Reducing the heavy metals toxicity in sludge

amended soil using VA mycorrhizae

 

R.A. Abdel-Aziz*; S.M.A. Radwan* and M.S. Dahdoh**.

*   Agricultural Microbiology Dept., National Research Center, Dokki, Cairo, Egypt.

**.Soil Science and Microbiology Dept., Desert Research Center, Cairo, Egypt.

 

Abstract

 

A pot experiment was conducted to evaluate the role of VA mycorrhizae as a biological agent in reducing the toxicity of heavy metals. A newly reclaimed soil from Ismaillia Governorate was supplemented with sewage sludge from two sources at five different rates 0.0, 0.5, 1.0, 2.0 and 4.0%. One half of each treatment was inoculated with VA mycorrhizae. Application of sewage sludge up to 2% increased faba bean growth, nodule number and weight, and phosphorus and nitrogen contents. Inoculation with VA mycorrhizae also induced significant increase in these parameters as compared with the uninoculated treatments. Plant Zn, Mn, Cu, Ni, Cd, Pb, and Co contents were increased with the application of sewage sludge. However, in the sewage sludge treated soil where the heavy metals were present in high concentrations, inoculation with VA mycorrhizae reduced the concentration of heavy metals. This indicates the role of VA mycorrhizae in reducing the hazardous effect of heavy metals when present in high levels in the media of growing plants.

Keywords: heavy metals, sludge, VA mycorrhizae

 

Introduction

 

Utilization of sewage sludge on agricultural land increases the concentration of heavy metals in soil. Studies suggest that leguminous crops dependent on symbiotic N2- fixation may be sensitive to the toxic effects of heavy metals present in sludge. Thus reduction in nodulation, plant size, and nitrogenase activity has been observed in white clover plants grown on soil highly contaminated with Cd, Pb, and Zn (Rother et al., 1983). Other studies have demonstrated the toxic effects of heavy metals on nodulation and nitrogenase activity (Vigue et al., 1981). In soil newly amended with sludge, Ham and Dowdy (1978) reported increased uptake of Zn and Cd, decreased nodulation, but improved soybean yield up to 2 years after the initial application. On the other hand, many workers point out that metal-rich sewage sludge, drastically reduced the yield of some crops after a critical amount of that sludge was added to the soil. These critical limits depend on the source of the sewage sludge, the application rate and its frequency, the soil removal mechanisms and the removal capabilities of palnt species. Little attention has been given to the role of vesicular- arbuscular mycorrhizae (VAM) in environments containing elevated and potentially toxic level of mineral elements,  though inoculation with VA mycorrhizae reduced Zn and Cd concentration is plant leaves grown in soil with high concentrations of these metals (Heggo et al., 1990) Denny and wilkins (1987) mentioned that Zn toxicity to Betula pendula Roth was ameliorated by colonization with mycorrhizal fungi and the ameliorating influence was positively correlated with the of compatibility between the fungal strain and the plant.

A similar amelioration has been noted in Betula papyrifela associated with various mycorrhizal symbionts exposed to Cu and Ni (Jones and Hutchinson, 1986). Gildon and Tinker (1982) have shown that VAM may also affect heavy metal uptake by plants and that VAM fungi are tolerant to high soil concentrations of metals.Colonization with metal- tolerant isolates of VAM fungi were further shown to protect plants against the toxic effects of excessive concentrations of heavy metals.

However, not all data have shown that VAM fungi reduce the uptake of heavy metals. It was found that VAM fungi enhanced metal uptake from deficient soils; e.g. VAM fungi enhance the uptake of Zn by peach in Zn- deficient California soils (Lrue et al., 1975) and by maize (Zea mays L.) and wheat (triticum aestivum L.) In some Zn- deficient soils of India.

The aim of the present work was to examine the effect of VAM fungi on growth and heavy metal uptake of faba plants grown in sewage sludge amended soil. Reducing the heavy metal toxicity using the VA mycorrhizae and its effect on nodulation and N2- flixation were also determined.

 

Materials and methods

 

A pot experiment was carried out in the greenhouse of the National Research Center, Dokki, Cairo, Egypt. Sandy soil from Ismaillia and sewage sludge from two different sewage treatment plants at Abou-Rawash and Al-Gabal Al- Asfar were used. The physical and chemical characteristics of the soil and sewage sludge  were reported in Tables, 1 and 2 . The analysis was done as described by Black (1983).

Soil was packed in earthenware pots 30 cm in diameter and 40 cm in depth. Air dried sewage sludge from each source was added to soil at rates of  0,0.5, 1.0, 2.0 and 4.0%. The soil was well mixed with the appropriate amount of sludge and uniformly packed in the pots. Half of the pots of each treatment were inoculated with mixed VA mycorrhizal spores (600 spores of Glomus clarum; Glomus mosseae and Glomus fasciculatum per pot). The VA mycorrhizal spores wer isolated from the rhizosphere of Egyptian clover grown in Al- Gabal Al- Asfar soil which has been treated with sewage sludge for more than 50 years, using the wet sieving and decanting method     (Gerdemann and Nicolson, 1963). The mycorrhizal spores were added 3 days before planting. Five seeds of faba been (Viceae faba) variety Giza 2, were sown into each pot and the pots were watered as needed. In all treatments faba bean seeds were inoculated with Rhizobium leguminosarum bv. Viceae just prior to sowing. Plants were thinned to two plants per pot after germination, then grown for 8 weeks in the greenhouse in a randomized design. All treatments were replicated five times.

At harvest, shoots were separated from roots, then the roots washed three times in water and nodule number and weight determined. Shoots were dried at 70 C and ground in a stainless steel wiley mill. The shoots (0.2 g) were digested in sulfuric and perchloric acids and heavy metals (Zn, Mn, Cu,Cd, Ni, Pb and Co) were measured in the digest. Total nitrogen in the digest was determined by Kjeldahl analysis and total phosphorus was determined colorimetrically (Black, 1983). Percent of mycorrhial rrot infection was estimated as described by phillips and Hayman (1970).

 

 

Table (1) Some physical and chemical characteristics of the soil.

Soil character

Values

Sand %

Silt %

Clay

Soil textural class

PH (Soil paste)

EC mmhos/ cm.

O.M.%

CaCo3%

CEC meq/100g soil

W.H.C.%

Total N (ppm)

Available P(ppm)

Total heavy metals (ppm)

Zn

Mn

Cu

Cd

Ni

Pb

Co

Available heavy metals (ppm)

Zn

Mn

Cu

Cd

Ni

Pb

Co

95.00

4.20

0.80

sandy

8.32

0.74

0.21

0.53

0.50

18.30

200.00

4.00

 

8.13

4.17

1.00

0.86

1.40

1.64

1.93

 

0.61

1.20

0.10

0.10

0.20

0.17

0.08

                      

                    Table (2) : Some chemical characteristics and elements content of sewage sludge.

Character

Al- Gabal Al-Asfer

Abuo-Rawash

PH

6.71

6.76

Ec(mmhos/cm)

5.66

7.42

O.C.%

18.19

17.24

Total elements

N %

1.65

2.50

P %

1.13

1.54

Zn  ppm

1215.00

1467.00

Mn  ppm

375.00

326.00

Cu  ppm

512.00

346.00

Ni  ppm

89.00

72.00

Cd  ppm

12.50

16.20

Pb  ppm

580.00

423.00

Co  ppm

27.00

35.50

Available elements (ppm)

N

22.00

40.00

P

19.00

26.00

Zn

259.00

287.00

Mn

43.00

29.00

Cu

41.00

32.00

Ni

3.70

2.90

Cd

0.36

0.44

Pb

28.00

23.00

Co

0.84

0.62

 

 

 

Results and discussion

 

Response of faba bean plant to sewage sludge application and inoculation with VA mycorrhizae

 

Faba bean shoots dry weight, nodules number & weight, and nitrogen and phosphorus contents responded significantly to sewage sludge application and VA mycorrhizae inoculation (Table,3), where best results obtained with the addition of 2% sewage sludge, irrespective to the source. The addition of 4% sewage sludge decreased the studied parameters. The increase of shoot dry weight due to sewage sludge application is a true reflection of its effect on the physical and chemical properties, as well as the nutritional status of the soil. This may result from decrease of soil pH and to the solubilization of nutrients and enhanced nutrient availability (Hue, 1988; Dahdoh et al., 1994 and El- Demerdash et al., 1994), or the stimulation of biodegradation through increased the populations and activities of soil microorganisms (Lowendrof, 1980). Faba bean plants grown in soil treated with sewage sludge from Abou- Rawash always gage higher yield than those grown in Al- Gabal Al- Asfar sewage gave higher yield than those grown in Al- Gabal Alsfar sewage- sludge amended soil. This may be due to the high content of essential elements in Abou-Rawash sewage sludge. Similar trends were found for nodule number and weight, with best results again obtained with 2% sewage sludge amendment. These results are in agreement with those of Vigue et al., (1981) who found that nodulation of various legumes may be inhibited by the presence of soluble Cd, Ni and Zn.

Faba bean plants grown in soil inoculated with VA mycorrhizae showed better growth than those which were not. This increase reflects the importance of VA mycorrhizae in enhancement of plant growth and possible N2-fixation as well the better utilization of essential macro and micronutrients. Other papers have reported similar results. Thus, VA mycorrhizae was found to enhance foliar dry matter yield of different plants (Heggo et al., 1990). It was shown that VA mycorrhizae could increase the uptake of insoluble nutrients such as phosphate, sulphate and microelements (Mosse, 1981, ameliorate water stress (Allen, 1982), produce plant growth hormones (Allen et al., 1980). It was also found that, nodule weight was greater in mycorrhizal plant then non- mycorrhizal ones (Saad, 1990).

Mycorrhizal root infection by indigenous mycorrhizae was low (Table,3), and was enhanced by the inoculation with VA mycorrhizae. Addition of sewage sludge up to 2% had no effect on mycorrhizal root infection but when sewage sludge was applied at the rate of 4% the percentage of mycorrhizal root infection decreased. This could be attributed to the effect of high level of heavy metals at that rate of application on the mycorrhizal spore germination, hyphal growth and branching (Gildon and Tinker, 1982).

Application of sewage sludge increased the phosphorus and nitrogen contents of faba bean plants irrespective to the source of sludge. This increase was recorded up to 2% of sewage sludge, while 4% sludge addition decreased the uptake of both  elements. The decrease in N and P- contents associated with 4% sewage sludge rate might be due to the inhibition effect of that rate on the growth of faba bean which diminish its capability to absorb those elements from soil.


 

 

Table (3): Effect of sewage sludge application and VA mycorrhizae on nodulation, shoots dry weight, nitrogen and phosphorus contents of faba bean plants.

 

 

Treatments

Nodules No./plant

Nodules dry weight (mg/plant)

Mycrrhizal root infection %

Shoots dry weight (g/plant)

N-Content mg/plant

P-Content mg/plant

-

VAM

+

VAM

-

VAM

+

VAM

-

VAM

+

VAM

-

VAM

+

VAM

-

VAM

+

VAM

-

VAM

+

VAM

Control

0.0%

36

39

153

160

12

64

7.15

7.62

150

178

14.3

17.5

Abou Rawash

0.5%

37

42

151

182

11

60

7.24

7.73

155

177

18.1

23.2

1.0%

43

64

178

191

11

62

8.61

8.92

215

223

25.8

31.2

2.0%

54

60

240

273

12

60

9.74

11.35

253

295

34.1

45.4

4.0%

31

33

142

151

9

59

6.45

7.32

135

161

15.4

16.9

Al-Gabal-Al-Asfar

0.5%

32

37

147

153

11

68

6.84

7.51

143

165

16.5

21.4

1.0%

39

45

162

179

12

65

7.92

8.74

182

201

22.6

26.7

2.0%

48

56

203

262

12

66

9.15

10.65

238

266

29.3

37.5

4.0%

30

34

137

139

10

60

6.61

6.94

146

152

13.7

15.2

L.S.D at 5% for treatments

3.86

3.88

8.17

0.60

4.81

2.09

L.S.D.at 5% for VAM inoculation

1.83

1.82

5.26

0.29

2.27

0.40

L.S.D.at 5% for interactaion

5.50

5.46

17.83

0.86

6.80

1.20

 

The present results support the finding of many investigators; Hue (1988); Dahdoh et al., (1994) and El- Demerdash et al., (1994) who found that plants grown in sludge amended soils accumulated higher amounts of P relative to plant grown in untreated soils. The present work also indicated that indicated that sewage sludge has a positive influence as evidenced by increased faba bean growth, P and N- Contents. A greater availability of  N on sludge-amended soil was indicated by greater total N accumulation; however, this N did not limit- fixation.

Freier (1984) suggest that small amounts of N mineralized from organic matter may enhance nodulation by stimulaating plant growth before onset of N2-fixation. Likewise, N2-fixation was increased as a result of increasing Ca release from sewage sludge-(Heckman et al., 1986). The favourable influence on plant growth and N2-fixation may have resulted from the direct effect of sludge on growth and nodulation and / or the improving of Rhizobium survival in soil (Lowendrof, 1980).

The role of VA mycorrhizae in improving P-uptake is widely recognized. VA mycorrhizae can improve plant growth, especially where soil P is limiting (Mosse and Hayman, 1980). VA mycorrhizae increase the root surface area and thus enhance P- uptake. VA mycorrhizae also have the ability to exploit non- labile forms of soil P such as tricalcium phosphate and rock phosphate (smith 1980). Data in Table 3, show that inoculation with VA mycorrhizae led to and overall significant increase in N-content which may probable resulted from soil. VA mycorrhizae may also increase the rate of N2-fixation and iit can assimilate and translocate ammonium (Van Kessel et al.,1975). Enhanced uptake of mineral elements in general by mycorrhizal plants compared to non-mycorrhizal ones may be due to the observation that VA mycorrhizae reduce distance that nutrients must diffuse to plant roots (Abbott ant Robson, 1982). It is worthy to mention that the effect of VA mycorrhizae on the uptake of N and P was quite similar regardless to sewage sludge source.

VA mycorrhizae and heavy metals toxicity:

Considering the effect of sewage sludge application on heavy metals concentration of  faba bean shoots, data in Table 4, show that plant Zn and Ni concentration were increased with increasing sludge application rate while, plant Mn and Cu were decreased. Similar Results were reported by Hue (1988). In the present work, the decrease in plant Mn concentration with increasing sludge application rates may be attributed to the lower availability of soil Mn and higher soil pH together with the greater growth of Mn oxidizing bacteria associated with added sludge. On the other hand, the decrease of plant Cu concentration may also be due to the strong binding between Cu and substances released from organic materials which reuses the Cu availability to plants (Kiekens et al., 1984). In this respect, it is known that the soil organic matter can regulate the availablity of metals through chelation reaction (Gamble et al., 1984) in which the metals from stable 5 and 6- membered ring structure have carboxy 1 and hydroxy1 functional groups in organic aggregation, thus becoming a part of the solid phase, mostly unavailable to plants. On the other hand,  soluble organic molecules especially low molecular weight organic acids produced during the microbial decomposition of added sludge, can complex Zn and other elements and make them more available and toxic to plants (Hue, 1988). Also the antagonistic relationship between Zn and Cu (El-Kadi et al., 1990) may be one of the reason which led to the increase of plant Zn concentration and decreases plant Cu. Ingeneral, the concentrations of Zn, Mn, Cu, and Ni in faba bean plants grown in soil amended with Abou- Rawash sewage sludge were higher than those grown in soil amended with Al- Gabal Al- Asfar sewage sludge.

 

Table (4) : Effect of sewage sludge application and inoculation with VA mycorrhizae on the concentration of heavy metals in faba bean shoots (ug/g).

 

 

Treatments

 

Mn

 

Zn

 

Cu

 

Ni

 

Cd

 

Pb

 

Co

Zn

Equivalent

(ppm)

-

VAM

 

+

VAM

-

VAM

 

+

VAM

-

VAM

 

+

VAM

-

VAM

 

+

VAM

-

VAM

 

+

VAM

-

VAM

 

+

VAM

-

VAM

 

+

VAM

-

VAM

 

+

VAM

Control

0.0%

16.1

20.9

19.0

22.8

3.22

4.72

0.70

1.05

0.07

0.08

2.38

3.15

0.60

0.71

30.1

41.5

Abou-Rawash

0.5%

72.9

47.5

43.0

32.2

8.84

7.36

2.73

2.27

0.41

0.32

14.2

10.7

1.77

1.44

66.1

51.5

1.0%

61.4

43.1

45.8

30.3

7.52

7.04

3.41

2.83

0.56

0.41

16.0

12.1

2.61

1.84

88.1

67.0

2.0%

55.2

41.2

53.7

35.4

7.45

6.91

3.65

2.94

0.67

0.52

18.3

15.2

3.09

2.09

97.8

72.7

4.0%

52.1

40.1

62.6

36.8

7.19

6.55

3.72

3.28

0.67

0.50

19.1

12.3

3.54

2.72

106.7

76.1

Al-Gabal Al-Asfar

0.5%

75.4

50.5

39.1

29.3

8.18

7.15

2.91

2.39

0.52

0.40

12.8

10.4

2.25

1.42

78.8

62.7

1.0%

65.6

54.1

43.4

31.2

7.66

6.74

3.68

2.71

0.74

0.51

14.5

11.3

2.61

1.80

88.2

66.4

2.0%

63.2

42.3

49.1

37.5

7.08

6.25

3.82

3.12

0.69

0.56

15.3

12.1

2.74

2.39

94.6

75.5

4.0%

60.1

41.1

55.7

36.2

6.81

5.81

3.90

3.26

0.70

0.56

22.2

14.7

2.91

2.85

100.5

73.9

L.S.D at 5% for treatments

2.12

3.01

0.54

0.28

0.04

0.25

0.05

1.17

L.S.D at 5% for VAM inoculation

1.00

1.40

0.25

0.13

0.02

0.12

0.03

0.55

L.S.D. at

5% for

interaction

3.00

4.26

0.76

0.40

0.05

0.35

0.07

1.65

 

 

Nickel is considered on of the essential elements (Welsh et al., 1981) and its toxic effectis 8 times as high as that of Zn while Cu is twice as toxic as Zn. For this reason, there is an equation to express the toxicity to specific plant in specific soils. In this respect, Chumbley (1971) introduced the “Zn equivalent” factor, where ppm Zn equivalent = ppm Zn+ 2 ppm Cu+ 8 ppm Ni, to take into account the differences among metals and suggested that more than 250 ppm Zn equivalent as a critical limit to be added to agricultral Soils (with pH maintained < 6.5). Whth regard to the obtained data it was noticed that Zn equivalent is less than the 250 ppm value even at the highest rate of the added sludge. This means that the so called Zn equivalent is not the only reason responsible for decreasing the plant growth especially when 4% of sludge was added, but there were other reasons, such as the high concentratin of the other heavy metals like Cd,Pb, Co and the negative effect on soil physical properties which led to unsuitable conditions for root respiration and possible to the indirect effect on the absorption of the nutritive elements. Data also show that the concentration of Cd, Pb and Co in faba bean shoots (Table, 4) was increased with increasing sewage sludge rate.

The heavy metals uptake was increased significantly to reach their maximum at 2% sewage addition while somewhat less pronounced at 4% sludge addition control (Table, 5). The relative decrease of uptake may be due to the reduction in the dry matter yield at this specific rate of application). This means that the heavy metals uptake was paralleled to the dry matter yield. These results arei in line with those of Dahdoh et al., (1994) and El- Demerdash et al.(1994).

Results in table, 4 show that in sludge unamended soil, the inoculation with VA mycorrhizae increased the heavy metals concentration of shoots as compared with the uninoculated one.

These results are in accordance with the finding of Swaminathan and Verma (1979) who observed an increase in uptake of  Zn, Cu  and  Cd .when  deficient  soils  were

with VA mycorrhizae. The increase of mineral elements by mycorrhizal plant my be due to the action of VA mycorrhizae in increasing nutrient uptake by reducing the distance that nutrients must diffuse to plant roots (Abbott ant Robson, 1982).

In addition to the advantages associated with enhanced capture of major nutrients such as N and P, VA mycorrhizae provide the capacity to reduce the accumulation of heavy metals in the shoots when these elements are present in potentially toxic concentrations in the external medium (Heggo et al, 1990). Results presented in Table,4 showed that, in the sewage sludge treated soil where the heavy metals were present in high concentrations, the inoculation with VA mycorrhizae reduced the concentration if the studied heavy metals in faba bean shoots as compared to uninoculated treatments. This reduction in shoots heavy metals concentration indicated the role of VA mycorrhizae in reducing the hazardous effect of elements when  present in high levels in the media of growing plants. the reduction in shoots heavy metals concentation may be due to the ability of mycorrhizal roots to concentrate these contaminatnts on their hypha and reduce the heavy metals translocation into the shoots. In this respect Hilary et al., (1987) have suggesed that the mechanism of amelioration of Zn toxicity may be due to Zn adsorption on the surface of mycorrhizal hyphae or metal adsorption to the electronegative sites in the hyphal cell wall and extrahyphal polysaccharide slime. Bradley et al., (1982) proposed also tht the endophyte mycorrhizae may provide adsorptive surface within the cortical cells of the host roots, thus excluding metal from shoots and avoidance of metal toxicity.

 

Table (5): Effect of sewage sludge application and inoculation with VA mycorrhizae of the uptake of heavy metals in faba bean shoots (ug/g).

 

 

Treatments

Mn

Zn

Cu

Ni

Cd

Pb

Co

-

VAM

+

VAM

-

VAM

+

VAM

-

VAM

+

VAM

-

VAM

+

VAM

-

VAM

+

VAM

-

VAM

+

VAM

-

VAM

+

VAM

Control

0.0%

115

159

136

174

23

36

5

8

0.53

0.64

17

24

4.3

5.4

Abou-Rawash

0.5%

528

528

311

249

64

57

20

18

2.47

103

83

13.0

11.0

 

1.0%

529

529

394

270

65

63

29

25

3.65

138

108

22.0

16.0

 

2.0%

538

468

523

402

73

78

36

33

5.90

179

173

30.0

24.0

 

4.0%

336

294

404

270

46

48

24

24

3.66

123

127

22.0

20.0

 

Al-Gabal Al-Asfar

0.5%

516

379

268

220

58

54

20

18

3.56

3.00

88

78

15.0

11.0

1.0%

320

394

343

273

61

54

29

24

5.07

4.46

115

99

21.0

16.0

2.0%

579

450

456

400

65

67

35

33

6.31

5.96

140

126

25.0

25.0

4.0%

398

285

368

251

45

40

26

23

4.63

3.89

147

137

19.0

20.0

L.S.D at 5% for VAM inoculation

15.65

13.58

5.28

3.42

0.19

6.88

1.61

L.S.D at 5% for VAM inoculation

7.38

6.40

2.49

1.61

0.09

3.24

0.76

L.S.D at 5% for VAM inoculation

22.13

19.21

7.64

9.81

0.26

9.73

2.27

 

Therefore, it is necessary that the mycorrhizal fungi are itself resistant to heavy metals contamination over the range of concentrations to be expected in the natural environment. Mathys (1977) suggested that heavy metals may be chelated by malate in toterant plants (mycorrhizal plants) and Harley (1969) found that the VA mycorrhizae increased the organic acid content in mycorrhizal roots.

From the aformentioned results, it could be concluded that inoculation with VA mycorrhizae could potentially ameliorate the heavy metal toxicity associated with the application of sewage sludge especially in the newly reclaimed soils. This  role of VA mycorrhizae in avoidance of heavy metal toxicity requires a full understanding of all the possible interactions between host, fungus and habitat.

 

References

 


Abbot, L.K. and A.D. Robson (1982). The role of vesicular- arbuscular mycrrohizal  fungi in agriculture and the selection of fungi for inoculation. J. Agric. Res.         33:89- 408.

Allen, M.F. (1982). Influence of vesicular arbuscular mycorrhizae on water movement through Bouteloua gracilis (H.B.K) lag ex Steud. New phytologist, 91: 191-196.

Allen, M.F.; T.S. Moore and M. Christensen (1980). Phytohormone changes in uteloua gracilis infected by vesicular arbscular mycorrhizae. I- Cytokinin          increases the host plant Canadian J. Botony 58:371- 374.

Black, C.A. (1983). Methods of Soil Analysis” Soil Sci. Soc. Am., Inc Publ. Madison Wisc. U.S.A.

Bradley R.; A. J. Burt and. D.J. Read (1982). The biology of mycorrhiza in the Eicaceae. VII. The role of mycorhizal infection in heavy metal resistance New           Phyologist 91: 197-209.

Chumbley, G. G. (1971). Permissible levels of toxic metals in sewage sludge used in agicultural land, Agiculture Development and Advisor service. Report No. 10,           Ministry of Agriculture, Fisheries and Food, London.

Dahdoh, M.S.A.; S. El-Demerdash and H.H. El-Mashady (1994). Corn growth and elements uptake as functioned by sludge source. 6th International Conference-           Delphi, Greece.

Denny, H. J. and D.A. Wilkins (1987). Zinc tolerance in Betula spp. Variation in resuponse to zine among ectomycorrhizal associates. New phyt. 106: 535-544.

El- Demerdash, S.; M.S.A. Dahdoh and F.A Hassan (1994). Residual effects of sludge application on corn growth, nutrients and heavy metals uptake. Fertilizer and Environment, VII International symposium, C.I.EC., IRNA- CSIC. Salamanc Spain.

El- Kadi, A.H.; M.S.A Dahdoh and H.H. Hatem (1990). Interaction between Zn, and Cu on yield and protein content of maize grown in calcareous soil. Egypt. J.            Appl. Sci., S, Z, 11-19. 535-544.

Freire, J.R. (1984). Important limiting factors in soil for the Rhizobium legume symbiosis. In : Alexander M., (ed). Biological nitrogen fixation. Plenum, New            York, pp. 51-73.

Gamble, D.S., C.H. Landfordand A. W. Underdown (1984). The interrelationship of aggregation and cation binding of fulvic acid. P. 349-356. In C. J.H. Krameran J. C. Dainker (eds.) comlexation of trace metals in natural waters. Martinus Nighoff Junk Publ., the Hafue, Netherland.

Gerdemann, J.W. and T.H. Nicolson (1963). Spores of mycorrhizal Endogone species extracted from soil by wet-sieving and decanting.

           Transactions of the Britis Mycological Society 46: 235-244.

Gildon, A. and P.B. Tinker (1982). Interactions of vesicular – arbuscular  mycorrhizal infection and heavy metals in plants. I. The effect of heavy metals on the            development of VA mycorrhizas. New phytologist. 94: 247-263.

Ham, G.E. and R.H. Dowdy (1978). Soybean growth and composition as influenced by soil amendments of sewage sludge and heavy metals: Field studies.Agron            J.70 : 326-330.

Harley, J.L (1969). The Biology of Mycorrhizae. Leonard Hill, London.

Heckman, J.R. : J. S. Angle and R.H. Chaney (1986). Soybean nodulation and nitrogen fixation on soil previously amended with sewage sludge. Biol. Fertil.            Soils 2: 181-185.

Heggo, A.; J.S. Angle and R. Hchany (1990). Effects of vesicular arbuscularmycorrhizal fungi on heavy metal uptake by soybean. Soil Biol.             Biochem. 22(6): 865-869.

Hilary, J.; H. Denny and D.A. Wilkins (1987). Zinc tolerance in Betula spp. IV. The mechanism of ectomycorrhizal amelioration of zinc toxicity. New phytol 106 :             545-553.

Hirrel. M.C. and J.W. Gerdemann (1980). Improved growth of onion and bell pepperin saline soils by two vesicular – arbuscular mycorrhizal fungi.             Proceeding of the Soil Sci. Soc. Of Americal 44: 654-655.

Hue, N.V. (1988). Residual effect of sewage sludge application on plant and soil profil chemical composition. Soil Sci. Plant Anal., 19: 1633- 1643.

Jones, M.D. and T.C. Hutchinson (1986). The effect of mycorrhizal infection on the responses of Betula papyrifera to nickel and copper. New Phytol. 102 : 429-           442.

Kiekens, L.; A Cottenie and G.Vanlondsc hoot (1984). Chemical activity andbiological effect of studge-borne heavy metals and inorganic metal salts added            to the soils. Plant and soil 79, 89-100.

Larue , J. H.; W. D. McClellan and W. L. Peacock (1975). Mycorrhizal fungi and peach nursery nutrition. Colifornia Agriculture 29:5-7.

            Liebhardt, T.W.C. and T.J. Koske (1974). The lead content of various plant species as affected bys cycle-lite hamus caninum. Soil Sci, Plant Anal. 5,             8092.

Lowendrof H.S. (1980). Factors affecting survival of Rhizobium in soil. In:  Alexander, M. (ed) Advances in microbial ecology. Plenum Press, Ne 87-123.

Mathys, W. (1977). The role of malate, oxalate and mustard oil glueosides in the evolution of zinc resistance in herbage plants. Physiol. Plantarium 40:130-136.

Mosses, B. (1981). Vesicular Arbuscular Mycorrhizae Research for Tropical  Agriculture University of Hawaii Press, Honolulu.

Mosse, B. and D.S. Hayman (198). Mycorrhizae in agricultural plants. In Tropical Mycorrhjizae Research (P. Mikola, Ed.), pp. 213-230. University press,             Oxford.

Phillips, J. M. and D.S. Hayman (1970). Improved procedures for clearing roots and staining parasitic and vesicular –arbscular mycorrhizal fungi for rapid             assessment of infection. Trans. Br. Mycol. Soc. 55: 159-161.

Rother, J.A.; J.W. Millbank and I. Thorntonb (1983). Nitrogen fixation by white clower (Trifolium repens) in grassland on soils contaminated with cadmium,             to lead and zinc J. Soil Sci. 34:127-136.

Saad, R.N. (1990). Studies on legumes- mycorrhiza- rhizobia symbiotic system in newly reclaimed soil. Ph. D. thesis, Fac. Agric. Ain shams Univ.

Smith, S.S.E. (1980). Mycorrhizas of outotrophic higher plants. Biological review of  the Cambridge philosoptical society 55:75-510.

Stevenson, F.J. (1986). Cycles of soils pp. 231. John Wiley and Sons New York, USA.

Swaminaathan, K. and B.C. Verma (1979). Responses of theree crop pecies to vesicular arbuscular- mycorrhizal infection in zinc- effcient Indian soils New              Phytol. 82: 481-487.

Van Kessel, C.; P.W. Singleton and H.T. Hoben (1975) Enhanced N- transfer from soybean to maize by vesicular- arbuscular mycorrhizal VAM) fungi. Plan              Physiol. 79:562-563.

Vigue, G.T.; I.L. Pepper and D.F. Bendick (1981). The effect of cadmium on nodulation and nitrogen fixtion by dry beans. J. Environ Qual 10 : 87-98.

Welsh, R.M. (1981). The biological significance of nickel.J. Plant Natr. 3, 345.