INTRODUCTION
Since the development of coal mines, coal waste dumps and acid leachate due to abandoned mines has caused secondary damages to the forest environment; waste is washed away and the water becomes polluted. (Jeong et al., 2010). Most closed mines in Korea have been neglected without any disciplinary measures, and just a few basic studies on coal wastes was initiated at the end of the 1980s when damage due to those wastes became a social issue. One method that is currently used to grow vegetation in coal waste piles in Korea is to fill an area of about 60 cm of general soil as collected from cut slopes, and to seed or plant the top. Meanwhile, due to requests to solve contaminants from closed mines with eco-friendly technologies, phytoremediation, a method using plants, is receiving attention, and research related to this, research on hyper-accumulators and tolerant species have been conducted in Korea and abroad. However, most of those research papers do not seem to be very relevant to the fact that mine protection could prevent heavy metals from leaching out (Jeong, 2012 ; Yi et al., 2013). Also, chemical additives that are used as enhancers could cause potential toxicity and heavy-metal elutions to plants, which could induce secondary pollution. Accordingly, proper measures and systematic study are required at this point.
To address such problems, phytostabilization, which decreases secondary pollution possibile due to heavy metals, was introduced. Phytostabilization is a method of suppressing the vertical and horizontal movement of pollutants that exists in the soil through the absorbing, accumulating or adhering of pollutants to plant roots or congealing around roots (Suh, 2003). This method aims to reduce the mobility of pollutants, prevent them from reaching ground water and minimize the contamination of the food chain. The species of trees that are mainly used are Robinia pseudoacacia and lespedeza spp., and Robinia pseudoacacia, which are well-known for their symbiotic relationship with root nodule bacteria in coal mine tailings, which lack nutrients for plants, and its ability to fix nitrogen from the air which directly affects root development (Lee, 2010). On the other hand, Robinia pseudoacacia is known to form a symbiotic relationship with arbuscular mycorrhizal fungi and create hyphae; and those hyphae facilitate soil aggregation (Miller and Jastrow, 2000). However, barely any study has been undertaken regarding the effects of arbuscular mycorrhizal fungi on the soil aggregation of coal mine tailings. Therefore, the purpose of this study is to transplant Robinia pseudoacacia seedlings infected by arbuscular mycorrhizal fungus Glomus etunicatum, and verify mycorrhizal infection rates and infection types; ergosterol content within soil aggregates; and the effects on soil aggregate formation.
MATERIALS AND METHODS
1.Experiment Materials
The Robinia pseudoacacia seeds used in the experiment were treated with 70 % alcohol for one minute; washed three times with sterile water; treated with 2 % sodium chlorite for 15 minutes; and sowed on a seedling plate. Soil that was put on the seedling plate was sterilized at 121 °C for two hours under high pressure. In case of Glomus etunicatum spores that would be inoculated to Robinia pseudoacacia's roots, soil from a Miscanthus habitat in Mihocheon, Cheongwon-gun, Chungcheongbuk-do was incubated and proliferated in millet(Figure 1). Proliferated spores were collected from the soil; their morphological characteristics including size, color and the number of spore walls were studied; and the finally identified G. etunicatum was separately classified by referring to Schenck and Perez (1987) and INVAM (http://invam.caf.wvu.edu/) (Figure 2).
G. etunicatum was inoculated using the following method: after transplanting to pots (diameter 16 cm × height 15 cm) the root-grown Robinia pseudoacacias, which were sowed on the seedling plate was filled with forest soil, 100 g soil containing 23.2 spores per 10 g was put into the pots to come into contact with the roots. After checking whether Robinia pseudoacacia seedlings, which were inoculated three months ago, were infected, they were classified into inoculated and noninoculated mycorrhizas, and were transplanted to pots (soil volume 1600 cm3, diameter 16 cm × height 15 cm) filled with coal mine tailings (Table 1) that were sterilized at 121 °C for two hours under high pressure. For comparison, the same process was carried out using forest soil. Coal mine tailings that were used for the experiment were collected from a closed coal mine near Baegun Temple at Mt. Seongjusan in Seongju-myeon, Boryeong-si, Chungcheongnam-do, and filtered with a 2 mm sieve. Forest soil was collected from a pine forest in Chungbuk University, filtered with a 2 mm sieve and mixed 1:1 with sand for ventilation and water movement.
2.Experiment Design
There were a total of four experimental plots: mycorrhiza-inoculated coal mine tailings and forest soil, and noninoculated coal mine tailings and forest soil. The number of repetitions by each experimental plot were 11; three seedlings were planted per pot; and they were incubated from May 2011 to June 2012 for 12 months in a green house at Chungbuk University.
3.Measurement
1)Mycorrhizal Infection Rate Analysis
To verify the mycorrhizal infection rates, samples were dyed with 0.03 % of trypan blue solution according to Brundrette et al. (1996)'s method; the dyed samples were destained with 50 % glycerol for 2~3 days; and observed with a dissecting microscope. In case of the measurement of mycorrhizal infection rates within roots, dyed root pieces were observed under a optical microscope, and the number of hyphae, arbuscules and vesicles that form arbuscular mycorrhizal fungi were measured. An average value, which was obtained by repeating the foregoing process three times was used (Mcgonigle et al., 1990).
2)Measuring Ergosterol Content In Soil
With regards to extracting and measuring ergosterol content in soil, methanol was used for extraction and HPLC (Hewlett Packard 1100 series) was used for quantitative and qualitative analysis. In case of HPLC analysis, the C18 reverse phase column (4.6 mm × 250 mm) was used, and as for solvent, 98 % methanol was evenly applied at a speed of 1 mℓ/min. As for the UV wavelength, 280 nm was used. In case of a calibration curve for quantitative analysis, it was obtained using solutions of 1 ppm, 5 ppm, 10 ppm, 20 ppm, 50 ppm and 100 ppm using ergosterol standard (Sigma, E6510).
3)Measuring the Weight and Ratio of Soil Aggregate
With regard to measuring the weight and ratio of soil aggregates (>0.1 mm), after air-drying soil which was separated from a pot according to Emerson and Foster (1985) (Figure 3), 50 g of soil sample in which aggregates (2-4 mm) were formed was soaked in 300 mℓ of distilled water for 16 hours. Then, the sample was poured into sieves at sizes of 2 mm, 1 mm, 0.5 mm, 0.25 mm and 0.1 mm, and a cylinder (PVC) was filled with 30 cm water (height 60 cm × width 23 cm). The materials caught in the sieves were put into the cylinder and slowly moved up and down for about 5 seconds per shake, and dried using a dry oven. A waterstable aggregate value excluding sand was measured by eye and substituted the following formula, and the waterstable aggregate ratio was measured.
4.Statistics Process
As for the statistics analysis of experiment results, ANOVA analysis was conducted using SPSS 12.0 at a significance level of 5 % for the measurement value of each process plot, and Duncan analysis used for post analysis.
RESULTS AND DISCUSSION
1.Mycorrhizal Infection Rate and Infection Characteristics by Soil
The mycorrhizal infection rate of mycorrhiza-inoculated coal mine tailings was 35.1 % and that of the forest soil was 48.9 %, which did not show a difference in arbuscule and hypha formation rates within roots. However, a vesicle formation rate of forest soil was significantly higher by about 7.2 % (Table 2). In this context, Del Val et al. (1999) and Chao and Wang (1990) reported in their papers that mycorrhizal infection rates and external hypha formation rates of plants that grow on sludge containing cadmium (Cd), lead (Pb) and zinc (Zn) were lower than that of sludge-noninoculated plants; and when Cd, Cu, Cr, Ni, Pb and Zn are added to mycorrhiza-inoculated soil, mycorrhizal infection rates would dramatically drop.
Also, depending on the types of heavy metals, the effects on the infection of arbuscular mycorrhizal fungi were different. According to Griffioen et al. (1994)'s paper, they planted arbuscular mycorrhizal fungus-inoculated perennial artemisia spp. on soil plots for each one containing copper, zinc, cadmium, respectively, and non-polluted soil, and checked the mycorrhizal infection rates after five years. As a result, soil containing copper was barely contaminated, but the mycorrhizal infection rate of soil from an area not polluted by heavy metals was about 60 %, and that of soil containing zinc and cadmium, respectively, was about 40 %. Meanwhile, the reason why 2.5 % of hypha infection rate was measured at noninoculated forest soil is thought to be due to other bacterium within root as the structures of arbuscules and vesicles were not found (Table 2).
Regarding the infection form of arbuscular mycorrhizal fungi within roots, the hyphae spread along the cortex, arbuscules were formed sparsely and hyphae branched at the part where arbuscules and the cortex contact. In this respect, the infection types of G. etunicatum at Robinia pseudoacacia's root were generally classified into the Arum-type and Paris-type. In case of the Arum-type, hyphae spread to the cortex, and branch and create arbuscules(Figure 4). And in case of the Paris-type, coiling hyphae are extensively developed within the cortex cells, and arbuscules are not developed from coiling hyphae (Smith and Read, 2008).
2.Ergosterol Content within Soil due to Mycorrhizal Inoculation
Ergosterol content within soil due to mycorrhizal inoculation was 1.92 ppm/g and 3.20 ppm/g in mycorrhiza-inoculated coal mine tailings and forest soil, respectively. The content in the latter was significantly higher by about 65.8 %, and almost no ergosterol was found in noninoculated coal mine tailings and forest soil (Figure 5). Ergosterol is a sterol existing in the cell membrane of ascomycetes and basidiomycetes, and it barely exists in plants or other microorganisms, but rather presents in live fungal cells. It is useful when estimating the growth volume of fungi that grows in soil and sawdust. Therefore, high ergosterol content means there are a lot of live hyphae (Koo et al., 2000 ; Kim et al., 2004 ; Jang et al., 2011). Essential parasitic nutrition of arbuscular mycorrhizal fungus life history could be classified into two stages: first, spore germination and hypha expansion before forming a symbiotic relationship; and second, the symbiotic stage, the settling of the expanded hyphae inside a host plant's root, and the hyphae expansion stage, in which the hyphae expanded to the outside of the plant's roots (Tommerup and Briggs, 1981). In other words, in order for arbuscular mycorrhizal fungi to grow well in a certain soil, the first stage, spore germination and hypha growth and development, needs to be realized smoothly. According to Hepper and Smith (1976), arbuscular mycorrhizal fungi living in low metal content soil are more sensitive to metals than those living in high metal content soil in terms of spore germination. Also, G. etunicatum showed a more sensitive reaction than G. intraradices, and the latter was more effective when handling stress due to heavy metals. Through this experiment, it was surmised that the lower ergosterol content in coal mine tailings compared to forest soil was due to the soil's suppression of the growth and development of hyphae.
3.Soil Aggregate Formation due to Mycorrhizal Inoculation
The soil aggregate formation rates due to mycorrhizal inoculation were 33.0 % and 39.1 % in mycorrhizainoculated coal mine tailings and forest soil, respectively, and 22.0 % and 19.0 % in mycorrhiza-noninoculated coal mine tailings and forest soil, respectively, showing significantly lower rates (Table 3). The difference between aggregate formation rates between mycorrhiza-inoculated and -noninoculated plots by soil were 11% and 19.9 % in coal mine tailings and forest soil, respectively. Result showed that soil aggregate formation was more affected by mycorrhizas in forest soil than coal mine tailings. This means mycorrhizal inoculation increased the soil aggregate formation in both soil, but there was a distinction in the effect by soil (Table 3).
The importance of the symbiotic relationship between plant body and arbuscular mycorrhizal fungi is not only limited to the increase in nutritional benefits, but to the facilitation of soil aggregation by outside hyphae. Soil aggregates play an important role of storing soil carbon (Degens et al., 1996; Rillig, 2004). Glomalin, a glycoprotein produced on a hypha cell wall, plays part of the facilitation of soil aggregation due to arbuscular mycorrhizal fungi. This substance does not dissolve in water, and exists in soil even when a hypha become extinct (Wright and upahyaya, 1996 ; Driver et al., 2005). Therefore, the amount of glomalin increases as the growth and development of hyphae thrive. In past research papers it was revealed that soil aggregation was facilitated by the outside hyphae of arbuscular mycorrhizal fungi through forest soil or farmland soil (Jakobsen et al., 1992). And in this study, it was identified that soil aggregates also increase in coal mine tailings depending on the growth and development of the hyphae of arbuscular mycorrhizal fungi. In conclusion, the inoculation of arbuscular mycorrhizal fungus G. etunicatum did facilitate the aggregation of coal mine tailings and forest soil, but the former showed a lower effect.
