Scientia Horticulturae 92 (2002) 329±338
Enzyme activity assays for nutrient solutions from closed irrigation systems Thomas Branda,*, Walter Wohankab, Beatrix W. Alsaniusa a
Department of Crop Science, Swedish University of Agricultural Sciences, P.O. Box 44, SE-230 53 Alnarp, Sweden b Department of Phytomedicine, State Research Institute Geisenheim, Von-Lade-Str. 1, DE-65366 Geisenheim, Germany Accepted 31 May 2001
Abstract Exo-enzymes degrading cell wall components of fungal pathogens may play a role in the biological control of plant diseases in hydroponic systems. Therefore, protease, cellulase and chitinase are of special interest. The main objective of this work was the development of enzyme assays suitable for the conditions found in nutrient solutions. Furthermore, inducibility of enzyme activities by amendment of organic substrates to nutrient solutions and the effect of storage on enzyme activities in nutrient solutions were investigated. Enzyme tests based on the rate of release of soluble dye-labeled fragments of chromogenic substrates were adapted for nutrient solutions and were evaluated as suitable for the purpose. Using Hide-Remazol Brilliant Blue R as substrate for protein degrading enzymes, a stable activity of protease for up to 16 h was found. In order to detect the activity of chitinolytic enzymes, Carboxymethyl-Chitin Remazol Brilliant Violet was utilized as substrate. Results indicated good performance of the test, although chitinase activity was not observed in the beginning but later in the crop season. Furthermore, an approach to detect cellulase activity using Carboxymethyl-Cellulose Remazol Brilliant Blue as substrate yielded satisfying results. Amendment of nutrient solutions with chitin and cellulose (1%) incubated for 3 days led to signi®cantly increased activity of chitinase and cellulase, respectively. For all of the studied enzymes, storage at 2 8C did not affect the activity for at least 1 week. In contrast, storage of samples of nutrient solution at room temperature led to a rapid decrease in activity of chitinase and cellulase, whereas protease activity was negatively affected by freezing ( 26 8C) the nutrient solution. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cellulase; Cellulose; Chitinase; Chitin; Induction; Lycopersicon esculentum L.; Protease; Storage; Tomato
* Corresponding author. Tel.: 46-40-415343; fax: 46-40-465590. E-mail address:
[email protected] (T. Brand).
0304-4238/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 3 0 4 - 1
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1. Introduction For environmental and economical reasons, the use of closed cultivation systems for production of greenhouse crops has been recommended and legally enforced by the scienti®c and political communities, respectively, in many European countries. However, concerns have been expressed for their execution with respect to substantial risk for spread of plant pathogens (McPherson et al., 1995), and accumulation of organisms and organic compounds adverse to plant growth (Van Peer and Schippers, 1989; Yu and Matsui, 1994). During recent years, the bene®cial impact of microorganisms inhabiting closed systems has been brought into light. In this context, development of disease suppressiveness in closed growing system has been shown by Postma et al. (2000) and McPherson (1998) for Pythium aphanidermatum and Phytophthora cryptogea, respectively. Different mechanisms might be involved in the expression of disease suppressiveness (Postma, 1996). As disease suppressive compounds, siderophores, antibiotic compounds of phenolic nature or exo-enzymes have been extensively discussed for soil systems (Swinburne, 1986; Inbar and Chet, 1991; Ryder and McClure, 1997). Hydroponic systems, however, have been the subject of only a few studies. Although there are speculations on the possible role of siderophores (Van Peer et al., 1989), their occurrence has not been examined in nutrient solutions of closed systems. Incidence of antibiotic compounds such as phenazine-1-carboxylic acid and pyoluteorin was veri®ed in the nutrient solution of closed systems growing tomato (Jung et al., 2001). Enzyme activity in the nutrient solution of closed systems, however, is a novel area of research. Polysaccharide lyases and proteolytic enzymes might be of considerable interest because of their ability to degrade cell wall components of phytopathogenic microorganisms either independently or synergistically (Sivan and Chet, 1988; Lorito et al., 1993). In closed systems, zoosporic fungi (Pythium spp., Phytophthora spp., Olpidium spp.) as well as Fusarium oxysporum are prevalent pathogens of economic importance (Ehret et al., 2001). The ®brillar components of cell walls from fungi belonging to Pythiaceae consist mainly of cellulose and glucans, whereas those from other fungi are chitin and glucans. Furthermore, proteins are substantial matrix components (Bartnicki-Garcia, 1968). Therefore, activity of proteases, chitinases, cellulases and glucanases in nutrient solutions may have impact on biologically mediated control of plant pathogens within closed growing systems. The objective of the present study was to elaborate methods for estimating the activities of enzyme complexes in nutrient solutions from hydroponic growing systems with particular emphasis on protease, chitinase and cellulase. The present study was focused on four main questions: 1. How can the activities of these enzyme complexes named above be measured in the nutrient solution from closed hydroponic greenhouse systems? 2. What is the optimal length of incubation? 3. Can the activities of the three enzyme complexes be induced? 4. How does storage affect the activities of the three complexes?
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2. Materials and methods 2.1. Sample collection Nutrient solution was sampled from a commercial closed hydroponic greenhouse system (7800 m2, Ingeltorps traÈdgaÊrdar AB, Glimmingebro, Sweden) growing tomato (Lycopersicon esculentum L., cvs. Aromata and Armada). The greenhouse complex was divided into four major units. In unit 1, peat was used as a growing medium and each tomato plant was cultivated in a 10 l bucket with individual drip irrigation. In units 2, 3 and 4, tomato plants were grown in conventional rockwool slabs and supplied with nutrient solution. The ef¯uent from the peat buckets and rockwool slabs was collected in a covered gutter between the rows and was passively transported to recycling reservoirs. Each greenhouse unit was equipped with an individual recycling reservoir, each storing 100 l. Individual samples (500 ml) were taken from irrigation ef¯uent of greenhouse units (nutrient solution 1: peat; nutrient solutions 2, 3 and 4: rockwool) before release to the reservoir, and were transported to the laboratory. For each trial, nutrient solution was sampled in two independent repetitions during spring and summer 2000. 2.2. Enzyme assays For the detection of protease (EC 3.4) activity, a method ®rst described by Rinderknecht et al. (1968) was adapted using Hide-Remazol Brilliant Blue R (H-RBB; CAS: 37340-548; Fluka) as a substrate. Aliquots of 50 ml of nutrient solution were mixed with 125 mg (2 mg) of the substrate in 100 ml Erlenmeyer ¯asks. A chitinase (EC 3.2.1.14) assay developed by Wirth and Wolf (1990) was modi®ed to suit the conditions present in the nutrient solutions. Aliquots of 50 ml of the nutrient solutions were blended in Erlenmeyer ¯asks with 12.5 ml of the substrate for chitinase, Carboxymethyl-Chitin Remazol Brilliant Violet solution (CM-Chitin-RBV, 2 mg ml 1; Loewe Biochemica, 04106). The cellulase (EC 3.2.1.4) assay approach followed the same procedure as the chitinase assay, but Carboxymethyl-Cellulose Remazol Brilliant Blue solution (CM-Cellulose-RBB, 4 mg ml 1, Loewe Biochemica, 04100) was used as a substrate. As sample controls, nutrient solution without the respective enzyme substrate was tested. Further, Na-acetate buffer (50 mM, pH 5.3) with enzyme substrate served as a substrate control. Both the controls were treated like the samples. All the samples were incubated with two replicates for 2, 4, 6, 8, 10, 12, 16, 20, 24, 36 and 48 h, respectively, at 37 8C in a shaking water bath at 200 rpm. After incubation, aliquots of 2.0 ml of the incubated solutions were transferred to centrifugation tubes (1 min of sedimentation for the protease assay). The reaction of protease was terminated by adding 1 ml of 1 M H3PO4 and centrifugation at 4000g for 5 min. The reactions of chitinase and cellulase were quenched by cooling the solution on ice for 10 min after adding 0.5 ml of HCl (2 M), inducing precipitation of non-digested substrate. Finally, the mixtures were centrifuged for 15 min at 4000g.
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Aliquots of 200 ml of the supernatant were used for spectrophotometrical measurement of the extinction (DIGISCAN, AsysHitec, Linz, Austria, software: DIGIWIN; protease assay 650 nm, chitinase assay 550 nm, cellulase assay 590 nm). Enzyme activity, speci®ed as milliUnit (mU) (change of extinction at a given wavelength in proportion to incubation time) was calculated by subtraction of the values of the controls from the average of the sample (Eq. (1)). milliUnit
mU E1
E2
E3
E4 1000t
1
(1)
where E1 is the extinction of the nutrient solution substrate, E2 the extinction of the nutrient solution substrate, E3 the extinction of the Na-acetate buffer
50 mM; pH 5:3 substrate, E4 the extinction of the Na-acetate buffer
50 mM; pH 5:3 substrate, t the incubation time (min). 2.3. Inducibility of enzyme activity Nutrient solution from greenhouse units 2, 3 and 4 were sampled and activities of chitinase and cellulase were determined. In order to induce the associated enzyme activity, chitin (CAS No. 1398-61-4, SIGMA) and cellulose (Avicel PH-101, Fluka; ELINCS/EINECS No.: 2326749; particle size, 50 mm) were added to nutrient solutions 2, 3 and 4 (1%). The amended solutions were incubated in Erlenmeyer ¯asks for 3 days at room temperature on a rotary shaker at 200 rpm. After centrifugation for 1 min at 1000g, enzyme activity in the supernatants of the incubated solutions was analyzed. Double amount of enzyme substrate was used (1 ml enzyme substrate solution:2 ml nutrient solution). Measurements of extinction were done after 4 h of incubation at 37 8C, each replicate with two parallels. 2.4. Shelf life of enzymes in stored nutrient solutions In order to investigate the impact of storage on the enzyme activities, induced nutrient solutions were used for the investigations on chitinase and cellulase, whereas protease activity was measured in fresh nutrient solution. Aliquots of nutrient solutions 2, 3 and 4 were stored in brown glass bottles in the dark at three temperature regimes: room temperature, 2 and 26 8C. Prior to the tests all stored nutrient solutions were heated to 37 8C in order to provide the same conditions. At the start of the experiment (0 day) and after 1, 3 and 7 days of storage, enzyme activities were determined as mentioned above (Section 2.3). 2.5. Statistics All tests were repeated twice. Data of the investigations on the inducibility of enzymatic activity and the effect of storage of nutrient solutions were statistically analyzed (t-test for dependent samples) using the software ``Statistica'' (StatSoft).
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3. Results 3.1. Enzyme assays During the initial phase of incubation, protease activity was detected in all of the tested nutrient solutions (Fig. 1A). This initial phase lasted for 8±16 h, depending on the sample. Enzyme activities calculated as the change of extinction at 650 nm/min varied during this phase between samples (0.18±0.52 mU). Each sample showed nearly stable or merely slightly decreasing proteolytic activity. After the initial phase of incubation, extinction decreased rapidly due to development of a yellow instead of blue color after addition of H3PO4 (1 M). Apart from nutrient solution 2.2 standard deviations were low. No chitinase activity could be found in the nutrient solutions collected early in the cropping season (nutrient solutions 1.1 and 2.1; Fig. 1B), but was detected later in the cropping season. Although the extinction increased steadily (data not shown), the highest activity was calculated for the shortest incubation period in test, 2 h. Throughout the test, nutrient solution collected from the rockwool system showed higher chitinolytic activity than the one from the peat system. Very low standard deviations were calculated all through the tests. Using CM-Cellulose-RBB as substrate, it was possible to detect the activity of cellulolytic enzymes (Fig. 1C). In the nutrient solutions from the rockwool system (nutrient solutions 2.1 and 2.2) activity was detectable throughout the incubation period, except one single instance (nutrient solution 2.1; 12 h). The activity was rather low, but nearly stable. Highest cellulase activities were detected after 2 and 4 h. The standard deviations were high, especially at the beginning of the experiment. In the nutrient solutions from the peat system (nutrient solutions 1.1 and 1.2) almost no activity was found, leading to negative calculated values. 3.2. Induction of enzyme activity Compared to the measurements before the induction, chitinase and cellulase activity was signi®cantly higher after 3 days (p 0:015 and 0:0015, respectively; Table 1). Generally, the chitinolytic activity was one order of magnitude higher in induced than in non-induced nutrient solutions. But for nutrient solutions with very low chitinase activity before adding chitin, much higher rates were calculated (up to 300 times, data not shown). Due to the calculated negative average cellulase activity in the non-induced nutrient solution it was not possible to give a respective calculation for cellulose degrading enzymes. 3.3. Shelf life of enzymes in nutrient solutions Protease activity was not signi®cantly affected by storage at room temperature and 2 8C for up to 7 days (Table 2), although a trend to less activity was observed in nutrient solutions stored at room temperature. Storage of the nutrient solution samples at 26 8C led to signi®cantly diminished proteolytic enzyme activity after 1 day and nulli®ed it during longer storage periods.
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Fig. 1. Enzyme activity (A: proteolytic, B: chitinolytic, C: cellulolytic) in the nutrient solution from a closed hydroponic greenhouse system. Nutrient solution (Ns) 1 from a system using peat as a growing medium, Ns 2 from a system using rockwool (n 2; suffixes represent replication, bars S:D:). mU: milliUnit (change of extinction per minute).
No change in chitinase activity was found in nutrient solutions when stored at 2 and 26 8C for up to 1 week. In contrast, storage at room temperature led to a statistically signi®cant reduction of the chitinolytic activity after 1 day. Chitinase activity was further decreased after 3 days and undetectable after 1 week of storage at room temperature.
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Table 1 Activities (mU) of chitinase and cellulase in nutrient solutions before and after induction with chitin and cellulose (1%) for 3 days, as evaluated after 4 h of incubation at 37 8C Enzyme activity (mU)*
Enzyme
Before Chitinase Cellulase
After
0.037 a 0.022 a
0.380 b 1.079 b
*
Different letters indicate significant differences (p 0:015 for chitinase; p 0:0015 for cellulase) between activities of the respective enzyme
n 6.
Table 2 Impact of duration of storage (day) and temperature regime on enzyme activity (mU) in nutrient solutions from closed hydroponic greenhouse systems Enzyme
Temperature (8C)
Duration of storage (day) 0
1
3
7
Protease
Room temperature 2 26
0.241
0.196 0.215 0.072*
0.189 0.220 0.010*
0.163 0.229 0.013*
Chitinase
Room temperature 2 26
0.617
0.447* 0.613 0.695
0.157* 0.619 0.629
0.012* 0.605 0.652
Cellulase
Room temperature 2 26
1.079
0.566* 1.105 0.935
0.219* 1.031 0.832*
0.056* 0.956 0.795*
* Indicate significant difference
p < 0:05 between activities of the respective enzyme in stored nutrient solution compared to non-stored (0 day)
n 6.
As for chitinase, storage at room temperature caused a signi®cant decrease in cellulase activity, whereas storage at 2 8C did not affect cellulase activity for up to 7 days. Freezing of nutrient solution at 26 8C was signi®cantly detrimental to cellulase activity after 3 days of storage. 4. Discussion The encouraging reports on disease suppressiveness in closed hydroponic greenhouse systems have been primarily discussed on the basis of microbial communities in relation to disease conduciveness (McPherson, 1998; Postma et al., 2000). However, mechanisms involved are poorly explained. According to Lorito et al. (1993), functional proteins encoding for proteolytic, chitinolytic and cellulolytic activity are of considerable interest due to their ability to degrade components of outer structures of plant pathogens. Therefore, enzyme studies might give relevant information to researchers and growers as well as extension services on the microbial status of closed hydroponic growing systems.
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The present report is the ®rst of its kind on enzyme activity in nutrient solutions from closed irrigation systems. The main advantages of the tested dye-labeled substrates are their speci®city and high sensitivity (Rinderknecht et al., 1968) as well as the easy execution of the assays. They therefore seem to be suitable for analyzing nutrient solutions. However, only low or no activity was found for chitinase and cellulase in some cases. This might be due to the interactions between the dye to be detected and substances in the nutrient solution, which may affect the occurrence of non-precipitable dye-labeled fragments. Possibly, dye-labeled fragments were adsorbed to organic material in the nutrient solution and precipitated causing low extinction. Also, this might explain the calculated negative values, resulting from Eq. (1) when controls showed higher activity than samples. From the gathered data, we conclude that an incubation of 4 h is suf®cient for a reliable estimation of activities of proteolytic, chitinolytic and cellulolytic enzymes. Further, for being able to evaluate the measured enzyme activities in a broader context, basic knowledge about the dynamics in enzyme activity in closed hydroponic systems is needed. This will be conducted within the frame of future experiments. Comparisons between different samples (e.g. spatial, temporal) within a system may provide information about general conditions in the speci®c environment. Extinctions (and their development) differed sometimes considerably between the parallels. Therefore, at least double, preferably multiple, determinations of enzyme activity should be conducted for each sample. Further investigations are needed aiming for improvement of sensitivity, development of miniaturized lab tests (Wirth and Wolf, 1990) as well as test kits for practical use. An interesting ®nding is the improved activity of chitinolytic enzymes in nutrient solutions later in the crop season. This might be due to changes of the microbial composition in the nutrient solution during cultivation. Apart from this seasonal effect, choice of growing media appears to be decisive for activity of chitinolytic enzymes. Whether the activities of other relevant enzymes are affected by these factors is uncertain. This has to be investigated in future studies and is of speci®c interest for the development of disease suppressiveness in closed hydroponic greenhouse systems. Provided that there is a correlation between enzymatic and microbial status or suppressiveness, enzyme measurements may serve as a proper tool for evaluations. Increased knowledge of the ecological status may affect future crop management, particularly with regard to plant protection. By amending nutrient solution in vitro with chitin and cellulose, it was possible to enhance the respective enzyme activities and get positive proof in assessing suitability of enzyme assays. Stimulation of chitinase production by chitin or fungal cell walls is a known phenomenon (Sivan and Chet, 1988; Thrane et al., 2000). For cellulase and protease, similar relations might be expected. Production of cellulase, chitinase and protease by Trichoderma spp. were enhanced in vitro by amendments of cellulose, chitin and skim milk powder, respectively (Antal et al., 2000). Thrane et al. (1997) reported on induction of cellulase built by Trichoderma harzianum in vitro in the presence of Pythium. The practical value of enzyme induction in nutrient solutions by natural occurrence of or amendments with suitable substrates is not evaluated yet. Selectively inducing enzyme
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activities for crop production purposes like plant protection or nutrition is an attractive vision, which raises many questions and requires further investigations. The decline of detected enzyme activities in nutrient solutions stored at room temperature shows that samples should generally be as fresh as possible. During transport and unavoidable delay, samples of nutrient solution should be cooled, but not frozen. Obst (1995) mentioned the same experiences for samples of water and sludge. After storage at room temperature, the studied polysaccharide hydrolases showed reduced activity. This might be due to digestion by proteolytic enzymes, which were not substantially affected at room temperature. In contrast, freezing of nutrient solution caused low protease activity, but cellulase was affected to a far less degree and chitinase not at all. This indicates different mechanisms in¯uencing the shelf life of enzymes in nutrient solutions. By freezing, precipitation of, presumably, salts and organic material was induced. From soil science it is known that proteases are bound to clay and organic matter (Alef, 1991). Thus, precipitation might cause elimination of protease from nutrient solutions. With further knowledge of the microbial status and proper tools for evaluation of the prevailing situation, it will be possible to facilitate crop management with respect to plant protection and nutrition. The detection of relevant enzyme activity may be one step in a complex control and decision process. Acknowledgements The authors want to thank Mr. Johnny Nilsson, Ingeltorps traÈdgaÊrdar AB, for giving us the opportunity to collect samples during the growing period, and Dr. Renate Loewe, Loewe Biochemica, Sauerlach, Germany, for fruitful discussion. The project was funded by the Royal Academy of Forestry and Agriculture (KSLA), Stockholm, the Swedish Research Council for Forestry and Agriculture (SJFR), Stockholm, and the State Agricultural Authority (Jordbruksverket), JoÈnkoÈping, Sweden, who are gratefully acknowledged. Further, we owe thanks to the Swedish Institute, Stockholm, which funded the guest research program for Dr. Thomas Brand. References Alef, K., 1991. Methodenhandbuch Bodenmikrobiologie. Ecomed, Landsberg. Antal, Z., Manczinger, L., Szakacs, G., Tengerdy, R.P., Ferenczy, L., 2000. Colony growth, in vitro antagonism and secretion of extracellular enzymes in cold tolerant strains of Trichoderma. Mycol. Res. 104, 545±549. Bartnicki-Garcia, S., 1968. Cell wall chemistry, morphogenesis, and taxonomy of fungi. Ann. Rev. Microbiol. 22, 87±108. Ehret, D., Alsanius, B.W., Menzies, J., Wohanka, W., Utkhede, R., 2001. Disinfestation of recirculating nutrient solutions in greenhouse horticulture. Agronomie 21, 323±339. Inbar, J., Chet, I., 1991. Evidence that chitinase produced by Aeromonas caviae is involved in the biological control of soil-borne plant pathogens by this bacterium. Soil Biol. Biochem. 23, 973±978. Ê ., Niedack, N., Bowens, P., Alsanius, B.W., 2001. Supported liquid Jung, V., Chimuka, L., JoÈnsson, J.-A membrane extraction for identification of phenolic compounds in the nutrient solution of closed hydroponic growing systems for tomato, submitted for publication.
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