SEARCH

SEARCH BY CITATION

Keywords:

  • Agrobacterium radiobacter;
  • Humic acids;
  • Fulvic acids;
  • Pseudomonas aeruginosa;
  • Rhizobium radiobacter;
  • Siderophores;
  • Antagonism;
  • Iron;
  • Paper sludge compost;
  • Pythium ultimum

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We investigated the in vitro influence of humic substances (HS) extracted from de-inking paper sludge compost on the inhibition of Pythium ultimum by two compost bacteria, Rhizobium radiobacter (Agrobacterium radiobacter) and Pseudomonas aeruginosa. When low concentrations (5 or 50 mgzl−1) of HS were added to the culture medium, fungal inhibition by R. radiobacter significantly increased (P < 0.01) by 2–3%. In contrast, these low levels of HS had no effect on P. ultimum inhibition by P. aeruginosa. The Fe, chelated by HS, was in part responsible for the decrease of P. ultimum inhibition by the bacteria when increasing amounts of HS were added in the culture medium. The addition of 500 mgl−1 of humic acids isolated from de-inking paper sludge compost or from fossil origin completely eliminated the inhibition of P. ultimum by R. radiobacter. This Fe effect also stimulated growth of R. radiobacter and reduced its siderophore production in a minimal medium supplemented with HS as sole source of Fe. The results showed that HS influence microbial antagonism when added to a culture medium. However, this effect varies with different factors such as the type of bacteria, concentration of HS, molecular weight and Fe content.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Application of compost to soil affects fertility of soil and plant growth, mainly by improving soil physical and chemical properties [1]. Several authors [2–4] reported that compost amendment always affects soil or rhizosphere microbial composition and activity. Addition of compost to soil stimulates the proliferation of rhizobacteria producing siderophores or antagonists to many soil-borne phytopathogenic fungi [2]. However, the factors that stimulate such specific groups of rhizobacteria remain to be elucidated. Both the kind of organic matter and its state of decomposition seem to play major roles [2,5,6]. The ultimate products of composting are humic substances (HS). These relatively stable organic compounds are the major ones that microorganisms encounter within mature compost or when these organic amendments are added to soil. HS can influence microorganisms indirectly by their cationic exchange capacity, which is five times higher than in soil minerals [1]. With this characteristic, HS can supply essential cations such as chelated Fe [7,8] or can chelate toxic concentrations of Cu [9], thus facilitating microbial growth. HS may also directly affect microbial metabolism when their molecular size is adequate for uptake [10]. Fulvic acids (FA) have lower molecular weight than humic acids (HA) and they have a greater influence on growth and physiological activities of many organisms [11]. In fact, HS can influence microbial enzymatic activities [12–15], respiration [16] and nutrient uptake by enhancing membrane permeability [14,15]. Substantial amounts of de-inking paper sludge are produced annually by paper-mills. This sludge can be combined with poultry manure to produce compost [17]. When used in a mixture of compost and perlite (1:2 v/v) the de-inking paper sludge compost (DPSC) was suppressive to the cucumber (Cucumis sativa cv. Straight Eight) damping-off caused by Pythium ultimum[18]. In comparison with three other commercial composts, the high suppressive effect of the de-inking paper sludge compost was associated in part to its high microbial activity and low iron content [18]. The aim of the present work was to examine the in vitro influence of FA and HA from de-inking paper sludge compost on microbial antagonism. We also tested the effects of three fractions of DPSC-HA ranging between 1000 and 10000, 10000 and 100000 and >100000 Da. The soil root pathogen P. ultimum and two bacteria isolated from compost, Rhizobium radiobacter (Agrobacterium radiobacter) and Pseudomonas aeruginosa, were used to investigate the effect of HS on microbial antagonisms.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Properties of de-inking paper sludge compost

A 29 month old compost of de-inking paper sludge and chicken manure was used [17]. The compost was air-dried and completely pulverized and sieved (2 mm). The pH was determined by mixing 10 g of compost with 20 ml of deionized water [19]. Compost humidity was determined after drying 24 h at 105 °C. Ash content was determined after combustion of compost samples at 550 °C in a muffle furnace for 17 h. Organic matter content was obtained by subtracting ash content from compost dry matter. Total C and N of compost samples previously air-dried and milled at 2 mm were measured with a Leco CNS 1000 elemental analyser (CNS-1000, Leco, St. Joseph, MI, USA). These physico-chemical properties of DPSC were measured in triplicate (see Table 1).

Table 1.  Physical and chemical properties of de-inking paper sludge compost
  1. Values are means ± standard error (n= 3).

pH7.7 ± 0.1
Humidity (%)56.4 ± 0.4
Ash (gkg−1)602.0 ± 3.0
Organic matter (gkg−1)398.0 ± 3.0
Total C (gkg−1)220.0 ± 1.0
Total N (gkg−1)8.0 ± 0.1
C:N ratio27.5 ± 0.1

2.2Humic substances

DPSC-fulvic acids (DPSC-FA) and DPSC-humic acids (DPSC-HA) were extracted and purified as follows. A 50 g sample of DPSC was treated for 1 h with 0.05 N HCl and extracted with 0.5 N KOH for 24 h under an atmosphere of N2 at room temperature, in a 1/10 (w/v) ratio. The resulting suspension was centrifuged (1500g, 60 min), and the supernatant, neutralized to pH 7, was subjected to three consecutive freezing–thawing cycles [20] followed by centrifugation. The HA (precipitate) and FA (supernatant) were separated by acidification of the extract to pH 2.0. HA were then centrifuged off (1500g, 60 min), suspended in twice their volume of distilled water and re-dissolved at pH 9.0 by the addition of 1 N KOH. The precipitation–dissolution procedure was repeated until the supernatant at pH 2 was almost colorless, indicating the absence of FA. The average yield of HA obtained after seven extraction cycles was 1.8%. One half of the HA obtained by centrifugation at 1500g was then fractionated into three molecular weight fractions (see Section 2.3) and the other half of HA was thoroughly washed with distilled water by using Amicon YC05 Diaflo ultrafiltration membrane. These latter HA in K salts form were then freeze-dried and stored in the dark at −20 °C without any molecular weight fractionation.

The FA solution, after adjustment to pH 2.0, was passed through Supelite XAD-8 resin 250–400 μm (Sigma–Aldrich, Ont., Canada), previously purified and activated by refluxing in a Soxhlet apparatus [21]. The adsorbed fulvic matter was eluted with 0.1 N NaOH and was subsequently passed on a H+ resin 150–300 μm (Sigma–Aldrich, Ont., Canada). FA in their hydrogen form were then freeze-dried and stored in the dark at −20 °C.

Commercial fossil FA prepared as Na salt by acidification of alkali-extracted leonardite (fossil-HA), were purchased from Aldrich Chemical Company (Milwaukee, USA).

2.3Fractionation of humic acids from DPSC

The HA precipitated at pH 2.0 were resuspended in Tris buffer (30 mM trihydroxymethylaminomethane and 150 mM KCl, pH 8.4 and ionic strength I= 0.15) and fractionated using Amicon ultrafiltration membranes (Millipore, Bedford, MA, USA) fitted into continuously stirred filtration cells. Fractions were obtained in the molecular weight ranges between 1000 and 10000, 10000 and 100000 and >100000 Da. During the ultrafiltration, the pressure described by the manufacturer was applied and the pH and ionic strength of the solutions were maintained at the levels previously indicated. The concentrates of the three fractions, contained in Amicon ultrafiltration cells fitted with an YC05 Diaflo ultrafiltration membrane (nominal molecular weight cut-off 500 Da), were thoroughly washed with distilled water until the ultrafiltrates had a pH 7.0 and gave a negative chloride (Cl) test with AgNO3. The HA in K salts form were then freeze-dried and stored in the dark at −20 °C.

2.4Elemental analysis of humic substances

Table 2 shows the total contents of some elements present in HS determined in triplicate by Inductively Coupled Plasma (ICP) spectrometry following nitric/hydrochloric acid digestion [22].

Table 2.  Total content of some elements present in DPSC-FA, DPSC-HA and fossil-HA
ElementsDPSC-FADPSC-HA (mgkg−1)Fossil-HA
  1. Values are means ± standard error (n= 3).

Na344.3 ± 0.8905.6 ± 1.998.802.2 ± 1.5
K254.9 ± 0.650363.8 ± 1.9375.6 ± 1.0
Ca116.3 ± 1.0175.8 ± 2.73797.1 ± 0.7
Mg24.6 ± 0.334.4 ± 0.44122.3 ± 0.9
Fe1176.0 ± 1.81918.3 ± 0.312870.7± 1.4

2.5Microorganisms

The bacteria R. radiobacter (A. radiobacter) and P. aeruginosa and the soil root phytopathogenic fungus P. ultimum 2499 were used. The two bacteria had been isolated from de-inking paper sludge compost [23] and the fungus was obtained from Dr. Nicole Benhamou (Départment de Phytologie, Université Laval, Que., Canada). The bacterial strains were selected for their ability to clearly inhibit P. ultimum 2499 growth on the Rhizosphere agar medium (RSM) [24] free of Fe or supplemented with Fe (Table 3). Also, bacteria were tested for their capacity to produce siderophores [25] and cyanide (HCN) [26]. Bacteria were considered positive for the production of siderophores or HCN when an orange halo on the chrome azurol S (CAS) agar plates or a change in color from yellow to orange–brown on the filter paper were observed, respectively. These tests were done in triplicate (see Table 3).

Table 3.  Some in vitro characteristics of the two antagonistic bacteria isolated from de-inking paper sludge compost
CharacteristicR. radiobacterP. aeruginosa
  1. Value are means ± standard error (n= 3).

% inhibition of growth of P. ultimum on  
Rhizosphere agar medium (RSM)20.4 ± 0.146.8 ± 0.1
RSM without Fe41.9 ± 0.268.2 ± 0.3
RSM with 200 μM of FeCl30.026.0 ± 0.1
Production of siderophores++
Production of cyanide (HCN)+

2.6Effect of humic substances on microbial antagonism

In bacterial antifungal assays involving Fe competition, the glassware used had been soaked at least for 24 h in 6 N HCl to remove residual Fe and was rinsed with distilled H2O. Stock solutions of 50 mgl−1 of DPSC-FA, DPSC-HA and fossil-HA were prepared in distilled H2O. The pH of solutions, were adjusted to 7 with HCl or KOH before autoclaving (121 °C, 20 min). A stock solution of 10 mM FeCl3· 6H2O was prepared in 10 mM HCl and filter sterilized through a 0.22 μm Millipore membrane. Specific quantities of stock solutions were added to the autoclaved agar medium used for bacterial antifungal assays to obtain the target concentration wanted. Bacteria were grown on 10% TSA (tryptic soy agar, Difco) for two days before transferring one colony into 5 ml of RSM liquid medium. Inoculated tubes were incubated at 28 °C and were agitated at 250 rev min−1 until an optical density of 0.6 at 600 nm was reached. The cells were then harvested, washed three times and resuspended to an optical density of 0.3 at 600 nm. This final suspension was equivalent to approximately log 6 CFUml−1 and was used as inoculum. Plugs (0.5 cm diam) cut at the edge of a young colony of P. ultimum grown on RSM medium for two days were used as inoculum.

Bacterial antifungal assays were made on RSM agar [24]. Fe was added to this medium in concentration equal to 20 μM of FeCl3. In the bacterial antifungal assay related to Fe competition, no FeCl3 was added to RSM medium. Also the Fe, present in the casamino acid solution added to this culture medium, had been removed [27]. Two equidistant drops of 10 μl of bacterial inoculum were placed at 3.5 cm from the middle of the Petri dish. Bacteria were then incubated at 28 °C for two days before a 0.5 cm diameter plug of P. ultimum was placed in the middle of the dish. Control plates without bacteria were centrally inoculated with the fungus. Plates were incubated at 28 °C and the inhibition zones were measured after two days, i.e. after the time required for fungal radial growth to reach bacterial inoculation points in the control.

Fungal inhibition in % was calculated as follow:

  • image

Three different bacterial antifungal experiments were performed in the presence of various HS. All assays were performed in four replicates. In the first experiment, RSM medium was supplemented with different concentrations (5, 50 or 500 mgl−1) of HS (either DPSC-FA, DPSC-HA or fossil-HA). Controls consisted of RSM medium without HS. In the second experiment, RSM medium was supplemented with different concentrations (10, 20, 30, 40 or 50 mgl−1) of HS (either DPSC-FA, DPSC-HA or fossil-HA) or of DPSC-HA fractions (1000–10000, 10000–100000 and >100000 Da). Controls consisted of RSM medium free of HS. In the third experiment, the bacterial antifungal assays were made on RSM medium free of Fe but supplemented with different concentrations (5, 50 or 500 mgl−1) of HS (either DPSC-FA, DPSC-HA or fossil-HA) in the presence or absence of 200 μM of FeCl3. Controls consisted of RSM medium free of HS and Fe supplementation or RSM medium free of HS but supplemented with 200 μM of FeCl3.

2.7Effects of humic substances on growth of R. radiobacter and siderophores production

Because of the limited supply of HS extracted from DPSC, this experiment was only carried out with R. radiobacter. However, 10 mM of FeSO4· 7H2O in distilled H2O was used as Fe stock solution. The liquid AT [28] minimal medium free of Fe (per liter of H2O: 10.9 g of KH2PO4, 160 mg MgSO4· 7H2O, 10 mg CaCl2· 2H2O, 2 mg MnCl2· 4H2O, 1 g (NH4)2SO4, 2 g glucose) was used for growth experiments. To test the effect of inorganic Fe on bacterial growth, AT medium was supplemented with filter sterilized (0.22-μm) Fe stock solution to obtain the required tested concentration.

R. radiobacter was first grown on 10% tryptic soy agar for two days before transferring one colony into a tube containing 5 ml of liquid AT medium. Inoculated tubes were incubated at 28 °C and agitated at 250 revmin−1 on a rotary shaker until optical density of 0.6 at 600 nm was reached. The cells were then harvested by centrifugation (1500g, 4 min, 4 °C), washed three times and resuspended in liquid AT medium and then diluted 1000-fold in this medium. The final cell suspension was equivalent to approximately log 6 CFUml−1. Aliquots of 5 ml of this dilute suspension were used to inoculate growth treatment media. To determine the effect of HS on bacterial growth and siderophores production, bacteria were cultured in 500-ml Erlenmeyer flasks containing 250 ml of liquid AT medium. HS were added as follows: (1) 50 mgl−1of either DPSC-FA, DPSC-HA or fossil-HA, which were equivalent to 1.05, 1.72 and 11.52 μM of Fe, respectively; and (2) a specific quantity of either DPSC-FA, DPSC-HA or fossil-HA equivalent to 1.72 μM Fe. In the control flasks, the 250 ml AT medium received: (1) no inorganic Fe and no HS (free of any source of Fe) (2) the equivalent of 1.72 μM FeSO4 and (3) the equivalent of 20 μM FeSO4. After inoculation, cultures in Erlenmeyer flasks were incubated at 28 °C on a rotary shaker at 250 revmin−1 for three days. R. radiobacter growth and siderophores production were followed by retrieving aliquots of 1.5 ml from each flask at different times over 70 h. Each aliquot was divided as follows: 0.5 ml was used for reading optical density at 600 nm for growth and the remaining 1.0 ml was centrifuged (1500g, 4 min, 4 °C) and the supernatant stored at −20 °C for siderophore determination. Samples were thawed and units of siderophores produced were estimated at an optical density of 630 nm by using the CAS solution [27]. All assays were duplicated.

2.8Statistical analyses

The data of bacterial antifungal assays were subjected to a one-way completely randomized analysis of variance by considering each humic substance and concentration combination as one independent treatment. To compare treatment means to the control, the Dunnett test with P < 0.01 was used [29]. Values of the bacterial growth and siderophore assays were expressed as a mean of two replicates for each treatment. The data were subjected to a two-way factorial analysis of variance with time and treatments as factors. A significant interaction was obtained between the factors time and treatments. Thus, values of growth and siderophores assays were presented graphically as a function of time. Means were compared with the protected Fisher’ s Least significant difference (LSD) test at P < 0.01[29]. Variance was homogeneous for all data sets. All analyses were carried out using the Statistical Analysis System procedure [30].

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Effect of humic substances on microbial antagonism

When RSM medium was supplemented with 5 mg DPSC-FA per liter, P. ultimum inhibition significantly (P < 0.01) increased by 2% compared to the control (Fig. 1). In the presence of 50 mg l−1 of DPSC-FA or DPSC-HA, antifungal activity significantly increased by 2% and 3%, respectively, while 50 mg fossil-HA l−1 significantly decreased this activity by 3% (Fig. 1). In the presence of 500 mgl−1 of DPSC-FA, DPSC-HA or fossil-HA, there was no fungal inhibition (Fig. 1). In the presence of 5 and 50 mgl−1 of DPSC-FA, DPSC-HA or fossil-HA, P. ultimum inhibition by P. aeruginosa was similar to that observed in the control (Fig. 2). Only in the presence of 500 mgl−1 of DPSC-FA and DPSC-HA, fungal inhibition decreased significantly (P < 0.01) by 10.4% and 9.7%, respectively (Fig. 1).

image

Figure 1. Effect of fulvic acids (FA) and humic acids (HA) from de-inking paper sludge compost (DPSC) or from fossil origin (Aldrich) on the inhibition of growth of P. ultimum by R. radiobacter or P. aeruginosa on the RSM medium after two days of incubation at 28 °C. An asterisk indicates that the mean is significantly (P < 0.01) different from the control (0 mgl−1) according to the Dunnet test. Bars are standard errors of means (n= 4).

Download figure to PowerPoint

image

Figure 2. Effect of FA and HA from DPSC or from fossil origin on the inhibition of growth of P. ultimum by R. radiobacter or P. aeruginosa on the RSM medium free of Fe (A, C) or supplemented with 200 μM of FeCl3 (B, D). Fungal growth was measured after two days of incubation at 28 °C. An asterisk indicates that the mean is significantly (P < 0.01) different from the control (0 mgl−1) according to the Dunnet test. Bars are standard errors of means (n= 4).

Download figure to PowerPoint

3.2Effects of humic substances and of DPSC-HA fractions on microbial antagonism

In the presence of 10–50 mgl−1 of DPSC-HA fraction with the molecular weight ranging between 1000 and 10000 Da or of fossil-HA, P. ultimum inhibition by R. radiobacter decreased (P < 0.01) by 2.1–3.5% and by 3.7–6.5% respectively, as compared to the control (results not shown). Levels of DPSC-FA, DPSC-HA and of DPSC-HA fractions with the molecular weight ranging between 10000 to 100000 Da and >100000 Da had no significant effect on fungal inhibition. Inhibition of P. ultimum by P. aeruginosa showed comparable trends.

3.3Effects of humic substances on microbial antagonism in the presence of Fe

The inhibition of P. ultimum by R. radiobacter was similar in the control (RSM without HS or Fe) and in the presence of 5 mgl−1 of DPSC-FA, DPSC-HA or fossil-HA (Fig. 2). Fungal inhibition significantly (P < 0.01) decreased by 7% compared to control in the presence of 50 mgl−1 of fossil-HA (Fig. 2). The addition of 500 mgl−1 DPSC-FA significantly (P < 0.01) reduced fungal inhibition by 14.3% while DPSC-HA and fossil-HA completely eliminated the inhibitory effect of this bacterium compared to the control (Fig. 2). When FeCl3 was added in excess (200 μM) to RSM medium, the antifungal activity of R. radiobacter was completely eliminated in the presence of all tested concentrations of HS and in the control without HS (Fig. 2). The addition of 5 or 50 mgl−1 of DPSC-FA, DPSC-HA or fossil-HA did not affect the inhibition of P. ultimum by P. aeruginosa (Fig. 2). The highest concentration (500 mgl−1) of DPSC-FA, DPSC-HA or fossil-HA significantly decreased P. ultimum inhibition by 7%, 29% and 29%, respectively (Fig. 2). The inhibitory effect of P. aeruginosa was reduced by the addition of 200 μM of FeCl3 to 25.9%, 27.7% and 26.6%, in the presence of 5, 50 and 500 mgl−1 of DPSC-FA, DPSC-HA and fossil-HA, respectively (Fig 2). This reduction was comparable to that observed with the control (26%), which did not receive HS.

3.4Effect of humic substances on R. radiobacter growth and siderophores production

After 30 h of incubation and until the end of the incubation period, growth of R. radiobacter was significantly lower in treatments free of Fe compared to those supplemented with organic or inorganic Fe (Figs. 3(a), 4(a) and 5(a)). However, by comparison to FeSO4 used as sole source of Fe (1.72 or 20 μM), growth of R. radiobacter was always significantly faster in the presence of 50 mgl−1 of HS (DPSC-FA, DPSC-HA or fossil-HA, which corresponded to 1.05, 1.72 and 11.52 μM of Fe, respectively) or when these organic compounds were added to the medium in quantities equivalent to 1.72 μM of Fe after 40 h of incubation and until the end of the incubation.

image

Figure 3. Growth (a) and siderophore production (b) of R. radiobacter at 28 °C in AT minimal medium free of Fe supplemented with: 50 mgl−1 DPSC-FA containing 1.05 μM of Fe (•); 82 mgl−1 DPSC-FA containing 1.72 μM of Fe (○). Controls consisted of AT medium free of Fe (□) or supplemented with 1.72 μM (▵) or 20 μM (▴) of FeSO4. Error bars show standard errors of means (n= 2).

Download figure to PowerPoint

image

Figure 4. Growth (a) and siderophore production (b) of R. radiobacter at 28 °C in AT minimal medium free of Fe supplemented with: 50 mgl−1 DPSC-HA containing 1.72 μM of Fe (•). Controls consisted of AT medium free of Fe (□) or supplemented with 1.72 μM (▵) or 20 μM (▴) of FeSO4. Error bars show standard errors of means (n= 2).

Download figure to PowerPoint

image

Figure 5. Growth (a) and siderophore production (b) of R. radiobacter at 28 °C in AT minimal medium free of Fe supplemented with: 50 mgl−1 fossil-HA containing 11.52 μM of Fe (•); 7.5 mgl−1 fossil-HA containing 1.72 μM of Fe (○). Controls consisted of AT medium free of Fe (□) or supplemented with 1.72 μM (▵) or 20 μM (▴) of FeSO4. Error bars show standard errors of means (n= 2).

Download figure to PowerPoint

Siderophore production by R. radiobacter was significantly higher in treatments free of Fe compared to those supplemented with organic or inorganic Fe (Figs. 3(b), 4(b) and 5(b)). In treatments free of Fe, units of siderophore reached the level of 90% after a total period of 70 h. However, comparing to humic substance treatments as sole source of Fe, siderophore production by R. radiobacter was significantly (P < 0.01) lower in FeSO4 treatments after 40 h of incubation and until the end of the incubation period. In the presence of 1.72 and 20 μM of FeSO4, units of siderophore produced were equivalent to 20% and 0%, respectively, after a period of 70 h. In the presence of DPSC-FA, DPSC-HA and of fossil-HA added in quantities equivalent to 1.72 μM of Fe, units of siderophore produced were equivalent to 40%, 60% and 80%, respectively. In the presence of 50 mgl−1 of DPSC-FA, DPSC-HA and fossil-HA, which were equivalent to 1.05, 1.72 and 11.52 μM of Fe, units of siderophores produced were equivalent to 80%, 60% and 20%, respectively (Figs. 3(b), 4(b) and 5(b)).

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Our results showed that the presence of HS in the culture medium influences microbial antagonism. This is in accordance with previous observations made by Visser [14] who found that soil HS added to selective culture media influenced the number and activities of different physiological groups of microorganisms. Also, many studies [15,31–33] showed that FA have a greater effect on cells biological activities than HA compounds. In fact, a study with radiolabelled molecules indicated that due to their smaller molecular weight, FA entered plant cells while the larger aggregates of HA remained associated with the cell walls [32]. The observed antifungal activity of R. radiobacter slightly increased in the presence of 5 mgl−1 of DPSC-FA, however, a similar effect was observed only after the addition of 50 mgl−1 of DPSC-HA (Fig. 1). In the presence of 50 mgl−1 of fossil-HA, P. ultimum inhibition by this bacterium decreased. Our results thus support previous findings [34,35] indicating that HS of compost origin seems to be more biostimulating than those of fossil origin. Low concentrations (<50 mgl−1) of FA and HA from de-inking paper sludge compost had no effect on P. ultimum inhibition by P. aeruginosa. Low concentrations of fossil-HA also had no effect on this interaction. Bacterial antifungal activity was in general decreased or completely eliminated when the RSM medium was supplemented with 500 mgl−1 of HS of either DPSC or fossil origin (Fig. 1). This suggests that the influence of HS on microbial antagonism varies with the organism tested (R. radiobacter or P. aeruginosa) and also with the origin of the HS (DPSC or fossil), concentrations (5, 50 or 500 mgl−1) and molecular weight (i.e. DPSC-FA < DPSC-HA < fossil-HA). It is also possible that the method of extracting DPSC-HA compared to the one used for fossil-HA, may have influenced bacterial antifungal activities. Our results corroborate previous statement [33] that the effect of HS on growth of plants and microorganisms within different experimental contexts can vary according to all of these previously mentioned factors in addition to their structure. P. ultimum inhibition by bacteria generally tended to decrease when RSM medium was supplemented with increased concentrations of HS used as sole source of Fe. This negative effect was most pronounced with fossil-HA followed by DPSC-HA and then by DPSC-FA. This pattern is also in agreement with their Fe content (Table 2). The lower Fe content of DPSC-FA probably explains why 500 mgl−1 of these compounds did not completely cancel P. ultimum inhibition by R. radiobacter in RSM medium as observed with DPSC-HA and fossil-HA (Fig. 2). However, when inorganic Fe was added in excess to RSM medium containing HS, antifungal activity of R. radiobacter was completely cancelled while that of P. aeruginosa was similar to control. The fact that R. radiobacter could no more suppress P. ultimum growth under these conditions indicates that this bacterium inhibits the fungus mainly by being the most competitive for Fe via its siderophore production. In contrast, P. aeruginosa seems to possess more than one mechanism for limiting growth of P. ultimum. In fact, this bacterium also produces HCN (Table 3) and lytic enzymes-like chitinases [23] in addition of well known antifungal antibiotics [36].

Fe is essential for microbial growth and that is why R. radiobacter growth was the slowest in the AT minimal medium free of this element. It is also under these conditions, that R. radiobacter produced its highest level of siderophore in order to survive by capturing the remaining traces of Fe. However, when FeSO4 or FA and HA from DPSC or fossil origin were added as sole source of Fe to AT medium, bacterial growth was faster. Also, in parallel, siderophore production by R. radiobacter decreased mostly in the presence of FeSO4 but also with HS additions. These results indicate that as with FeSO4, the Fe, chelated to HS, clearly influenced R. radiobacter growth and siderophore production. Siderophores of this bacterium were thus probably capable of capturing the Fe chelated on FA and HA from DPSC or fossil origin. This also means that the stability constant of Fe complexed to R. radiobacter siderophores was probably higher than the one existing between this element and the organic compounds. In fact the stability constant of Fe complexes with siderophores are generally higher than those calculated for Fe-humate complexes [1,37]. No real differences were observed between R. radiobacter growth curves in the presence of HS equivalent to 50 mgl−1 and 1.72 μM of organic Fe or in the presence of 1.72 and 20 μM of FeSO4. However, growth of R. radiobacter in AT medium free of Fe was generally more stimulated by HS rather than by FeSO4 supplements. Effectively, by comparison, growth of R. radiobacter was enhanced much more in the presence of DPSC-FA, DPSC-HA and fossil-HA at concentrations equivalent to 1.72 μM of organic Fe than in the presence of 1.72 μM of inorganic Fe. However, siderophore production by R. radiobacter was higher in the presence of HS than with FeSO4 additions. In the presence of extra FeSO4 (20 μM), siderophore production was completely inhibited. Interestingly, these results therefore suggest that the Fe chelated to HS is not available as inorganic Fe. Thus, the biostimulating effect of HS may not be related only to its Fe content. HS have possibly enhanced R. radiobacter growth by other indirect (i.e. nutritional, CEC, etc.) or direct mechanisms (i.e. electron transfer in respiration, enhancement of membrane permeability, etc.) frequently reported in literature. However, another interesting observation can be made from these results. The availability of Fe, chelated to HS, seems to vary with their nature since after 70 h incubation approximately 80%, 60% and 40% units of siderophores were measured with fossil-HA, DPSC-HA and DPSC-FA, respectively, used at concentrations equivalent to 1.72 μM of Fe. Accordingly, this effect is possibly related to their stereochemical structure [38]. In effect, highly aromatic HS are stereochemically complex and tend to prevent easy exchange of Fe from the humic material to the naturally occurring Fe chelators. It is therefore not surprising that R. radiobacter siderophore production was more reduced with the FA compounds (DPSC-FA) than with HA compounds (DPSC-HA and fossil-HA). Also, these results again confirm that HA from DPSC are less humified or complexed than those from fossil origin. In the presence of 50 mgl−1 of DPSC-FA, DPSC-HA and fossil-HA, about 80%, 60% and 20% siderophore units were detected. This variation in siderophore production may be easily explained by the fact that these specific quantities of DPSC-FA, DPSC-HA and fossil-HA were equivalent to 1.05, 1.72 and 11.52 μM of organic Fe, respectively. These results indicate that as the organic Fe content increases, bacterial siderophore production decreases, confirming the important influence of Fe content in HS on growth of R. radiobacter and siderophore production and also on microbial antagonism mediated by Fe competition. Soil HS are usually present in physiologically inactive forms. However, chemical, biochemical and microbiological conditions of rhizosphere differ widely from those of bulk soil [39], and this can lead to changes in the dynamics and structure of humified organic matter. In fact, when humic macromolecules were treated with acetic acid, small-size humic fractions were obtained and thus also stimulated specific biological properties in plants [40,41]. Therefore, in rhizosphere, soil HS or those originating from addition of compost possibly play an important role in microbial activities such as antagonism and siderophore production.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We thank Dr. Josée Fortin, Jean Martin and Geneviève Couture for their help, constructive comments and discussions. This work was supported in part by grants from the National Sciences and Engineering Research Council of Canada (NSERC), and from the “Fonds pour la Formation de Chercheurs et l’ Aide à la Recherche” (FCAR) to H.A. and by Stadacona (Que., Canada) to C.J.B.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
  • [1]
    Stevenson, F.J. Extraction, Fractionation, and General Chemical Composition of Soil Organic Matter. Humus Chemistry: Genesis, Composition, Reactions 1994, Wiley-Interscience, New Yorkpp. 26–54.
  • [2]
    de Brito Alvarez, M.A., Gagné, S., Antoun, H. (1995) Effect of compost on rhizosphere microflora of the tomato and on the incidence of plant growth-promoting rhizobacteria. Appl. Environ. Microbiol. 61, 194199.
  • [3]
    Pera, A., Vallini, G., Sireno, I., Lorella Bianchin, M., de Bertoldi, M. (1983) Effect of organic matter on rhizosphere microorganisms and root development of sorghum plants in two different soils. Plant Soil 74, 318.
  • [4]
    Schisler, D.A., Linderman, R.G. (1989) Influence of humus-rich organic amendments to coniferous nursery soils on douglas-fir growth, damping-off and associated soil microorganisms. Soil Biol. Biochem. 21, 403408.
  • [5]
    Boehm, M.J., Madden, L.V., Hoitink, H.A.J. (1993) Effect of organic matter decomposition level on bacterial species diversity and composition in relationship to pythium damping-off severity. Appl. Environ. Microbiol. 59, 41714179.
  • [6]
    Linderman, R.G. (1989) Organic amendments and soil-borne diseases. Can. J. Plant Pathol. 11, 180183.
  • [7]
    Burk, D., Lineweaver, H., Horner, K. (1932) Iron in relation to the stimulation of growth by humic acid. Soil Sci. 33, 413435.
  • [8]
    Burk, D., Lineweaver, H., Horner, K. (1932) The physiological nature of humic acid stimulation of Azotobacter growth. Soil Sci. 33, 455487.
  • [9]
    Toledo, A.P.P., Tundisi, J.G., D’ Aquire, V.A. (1980) Humic acid influence on the growth and copper tolerance of Chlorella sp. Hydrobiologia 71, 261263.
  • [10]
    A. Albuzio G. Dell’ Agnola D. Dibona G. Concheri S. Nardi Humic constituent of forest soils as plant growth regulating substances M.G. Paoletti W. Foissner D.C. Coleman Soil Biota, Nutrient Cycling, and Farming Systems 1993 Lewis publishers Boca Raton, FL 15 25.
  • [11]
    D. Vaughan R.E. Malcolm Influence of humic substances on growth and physiological processes D. Vaughan R.E. Malcolm Soil Organic Matter and Biological Activity 1985 Kluwer Academic Publishers Dordrecht 37 75.
  • [12]
    de Haan, H. (1976) Evidence for the induction of catechol-1,2-oxygenase by fulvic acid. Plant Soil 45, 129138.
  • [13]
    Hwang, S. R.L. Tate III (1997) Humic acid effects on 2-hydroxypyridine metabolism by starving Arthrobacter crystallopoietes cells. Biol. Fertil. Soils 25, 3640.
  • [14]
    Visser, S.A. (1985) Effect of humic acids on numbers and activities of microorganisms within physiological groups. Org. Geochem. 8, 8185.
  • [15]
    Visser, S.A. (1985) Physiological action of humic substances on microbial cells. Soil Biol. Biochem. 17, 457462.
  • [16]
    Lovley, D.R., Coates, J.D., Blunt-Harris, E.L., Phillips, E.J.P., Woodward, J.C. (1996) Humic substances as electron acceptors for microbial respiration. Nature 382, 445448.
  • [17]
    Charest, M.-H., Beauchamp, C.J. (2002) Composting of de-inking paper sludge with poultry manure at three nitrogen levels using mechanical turning: behavior of physico-chemical parameters. Bioresource Technol. 81, 717.
  • [18]
    Lessard, A. (1999) Les caractéristiques chimiques et biologiques des composts bénéfiques à la croissance du concombre (Cucumis spp.) et suppressifs à la fonte du semis causée par le Pythium ultimum. Mémoire de maîtrise, Université Laval, Qué., Canada
  • [19]
    A. Karam Chemical properties of organic soils M.R. Carter Soil Sampling and Methods of Analysis 1993 Lewis Publishers Boca Raton 469 471.
  • [20]
    Forsyth, W.G.C., Fraser, G.K. Freezing as an aid in the drying and purification of humus and allied materials. Nature. 1, 1947, 607.
  • [21]
    Thurman, E.M., Malcolm, R.L. (1981) Preparative isolation of aquatic humic substances. Environ. Sci. Technol. 15, 463466.
  • [22]
    Barnhisel, R., Bertsch, P.M. Aluminum, Page, A.L., Ed. Methods of Soil Analysis part 2, Chemical and Microbiological Propertiessecond ed. 1982, Soil Science Society of America, Madison, 275–300.
  • [23]
    Charest, M-H., Antoun, H., Beauchamp, C.J. (2004) Dynamics of water-soluble carbon substances and microbial populations during the composting of de-inking paper sludge. Bioresource Technol. 91, 5367.
  • [24]
    Buyer, J.S., Sikora, L.J., Chaney, R.L. (1989) A new growth medium for the study of siderophore-mediated interaction. Biol. Fertil. Soils 8, 97101.
  • [25]
    Alexander, D.B., Zuberer, D.A. (1991) Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol. Fertil. Soils 12, 3945.
  • [26]
    Bakker, A.W., Schippers, B. (1987) Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas spp.-mediated plant growth-stimulation. Soil Biol. Biochem. 19, 451457.
  • [27]
    Schwyn, B., Neilands, J.B. (1987) Universal chemical assay for detection and determination of siderophores. Anal. Biochem. 160, 4756.
  • [28]
    Dion, P., Bélanger, C., Xu, D., Mohammadi, M. Effect of acetosyringone on growth and oncogenic potential of Agrobacterium tumefaciens.
  • [29]
    Steel, R.G.D., Torrie, J.H. Multiple comparisons. Principles and Procedures of Statistics: a biometrical approach 1980, McGraw-Hill, New York pp. 172–191.
  • [30]
    SAS Institute, (1988) SAS/STAT User’ s Guide, Release 6.03, SAS Institute Inc., Cary
  • [31]
    Prakash, A, Rashid, M.A., Jensen, A., Subba Rao, D.V. (1973) Influence of humic substances on the growth of marine phytoplankton: diatoms. Limnol. Oceanogr. 18, 516524.
  • [32]
    Vaughan, D. (1974) Some effects of humic acid on two different biological systems. Plant Soil 40, 429434.
  • [33]
    D. Vaughan R.E. Malcolm B.G. Ord Influence of humic substances on biochemical processes in plants D. Vaughan R.E. Malcolm Soil Organic Matter and Biological Activity 1985 Kluwer Academic Publishers Dordrecht 78 108.
  • [34]
    Valdrighi, M.M., Pera, A., Scatena, S., Agnolucci, M., Vallini, G. (1995) Effects of humic acids extracted from mined lignite or composted vegetable residues on plant growth and soil microbial populations. Compost. Sci. Util. 3, 3038.
  • [35]
    Valdrighi, M.M., Pera, A., Agnolucci, M., Frassinetti, S., Lunardi, D., Vallini, G. (1996) Effects of compost-derived humic acids on vegetable biomass production and microbial growth within a plant (Cichorium intybus)-soil system: a comparative study. Agr. Ecosyst. Environ. 58, 133144.
  • [36]
    Défago, G., Haas, D. Pseudomonads as antagonists of soilborne plant pathogens: modes of action and genetic analysis.
  • [37]
    Cline, G.R., Powell, P.E., Szaniszlo, P.J., Reid, C.P.P. (1982) Comparison of the abilities of hydroxamic acid, synthetic and other natural organic acids to chelate iron and other ions in nutrient solution. Soil Sci. Soc. Am. J. 46, 11581164.
  • [38]
    Piccolo, A., Pietramellara, G., Celano, G. (1993) Iron extractability from iron-humate complexes by a siderophore and a mixture of organic acids. Can. J. Soil Sci. 73, 293298.
  • [39]
    Z. Varanini R. Pinton Direct versus indirect effects of soil humic substances on plant growth and nutrition R. Pinton Z. Varanini P. Nannipieri The Rhizosphere: Biochemistry and Organic Substances at the Soil–Plant Interface 2001 Marcel Deckker New York 141 157.
  • [40]
    Nardi, S., Arnoldi, G., Dell’ Agnola, G. (1988) Release of the hormone-like activities from Allobophora rosea (Sav.) and Allobophora caliginosa (Sav.) feces. Can. J. Soil Sci. 68, 563567.
  • [41]
    Nardi, S., Concheri, G., Dell’ Agnola, G., Scrimin, P. (1991) Nitrate uptake and ATPase activity in oat seedlings in the presence of two humic fractions. Soil Biol. Biochem. 23, 833836.