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Journal of Environmental Quality 31:1339-1348 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Waste Management

Growth of Alfalfa in Sludge-Amended Soils and Inoculated with Rhizobia Produced in Sludge

F. Ben Rebaha, D. Prévostb and R.D. Tyagi*,a

a Université du Québec, Institut national de la recherche scientifique (INRS-Eau), 2700 rue Einstein, Sainte Foy, QC, Canada G1V 4C7
b Centre de recherche et de dévolppement sur les sols et les grandes culture, Agriculture et Agraoalimentaire Canada, Sainte Foy, QC, Canda G1V 2J3

* Corresponding author (tyagi{at}inrs-eau.uquebec.ca)

Received for publication July 6, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The efficiency of rhizobial inoculants produced in wastewater sludge used as a growth medium and as a carrier was compared with that of inoculants produced in yeast mannitol broth (YMB) medium and by using peat as a carrier. Alfalfa (Medicago sativa L.) plants were inoculated with solid and liquid Sinorhizobium meliloti inoculants and grown in pots containing two soil types (Kamouraska clay soil and Saint-André sandy soil). The effect of various levels of sludge amendment (60 and 120 kg N/ha) and nitrogen fertilizer (60 kg N/ha) was also studied. The sludge-based inoculants showed the same symbiotic efficiency (nodulation and plant yield) as YMB-based inoculants. The inoculation increased the nodulation indexes from 4–6 to 8–12, and the rhizobial number from 103 (uninoculated soils) to 106–107 cells/g in inoculated soils. However, the shoot dry weights and the nitrogen contents were not increased significantly by the inoculation. Applying sludge as an amendment enhanced the rhizobial number in soils from 103 to 104 cells/g and improved significantly the plant growth (shoot dry weights and nitrogen contents). This improvement increased with sludge rate and with the cut (three cuts). Compared with sludge, N fertilizer gave lower plant yields. The nodulation was not affected by sludge and N-fertilizer application. The texture and physico–chemical properties of soil were found to affect the yield and nitrogen content of the plants. In this study, macroelements and heavy metals were at acceptable levels and were not considered to be negative factors.

Abbreviations: MPN, most probable number • YMB, yeast mannitol broth


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
WASTEWATER TREATMENT produces a large amount of sludge. The disposal and/or utilization of wastewater sludge is an increasing environmental problem. The sludge handling and disposal cost varies from 30 to 40% of the capital cost and represents about 50% of the operating costs of a typical wastewater treatment facility (Vesilind, 1974). Landfilling (Bradley et al., 1992; USEPA, 1993), ocean discharge (Gross, 1993), and land application (Golueke, 1992) are conventional sludge disposal methods. Applying sludge to agricultural soils improves the soil physical and biological properties because it contains organic matter and plant nutrients (Pagliai et al., 1981; Wei et al., 1985). In Canada, 29% of municipal sludge is used in agricultural soils (Webber, 1988). It can enhance microbial growth in sols (Pichtel and Hayes, 1990) such as the rhizobia (Kinkle et al., 1987; Heckman et al., 1987a,b). Rhizobia are important soil bacteria because of their ability to supply legumes with nitrogen, through the process of nodulation and symbiotic nitrogen fixation. However, wastewater sludge often contains materials potentially toxic for rhizobia, such as heavy metals and soluble salts (Madariaga and Angle, 1992; McGrath et al., 1988; Giller et al., 1989). According to Huang et al. (1974), low metal concentration stimulates slightly the nodulation, but higher concentrations can affect the nodule number and reduce the root and shoot weight. McGrath et al. (1988), reported that sludge application reduced nitrogen fixation and white clover (Trifolium repens L.) growth. In order to reduce the toxicity in agriculture practice, the heavy metal concentration should not exceed the recommended levels (Gouvernement du Québec, 1991).

Inoculating legumes with commercial preparation of rhizobia is a common agriculture practice. Recently, we demonstrated that wastewater sludge can be used as an effective medium for rhizobial growth (Ben Rebah et al., 2001, 2002a). The dehydrated sludge was also tested as a carrier that can sustain high numbers of S. meliloti, the microsymbiont of the legume alfalfa (Ben Rebah et al., 2002b). This new application constitutes an additional and suitable alternative for wastewater sludge recycling.

Legume inoculant products should ensure the nodulation and the capacity of nitrogen fixation efficiency of rhizobia during plant growth. Therefore, in order to evaluate the quality of sludge-produced inoculants, the effects of inoculation with sludge-grown rhizobia and soil amendment with sludge were investigated on nodulation and alfalfa growth.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soil and Sludge Origins
Two soil samples were used in this study. The soils were from Saint-André (sandy soil) and Kamouraska (clay soil), located in the province of Quebec. The sludge was an aerobically digested secondary sludge from the Communauté Urbaine de Québec (CUQ) wastewater treatment plant.

Chemical Analysis
Sludge Analysis
Total organic carbon, total nitrogen, total phosphorus, ammonium (NH4–N), and nitrate (NO-3) were determined according to the standard methods of the American Public Health Association (1992). Heavy metals (Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) and mineral salts (Mg, Ca, and K) were determined after digestion using Thermo Jarell Ash (Franklin, MA) inductively coupled plasma (American Public Health Association, 1992).

Soil Analysis
The soil pH was measured in water and characteristics (total nitrogen, total phosphorus, and total organic carbon) were determined using the analytical methods as indicated above for the sludge. However, heavy metals and mineral salts were determined according to the Mehlich III method (Tran and Simard, 1993).

Estimation of Rhizobia Population Size
The most probable number (MPN) method (Vincent, 1970) was used to estimate the number of rhizobia in soils by using dilution series and alfalfa as a host. Five replicate plant infection growth pouches (Mega International, Minneapolis, MN) were inoculated with 1-mL aliquots at each dilution step and pouches placed in a controlled growth cabinet (with 16-h days at 20°C and 15°C nights). After 28 d of growth, roots were observed for nodulation. The numbers of rhizobia were calculated using a MPNES computer program (Woomer et al., 1990).

Procedures and Experimental Design
Soil Preparation
Soils were prepared as recommended by Conseil des productions végétales du Québec (1994) for alfalfa growth. Number of indigenous rhizobia was determined by the MPN method. Both soils were amended with lime equivalent to 14 and 12.2 Mg/ha, respectively, for Kamouraska and Saint-André to increase the pH to 6.5. Standard mineral fertilizer (46% P2O5) equivalent to 40 and 20 kg P2O5/ha was added, respectively, for Kamouraska and Saint-André soil. Because of the soil composition, no K was added in this experiment. For some treatments, soils were amended with secondary sludge applied at levels of 60 and 120 kg of available N/ha, calculated according to USEPA (1983). Another soil treatment was N fertilizer application (27% N) equivalent to 60 kg N/ha. The 60 kg N/ha level was chosen as an acceptable level for nitrogen amendment for alfalfa growth recommended by Conseil des productions végétales du Québec (1994).

Rhizobial Inoculants
The fast-growing Sinorhizobium meliloti strain A2 (Agriculture and Agri-food Canada, Sainte-Foy, QC, Canada) was used for the inoculant preparation. Two types of inoculants (liquid and solid inoculants) were used. Liquid inoculants were prepared by growing strain in standard medium yeast mannitol broth (YMB) and in sterile secondary sludge as described by Ben Rebah et al. (2001)(2002a,b). The YMB medium contained the following constituents in grams per liter: K2HPO4, 0.5; MgSO4·7H2O, 0.2; NaCl, 0.1; yeast extract, 1; and mannitol, 10. Solid inoculants were obtained by mixing culture of S. meliloti grown in YMB or in secondary sludge with sterile peat (obtained from Microbio Rhizogen Corporation, Saskatoon, SK, Canada) and sterile dried sludge, respectively, as described by Ben Rebah et al. (2002a).

Plant Growth and Nodulation Assessment
Three germinated alfalfa seeds were sown in each 15-cm (6-in) pot (Riviera Pot, Marseille, France). Seedlings from all pots of the two soil types (unamended, sludge 60 and 120 kg N/ha, N fertilizer 60 kg N/ha) were inoculated with liquid inoculant (YMB-grown rhizobia and secondary sludge-grown rhizobia) to give an equivalent of 108 cells/seed. However, solid inoculants (108 cells/seed) were used only for unamended soils (without sludge or nitrogen fertilization). For each treatment, uninoculated pots were used as a control. Plants were kept in a growth room (16-h days at 20°C and 15°C nights) and irrigated with deionized water as needed. Pots were placed in a randomized complete block design with five replicates for all treatments. Plant growth yields were determined on three cuttings at the beginning of flowering (10–20%). The first cut was done after 60 d of growth, the second cut was done 26 d later, and the third cut was done 35 d after the second cut. Shoot samples were collected, dried at 70°C for 48 h, weighed, and ground for chemical analysis. Total nitrogen was determined on the five plant replicates and heavy metal and mineral salts were determined on plant composite samples by using the same methodology as indicated for sludge. At the end of experiment, roots were washed free of soil and the number, color, and size of root nodules were recorded to calculate the nodulation index as indicated in Table 1 . The MPN evaluation was also done in soils at the end of experiments.


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  		Table 1. Determination of the nodulation index. Nodulation index = A x B x C <= 18.

 
Except for MPN, heavy metals, and salts analysis, data were subjected to analyses by using the least significant difference (LSD) test. Orthogonal contrasts were used to detect differences among treatments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Sludge and Soil Chemical Characteristics
Table 2 lists the characteristics of the original soils. For both soils, the pH values were similar (around pH 6). Total nitrogen and total carbon were slightly higher in Kamouraska soil, but C to N ratios were similar in both soils (12.1 and 12.2, respectively, for Kamouraska and Saint-André). However, total phosphorus in Saint-André soil was almost two times higher than in Kamouraska. Heavy metal concentrations were similar for both soils except for Al and Fe. The Al concentration was lower in Kamouraska (1036 mg/kg) than in Saint-André (1490 mg/kg), while the Fe concentration was lower in Saint-André (217 mg/kg) than in Kamouraska (347 mg/kg). The numbers of rhizobia in the soil samples estimated by the most probable number were similar in both soils (2.26 x 103 and 1.5 x 103, respectively, for Kamouraska and Saint-André soils).


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  		Table 2. Soil characteristics.

 
The secondary sludge from Communauté Urbaine de Québec contained higher concentrations of carbon, nitrogen, phosphorus, and mineral salts than those found in soils (Table 3) , but the C to N ratio was lower. The concentrations of Ca, Mg, and K were also higher than those in soils. Moreover, sludge contained higher heavy metal concentrations but at acceptable levels for agricultural use as prescribed by the environment of Québec and the European Economic Community (Gouvernement du Québec, 1991; McGrath et al., 1994).


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  		Table 3. Total composition of sludge, quantity added to soil, and recommended levels of heavy metals for agricultural practices.

 
Assessment of Alfalfa Nodulation and S. meliloti Population Size
Table 4 shows the nodulation characteristics and the number of rhizobia evaluated at the end of the experiment. Plants roots from inoculated and uninoculated treatments were nodulated at different levels and all nodules were pink, indicating active N2 fixation. As evaluated by the nodulation index, alfalfa nodulation was affected by soil type. Generally, nodulation index was more important for Kamouraska soil (6 to 12) than Saint-André soil (2 to 12). Plants from uninoculated and unamended treatments showed only a few large nodule clusters located along the secondary roots. Moreover, plants in sludge-amended soils showed more large nodules than in the unamended soil and this was reflected in the higher nodulation index. The inoculation was successful as shown by the large or medium pink nodules spread throughout the entire root system of plants from inoculated treatments. However, the N fertilization decreased the nodulation index in both inoculated and uninoculated plants grown in Saint-André soil.


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  		Table 4. Nodulation index (NI) of alfalfa and rhizobial number in soils at the end of the experiment.

 
The numbers of indigenous rhizobia were similar in both Kamouraska and Sainte-André soils (4.83 x 103 and 3.25 x 103 cfu/g dry soil, respectively) and those numbers were only slightly higher than those found in original soil samples (2.26 x 103 and 1.5 x 103 cfu/g dry soil, respectively). However, the amendment with 60 kg N/ha of sludge increased the population size by one log (2.57 x 104 and 1.72 x 104 cfu/g dry soil, respectively, for both soils). Inoculation with liquid inoculants (YMB or sludge) increased the population level to 107 cfu/g dry soil for Kamouraska and to 106 cfu/g dry soil for Sainte-André. Those numbers were not affected by the amendment with 60 kg N/ha of sludge.

Shoot Dry Weight and Nitrogen Content
Alfalfa Plants Growing in Kamouraska Soil
For all three cuttings, there was no significant effect of inoculation on shoot dry weight and nitrogen content of alfalfa plants (Table 5) . However, differences due to the type of inoculant were observed at the first cutting in the unamended treatment, where plants inoculated with YMB-grown rhizobia had a significantly higher nitrogen content (95 mg N/pot) than those inoculated with sludge-grown rhizobia (67 mg N/pot). At each cutting, shoot dry weight and nitrogen content increased with the rate of sludge applied, and the rate of 120 kg N/ha of sludge gave the highest shoot dry weight and nitrogen content. For example, the total means of shoot yield of alfalfa increased from 15 g/pot in unamended soils to 20 g/pot in soils amended with 120 kg N/ha of sludge. The 60 kg N/ha rate, either applied with fertilizer or with sludge, gave similar shoot dry weights and nitrogen contents, except at the third cutting, where nitrogen content of plants amended with sludge was significantly higher than those fertilized with nitrogen.


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  		Table 5. Shoot dry weight and nitrogen content for alfalfa cultivated in Kamouraska soil.

 
In general, the shoot dry weights and nitrogen contents of plants from all treatments increased by a factor of two to three from the first cut to the third cut. For example, the means of all treatments of nitrogen content increased from 98 mg N/pot (first cut) to 219 mg N/pot (third cut).

Alfalfa Plants Growing in Saint-André Soil
For Saint-André soil, contrasts indicated no significant differences between inoculated and uninoculated plants (Table 6) . Generally, all inoculants gave similar yields except differences in shoot dry weight between liquid inoculants (YMB and sludge) at the first cut and between solid inoculants (peat and sludge) at the third cut. The sludge amendment influenced the plant growth and the rate of 120 kg N/ha of sludge had the highest improvement for both dry weight and nitrogen content. The total means of nitrogen yield of alfalfa increased from 245 mg N/pot in unamended soils to 486 mg N/pot in soils amended with 120 kg N/ha of sludge. However, yields obtained with the N fertilizer remained significantly lower while compared with the sludge-amendment treatments.


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  		Table 6. Shoot dry weight and nitrogen content for alfalfa cultivated in Saint-André soil.

 
In general, the growth of alfalfa in Saint-André soil (Table 6) was lower than that observed in Kamouraska soil (Table 5). The means for all treatments and for the three cuttings were 17 g dry weight/pot and 445 mg N/pot in Kamouraska soil compared with 13 g dry weight/pot and 329 mg N/pot for Sainte-André soil.

Macronutrients and Heavy Metals Uptake by Alfalfa Plants
Macronutrient Uptake
Shoot concentrations of macronutrients (P, Mg, K, and Ca) and heavy metals are presented in Tables 7 and 8 , respectively, for Kamouraska and Saint-André soils. In both soils, phosphorus concentrations were similar among treatments at the first cut. However, the sludge amendment caused an increase of P uptake at the third cut. For example, in uninoculated Kamouraska soil for 120 kg N/ha of sludge, the phosphorus concentrations increased from 1531 mg/kg at the first cut to 2354 mg/kg at the third cut.


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  		Table 7. Phosphorus, mineral salts, and heavy metal concentrations in alfalfa shoots cultivated in Kamouraska soil.

 

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  		Table 8. Phosphorus, mineral salts, and heavy metal concentrations in alfalfa shoots cultivated in Saint-André soil.

 
For both soils, K, Ca, and Mg concentrations in alfalfa plants were generally similar among all treatments at each cut, but they were slightly lower at the third cut than at the first cut. However, K and Mg uptakes were higher in Kamouraska than in Saint-André soil. For example, at the first cut, K values for Kamouraska soil ranged between 22361 mg/kg (for 120 kg N/ha of sludge and inoculated by YMB-grown rhizobia) and 25094 mg/kg (for 60 kg N/ha of sludge and inoculated by sludge-grown rhizobia), while they were between 19995 mg/kg (for unamended) and 22756 mg/kg (for 60 kg N/ha of sludge and inoculated by sludge-grown rhizobia) for Saint-André soil. However, values were similar for inoculated and uninoculated soils and it seems that inoculation had no effect on nutrient uptake.

Heavy Metal Uptake
Generally, heavy metal (Cu, Zn, Fe, Al, and Mn) uptake by alfalfa was higher at the first cutting than at the third cutting in both soils (Table 7 and 8). Plants cultivated in Saint-André soil presented Cu uptakes slightly lower than in Kamouraska soil, but there was no significant effect of inoculation or sludge amendment in both soils. The Zn concentrations were two times higher for Kamouraska (31 to 34 mg/kg at the first cut) than in Saint-André soil (12 to 16 mg/kg at the first cut). For both soils, the values at the third cut decreased by about 50% in comparison with the first cut.

The highest metal concentrations in alfalfa shoots were noted for Fe, Al, and Mn. The Fe shoot concentrations were similar in both soils (Saint-André, 83–108 mg/kg and Kamouraska, 85–117 mg/kg at the first cut) and they slightly decreased at the third cut. Sludge amendment tended to enhance the Fe, Al, and Mn uptake in both soils at the two cuttings, as observed in uninoculated treatments. However, the overall metal concentrations were reduced at the third cut.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The main objective of this work was to evaluate the potential of using sludge in the legume inoculant production. As we reported earlier, sludge is a cheap and good raw material generated by industrial and municipal wastewater treatment plants, which can support the rhizobial growth and could substitute completely the standard medium (Ben Rebah et al., 2001, 2002a). Also, sludge has a good potential as a carrier for rhizobia (Ben Rebah et al., 2002b). Hence, the use of sludge as a substrate and carrier for rhizobia is a new alternative of waste recycling. This alternative, along with some other new applications such as converting sludge into building materials (Tay and Show, 1992) and oils (Campbell and Martinoli, 1991) and biopesticide production (Sachdeva et al., 2000; Vidyarthi et al., 2000), helps to reduce the environmental problem of sludge disposal. In this study we demonstrated that using sludge-based inoculants might have the same potential as standard YMB-based inoculants on alfalfa growing in unamended and sludge-amended soils. The inoculation was successful as demonstrated by the enhancement of nodulation and by the increase of rhizobial population in soils after inoculation. Solid or liquid sludge or YMB-based inoculant did not affect the efficiency of the symbiosis, as reflected by the shoot dry weight. However, the inoculation at the high level of 108 cells/seed seems to be beneficial because it increased the nodulation index and the rhizobial number. In all inoculated treatments the number of rhizobia was about one log higher in Kamouraska than in Saint-André soil. This may be attributed to soil composition. The clay soil (Kamouraska) may have a greater adsorption capacity than sandy soil such as Saint-André.

Independently of soil type, the amendment with secondary sludge had a beneficial effect on indigenous rhizobia and the enhancement of their numbers in both soils is confirmed by other workers (Kinkle et al., 1987; Heckman et al., 1987a,b). However, other studies reported that sludge might affect negatively indigenous rhizobia. For example, significant reduction in number of R. leguminousarum bv. trifolii was found in soils previously treated with sewage sludge containing heavy metals (Giller et al., 1989, 1993). These results cannot be compared with our results because it reported the problem of long-term sludge application while the present study deals with the short-term effect of sludge application.

Sludge application increased the indigenous rhizobia population and the nodulation index in uninoculated treatments while it increased only the nodulation index in inoculated treatments. Since the nodulation index is a qualitative value, we cannot conclude if there is a correlation between the number of rhizobia, the rate of sludge amendment, and the nodulation index. However, the most important conclusion that confirms the quality of the sludge-based inoculant (solid or liquid) is the similarity of its nodulation index to that of YMB-based inoculant. In a previous study using the same rhizobial strain A2 with alfalfa grown in seed growth pouches, the nodulation indices obtained with sludge-grown rhizobia were, in general, similar to that obtained with YMB-grown rhizobia (Ben Rebah et al., 2001, 2002a).

The equivalent quality of the sludge and YMB-based inoculants was also confirmed by the similar shoot dry weight and the plant nitrogen content, although the effect of inoculation was not reflected on plant shoot growth. Introducing 108 cells/seed in soils by inoculation did not significantly increase both biomass and nitrogen content. We can suppose that the presence of indigenous soil rhizobia was at an acceptable level (103 cells/g dry soil) to adequately nodulate the plants and assure optimal symbiotic nitrogen fixation. One important fact after inoculation is the nodulation of the plant by the introduced strain. This fact was observed by examining the nodule distribution in the root system. In uninoculated soils, the nodules formed some clusters along the secondary roots but in inoculated soils the nodules were spread throughout the entire root system, indicating a more homogenous distribution of rhizobia in soils close to the root system due to inoculation. It would be important to study the genetic diversity of some isolates of rhizobia from the root system of inoculated and uninoculated plants, which may possibly confirm this conclusion.

The results of the present research agree with those of Ferreira and Castro (1995) and Giller et al. (1989) about the effect of sludge amendment on soil rhizobial population. For example, Giller et al. (1989) demonstrated that the addition of R. leguminosarum bv. trifolli to sludge-amended soils enabled the survival of enough rhizobia to establish nodulation. Contrary to the fast-growing rhizobia, Bradyrhizobia strains were negatively affected by the application of high rates of sewage sludge to the soil over the short-term application (Madariaga and Angle, 1992; Reddy et al., 1983).

The increase in dry matter and nitrogen content of alfalfa growing in sludge-amended soils may be attributed to high organic matter content and high macro- and micronutrient concentrations. This enhancement also confirms the increase in shoot weight of legumes grown in sludge-amended soil, as previously reported by Heckman et al. (1987a)( b) and by Ibekwe et al. (1995). Despite the presence of available nitrogen in sludge and in standard fertilizer-amended soils, the nodulation was not inhibited, indicating that the available forms of nitrogen were not enough for supplying the alfalfa needs. The same tendency was observed with clover plants cultivated in soils amended with sludge rates fom 5 to 60 Mg/ha (Ferreira and Castro, 1995). The noninhibition of nodulation by available nitrogen especially high in aerobically digested sludge (rate of 120 kg N/ha) can be related to sludge maturity (Diaz-Burgos et al., 1991). The increase of both shoot dry weight and the nitrogen content in alfalfa plants from the first to the third cut was mainly due to the plant establishment. However, sludge maturity can offer also an explanation for the enhancement. In our study the sludge is nondigested secondary sludge applied directly in liquid form, and was used immediately after the samples were taken. Therefore, it may be too immature to be used as organic fertilizer (Chardas et al., 1991) and plants cannot benefit rapidly from its nutrient content. Moreover, sludge can lose nitrogen through evaporation, which can have a negative effect on plant growth due to a high salt concentration (which greatly increases the electrical conductivity of the soil solution; Cabral et al., 1991) and to the presence of reduced forms of nitrogen (Cabral et al., 1991; Diaz-Burgos et al., 1991). In the same respect, Ferreira and Castro (1995) indicated that the sludge application in the first year exerted harmful effects on the growth of clover plants at the highest rate (60 Mg/h of sludge), but the second year showed beneficial effects, increasing with the increasing sludge rate. In our study, the beneficial effect of sludge increased by increasing the application rate. For example, in Kamouraska soil, the total nitrogen enhancement (sum of three cuts) due to the application of 60 kg N/ha of sludge was only 8.1%, but was much higher at the 120 kg N/ha rate (34.6%). This proves the beneficial effects on legume of short-term sludge application.

The better plant yields obtained in Kamouraska soil (clay soil) may be explained by its higher water holding capacity than Saint-André soil (sandy soil). This may be also due to a greater leaching process of nutrients in Saint-André soil (sandy soil), which could have decreased the nutrient amount in the root zone.

Macroelements (P, Mg, Ca, and K) were within the normal range for most small legumes (Martin and Matocha, 1973). At the first harvest, it seems that phosphorus uptake was limited, but increased at the third cut and remained at the acceptable level for alfalfa. In our study, the sludge used for the amendment contained a high concentration of mineral salts, which could influence the behavior of phosphorus (Willson, 1970). Because salts are easily leached in soils from the root zone by irrigation, the plant salt uptakes should diminish with time, which could be responsible for the increasing P uptake. Moreover, soil clay with a fine texture (Kamouraska) is potentially able to hold more cations, favoring higher mineral uptakes (K and Mg) by the plant than in the sandy soil (Saint-André soil). However, the higher concentrations of Ca in plants cultivated in Saint-André indicated that other factors may influence cation uptakes such as the concentration, interaction, and distribution of each element in soils. For example, the concentrations of Al and K affect the uptakes of Ca by plants (Doll and Lucas, 1973).

The total metal contents of the soils and sludge used in our study were respectful of limits for agriculture application. Moreover, shoot analysis showed no toxicity because the concentrations of Cu, Zn, Fe, Al, and Mn were less than phytotoxic levels. According to Chaney et al. (1978), the phytotoxicity levels for small legumes are 500 for Mn, 500 for Zn, 40 for Cu, and 50 mg/kg dry weight for Ni. However, according to Angle et al. (1992), the negative responses for short-term application of sludge may be caused by sludge-borne salts and not by sludge-borne metals. Therefore, heavy metals were not considered to be a negative factor in this study. However, their concentrations in shoots seem to be influenced by the cut, the sludge rate, and by the soil types. The decrease of some metal shoot concentrations at the third cut can be attributed to the reduction of metal concentration in soils as a result of the leaching process by irrigation.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
This study showed that the inoculation of alfalfa with sludge-grown S. meliloti was beneficial for increasing nodulation at the same level as the inoculation with YMB-grown S. meliloti. Moreover, sludge-based inoculant can be used in liquid or solid form without affecting the nodulation efficiency. Sludge may also be used as an organic fertilizer with great beneficial effect on rhizobial number, nodulation, shoot dry weight, and nitrogen content of alfalfa. The current study showed that none of the parameters examined was negatively affected by sludge application (salts and heavy metal contents) or use of sludge-based inoculants.


   ACKNOWLEDGMENTS
 
The authors sincerely thank the Natural Sciences and Engineering Research Council of Canada (Grant A 4984) for providing financial assistance to conduct this research.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 





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