Isolation of Iindigenous arbuscular mycorrhizal fungi (AMF) to support revegetation on the nickel post-mining land

The revegetation on the nickel post-mining area requires symbiotic associations with mycorrhizal fungi to support the plant growth because it faces major obstacles of harsh soil condition. The study was conducted to identify the AMF status on the nickel post-mining land and its association with pioneer plant roots. Soil and root sampling werewas collected on 4 type areas of nickel post-mining land i.e TR (top soil + revegetation), TNR (top soil + no revegetation), NTR (no top soil + revegetation) and NTNR (no top soil + no revegetation) to determine spore density, soil chemical properties and AMF colonization. The results showed that the nickel mining activities interfere in soil fertility and soil microbial population. It was denoted by lack available nutrient and low AMF diversity. Population of AMF only 7 – 83 spore per gram soil. Glomus or Acaulospora is dominating genus in each area. However, AMF occurrence is a vital to help the pioneer plants growth. It was proven by 163 species of pioneer plants that colonize nickel post-mining land associated with the AMF in their root system. Grass species have highest AMF colonization level. Therefore, inoculation of AMF indigenous isolate is needed as an intervention ion post-mining reclamation effort, so the possibility of symbiosis between plant roots and AMF becomes increasing. Eventually the development of natural plants will be accelerated to catalytic the natural succession process.


Introduction
Nickel mine is one of the country's foreign exchange contributors, and Indonesia is ranked 4th of the world as a nickel producer. However, in addition to generating foreign exchange, the nickel mining industries also caused deforestation as they apply open-pit mining methods. Nickel mining leaves the post-mining lands with very low soil properties i.e. no organic matter, low pH, low nutrient availability and high soil temperatures. Moreover, according to [1] found that nickel mining led to heavy metal pollution i.e. Ni, Co, Fe, Mn, Cu, Pb and Zn in the soil. Nickel mining activities have also been shown to decrease the soil microbial communities [2]. The decline in soil microbial communities, especially beneficial soil microbes will further hamper the the development of natural plants.
AMF (Arbuscular Mycorrhizal Fungi) is one of the soil microbial components that have an important long-term role for the development of plant communities [3][4] [5]. AMF established mutualism symbiosis with vascular plant. The fungi get in touch with root plants, but the symbiosis is created with the plant, as the photosyntethic assimilates are delivered to roots system. Hence, the plant provides constant and direct access to carbohydrates, such as glucose and sucrose, for the fungus. In reply, the plant gets the benefits for water and mineral nutrients due to the large surface area of fungal  [6] and [7], The arbuscular mycorrhizal association able to increase P absorption and other nutrients, as well as drought tolerance. Moreover [3} [8] [9] [10], stated arbuscular mycorrhizae also improve the soil physical properties by the improvement of soil structure because one of the role of AMF hyphae contributes to the formation of soil aggregates. Therefore, the AMF associations play a role in accelerating plant survival and growth, especially in the marginal land that extremely low nutritional status.
The declining AMF population will cause the development of the vegetation community to be inhibited. According to [11], AMF has great potential in the development and survival of plants in natural conditions and environmental stresses on mined land. The characteristic of the nickel postmining lands are also suspected causing AMF population and infectivity decline due to low soil quality. Meanwhile AMF is vital for the successful reclamation of post-mining land [12] [13]. Low AMF population and decreased infectivity will hamper mineralization and immobilization of nutrients, thus inhibiting the development of native plants, and further affect the process of natural succession. In this research, isolation of AMF was conducted to analyze species diversity, species richness and spore density of AMF on the nickel post-mining area. In addition, measurements of AMF infection level in the pioneer plants were also undertaken to determine the significance of AMF's role in the successful of vegetation invasion and colonization on the area.

Study area and sampling method
Location of soil and root sampling was conducted in the mining area of Stargate Pacific Resources (SPR) company in Konawe Utara Regency, Southeast Sulawesi Province, Indonesia. Geographically the location of mining concession rights of PT. The SPR approved by the principle of forest use lies between 03 ° 17 '24 "LS -03 ° 22' 1.2" LS and 122 ° 14 '45.6 "BT -122 ° 17' 42" BT. The post-mining nickel area is 3 years old and has been arranged by overburden materials. Some of these areas have also been spread with topsoil and have been revegetated or no revegetated. The location of soil and root samplings in the post-mining area was carried out in 4 type areas TR (top soil + revegetation), TNR (top soil + no revegetation), NTR (no top soil + revegetation) and NTNR (no top soil + no revegetation).
Collecting soil samplings was performed to AMF spore isolation and soil chemical properties analysis. Soil sampling was done by subplot size 20 cm x 20 cm [13] in main plots 10 m x 10 m on dry season (September 2015). Preparation of main plots in each type of area is carried out by systematic with random start. Subplots are placed at 4 points of the main plot angle's and 1 point from the intersection of the main plot diagonal line. The soil samples were taken at a depth of 0-20 cm. Furthermore, soil samples were kept in a plastic bag and then composted according to its subplot. Root samplings were collected from all pioneer plants in each main plot. Sampling of pioneer plant specimens was also performed for its identification purposes.

AMF isolation and identification
The collected soil samples were processed for AMF spore isolation and identification. These process were done on October -Desember 2015. The spore extraction was conducted following wet sieving and decanting methods from Gerdemann and Nicolson in [14] and followed by centrifugation techniques from [15] that has been modified. Spores made of preparations are spores that have a complete structure. Preparation of spores using Polyvinyl Lacto Glycerol (PVLG) solution as a preservative and Meltzer's solution as a dye. The identification was done morphologically based on the spore shape and its reaction to Meltzer's solution. The spore color change in Meltzer's solution is one of the indicators for determining the genus [15].

Assessment of AMF colonization level
Roots were stored in 50 % (v/v) ethanol then cleared and stained using the method of [16]. The slide method of [17] was used observing the percentage of mycorrhizal colonization.

Soil chemical analysis
The evaluation of soil chemical properties consisting pH, C-org content, N total, P available (P2O5), and CEC (Cation Exchangeable Capacity).

Data analysis
The AMF community structures were analyzed using several diversity parameters including species richness and important value indexes [ [18], [19]], as well as the Shannon-Wiener diversity index. The soil chemical properties, AMF spore density, species diversity and level of AMF colonization in the pioneer root system were analyze by ANOVA in SPSS 25. The one way analysis of variance was carried out to compare the means of different treatments (area). Means separation tests were performed using Duncan Multiple Range Test (DMRT) at the P<0.05 level after significant F values were obtained.

Soil chemical properties
Statitistical F-tests revealed that among all soil parameters measured in nickel post-mining land, content of soil C-organik showed significant differences the areas. Meanwhile, N content, P available, K content and CEC were non-significant. However, most soil chemical parameters showed very low or low category in the category set by the Indonesian Centre for Agricultural Land Resource Research and Development (ICALRD) ( Table 1). This mean that the soil chemical properties of nickel postmining is harsh for plant growth even though after top soil spreading and revegetation. Available phosphorus concentration increased with top soil spreading and more enhancing with revegetation. Topsoil and vegetation restoration can significantly improve the content of soil available phosphorus [20] [21]. Different trend was showed by N and K content. Top soil amendment and revegetation have no influence to the availability this macronutrient. The organic C content of soil on nickel post-mining was very low (Table 2), which may account for the lack of improvement in the CEC. The increasing of organic C content was denoted by areas which have been spread with top soil and revegetated. According to [20], top soil spreading significantly increased soil organic matter and indicated increasing of organic C content of soil. Furthermore, trees can potentially improve soils through numerous processes, including -maintenance or increase of soil organic matter [12].

AMF spore abundance
The nickel mining process which is conducted by open-pit mining method thus removing vegetation and topsoil layer proved to have a very significant impact on the soil microbial community decreasing, i.e., AMF. A very noticeable decrease is the density of the spores. In Figure 1. the nickel post-mining area that has been stockpiled with an overburden material but not followed by topsoil spreading and revegetation (NTNR) has the lowest spore density and so does its species diversity and richness. The decrease of spore density of AMF due to mining activity also occurs on the post-mining land of limestone, tin, and gold [5] [13] [22][23] [24] that use open pit mining method. The AMF population on the nickel post-mining land increases when the area that has been dumped with the overburden material, then spread with topsoil, moreover when the revegetation was done in the area (Figure 1). The revegetation can increase AMF spore density, species diversity, and species richness. However, when the revegetation was not preceded by top soil spreading, the AMF spores IS BIOREV 2018 IOP Conf. Series: Earth and Environmental Science 308 (2019) 012038 IOP Publishing doi:10.1088/1755-1315/308/1/012038 5 density, species diversity and species richness were lower than area with top soil but not following by revegetation. It was showed by the comparison of NTR and TNR (see Figure 1.). This result indicate top soil spreading is important in post-mining reclamation. The top soil contain available nutrient that was required for plant growth and appropriate soil microbes for plant survival. According to [25] and [26] topsoil contains mycorrhizal or bacterial symbionts for promoting the establishment and persistence of plant species indigenous to the local environment. A total of 11 morphotypes of AMF spores isolated from nickel post-mining land were identified as 3 genus namely Acaulospora, Gigaspora and Glomus ( Figure 2). The presence and density of spores of each genus in each type of area are varied. In addition, not all genus are found in every type of area and the dominating genus is also different (Figure 3).
The genus Acaulospora, Gigaspora, and Glomus were found in the post-mining areas that have been spread by top soil and revegetated (TR). Meanwhile in the other area of nickel post-mining (NTNR, NTR, TNR) only genus Acaulospora and Glomus were found. The results of this study also indicate that higher density of certain genus spores in an area is also followed by a higher index value importance (IVI). According to [19] the AMF species or genus having INP ≥ 20 is the dominant species or genus. Glomus is the most dominant AMF genus on the area with top soil and then revegetated, while Acaulospora is dominated on the other area. Dominance of Acaulospora and Glomus are possible by shorter spore production cycles and smaller spore size than Gigasporaceae [27] and Acaulospora dominance is also found in limestone spoiled mine areas [13].
The results of this study prove that spreading of top soil can increase the population and the diversity of AMF, especially when followed by revegetation. However, top soil spreading is often constrained by the insufficiency of the top soil itself. Therefore, revegetation supplemented by mycorrhizal inoculum is an effort to catalyse increasing of AMF population and plant establishment. Revegetation of limestone quarry by mycorrhizal inoculated planting increase its indigenous AMF population 2 times higher than revegetation with non-mycorrhizal plants, even 4 times higher than no  [13]. AMF inoculation in revegetation of post-mining land is necessary because it ensures the possibility of arbuscular mycorrhizal associations in plant roots. In addition, the utilization of indigenous AMF isolates in revegetation of post-mining land is recommended as more adaptive to local edaphic conditions. Figure 3. Spore density and important value index (INP) of each genus AMF found in nickel post-mining area. NTNR = post-mining area already stockpiled with overburden not following top soil spreading and revegetation, NTR= post-mining area already stockpiled with overburden not following top soil spreading but revegetated;, RT = post-mining area already stockpiled with overburden + topsoil + revegetated.

AMF colonization
A total of 16 species of pioneer plants including the species of grasses, herbs, shrubs, and trees that naturally invade and colonize the nickel post-mining land associated with AMF in its root system (Table 2). Similarly, 2 species of trees namely Paraserienthes falcataria and Palaquium luzoniense Vid that are planted for revegetation of the land. The AMF level colonization of these plants is low because of ≤ 50%.