Bacterial amelioration of ferulic acid toxicity to hydroponically
grown lettuce (
Lactuca sativa
L.)
Siri Caspersen*, Beatrix Waechter Alsanius, Peter Sundin
1, Paul JenseÂn
Department of Horticulture, Swedish University of Agricultural Sciences, P.O. Box 55, SE-230 53 Alnarp, Sweden Accepted 21 December 1999
Abstract
Ferulic acid (FA) is released from plant roots and by decomposition of plant residues and may be involved in allelopathic interactions. We isolated bacteria from the recirculating nutrient solution of a closed, hydroponic lettuce culture using nutrient media supplemented with 1.0 mM FA. The isolates were tested for their capacity to degrade FA in concentrations up to 200
mM. Isolates p208, p210 and p307 showed the highest degradation rates and were therefore used for single- and multiple-strain inoculation in two factorial experiments where lettuce (Lactuca sativa L. cv. Grand Rapids) plants were grown gnotobiotically for 2 weeks in nutrient solution with or without 200mM FA. When isolate p208 or multiple strains were added, no FA was detectable at the end of the experiments. In the absence of FA, no signi®cant eects of the bacterial treatments could be found with respect to plant dry weight. However, in the presence of FA, isolate p210 increased shoot dry weight and the multiple-strain treatment increased root and shoot dry weights in the ®rst experiment. In the second experiment, isolate p210 neither aected the concentration of FA nor plant dry weights. Isolate p208 and the multiple-strain treatment reduced the negative eect of FA on lateral root lengths and root hair formation in both experiments. Finally, we conclude that bacteria with the capacity to degrade FA and to ameliorate phytotoxic eects of FA were present in the nutrient solution of a commercial hydroponic lettuce culture.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Bacteria; Gnotobiotic; Hydroponic culture;Lactuca sativaL; Phenolic acid
1. Introduction
Ferulic acid (FA) is a precursor in lignin formation and has been suggested to be involved in cell wall extensibility (Locher et al., 1994). Ferulic and other phenolic acids are released by living roots and by decomposing plant residues (Siqueira et al., 1991). While low concentrations of phenolic acids in the root environment may stimulate plant growth, phytotoxic eects often occur at higher concentrations (Rice, 1984; Siqueira et al., 1991). For lettuce plants grown
axenically, plant growth and root development were
inhibited at 200 mM FA in the nutrient solution
(Cas-persen et al., 1999).
It is well established that microorganisms are capable of metabolizing and producing a range of phe-nolic compounds in plant±soil systems (Siqueira et al., 1991). Rosazza et al. (1995) have reviewed microbial transformation and degradation of FA. Vanillin, vanil-lic and protocatechuic acids, guaiacol and 4-vinyl-guaiacol are common products of microbial conversion of FA (Turner and Rice, 1975; Rosazza et al., 1995).
It has been proposed that plants may be protected against potentially toxic compounds in soil by the metabolic detoxi®cation capabilities of the rhizosphere microbial community (Walton et al., 1994). The increased degradation of xenobiotics which is often observed in rhizosphere soil may be associated with
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* Corresponding author. Fax: +46-40-460-441. E-mail address:[email protected] (S. Caspersen).
1
the input of carbon compounds by plant roots (Shann and Boyle, 1994). Rapid decomposition of FA gener-ally prevents its accumulation in soils (Turner and Rice, 1975), and soil treatment with FA may result in the induction or selection of micro-organisms that can utilize FA as a carbon source (Sparling et al., 1981; Blum and Shafer, 1988).
When plants are produced hydroponically, ``closing'' of the system by recirculation of the nutrient solution is suggested as a way of reducing the release of excess water and nutrients to the environment. However, alle-lochemicals like phenolic acids could accumulate in the circulating nutrient solution (Yu and Matsui, 1993, 1994) or in solid substrates like peat or bark (Politycka et al., 1984; Ortega et al., 1996). The activity of micro-organisms may suppress the phytotoxic eects of phe-nolic acids also when plants are grown in soil-less culture (Vaughan et al., 1983, 1993; Caspersen et al., 1999). Eklund (1970) reported that bacteria isolated from the rhizosphere of peat-grown cucumber plants were capable of degrading phenolic acids. Additions of dierent phenolic acids to nutrient solution collected from hydroponic systems in commercial greenhouses showed that their concentration decreased to very low levels within 24 h, indicating that there is a natural po-tential in the nutrient solution of hydroponic cultures to degrade phenolic acids (Sundin et al., 1995). Waech-ter-Kristensen et al. (1994) reported that bacteria able
to degrade a mixture of 50 mM each of p
-hydroxyben-zoic, vanillic and caeic acids over 72 h were present in the irrigation water of closed, hydroponic tomato cultures. To avoid the spread of root-infecting patho-gens in closed, hydroponic systems, however, the nutri-ent solution is often disinfected, e.g. by treatmnutri-ent with heat, ozone or UV-light, or by ®ltration (Menzies and BeÂlanger, 1996).
Our objective was to determine the potential role of bacteria present in the nutrient solution of a closed, hydroponic lettuce culture in the degradation of FA, and thus aecting the in¯uence of FA on plant growth and development. Bacteria were isolated from the nutrient solution of a commercial, hydroponic lettuce culture and screened for their ability to degrade FA. The ability of selected isolates to ameliorate toxic eects of FA on lettuce plants were then investigated under gnotobiotic conditions.
2. Materials and methods
2.1. Isolation and propagation of bacteria
Bacteria were isolated from the circulating nutrient solution in a commercial greenhouse in PaÊarp, Sweden, where lettuce was grown in a closed, hydroponic sys-tem. Nutrient solution from the run-o of three gutters
was collected in sterile glass ¯asks and was chilled to
88C during the transport to the laboratory. The
samples were processed for isolation of bacteria as described by Waechter-Kristensen et al. (1994). For the isolation of bacteria capable of growth in the pre-sence of FA, bacterial cells from the pure culture were transferred to 10% Tryptic Soy Agar (TSA) or to King's Medium B (King et al., 1954), both amended
with 1 mM FA. trans-FA
(4-hydroxy-3-methoxycin-namic acid, Sigma) was dissolved separately and
steri-lized by membrane ®ltration (0.22 mm, Durapore
GVWP, Millipore). The FA solution was added to the
autoclaved (1218C, 20 min) media after cooling. The
isolates were propagated at room temperature for 48 h and then transferred to sterile Ringer solution
contain-ing 50 mg NaHCO3, 100 mg KCl, 2.5 g NaCl, 80 mg
CaCl2, and 1000 ml ultra-pure water (Elgastat
max-ima, Elga).
2.2. Selection of bacteria
Catabolism of FA by the isolated bacteria was stu-died over 72 h with three replicates following the pro-cedure of Waechter-Kristensen et al. (1994). All
isolates were examined using 25 and 50 mM FA. Four
isolates (p208, p210, p304, p307) were then selected to be studied in a repeated experiment where 100 and 200
mM of FA were used. Degradation of FA in original
concentrations of 100 and 200 mM was also examined
using a mixture containing equal cell concentrations of p208, p210 and p307. Three isolates (p208, p210, p307) were selected for use in the experiments with plants.
These isolates were identi®ed as Pseudomonas spp. by
API-test (bioMeÂrieux, France).
2.3. Plant experiments
A factorial experiment, with ®ve bacterial treatments (0, isolates p208, p210, p307, and a mixture of the three isolates) and two concentrations of FA (0, 200
mM), was conducted twice. There were seven replicate
culture ¯asks for each treatment, and three and four plants were used for the determination of root coloni-zation and dry weights, respectively. There were also three sterile control ¯asks containing FA but without plants.
Seeds of lettuce (Lactuca sativa L. cv. Grand
Rapids) were immersed in 10% H2O2for 30 min
fol-lowed by rinsing in sterile, ultra-pure water. The sur-face-sterilized seeds were placed on 10% TSA. Six days after sowing, surface sterile seedlings were
trans-ferred singly to autoclaved (1218C, 20 min) cultivation
systems consisting of a conical culture ¯ask (500 ml) closed with a cotton plug (Caspersen, 1997). Each
¯ask contained 200 ml of a ®lter sterilized (0.2mm
(Cas-persen, 1997) with or without 200 mM trans-FA. The
pH was adjusted to 6.0 by H2SO4, and the electrolytic
conductivity (EC) was 2.83 mS cmÿ1
.
The bacterial isolates were cultured on 10% TSA
amended with 1 mM FA for 48 h at 208C before
they were transferred to 10% Tryptic Soy Broth (TSB), propagated for 48 h (shaking at 200 rpm),
centrifuged for 30 min at 300g, and resuspended
in Ringer solution. The cell concentration in the sus-pension was adjusted (by addition of sterile Ringer sol-ution) spectrophotometrically (620 nm) in accordance with an earlier determined calibration curve. The mix-ture consisted of equal cell concentrations of the iso-lates p208, p210 and p307. One ml of bacterial suspension, or sterile Ringer solution for the axenic controls, was added to the nutrient solution of each culture ¯ask. The ®nal concentration of bacterial cells in the nutrient solution of the inoculated ¯asks was 5
104cfu mlÿ1.
The culture ¯asks were distributed randomly on trol-leys in a climate chamber with a photoperiod of 18 h provided by ¯uorescent tubes (VHA Sylvania Cool White 215 W). The irradiance was measured at the top of 24 of the ¯asks with a quantum sensor (Li190SA,
Lambda Instr. Lincoln, Neb., USA), and was (2SD)
330243mmol mÿ2 sÿ1 PAR (400±700 nm). Day
tem-perature was 20.820.48C and night temperature was
18.820.18C. When the experiment was repeated, the
irradiance was 283239 mmol mÿ2 sÿ1. Day
tempera-ture was 21.220.48C and night temperature was 19.5
20.58C. The atmosphere in each ¯ask was aerated by
sterile ®ltered (0.22mm, Millex FG, Millipore) air at a
¯ow rate (measured at the end of the experiment) of
17224 ml minÿ1
in Experiment I and 16526 ml
minÿ1
in Experiment II (mean of all cultivation units2
SD).
The plants were harvested after 2 weeks. For investi-gation of bacterial root colonization, three root sys-tems from each treatment were separately macerated, transferred to 50 ml detergent (0.2% Na-hexametapho-sphate and 0.1% peptone in ultra-pure water) and sha-ken for 20 min at 200 rpm. One ml of each suspension was serially diluted for viable count determination (cfu
gÿ1fresh weight). In Experiment II, 1 ml of the
nutri-ent solution was taken from each culture ¯ask and serially diluted for the assessment of the number of bacterial cells in the nutrient solution (cfu mlÿ1). The
number of viable cells was determined by spread plate technique and surface count method (Collins et al., 1995). Root systems were photographed before roots
and shoots were frozen at ÿ808C. Dry weights were
determined after freeze drying to constant weight. After sterile ®ltering (0.22mm, MinisartPlus, Sartorius)
of the nutrient solution, contents of cis- and trans-FA
were determined by high performance liquid chroma-tography (HPLC) (Caspersen et al., 1999).
2.4. Statistical methods
All results are presented as means 2 SEM. The
results were evaluated by a two-way analysis of var-iance (ANOVA) by the statistical program SAS (SAS Institute). Residuals were plotted to check the assump-tions for the analysis of variance, and bacterial coloni-zation data were log-transformed (Kloepper and Beauchamp, 1992). When there is a large number of treatments in the overall ANOVA, one signi®cant dierence may be ``hidden'' by the non-signi®cant (Chew, 1976). Therefore, the dry weight variables were evaluated further by analysis of simple comparisons (Keppel, 1991). Means of signi®cant eects with more than two levels were separated by Tukey's (bacterial colonization data) or Dunnet's (dry weight variables)
methods of multiple comparisons (P< 0.05).
3. Results
3.1. Selection of bacteria
Thirty bacterial isolates grew in the presence of 1 mM FA. Only a few of these isolates, however,
degraded 25 and 50 mM FA over 72 h. The four most
ecient isolates (p208, p210, p304 and p307) were iso-lated using the modi®ed 10% TSA and degraded 85± 100% of the FA at those concentrations. These iso-lates, as well as a mixture of p208, p210 and p307, were selected to study their ability to degrade higher concentrations of FA (Fig. 1). Isolate p304 did not
degrade FA at concentrations of 100 and 200 mM. At
these concentrations the mixture performed similarly to p210, while p307 alone degraded FA to a lesser
Fig. 1. Removal of ferulic acid (FA) (% remaining of initial concen-trations of 25, 50, 100 and 200 mM) by the bacterial isolates p208, p210, p304, p307, and by a mixture of the isolates p208, p210 and p307 (mix) over 72 h in KMB broth. = not detected. Means2
extent, and p208 was the most ecient isolate in this regard.
3.2. Plant experiments
3.2.1. Bacterial colonization
Addition of the bacterial isolates as single- and mul-tiple-strain treatments to the nutrient solution resulted in colonization of roots and nutrient solution of gnoto-biotically grown lettuce plants. In Experiment I, root colonization rates were generally higher (P< 0.01) in the presence of FA (Table 1). In Experiment II, how-ever, there was no signi®cant eect of FA on bacterial colonization of the roots (Table 1) or of the nutrient solution (data not shown). For the dierent bacterial treatments, there were no clear dierences in the degree of root colonization. In Experiment II, the colonization of the nutrient solution was in the order
of 106 cfu mlÿ1, but depended on bacterial treatment
(P< 0.001) and was higher for isolate p208 compared
to the other bacterial treatments.
3.2.2. Plant growth
3.2.2.1.Experiment I. The bacterial treatments had a
signi®cant (P< 0.05) main eect on shoot and total
dry weights, re¯ecting a signi®cant dierence only between plants grown in the presence of isolates p210 and p307. Further analysis of the eects of the bac-terial treament within each FA concentration showed that in the absence of FA, there were no signi®cant eects of the bacterial treatments on dry weights (Fig. 2). In the presence of FA, however, isolate p210 increased shoot dry weight, and the bacterial mix increased shoot and root dry weights, in comparison with the axenic control.
Analysis of the eect of FA within each bacterial treatment showed that in the axenic ¯asks, addition of
200 mM FA reduced shoot, root, and total dry weights
by 25, 37 and 30%, respectively, compared to the plants grown in the absence of FA. This reduction was statistically signi®cant for root dry weight only. The same tendency was observed in culture ¯asks where isolate p307 was present. In contrast, when the
bac-terial mix was added together with 200 mM FA, shoot
(P< 0.1) and root (P< 0.05) dry weights were higher by 31% and 48%, respectively, compared to the ¯asks containing the bacterial mix but no FA.
FA strongly inhibited root hair formation, and pri-mary and lateral root lengths were reduced by FA both for the axenic plants and when isolate p307 was added (visual observation). In the presence of the iso-lates p208, p210, and of the bacterial mix, there was no observable eect of FA on root lengths. Root hair formation of the FA-treated plants was stimulated by the presence of the bacterial isolates.
Fig. 2. Experiment I: shoot (a) and root (b) dry weights (mg) of let-tuce plants cultivated for 2 weeks with or without 200 mM ferulic acid (FA) and in the presence of ®ve bacterial treatments (axenic control, isolates p208, p210 and p307, and a mixture of the three iso-lates). Means of three or four replicates2SEM. FA reduced root dry weight of the axenic control (P< 0.05). For each of the FA treatments, bacterial treatment means were separated by Dunnett's test. = dierent from the axenic control at a 5% level of signi®-cance.
Table 1
Colonization of lettuce roots (cfu gÿ1
fresh weight) by ferulic acid (FA) degrading bacterial isolates (p208, p210, p307) added singly, or together (Mix)a
Isolate ÿFA +FA
Experiment I
Control 0 0
p208 (2.522.0)105 (2.920.4)105 p210 (5.122.0)104 (3.521.3)105 p307 (2.220.8)105 (2.721.5)106 Mix (2.620.8)105 (4.020.7)105
Experiment II
Control 0 0
p208 (5.322.0)107 (1.320.5)108 p210 (1.220.4)108 (2.421.0)108 p307 (7.123.1)107 (3.921.1)107 Mix (3.321.6)107 (3.421.0)107
a
3.2.2.2.Experiment II. When the experiment was repeated, the total dry weight of the plants grown in the axenic control was 91% of that observed in the ®rst experiment. The tendency to lower dry weights in the second experiment compared to the ®rst was even stronger when bacteria were present (Fig. 3).
Neither the bacterial treatment nor the addition of FA had any signi®cant main eect on plant dry weight. However, analysis of the eect of FA within each bacterial treatment showed that for the non-inoculated control, root dry weight was reduced by FA (Fig. 3). For plants exposed to FA, there was a tendency of increased root dry weight for plants grown in the presence of the bacterial mix compared to the axenic plants.
FA reduced primary and lateral root lengths of the axenic plants and also when isolate p210 or p307 was present (visual observation, Fig. 4). In the presence of isolate p208 or the bacterial mix, however, the eect of FA on root lengths was less pronounced. FA strongly inhibited root hair formation of plants grown axeni-cally or in the presence of isolate p210. Root hair for-mation of the FA-treated plants was stimulated by isolates p208, p307 and by the bacterial mix.
3.2.3. FA in the nutrient solution
3.2.3.1.Experiment I. The concentration of trans-FA left in the nutrient solution 2 weeks after the start of
the experiment was 8128 and 17421 mM,
respect-ively, in the axenic ¯asks with or without plants. Cal-culation of the total amounts of FA from the FA concentrations and the amounts of nutrient solution left in the ¯asks showed that 33% and 70% of the amount of FA initially added was still present as trans-FA in the ¯asks with or without plants after 2
weeks. For cis-FA the concentrations in the nutrient
solution of the axenic ¯asks after 2 weeks was 1121
(5%) and 2122 (8%)mM, respectively, in the presence
or absence of plants.
While no FA was left after 2 weeks in ¯asks where isolates p208, p210 or the bacterial mix had been added, trace amounts of FA were detected in four of the seven ¯asks containing isolate p307. No FA was detected in the axenic control ¯asks where no FA had been added at the start of the experiment.
3.2.3.2.Experiment II. The concentration (percentage of initially added amount of FA is shown in
parenth-esis) of trans-FA left in the nutrient solution 2 weeks
after the start of the experiment was 9228 (36%) and
19023 (75%) mM, respectively, in the axenic ¯asks
with or without plants. For cis-FA the concentrations
in the nutrient solution of the axenic ¯asks after 2
weeks was 1221 (5%) and 2022 mM (8%),
respect-ively, in the presence or absence of plants.
No FA was left after 2 weeks in ¯asks where isolate p208 or the bacterial mix had been added to the nutri-ent solution. In the presence of isolates p210 and p307, however, the concentrations in the nutrient solution
after 2 weeks were 103210 mM trans- and 1321 mM
cis-, and 54219 mM trans- and 723 mM cis-FA,
re-spectively. No FA was detected in the axenic control ¯asks where no FA had been added at the start of the experiment.
4. Discussion
When lettuce plants were cultivated for 2 weeks in the presence of an unspeci®ed bacterial population, we
could not detect any of the 200 mM FA initially
pre-sent in the nutrient solution (Caspersen et al., 1999). In the present study, we have shown that single bac-terial isolates, obtained from the nutrient solution of hydroponically cultivated lettuce, degraded FA. Many microorganisms degrade FA completely, as was shown for Pseudomonas cepacia (Andreoni et al., 1984), Fusarium solani (Nazareth and Mavinkurve, 1986) and Penicilliumsp. (Tillett and Walker, 1990). Interestingly,
Toms and Wood (1970) observed that cis-FA was
dized by washed cell suspensions ofPseudomonas acid-ovoransat 60% of the rate of thetrans-isomer.
The degree of trans±cisisomerization and the
disap-pearance of FA from the axenic ¯asks in the presence or absence of plants were comparable to that observed by Fenton et al. (1978), Caspersen et al. (1999). The amounts of FA remaining in the uninoculated ¯asks were slightly higher in the second compared to the ®rst experiment. This might be explained by a lower removal of FA by the plant at the lower light intensity, which is supported by results from an earlier study (Caspersen et al., 1999).
The reduction of lettuce root and shoot dry weights (Figs. 2 and 3) and the decreased root lengths and root hair formation (Fig. 4) of axenic plants by 200
mM FA in the present experiments were also
compar-able to our earlier observations (Caspersen et al., 1999).
The bacterial mix reduced the negative eects of FA on plant growth in both experiments, while isolate p210 alleviated the negative eects of FA in the ®rst experiment only. The reduction of primary and lateral
root lengths and of root hair formation by FA was counteracted by isolate p208 and by the bacterial mix in both experiments, and by isolate p210 in the ®rst ex-periment. It seems likely that bacterial removal of FA from the nutrient solution is involved in the ability to ameliorate the eects of FA on plant growth and development. Similarly, by reducing the concentrations
of the phenolic acids in the nutrient solution,
Pseudo-monasp sp. and Syncephalastrum racemosus reduced
the toxicity of 1000 mM of ferulic, p-coumaric or p
-hydroxybenzoic acids towards wheat (Vaughan et al.,
1983) and Volutella ciliatareduced the toxicity of 1000
mM vanillic acid towards Pisum sativum (Vaughan et
al., 1993).
The stimulation of lettuce shoot growth by isolate p210 in Experiment I could be related to the
pro-duction of phytohormones or other metabolites.
There is evidence for secretion of IAA and other plant growth regulators as a mechanism for stimu-lation of plant growth and for eects on root elongation and lateral root development by rhizo-bacteria (MuÈller et al., 1989; Frankenberger and
Arshad, 1995). Changes in plant growth regulator activity might also have been involved in the stimu-lation of plant growth by the bacterial mix in the presence of FA (Eklund, 1970).
It is possible that the reduction of the total dry weight of the axenic control plants by 9% in the second experiment (Fig. 3) compared with the ®rst (Fig. 2) was related to the lower light intensity in the second experiment. The relative reduction of plant growth in Experiment II was even more evi-dent in the presence of the bacterial isolates, which might have been related to a higher bacterial pro-liferation in combination with the lower light inten-sity. Vaughan et al. (1983) noted that vigorous microbial growth in nutrient solutions amended with phenolic acid was inhibitory to plant growth. In Experiment II, changes in the qualitative compo-sition of root exudates at a lower light intensity (Rovira, 1969) may have contributed to the increase
in bacterial colonization per gram fresh weight
(Table 1) and in the viable count (cfu) on roots. The denser bacterial colonization of the roots may have contributed to the growth depression of the colonized plants by microbial competition for oxy-gen and nutrients, a mechanism earlier suggested by Lynch and White (1977). This may also explain the absence of a growth stimulation by isolate p210 and by the bacterial mix in this experiment. Fran-kenberger and Arshad (1995) stated that less than optimal or unfavorable conditions may lead to little or no synthesis of phytohormones in the root zone, resulting in the failure of inocula to promote plant growth.
In light of the increase in bacterial root coloniza-tion, the reduced degradation of FA by isolates p210 and p307 in Experiment II is somewhat sur-prising, but might be related to preferential metab-olism of other substrates than FA. Our isolates were all selected in the presence of other carbon sources (10% TSA) in addition to FA. Even if many microorganisms are able to grow with FA as
a sole carbon source (Turner and Rice, 1975;
Andreoni et al., 1984; Jurkova and Wurst, 1993),
Jurkova and Wurst (1993) observed that the
decrease in concentration of ferulic and sinapic
acids by Pseudomonas mira V2 was slowed down
when glucose was added to the medium.
We conclude that also bacteria isolated from the nutrient solution of hydroponically grown lettuce plants may remove FA from the nutrient solution and ameliorate the negative eects of FA on plant growth and development. Plant growth was generally higher in the presence of the bacterial mix, supporting the sug-gestion by Waechter-Kristensen et al. (1994) that a composite bacterial community is more eective than single isolates in degrading phenolic acids.
Acknowledgements
We are grateful to Erik Hansen, PaÊarp, for allowing us to take nutrient solution samples from his lettuce culture. We also thank Eva Olsson for skilful technical assistance, Dr Jan-Eric Englund for statistical advice, and Markus Andersson for assistance with manuscript preparation. This study was supported by grants from the Swedish Council for Forestry and Agricultural Research.
References
Andreoni, V., Galli, E., Galliani, G., 1984. Metabolism of ferulic acid by a facultatively anaerobic strain ofPseudomonas cepacia. Systematic and Applied Microbiology 5, 299±304.
Blum, U., Shafer, S.R., 1988. Microbial populations and phenolic acids in soil. Soil Biology & Biochemistry 20, 793±800.
Caspersen, S., 1997. Ferulic acid interactions and root exudation by hydroponically grown lettuce (Lactuca sativaL.) as in¯uenced by external factors. In: Acta Universitatis Agriculturae Sueciae. Agraria, vol. 45. Swedish University of Agricultural Sciences, Alnarp.
Caspersen, S., Sundin, P., Munro, M., A+alsteinsson, S., Hooker, J.E., JenseÂn, P., 1999. Interactive eects of lettuce (Lactuca sativa L.), irradiance, and ferulic acid in axenic, hydroponic culture. Plant and Soil 210, 115±126.
Chew, V., 1976. Comparing treatment means: a compendium. HortScience 11, 348±357.
Collins, C.H., Lyne, P.M., Grange, J.M., 1995. Microbiological Methods, 7th ed. Butterworth and Heinemann, Oxford.
Eklund, E., 1970. Secondary eects of some pseudomonads in the rhizoplane of peat grown cucumber plants. Acta Agriculturae Scandinavica 17, 1±57.
Fenton, T.W., Mueller, M.M., Clandinin, D.R., 1978. Isomerization of some cinnamic acid derivatives. Journal of Chromatography 152, 517±522.
Frankenberger Jr., W.T., Arshad, M., 1995. Phytohormones in Soils. Marcel Dekker, New York.
JurkovaÂ, M., Wurst, M., 1993. Biodegradation of aromatic car-boxylic acids byPseudomonas mira. FEMS Microbiology Letters 111, 245±250.
Keppel, G., 1991. Design and Analysis, 3rd ed. Prentice Hall, NJ. King, E.O., Ward, M.K., Randey, D.E., 1954. Two simple media for
the demonstration of pyocyanin and ¯uorescin. Journal of Laboratory Clinical Medicine 44, 301±307.
Kloepper, J.W., Beauchamp, C.J., 1992. A review of issues related to measuring colonization of plant roots by bacteria. Canadian Journal of Microbiology 38, 1219±1232.
Locher, R., Martin, H.V., Grison, R., Pilet, P.E., 1994. Cell wall-bound trans- and cis-ferulic acids in growing maize roots. Physiologia Plantarum 90, 734±738.
Lynch, J.M., White, N., 1977. Eects of some non-pathogenic micro-organisms on the growth of gnotobiotic barley plants. Plant and Soil 47, 161±170.
Menzies, J.G., Belanger, R.R., 1996. Recent advances in cultural management of diseases of greenhouse crops. Canadian Journal of Plant Pathology 18, 186±193.
MuÈller, M., Deigele, C., Ziegler, H., 1989. Hormonal interactions in the rhizosphere of maize (Zea maysL.) and their eects on plant development. Zeitschrift fuÈr P¯anzenernaÈhrung und Bodenkunde 152, 247±254.
4-vinylguaiacol by Fusarium solani (Mart.) Sacc. Canadian Journal of Microbiology 32, 494±497.
Ortega, M.C., Moreno, M.T., OrdovaÂs, J., Aguado, M.T., 1996. Behaviour of dierent horticultural species in phytotoxic bioas-says of bark substrates. Scientia Horticulturae 66, 125±132. Politycka, B., WoÂjcik-Wojtkowiak, D., Pudelski, T., 1984. Phenolic
compounds as a cause of phytotoxicity in greenhouse substrates used in cucumber growing. Acta Horticulturae 156, 89±94. Rice, E.L., 1984. Allelopathy. Academic Press, Orlando, Florida. Rosazza, J.P.N., Huang, Z., Dostal, L., Volm, T., Rousseau, B.,
1995. Review: biocatalytic transformations of ferulic acid: an abundant aromatic natural product. Journal of Industrial Microbiology 15, 457±471.
Rovira, A.D., 1969. Plant root exudates. The Botanical Review 35, 35±57.
Shann, J.R., Boyle, J.J., 1994. In¯uence of plant species on in situ rhizosphere degradation. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through Rhizosphere Technology. ACS Symposium Series, vol. 563. American Chemical Society, Washington, DC, pp. 70±81.
Siqueira, J.O., Nair, M.G., Hammerschmidt, R., Sa®r, G.R., 1991. Signi®cance of phenolic compounds in plant±soil±microbial sys-tems. Critical Reviews in Plant Sciences 10, 63±121.
Sparling, G.P., Ord, B.G., Vaughan, D., 1981. Changes in microbial biomass and activity in soils amended with phenolic acids. Soil Biology and Biochemistry 13, 455±460.
Sundin, P., Waechter-Kristensen, B., JenseÂn, P., 1995. Eects of saprophytic bacteria in the closed hydroponic culture of green-house crops. Acta Horticulturae 396, 243±250.
Tillett, R., Walker, J.R.L., 1990. Metabolism of ferulic acid by a Penicilliumsp. Archives of Microbiology 154, 206±208.
Toms, A., Wood, J.M., 1970. The degradation oftrans-ferulic acid byPseudomonas acidovorans. Biochemistry 9, 337±343.
Turner, J.A., Rice, E.L., 1975. Microbial decomposition of ferulic acid in soil. Journal of Chemical Ecology 1, 41±58.
Vaughan, D., Jones, D., Ord, B.G., 1993. Amelioration byVolutella ciliataof the phytotoxicity of vanillic acid towards the growth of Pisum sativumL. Soil Biology and Biochemistry 25, 11±17. Vaughan, D., Sparling, G.P., Ord, B.G., 1983. Amelioration of the
phytotoxicity of phenolic acids by some soil microbes. Soil Biology & Biochemistry 15, 613±614.
Waechter-Kristensen, B., Sundin, P., JenseÂn, P., 1994. Degradation of phenolic acids by bacteria isolated from hydroponic tomato culture with circulating nutrient solution. Acta Horticulturae 381, 611±614.
Walton, B.T., Hoylman, A.M., Perez, M.M., Anderson, T.A., Johnson, T.R., Guthrie, E.A., Christman, R.F., 1994. Rhizosphere microbial communities as a plant defense against toxic substances in soils. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through Rhizosphere Technology, ACS Symposium Series, vol. 563. American Chemical Society, Washington, DC, pp. 82±92.
Yu, J.Q., Matsui, Y., 1993. Extraction and identi®cation of phyto-toxic substances accumulated in nutrient solution for the hydro-ponic culture of tomato. Soil Science and Plant Nutrition 39, 691±700.
