectin, xylan or cellulose. Further, because Fol strains are able to invade all the root tissues of the host plants including xylem vessels, which the non-pathogenic strain does not (Olivain & Alabouvette, 1997) it was expected that variations in the growth parameters of the pathogenic strains would be smaller than those of the non-pathogenic strain on glucose to the other C sources. Polygalacturonases and pectates lysases from strains of F.

1215 oxysporum f. sp. lycopersici (Di Pietro & Roncero, 1996 a, b) and from strains of F. oxysporum f. sp. radicis-lycopersici (Patino et al., 1997) have been puried and characterized although their role in pathogenesis has not yet been elucidated. In contrast, the amount of polygalacturonase appeared higher with the F. oxysporum f. sp. radicis-lycopersici than with f. sp. lycopersici, while the reverse was observed with pectin lyase production in media supplemented with DGA. (Blais, Rogers & Charest, 1992). In these studies, however, pathogenic strains were studied in isolation and not compared with non-pathogenic strains. The results of the present study clearly indicate that variations of growth parameters among substrates were not related to pathogenicity. Growth parameters are traits which are unique to each strain, therefore, whatever the substrate, strains can be accurately characterised on the basis of growth parameters but pathogenicity can not. Based on the ndings in this study together with previous ones on fungal development in the root vicinity of the host plant, it is concluded that outside the root all F. oxysporum strains compete for substrates and root colonisation, as suggested by Couteaudier (1992) and Eparvier, Gautheron & Alabouvette (1993) respectively but such competition is not related to pathogenicity. Recognition mechanisms, therefore, if any exist between the host plant and the invading F. oxysporum strain, must occur within the root tissues once the strain has penetrated. It is, therefore, possible, as suggested by Benhamou (1992) and Lairini, Perez-Espinosa & Ruiz-Robio (1997) that non-pathogenic, Forl and Fol strains may be dierentiated on the basis of their susceptibility to the plant defence reactions. The rst author was nancially supported by a grant from the INRA-BBSRC agreement. The authors are grateful to Dr P. Mills, Head of the Plant Pathology and Microbiology Department, H. R. I., for providing facilities for this work, to N. Gautheron in France and S. Budge in England for help in many ways, and to L. Caine for typing.

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