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Songül ÇELİK (DALCI) 1 , Sevinç ARCAK 2 1 Soil and Fertiliser Research Institute, Ankara, Turkey 2 University of Ankara, Faculty of Agriculture Department of Soil Science Abstract In this study, the effects of VA mycorrhizae on the growth of oat and the uptake of some heavy metals by the plant were studied. Within this context, oat roots were inoculated with VAM fungus and the rate of infection on the roots was determined. Heavy metal contents increased in soil, root, stem and leaves, depending on the increased doses of Cu, Zn and Cd being applied to both inoculated and non-inoculated treatments. A decrease was observed in the rate of mycorrhizal infection associated with the increased doses of heavy metals. Increased doses of Cu, Zn and Cd resulted in more accumulation in oat root, stem and leaf systems in the non-infected treatments. However , a large part of the metals were retained by roots and thereby less translocation to stems and leaves occurred in the infected treatments. Consequently, VA mycorrhiza hindered metal movements to stem and leaves. Introduction With the advent of environmental problems originating from pollutants having been transmitted up to the consumer through food chain, the concept of ecological pollution has been the focal issue. Today, agriculture practices being full filled by taking environmental concerns into account are of great importance. This phenomena has urged consistent cultivation and production systems to be developed and implemented. The purpose of the agriecosystem is to ensure alternatives with the concern that population of natural species is to be sustained and the likelihood hazardous effects is to be minimized. One of these alternatives proposed is that soil microorganisms might be used in agriculture, thereby a natural protection management might be promoted. As known, mycorrhizal fungus gives the plants an ability to resist heavy metal stress and enviromentally pollution agents. Interest in Cd as an enviromental pollutant has arisen because of its potentially harmful effects on human health.There is concern that , as a result of additions to soil in sewage sludge and in other ways, increasing amounts of Cd are entering the food chain through uptake by plants ( Hatch at all,1988). Several authors reported that shoot concentrations of Zn, Cu, Pb and Cd decreased compared with AM colonization at high levels of available metals, whereas at lower levels metal uptake increased compared with non-mycorrhizal plants (Weissenhorn at all,1995). High concentrations of heavy metals in soil have an adverse effect on micro-organisms and microbial processes. Among soil microorganisms, mycorrhizal fungi are the only ones providing a direct link between soil and roots, and can therefore be of great importance in heavy metal availability and toxicity to plants (Leyval at all.1997) The objective of the study is to investigate the possibilities of utilisation of VAM in mitigating pollution in soil. In this context, the uptake of heavy metals by plants, the effects of mycorrhiza on heavy metal absorbed by plants and mycorrhizal tolerance to heavy metals were tried to explain. Material and Methods Soil samples used in the research were taken from (Lodumlu) Research Institute at the depth of 0-20 cm. The soil was sieved (4mm) and mixed with sand (3:1)(w/w) ratio. The mixture was sterilised (120° C for 2 h on three consecutive days) to eliminate native AM propagüles. Four species of AM fungi were used as mycorrhizal inoculum: Glomus mossea, G.fasciculatum, G.etunicatum, Gigaspora margarita. The host plants was onion (Allium cepa); the test plants was oat (Avena sativa L.) (native variety : Yeşilköy-Y 1779). The different metal contents in the soil were obtained by adding aqueous solutions of ZnSO4. 7H2O, at the rates of 0-50-100-200-400 mg Zn kg-1 soil ,Cu SO4. 5H2O (0-5-10-20-40 mg Cu kg-1 soil and 3Cd(SO4). 8H2O at the rates of 0-1-5-10-20 mg Cd kg-1 soil. After carefully mixing the metal solutions with the soil, this was allowed to stabilise for 15 days before using. All treatments were applied with and without mycorrhizal inoculum and there were four replicates. In order for infection material to be reproduced, AM fungus-infected onion was used for the infection process of oat roots. One g of inoculum consisting of soil with spores, infected roots and hyphae was added to each pot at 3 cm deep and mixed with the soil. PVC pots of 8 kg were used in the experiment. Oat seeds were sterilised with hydrogen peroxide (10 vol) for 30 minutes, washed with sterile water. The seeds were sown into pots (30 in each) and thinned to 20 per pot after germination. After a growth period of 8 weeks in the greenhouse all plants were harvested. Inoculated root samples (1 in each) were stained with lactoglicerine-Trypan blue (by Philips and Hayman 1970) to determine the extent of mycorrhizal infection. Percentage infection was calculated as the light microscope fields per 100 containing any mycorrhizal fungus structure. The plants were saperated into root and shoot dried to constant weight at 65° C. Dry plant material was digested in HNO3: HClO4 (3:1). Soil samples extracted with DTPA (pH:7.3). Zn, Cu concentration were measured by flame AAS (Perkin-Elmer 2100) and Cd concentration was measured by flame AAS(Perkin-Elmer AS 800). Statistical tests (Analysis of variance, regression , Pearson correlation) were made by the statistical Package for the social Sciences(SPSS). Results and Discussion VA mycorrhizae infection : Infection rate of 36% was determined in infected oat roots (Table l). Infection rate decreased in parallel with the increased doses of heavy metals (Cu, Zn and Cd). The fact that an infection rate of 36% took place in infected plants alone may be due to the available phosphorus content of 5 kg da-1 of the soil samples and the slightly increase in the available phosphorus content after sterilisation. Generally, soil nutrients are relaced through the decomposition of organic components by sterilisation and died organisms( Walker 1985, Stribley 1987). A known fact is that heavy metal uptake by plant and the plant tolerance to heavy metals may contribute to the changes in infection rate (Olsen 1972, Baker 1978).  The effect of VAM on plant and root dry weight : Plant dry weight of 6.605 g pot-1 in control and 5.970 g pot-1 in mycorrhizal infected plants were recorded. Mycorrhizal infection did not contribute to plant dry weight. As mycorrhizal infection ratio decreased so did the plant dry weight. A positive correlation was found among mycorrhizae, plant and root dry weight in each three heavy metal treatments. Graham and Fardelman (1986) conducted a research of similar nature and found no correlation between root infection and mycorrhizae effectiveness. Heavy metal tolerance of VAM infection : A negative correlation was found between Cu doses and mycorrhizal infected roots (Y= -0.318x+ 31.662, r = -0.82**,P<0.01). As Cu doses increased, mycorrhizal infection decreased. A negative correlation was also found between Zn doses and mycorrhizal infected oat roots in Zn application (Y=-0.0403x + 28.358 r=-78**,P<0.01). As Zn doses increased, mycorrhizal infection decreased. As to the effect of Cd, a negative correlation was observed between mycorrhizal infected plants and Cd doses. Increased Cd doses caused mycorrhizal infection to decrease (Y=-1.424x+ 28.255, r= -0.88**, P< 0.01). Similarly, increased doses of heavy metals (Cu, Zn and Cd) brought about decrease in mycorrhizal infection rate. Heavy metals concentrations in soil, root and shoot : Cu uptake by mycorrhizal infected roots (24.80 ppm) was higher than that of mycorrhiza-free roots (14.10 ppm). Heavy metal contents of the soil increased depending on the applications of heavy metals. Cu content of mycorrhizal infected roots was observed higher than that of non-infected roots. Increased doses of Cu caused Cu uptake by both applications to increase (Fig.1). Infected roots absorbed much less Zn in comparison with the control application. As Cu doses increased in both application so did Zn concentration in roots. However, Zn content in mycorrhizal application was recorded lower than that in non-mycorrhizal application. Due to the high level of available phosphorus in the soil (5.0 kg da-1 P2O5) and its negative effect on Zn, less Zn uptake occurred in mycorrhizal applications (Fig.2). Dueck at all (1986), expressed that although VAM had no complete hindrance effect on Zn uptake, VAM colonization mitigated Zn effect on the growth of meadow grass. Cd content in infected roots was found less than that in control application. With the increased Cd doses, Cd content of the mycorrhiza free samples increased as well. The highest Cd value was recorded in the application of Cd20 (M: 13.43 ppm, NM: 24.28ppm) . This revealed the positive effect of the mycorrhizal infection and the less Cd uptake by roots through hyphae of VAM hindering the translocation of the elements (Fig.3).  The comparison between mycorrhizal (M) and non- mycorrhizal (NM) application showed that high level of Cu in the root of the infected plants was hold by mycorrhiza, thereby the translocation of the elements to leaves was hindered resulting in less Cu movement to the leaves. The highest Zn accumulation was found in root system and Zn content in non-infected plants was higher than that recorded in infected plants. Due to the retention by hyphae depending on the uptake by the plant, Zn content in the infected plants was found less than that in non-infected plants. Zn translocation to the leaves in infected plants was not completely hampered. Much less Cd uptake took place in mycorrhizal infected plants in comparison with the non-infected plants. Cd application affected mycorrhizal infection negatively; however, Cd uptaken by infected plants was low and was retained in root systems allowing not a complete translocation to leaves. Gildon and Tinker (1983b), conducted a research by applying Cu, Zn, Ni and Cd to trifolium and found no significant effect on plant dry weight in VAM infected plants. In addition, they expressed that the addition of high level of Zn and Ni caused decrease in plant dry weight. Conclusion Infection rate was found 36 % in VAM infected oat roots. As a result of increased heavy metal application (Cu, Zn and Cd) VAM infection rate lessened [ Cu (36-21%), Zn (36-15%) and Cd (36-4%) ]. As heavy metal doses increased oat root dry weight and leaf dry weight decreased. VAM had nothing to do with the increasing the root and plant dry weight and increased heavy metal doses enhanced the soil heavy metal content. The effect of heavy metals on root and plant dry weight : No difference was observed between the root dry weight of infected and non-infected plants, both of which received Cu, Zn and Cd applications. Increased doses of heavy metals brought about decrease in both infected and non-infected plants (Fig.4, Fig.5, and Fig.6). Smilde (1981), examined the effect of increased Cu, Zn and Cd application on the growth of oat and maize and found less oat weight in response to the increased Cu and Cd doses and no toxicity in the plant. When Cd was added to the soil, dry weight and grain yield decreased (Allinson and Dzialo 1981). Considerable decrease occurred in dry weight, when high doses of zinc were added (Gildon and Tinker 1983 b). Researches of similar nature conducted show that mycorrhizal symbiosis has no notable effect on plant dry weight.  It can be expressed that VAM infection enables the most of Cu, Zn and Cd to be retained by roots, allowing less heavy metal translocation to leaves. The filtering property of mycorrhiza may contribute to the efforts to mitigate high levels of heavy metals in soils which is direct linkage with the food chain.  References . Allınson , D.W. and Dzıalo, C.1981.The Influence of lead, cadmium and nickel on the growth of ryegrass and oats. Plant and Soil 62:81-89. . Baker, A.J.M.1978. Zinc-Phosphorus interactions in a zinc-tolerant and non-tolerant population of Silene maritime Wilt.New Phytologist. 81,331-340. . Dueck, Th.A., Visser, P., Ernst, W.H.O. and Schat, H. 1986. Vesicular Arbuscular Mycorrhizae decrease zinc-toxicity to grasses growing ın zinc-polluted soil. Soil Biol. Biochem. 18 (3), 331-333. . Gildon, A., Tinker P.B. 1983 b. Interactions of vesicular- arbuscular mycorrhizal infection and heavy metals in plants. New Phytol., 95: 247-261. . Graham, J.H. and Fardelmann, D.1986. Inoculation of citrus with root fragments containing chlamydospores of the mycorrhizal fungus Glomus intraradices. Can.Jour.of Botany 64:1739-1744. . Hatch D.J., Jones, L.H.P. and Burau R.G.1988.The effects of pH on the uptake of cadmium by four plant species grown in flowing solution culture. Plant and Soil 105:121-126. . Leyval, C. Turnau, K. Haselwandter, and K.1997.Effect of heavy metal pollution on mycorrhizal colonization and function: physiological, ecological and applied aspects. Mycorrhiza 7(3): 139-153. . Olsen, S.R.1972. Micronutrient interactions. In: Micronutrients in Agriculture pp.243-264. Soil Science Society of America, Madison. . Phillips, J.M. and Hayman, D.S.1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society.55, 158-161. . Smılde.K.W. 1981.Heavy metal accumulation in crops grown on sewage sludge amended with metal salts. Plant and Soil, 62:3-14. . Stribley, D.1987. Mineral nutrition. In Ecophysiology of VA mycorrhizal plants. (Ed). G.R.Safir, pp 59-69.CRC press. Boco Raton. FL . Walker, C.1985. Mycorrhizal growth enhancement in sitka spruce seedling differs in non-sterile compared to sterilised soil. In proceedings of 6th North American Conference on Mycorrhizae.(Ed) R.Molina.Oregon. . Weissenhorn, I., Leyval, C.,Belgy, G. and Berthelin, J. 1995. Arbuscular mycorrhizal contribution to heavy metal uptake by maize (Zea Mays L.) In pot culture with contaminated soil. Mycorrhizae 5: 245-251. |