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Kemal GÜR, Mehmet Emin AYDIN, Senar ÖZCAN, Zehra YILMAZ Selcuk University, Environmental Engineering Department, Konya, Turkiye Abstract The present work was undertaken to evaluate functions of a VAM fungus species (Glomus mossoae) and two N2-fixing bacteria (Rhizobium trifolii and Azotobacter indicum) in a coarse sandy loam mine soil and to assess their possible aggregating potentials of the soil particles in a predominantly herbaceous legume and graminae ground covers supplied with some organic wastes. The results can be summarized as follows; 1) The external mycelium of the VAM fungus and also the polysoccharides were secreted by both fungus and bacteria on the surfaces of coarse sand particles in contact with the fungal hyphae and bacterial cell fo a sufficent aggregation of the mine soil. 2) The fungal and bacterial inoculations both increased signifacantly (P< 0.01) the aggregate stability while decreased the modulus of rupture values of the mine soil samples. 3) It was found that aggregate stability values of the soil samples were positively and significantly correlated to the VA. Mycorrhizal, azobacterial and rhizobial infection levels. 4) Negative and significant relations (P< 0.01) were found between the water-stable aggregate percentages and the modulus of rapture values of the soil samples. 5) It was concluded that, VAM fungal and bacterial inoculation amended with various kind of organic wastes could help to remediate the mine soil to some extend and thus to reduce the crust formation on the soil surface, the propable water runoff and surface erosion by binding of the soil particles into stable aggregates. Introduction Vegetation types are recognized factors in mine soil aggregation (Allen,1980). Since Vesicular-arbuscular Mycorrhizal (VAM) fungi derive most of their energy directly from the plant roots, the host plant may greatly influence the physiological response of the fungal associate in mycorrhiza-soil interactions on surface-mine sites. The (VAM) fungi are involved in many well-known beneficial soil reactions (Allen,1980; Fresques, 1982) including the binding of soil particles into stable aggregates. Little information is available, however, on their possible role in mine soil aggregation. The main aim of this work was to assess the aggregating potential in a predominantly herbaceous legume and gramimaeground cover (Allen, 1980; Fresques, 1982). VAM fungi have been known to be ubuquitous, stimulate growth of several plant species including leguminous and graminous plants. On the other hand , it has been supposed that VAM fungi and some N2-fixing bacteria (Azotobacter spp. and Rhizobium spp.) could play several important roles including the binding of the soil particles into stable aggregates (Gur, 1974; Gur, 1976; Allen, 1980; Fresques, 1982). Limited information is available, however, in the literature, on the possible roles of VAM fungal and the bacterial inocula in mine soil (Allen, 1980; Trappe, 1981; Fresques, 1982). It is generally known that, there are several factors that influence the formation of water stable aggregates as well as the crust formation and thus the modulus of rupture values, of soils. Again, as stated by several workers in the literature, there have been negative correlations between the percentage of water stable aggregate and the modulus of rupture values representing the degree of the surface soil crust formation avoiding the germinating the plant seeds to emerge on the surface of the agricultural lands (Kemper, 1965; Reeve, 1965; Gur, 1987 ). The purpose of this study was to investigate the effects of the Vesicular-Arbuscular Mycorrhizal (VAM) fungus (i.e. Glomus mosseae L2) and two N2-fixing bacteria R. Trifolii and A. indicum) with applications of different levels some organic wastes (i.e. sawdust and ground straw) on the aggregate stability and modulus of rupture values of a mine soil with a coarse sandy loam textured collected from the vicinity of Ilgın town. Materials and Methods The soil sample: For the study, the soil sample was collected from a surface mined coal site in the vicinity of Ilgın town belonging to the Konya Province in the Central Anatolia of Turkiye. For experiments, freshly collected soil samples were air-dried, sieved to pass 4 mm mesh immediately after they were brought to the laboratory and then they were steam sterilized at 100 0C for one hour. The soil of the sampling site was a coarse-sandy loam texture with the following properties; pH 7.10 (1:2.5 soil-water) organic matter 0.28 %, CEC 23.4 me/100 g, exchangeable sodium 0.24 me/100 g and the available phosphorus 5.85 ppm respectively. Water stable aggregate percantages and modulus of rupture values were determined by modifying the methods of Kamper (10) and Reeve (12). The microbial cultures: For the microbiological inoculation, the cultures of Rhizobium trifolii (symbiotic, N2-fixing bacteria) and Azotobacter indicum ( non-symbiotic, N2-fixing bacteria) giving about 106 and 108 organisms per plant seed respectively when applied by the peat inoculum techniques, were used in all experiments. The number and size of rhizobial nodules on the experimental legume plant (clover) roots were also assessed and recorded, at harvest. In addition, the number of Azotobacter indicum in the experimental soils were also determined by the dilution and plate count techniques (using the most probable number assesment method, at the end of the experiment (Gur, 1976; Gur, 1984; Gur, 1987; Gur, 1993). VAM fungal inoculum: The indigenous strain of (Glomus mosseae L4) with predominantly coarse hyphae was isolated and maintained on onion roots was used for the VAM fungal inoculation. The endophty originally was isolated and identified from the agricultural soils of Konya Province, prior to the preparation of the VAM fungal culture. The mycorrhizal inoculum was a mixture of spores and infected root segments of onion plants and the mixed culture of the VAM fungus were diluted in the sterile water prior to the inoculation. At harvest, the roots of the experimental plants were washed free of soil particles, cleaned and stained then the level of VAM fungal infection was determined using the grid intersect method (Phillips, 1970; Gur, 1974; Gur, 1976; Clough, 1978). Organic waste amendment: Saw dust (C:N=187:1) and ground straw (C:N=98:1) materials were used as organic wastes at the application rates of 0.0; 0.25 % and 0.50 % (w/w). They were mixed throughly with the experimental soil samples prior to the seeding. Plant culture and experimental design: Experimental plants were grown in a 16 cm pots lined with plastic bags which contained 3 kg soil. Pots were watered to field capacity and left for two weeks at 25 0C (in root cooking tanks) prior to planting. Each pot received NPK ferilizer and was seeded with Trifolium repense, Agropyron cristatum and Festuca ovina representing leguminoceae and graminacene families respectively. The seeds were germinated (one day) prior to planting and inoculated with one ml of dense suspension of the Rhizobium and Azobacter species of planting. This work was planned as a pot experiment under the glass house conditions with an (plant x inoculation x organic waste x replicate : 8 x 8 x 7 x 3) randomized experimental design with three replications. The soil sample was coarse sandy loam with 0.28 percent organic matter and average pH of 7.2 (1:2 soil water mix). Each pot received NPK fertilizer and was seeded with three plant species such as, Trifolium repense, Agropyrun cristatum and Festuca ovina. Every two monts during the second and third growing seasons roots of the experimental plants with rhizosphore soil intact were sampled from the experimental pots. Root samles were immersed in water overnight and carefully washed free of adhering soil with a fine spray rinse. Young growth portions of the root system were cut into segments approximately 2.5 cm and stained with lactophenol-anilize blue to detect the presence of VAM fungi. Polysaccharide materials on the fungal hyphae and internal surfaces of sand grains making up the aggregates were determined by the periodic acid schiff reagent staining procedure of Clough and Sutton. Additional rhizosphere samples for sericea lespedeza were collected near the and of the fifth and sixth growing season (2000). The soil was airdried until friable, seperated from the root material by gentle shaking and sieved manually to recover the 4 mm aggregate fraction. Typical granular type aggregates selected from the sieved fraction were dispersed carefully in a staining solution and observed microscopically to determine the percent maycorhizas with these structures. Root samles were treated as noted previously (Kemper, 1965; Reeve, 1965; Clough, 1978; Gur, 1987). Results and Discussion  As it is shown in Table 1, the microbial inoculums have significantly increased the percentage of water stable aggregates whereas decreased the modulus of rupture values of the mine soil samples, where the mixed inocula of "VAM + R. trifolii + A. indicum" was found to be the most effective treatment and it was followed by "VAM + R. trifolii", "VAM + A. indicum", "R.trifolii + A. indicum", "VAM", "Rhizobium trifolii" and "Azotobacter indicum" inoculum respectively, in comparison to the control (non-inoculated) treatment (Table 1).  It was found that, additions of the organic wastes have significantly increased the percentage of the water stable aggregate while decreased the modulus of rubture values of the mine soil samples. Among the organic wastes added, the treatment with the mixture of "saw dust and straw" at 0.50 % level was found to be the most effective addition and then it was followed by the treatments of "0.25 % saw dust + 0.25 % straw", "0.50 % ground straw", "0.50 % saw dust", "0.25 % ground straw, 0.25 % saw dust" respectively (Table 2).  When the plant varities were compared to each other, in terms of increasing the percentage of water stable aggregate and decreasing the modulus of rupture values of the mine soil sample, the mixture of "P7: T. repense + A. cristatum + F. ovina" was found to be the most effective treatment and then it was followed by the treatments of P4, P5, P6, P, P3, P2 respectively with compare to the control (without plant) treatment (Table 3). It has been suggested that while pollysaccharides are effective in the organization of small aggregates, they are generally less significant as primary binding agents as soil level of humic materials increased (6,22). Humic materials effective in stabilizating aggregates are thought to function primarily in the organization and strenghening of bonds between quartz and clay particles, though the reaction sites of humus also may bind sand particles directly (5,10). While the stabiliszating mechanism has yet to be determined for the granular aggregates in this work, and the bonding sites of the clay fraction is undoubtedly involved to some extent, thus it was suggested that the abundant fibrous roots and fungal hyphae were significant factors contributing to minesoil stabilization as a result of humic substances formed in the interaction between the experimental plants and the VAM fungal inoculum (Clough, 1978; Fresque, 1982). Under the minesoil conditions in this, Glomus mosseae (VAM) formed an especially effective mycorrhizal association with the experimental plants typically produacing great quantities of both internal and external hyphae. Histochemical techniques have demonstrated the presence of chitoson, polymer composed of aminosugar unit (17) in the hyphal wall of a closely related species in the genus, and it is resonable to assume that there is a comparable morphogenetic pattern in Glomus mossea. Because of the proximety of the mycorrizal association, it is likely that the phenolic compounds released during lignin degratation in the plant tissues react with the glucoamine units in the cell wall of the VAM fungus, i.e; Glomus mossea. These conjugnted sites are proposed as the effective centers in the organization and subsequent stabilization of granular type aggregates in the sandy loam (Clough, 1978; Allen, 1980). Lignin represent an important source of aromatic compounds "for humus formation" (10) and as suggested by Kirk, the structural differences of lignin in different plants may, to a great extent, influence their degradation potential. While the slow degratation rate of the experimental plants may be an undesirable agronomic ferture, the longterm availability of substainces important in soil structure devlopment and the effectiveness of these legume plants as a mycorrhiza host appear to be contributing factors n its success in revegetation surface mines and controlling minesoil (Trappe, 1981; Fresques, 1982). Conclusions The results can be elucidated as follows: 1) The external mycelium of the VAM fungus and also the polysoccharides were secreted by both fungus and bacteria on the surfaces of coarse sand particles in contact with the fungal hyphae and bacterial cells. 2) The fungal and bacterial inoculations both increased signifacantly (P< 0.01) the aggregate stability while decreased the modulus of rupture values of the mine soil samples. 3) It was found that aggregate stability values of the soil samples were positively and significantly correlated to the VA. Mycorrhizal, azobacterial and rhizobial infection levels. 4) Negative relationships (P< 0.01) were found between the water-stable aggregate percentages and the modulus of rapture values of the soil samples. 5) It was concluded that, VAM fungal and bacterial inoculation amended with various organic wastes could help to remediate the mine soil to some extend and thus to reduce the crust formation on the soil surface, the propable water runoff and surface erosion by binding of the soil particles into stable aggregates. However, it would be much better and necessary to do a number experiments under the greenhouse conditions by using different types of herbaceous plants as a host and organic wastes in association with the microbial inoculation of VAM fungi and the organic waste-decomposing bacteria prior to any practical application in a large scale to the field. References . Allen, E.B. & Allen, M.F., 1980. Natural Re-establishment of Vesicular-arbuscular Mycorrhizae Following Stripmine Reclamation in Wyoming. J. Appl. Ecol. 17, 139-147. . Clough, K.S. & Sutton, J.C., 1978. Direct Observation of Fungal Aggregates in Sand Dune Soil. Can. J. Microbiol. 24, 333-335. . Fresques, P.R. & Lindemann, W.C., 1982. Soil and Rhizosphere Microorganisms in amended Coal Mine Spoils. Soil Sci. Soc. Am. J. 46, 751-755. . Gur, K., 1974. Studies on the Distribution and Activities of Vesicular-arbuscular Mycorrhizae. Master of Agric. Science Thesis. Reading University Library. No. 3245., Reding, England. . Gur, K., 1976. The Effects of VAM Fungal Inoculant Upon the Growth and P-uptake of the Onion Plants Grown in Two Soil Types Under Laboratory Conditions (Ph. D. Thesis) With Summary Paper in the Agricultural Journal of Ataturk University, Erzurum, Turkiye, Vol: 7(03), pp.13-21. . Gur, K., 1976. A Study on Interaction Between VAM Fungi and the Bacteria of Rhizobium Trifolii in Relation to Their Influences upon the Plant Growth. The XVII.th National Turkish Scientific Congress (5-8 October, 1976, Girne, Cyprus), On The Applied Microbiology Proceedings, Cyprus, pp. 434-443. . Gur, K., 1984. Effects of Detergents upon the Symbiotic Nitrogen Fixation and the VAM Fungal Activities in the Roots of Legumunious Plants. National Symposium of the Turkish Scientific and Techniquel Research Organization (TUBITAK) Comitee (12-15 November 1984), Adana, Turkiye, Proceedings. . Gur, K., 1987. A Work upon the Effects of Microbial Inoculums of Some Fungal Species (i.e., Aspergillus versicolor, and Penicillium chrysogenum) on the Reducing the Soil Crust Formation Due to the Increasing the Percentage of Water-stable Aggregates of the Soil. The 10th Scientific and National Soil Science Conferance of the Turkish Soil Science Society Proceedings (30 June-4 July 1987), Kırklareli, Turkiye. . Gur, K., Uyanoz, R., Akın, M. & Ozkan, V. K., 1993. A Work on the Distribution and Activities of VAM Fungi in the Soils Collected From the Vicinity of Konya Province. The Scientific Congres of the Turkish Microbial Culture Collection Centre (KUKEM) Upon the Applied Microbiology (20-23 September 1993), Ankara, Turkiye, Proceedings, Vol:16(02), pp. 126. . Kemper, W. D., 1965. Aggregate Stability. In C. A. Black (ed.) Methods of Soil Analysis, Part. I. ASA, Madison, wis., USA., 511-519. . Phillips, J. M & Hayman, D. S., 1970. Improved Procedures for Clearing Roots and Staining Parasitic and Vesicular-arbuscular Mycorhizal Fungi for Rapid Assessment of Infection. Trans. Br. Mycol. Soc. 55, 158-161. . Reeve, R. C., 1965. Modulus of Rubture. In C. A. Black (ed.) Methods of Soil Analysis. Part. I. ASA Madison, Wis., USA., 466-471. . Trappe, J.M., 1981. Mycorrhizae and Productivity of Arid and Semiarid Rangelands. In Advences in Food Producing Systems for Arid and Semiarid Lands. Amcademic Press, New York. pp 518-599. |