Interactions between Fusarium culmorum and its potential biocontrol agent, Trichoderma harzianum, in a packed-bed, continuous-flow column reactor

ELSEVIER

Interactions between Fusarium culmorum and its potential biocontrol agent, Trichoderma harzianum, in a packed-bed, continuous-flow column reactor Janet L. Cheetham,* Michael J. Bazin,* and James M. Lynch+ *Division of Life Sciences, King’s College London, London, United Kingdom Sciences, University of Surrey, Guildford, Surrey, United Kingdom

‘School of Biological

The interaction between the filamentous fungi Fusarium culmorum and Trichoderma harzianum was studied. A conrinuous-jlow column reactor analogous to a soil column was used to provide a unidirectional flow of nutrients and solid support particles to which the fungi could attach. Such a system gave rise to a heterogeneous distribution of microorganisms and concentration gradients, and thus exhibited the most signijicant physical characteristics of rhe soil. In columns inoculated with both fungal species, a succession in population dominance was observed. F. culmorum was the dominant organism at the beginning of the experiment; however, it was gradually replaced by T. harzianum. To analyze the results obtained from the dual culture columns, a mathemarical model was developed. Three functions representing inzeraction between the two species were tested to determine which represented the experimental results most accurately. The best qualitative fit was obtained with a model incorporating Monod kinetics and making logistic limitation of F. culmorum a linear funcrion of the population density of T. harzianum. The implication of combined experimental and mathematical evidence was that the interaction between the two species was not solely competition for a single resource: the popularion sf T. harzianum had a direct effect on that of F. culmorum. 0 1997 Elsevier Science Inc. Keywords: Trichodermu hnrzianum; Fusarium culmorum; mathematical model

continuous-flow column reactor; fungal interactions;

Introduction Fusarium culmorum is the causative agent of foot rot in cereal plants, in particular wheat.’ Glasshouse trials have shown Trichoderma harzianum to be an effective biocontrol agent for F. culmorum* but little information on the interaction between the two fungal species is available.3 Here we report experimental and theoretical studies on this interaction. Theodorou et a1.4 employed a packed-bed, continuousflow column reactor to simulate experimentally a soil ecosystem containing the cellulolytic fungus, Trichoderma

Address reprint requeststo Dr. James M. Lynch, Professor/Head of School, Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, United Kingdom Received 24 July 1996; accepted 14 January 1997

Enzyme and Microbial Technology 21:321-326, 1997 0 1997 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

reesei. They justified the use of such an experimental system on the grounds that it, in common with natural ecosystems, is thermodynamically open. In their study, they assayed the effluent solution from columns and compared their results to the predictions of a mathetiatical model in an attempt to characterize growth and production of cellulase with respect to time. They were unable to estimate biomass directly or to measure activity as a function of depth down the column. We have adopted an approach similar to that of Theodorou et aL4 in order to investigate growth and interaction between F. culmorum and T. harzianum. In our study, however, it was essential to estimate biomass densities of both fungal species with respect to time and depth in the column reactor. We achieved this by employing a novel method for estimating the dry weight of filamentous fungi based on fragmentation of the mycelia and subsequent plating on solid medium containing sodium deoxycholate to inhibit hyphal extension,5 and destructive sampling of

0141-0229/971$17.00 PII SOl41-0229(97)00046-X

Papers columns at timed intervals. These methods allowed us to follow, quantitatively, succession of the two fungal species under carbon-limited conditions. We used a layer model6 similar to that used previously elsewhere’ in order to qualitatively compare our results to functions representing three different types of interactions between the two fungal species. This analysis indicated that not only did competition for the growth-limiting carbon source occur, but that T. harzianum is able to limit the growth of F. culmorum by more direct system.

means

and

eventually

eliminate

it from

the

Materials and methods Organisms Trichoderma harzianum Rifai strain THl (IMI 275950) was isolated from wheat straw.’ Fusarium culmorum (IMI 239950) was isolated from ryegrass roots3 Both organisms were cultured routinely on potato dextrose agar (PDA; Oxoid) and after 5 days incubation at 25°C were transferred to 4°C for storage. For experimental purposes, stock material was transferred to fresh PDA and incubated at 25°C.

Preparation

of spore suspensions

Culture medium Modified Vogel’s medium’ was used for experimental purposes. The final composition of the medium (gl-‘) was: glucose, 1.0; KH,PO,, 5.0; NH,NO,, 5.0; MgSO, - 7H,O, 0.2; CaCl, - 2H,O, 0.1; ZnSO, * 7H,O, 5.3 X 10p3; Fe(NH,),(SO,), * 6H,O, 1.1 X 10p3; CuSO, - 5H,O, 2.6 X 10p4; MnSO, * 4H,O, 5.0 X 10p4; H,BO,, 5.0 X 10m5; N+MoO, * 2H,O, 2.5 X 10p4; thiamine hydrochloride, 2.5 X 10e4; biotin, 5.0 X 10p5; distilled water, 1 1; pH 4.7.

column design and operation

The continuous-flow columns employed were similar to those used for the study of cellulose degradation.4 Each consisted of a vertical glass column with an internal diameter of 1.3 cm filled with 36.5 g of glass beads (0.4 cm diameter); the void volume of each column was approximately 10 ml. Inflowing and outflowing solutions passed through stainless steel tubes set in silicon rubber bungs at the top and bottom of each column. A plug of glass wool (depth 5 mm) was placed at the bottom of a column and a space of approximately 1 cm left free of beads at the top. These precautionary measures were effective in preventing the inlet and outlet tubes from becoming blocked with fungal mycelium. Sterile filtered air (10 ml hh’) was passed into the column using a fish tank aerator pump (Second Nature, Whisper 400). Sterile medium was passed into the top of the columns at a rate of 1.O ml h- ’ . The flow rate of the medium to the columns was maintained using a Watson-Marlow 501 U peristaltic pump with a multichannel head adaptor. Each column was inoculated at the top with 10’ spores in a

322

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Assay for fungal biomass A quantitative estimate of the total fungal biomass contained within the columns was achieved with the aid of a destructive sampling technique. Glass beads contained within successive 2.5 cm depths of the columns were removed with an alcohol-sterilized spatula and placed into sterile plastic Petri dishes. The fungal mycelium attached to the beads was removed by adding steriledistilled water to the Petri dish and swirling gently (three washes, total volume of water = 10 ml). The resulting mycelial suspensions were transferred to sterile Universal bottles and processed by fragmenting, diluting, and plating the mycelial fragments onto colony extension-limiting medium as described elsewhere.5 Five estimates of biomass were undertaken for each sample.

Experimental

1997, vol. 21, October

design

Each species was grown in separate columns referred to as monocultures. These were incubated for 300 h and then destructively sampled. Nine columns were inoculated with a 1: 1 mixture of F. culmorum and T. harzianum (dual cultures) and sampled destructively at timed intervals up to 456 h.

Mathematical

Spore suspensions were prepared in sterile 0.1% (v/v) Tween 80 (Sigma Chemical Co., St. Louis, MO) from cultures grown on PDA. Each suspension was centrifuged and sterile-distilled water added to the pellet to give a final concentration of 105-lo6 spores ml-‘. The spore concentration of each suspension was determined using a hemocytometer grid observed under a light microscope at 200X magnification.

Continuous-flow

suspension approximately equal to its void volume (10 ml). The columns were kept in a constant temperature room at 25°C.

model

In order to analyze the results from the dual culture columns, we used a layer model6 to simulate a continuous-flow column. We then tested three functions representing interaction between the two species to determine which best represented our experimental results. Although previous studies have used similar techniques, ‘,‘O.” their approach was slightly different to ours. These authors constructed mathematical models on the basis of specific hypotheses and then generated experimentally testable predictions from them. We used models in an attempt to explain our results after we had obtained them. In order to construct the layer model, a column was considered to consist of a number of theoretical compartments sufficiently small to approximate a continuous system. Each compartment was considered to be well mixed. Each compartment was assumed to: Receive nutrient from the compartment above (except the top compartment to which nutrient was supplied directly); Receive a fraction of the biomass from the compartment above; Lose nutrient and a fraction of biomass to the compartment below. The theoretical compartments designated j where j = 1, . . , 10 are considered to be of equal volume. The constant effective dilution rate D is defined as the nutrient flow rate per unit volume. S, represents the initial concentration of the growth-limiting nutrient (glucose). The nutrient balance for compartment 1 (j = 1) is described by the equation:

dS,

-r

dt

D(S,

_ s,)

-

+

_ rl

2

(1)

.I

where Sj = concentration of glucose; Xj = biomass concentration of F. culmorum; Z, = biomass concentration of T. harzianum; Y_1, = yield of F. culmorum at biomass concentration X; Yzj = yield of T. harzianum at biomass concentration Z; Aj = growth rate of F. culmorum; and Bj = growth rate of T. harzianum. If a fraction of the fungal biomass washed down from compartment j to compartment (j + 1) is designated Fxj and F, for F. culmorum and T. harzianum, respectively, then the biomass balance for compartment one becomes

lnferections

dx, dt

d-5 dt

=

between

Fusarium

culmorum

A, - Fr,D,,

and Trichoderma

harzianum:

J. L. Cheetham

et al.

(2)

= 3, - F,,D,,.

(3) 12 -

For the jth compartment, j # 1, the equations of balance are: 10 dSj dr=

D(S,_,

-S,)

-$-g Xl

J

(4)

8-

dX L=A,+DF,,(X;_,-Xj) dt

6-

dt = B, + DF,(Z,_ , - Z,).

2-

4-

03

The functions A and B represent the way in which the two species of fungi grow and interact with each other. After specifying these functions, a FORTRAN program incorporating a fourth-order Runge-Kutta routine was written and used to solve the differential equations qualitative no attempt Variables previously

numerically. Since our intention was to seek only a correlation between simulated and experimental results, was made to fit the data and units for the parameters. were assigned arbitrarily, and were consistent with published information.4.7

Results The relationship between the biomass density of each species grown in monoculture and column depth at the end of the experiment (300 h) is shown in Figure 1. Biomass density decreased as column depth increased for both species. The T. harzianum population was always at a higher concentration than that of F. culmarum. The sampling method was equally effective at recovering mycelia of both species; therefore, F. culmorum appeared to produce less biomass than T. harzianum under these conditions. T. harzianum biomass density decreased from 2.8 X lo3 p,g -3 at a column depth of O-2.5 cm to 76 kg cm-3 at a irpth of 17.5-20.0 cm. Similarly, the biomass density of F. culmorum decreased from 1.6 X lo2 kg cmP3 at O-2.5 cm to 1 pg crne7 at 17.5-20.0 cm. Biomass estimates for the dual cultures are shown in Figure 2. F. culmorum was the dominant species after 72 h. Conversely, T. harzianum had become the dominant species by the end of the experiment (456 h). This change could be detected visually by the progression of color within the columns from pink to green, reflecting the difference in pigmentation between the two species. Figure 2 essentially represents a series of snapshots of the distribution of fungal biomass contained within the columns over the course of the experiment. At 72 h, F. culmorum was the dominant species to a depth of about 15 cm. At greater depths, T. harzianum was present at a higher concentration than F. culmorum. At 120 h, both species had increased their concentrations. The switch in dominance appeared to occur higher in the column. This trend continued at 168 h with the switch occurring at about 8.75 cm. By 216 h, the F. culmorum concentration exceeded that of T. harzianum only at the top of the column. From this time (216 h), F. culmorum concentrations decreased. At 360 and 408 h, this species

9

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Figure 1 Natural logarithm (Ln) of biomass concentrations (pg cmm3) of (a) Trichoderma harzianom (!I) and (b) Fusarium culmorum (+) monocultures in columns destructively sampled after 300 h. The data points represent the mean of five replicates 2 SE. Where the error bars cannot be seen, the error is less than the size of the symbol

was detected only toward the bottom of the column and by 456 h appeared to be eliminated altogether.

Discussion The biomass in the monoculture columns appeared to decrease exponentially. The data shown in Figure 1 were fitted to the equation: log y = log yO-kx

(7)

where y = biomass concentration (p.g cm-“), y0 = biomass concentration at the top of the column (pg cme3), x = depth down the column (cm), and k is the specific rate of biomass decrease (cm-‘). The k values for the two species were 0.089 cm-’ for T. harzianum and 0.13 cm-’ for F. culmorum. Enzyme Microb.

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Papers

Column depth (cm)

Column depth (cm)

._

mL

0

..I..

0

,..,.‘,..,.

2

4

6

6

10

20 1

lo1 Jpqq t=5

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. . . . . .

0

0

2

4

6

6

10

0

2

4

6

8

10

Column depth (cm)

Column depth (cm)

Figure 2 Natural logarithm (Ln) of biomass concentrations (kg cmm3) obtained by destructive sampling of columns containing a dual culture of T. harzianum (0) and F. culmorum (+) (I:1 initial inoculum ratio) at the times indicated. The data points represent the mean of five replicates _’ SE

In the dual culture columns, F. culmorum was the dominant organism at the beginning of the experiment; however, it was gradually replaced starting at the top of the columns by T. harzianum. At the end of the experiment (456 h), the F. culmorum population could not be detected. Three different functions for A and B in the layer model were used in an attempt to simulate this change in dominance of the two species during the course of the experiment. These were: 1. Monad’* functions implying that the interaction between the two species was an indirect resource-type competition; I3 thus,

Compartment number

(9) where prnr and P,,,~ are the maximum specific growth rates of the two species, and K, and KS, are their saturation constants. 2. Monod functions with constant surface area availability; l4 thus, 324

Enzyme Microb.

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logistic

limitation

of

Figure 3 Simulated interaction between T. harzianum (0) F. cufmorum (+) assuming constant logistic limitation. following arbitrary parameter values were used: D = 1.8, 10.0, krnx = 0.4, Prnz = 0.3, KS, = 1.0, KS, = 0.5, c, = 0.3, 0.8. Arbitrary time units

Ai=--

Fmx xjsj

Ks, + Sj 1997, vol. 21, October

c,x,’

and The S, = c2: =

Interactions

between

Fusarium

culmorum

and Trichoderma Aj = *

0

2

4

6

6

10

6-

t=5

42-

00 0

2

4

6

6

10

2

4

6

6

10

6-

6-

22 0

Figure 4

Simulated interaction between T. harzianum (0) and F. culmorum (a) assuming that logistic growth of the latter is a linear function of the former. Symbols and parameter values as in Figure 3. C, = 0.05

zjsj

4, + S,

qz,’

(11)

where c, and c2 are constants. of the logistic term for F. culmorum such that it becomes a linear function of the population density of T. harzianum; thus, one fungal species has a direct limiting effect on the other and

3. A modification

(12)

I

This research was made possible by an AFRC Linked Research Grant to MJB and JML and a SERC CASE studentship (with Horticulture Research International, Littlehampton) to JLC.

References I.

J

- (c, + C~Zj,X,z

et al.

Acknowledgments

Compartment number

B. = -bk

J. L. Cheetham

where cj is a constant. Simulations employing Monod functions to represent competition for a single growth-limiting nutrient did not offer a good qualitative fit to the experimental data. T. harzianum was eliminated in all compartments and the two species were not observed to coexist. This seems to suggest that the interaction between the two species was not only indirect competition for a single resource. Incorporating a logistic term for the growth and interaction functions gave a better representation of the experimental data. Figure 3 shows the relationship between the biomass concentrations of the two species and the column compartment obtained using these functions. The two species coexisted in all compartments; however, T. harzianum was always present at a higher concentration than F. culmorum. The two species eventually reached steady state but no switch in dominance occurred. Figure 4 shows the simulated relationship between species concentration and compartment number obtained when Eq. (12) was used to represent growth and interaction of F. culmorum. In this case, the two species coexisted in all compartments over the time period examined and a change in dominance occurred. At the start of the simulation, F. culmorum had the higher population density whereas T. harzianum dominated at the end. The two species reached steady state as with the constant logistic model, and at this time, the biomass concentration of T. harzianum was slightly higher than that of F. culmorum in all compartments except compartment one. The best qualitative fit to the experimental data, thus, was obtained with a model incorporating Monod kinetics and making logistic limitation of F. culmorum a linear function of the population density of T. harzianum. This implies that the interaction between the two species was not simply competition for a single resource; the population of one species had a more direct effect on the other. T. harzianum could either be parasitic on F. culmorum or alternatively, produce an antibiotic or other such secondary metabolite which inhibited the population of the pathogen.

10 -

6-

5’

harzianum:

2.

.1 4.

5.

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