Potential use of indigenous arbuscular mycorrhizal fungi to improve soil productivity in tailing of tin mining: a greenhouse study scale

Tin mining is as a critical factor destroying ecosystems and challenging for reclamation. Indigenous arbuscular mycorrhizal fungi (AMF) are well-known for assisting plants growing in harsh environments. This study aimed to (1) isolate indigenous AMF; (2) screen AMF for nutrient uptake and plant biomass; (3) investigate the screened AMF in supporting plant growing in tin tailing. Soils were collected from natural forest and post-tin mining in Bangka. Wet sieving was conducted to obtain spore of AMF. Collected AMF were propagated and screened for phosphorus uptake and plant biomass of tested Sorghum bicolor growing in sterilized sand at greenhouse. Eight best-screened AMF was evaluated using Piper nigrum growing in sterilized tin tailing+10% compost at greenhouse. The new leaf number, phosphorus uptake, and flowering emergence were measured. Trap culture obtained 16 Acaulospora sp., 1 Glomus sp., and 1 Scutelospora sp, in post-tin mining, and 9 Acaulospora sp. in natural forest. Screening of AMF resulted in higher shoot P uptake and biomass than controls. Application of screened AMF on tin-talling showed that four AMF had higher new leaf number, phosphorus uptake, and flower emergence of P. nigrum. These results imply that selected indigenous AMF are potential for future land productivity of post-tin mining area.


Introduction
Tin mining is one of important industry in Indonesia.However, this mining activity contributes on environmental degradation.The process of tin mining involves several stages: exploration, clearing and land preparation, excavation and extraction, ore processing and beneficiation, each of which can contribute to environmental degradation if not properly managed [1].Tin mining may associated with deforestation [2], soil erosion and degradation [3], water pollution, habitat destruction and biodiversity loss [4].The exposed soil due to excavation and removal of topsoil is vulnerable to erosion by wind and water, leading to the loss of fertile topsoil and the degradation of soil quality [5].As a result, it reduced soil organic carbon, CEC, N, P, and K levels, causing the soil to become nutrient-deficient [6].Plants will be extremely difficult to grow in this condition; therefore, rehabilitation to improve soil productivity is critical.Physiochemical methods can be used to improve tin tailing productivity by enhancing the recovery of tin and reducing environmental impacts [5,7].However, those methods are costly.Furthermore, high failure of rehabilitation in post-tin mining is still challenge.Arbuscular mycorrhizal fungi (AMF) are microorganism forming a mutualistic symbiotic association with plant roots, benefiting both the fungi and the plants.They are wellknown in nutrient uptake and plant growth promotion [8], soil structure improvement [9], erosion control [10] and soil stabilization [11], and supporting plant stress tolerance [12].AMF enhance nutrient uptake by plants, especially phosphorus, which is often limited in postmining soils.AMF extend their hyphae into the soil, increasing the root surface area for nutrient absorption and facilitating the uptake of essential nutrients.This promotes plant growth and improves the establishment of vegetation in rehabilitated areas.AMF play a role in improving soil structure by producing a network of hyphae that enhance soil aggregation, porosity, and water-holding capacity.This helps to improve soil aeration, water drainage, and root penetration, creating a more favourable environment for plant growth.The extensive network of AMF hyphae helps bind soil particles together, reducing soil erosion and the loss of topsoil.By enhancing soil stability and preventing erosion, AMF contribute to the overall stability and resilience of rehabilitated areas.
Regarding to the role of AMF for rehabilitation of degraded area, such as post mining area, therefore in this study we aimed to investigate the potential use of indigenous arbuscular mycorrhizal fungi to improve soil productivity in tailing of tin mining, a crucial aspect of post-mining land rehabilitation.In this study we used Piper nigrum as the tested plants growing in tin tailing.After tin mining, P. nigrum is an economically important crop commodity for local people in Bangka Island.

Soil sampling
Soils were collected from the natural forest and post-tin mining area in Bangka Island, Indonesia.Each soil was collected about 1 kg in 0-20 depth from five different sites from each area with five replications for each sample.Soils from each point sampling were kept in a plastic bag.There were 25 soils in total.

Isolation and propagation of AMF
Spores of AMF were isolated from soils collected from natural forest and post tin mining by wet sieving and decanting method [13].The sieved spore than observed, accounted, and morphological identified under microscope based mainly on spore size, color, wall structure and hyphal attachment to the genus level [14].Collected identified spore then propagated for each genus of AMF using Pueraria javanica as the hostplant growing in sterilized sand (autocalve 80 o C for 14 minutes).Plants were grown for three months under greenhouse condition.After three months, plants were drought stress to maximize the sporulation of AMF.Thus spore AMF were used for screeing of AMF.

Screening of AMF
Because AMF ability are varies, screening of AMF was carried out by pot experiment to select the good AMF in promoting plant growth particularly in improving nutrient uptake and yield biomass.This screening was started by inoculating 50 gram propagated spore of AMF from previous step research into Sorghum bicolor.The plants were grown in 200 g sterilized sand.The sand was sterilized using autoclave at 85 o C for 15 minutes.After inoculation, the plants were allowed to grow for three months under greenhouse condition.The sterilized sand used in this step research was to minimize the nutrient effect in soil for AMF selection purpose.Non-inoculated plants were prepared as control as well.Plants were watered once in two days.Meanwhile, modified Hoagland solution containing 1 ppm of P [15] was applied every two weeks.After three months, plants were harvested.Shoot dry weight, shoot phosphorus (P) concentration (Morgan method), and AMF colonization were analyzed.

Inoculation of AMF into Piper nigrum growing in tin tailing
AMF used in this step study was selected AMF resulted from the previous screening of AMF.This step research was studied to get the best AMF that could enhance plant growth performance by improving nutrient uptake through their colonization into the plants growing in tin tailing.Tin tailing mixed with 10% compost was used as growth substrate.About 50 gram of propagated selected AMF was inoculated into sterilized tin tailing mixed with 10% sterilized compost.Non-inoculated substrate was prepared as well as control.Seedling of Piper nigrum was used as testing plant.These seedlings were obtained from stem cutting of P. nigrum which was grown in sterilized sand containing 10% compost under moist shaded condition.After two months, stem cutting of P. nigrum showed hairy roots with two leaves appeared.This condition allowed the stem cutting of P. nigrum to be transferred into tin tailing containing 10% compost substrate media.Stem cutting of P. nigrum were allowed to grow with or without inoculation of AMF for three months at greenhouse.After three months, the new leave number, flowering emergence, AMF colonization, and shoot P concentration (Morgan method) was measured.

Analysis of AMF colonization
The roots of tested seedlings were washed with tap water to clean it up from debris particles.The roots were cleared in KOH (100 g/L) at 80 °C for 15 min, acidified with1% HCl, and stained with 500 mg/L aniline blue [16].AM fungal colonization was quantified under a compound microscopeat 40-200x magnification.

Statistical analysis
Statistical significance of between treatments was analyzed using KaleidaGraph 4.1 software (Synergy Software 2012, USA) for analysis of variance (ANOVA).Post hoc analysis used was the Tukey HSD test (P < 0.05).

Results and Discussions
Spore of AMF were successfully obtained from both natural forest and post-tin mining area (Table 1).There sp., 1 Glomus sp., and 1 Scutellospora sp.Spore number between AMF species and site origin of AMF were varies.Generally spore number obtained from post-tin mining was higher than natural forest.The low number and type of mycorrhizal fungi obtained from natural forest may be because of various factors, including the specific forest ecosystem, soil conditions, tree species present, and environmental conditions.Natural forests in their climax condition may have a relatively lower abundance of AMF compared to disturbed forest.The climax condition of a forest refers to a stable, self-regenerating stage where the forest has reached its maximum ecological potential.In such forests, trees have typically developed complex root systems that allow them to access soil nutrients efficiently.As a result, the dependence on AMF for nutrient uptake may be relatively reduced compared to early successional stages when the root systems are still developing.Other possibility is that AMF prefer to live in forest litter, and promote litter decomposition [17].Furthermore, the high soil organic carbon may reduce the spore number, which may related to the availability of P [18].
The higher number and variation type of AMF in post-tin mining area may be come from the soil erosion from surrounded area such as agroforestry area.Furthermore, the existence Imperata cylindrica found in some spot area in post-tin mining site may become hostplant for AMF.I. cylindrica is a perennial grass species that is known for its ability to form extensive underground networks of symbiotic AMF [19].Acaulospora sp. were found to be dominated in both area, particularly in post-tin mining area (Table 1).The dominance of this Acaulospora sp. could be due to their short cycle of spore production and smaller spore size [20].The domination of Acaulospora sp. was also found in limestone mine area [21].The high number of spore of Acaulospora sp.might be related to the low soil organic carbon (SOC) contain in post-tin mining [6].Relation of SOC and sporulation of AMF was described by [22].All inoculation of AMF had variations in colonizing Sorghum bicolor (Figure 2).Colonization of AMF ranged from 25 % -85% both for AMF collected from natural forest and post-tin mining area.There was no colonization of AMF found in control seedling.

Figure 2. Colonization of arbuscular mycorrhizal fungi after inoculated into Sorghum bicolor
growing in sterilized sand for screening purpose.
Inoculation of AMF resulted on variation of shoot P concentration (Figure 3).There were five AMF collected from post-tin mining: Acaulospora sp.  4).Most of inoculated plants tended to have better biomass yield than control plants.Acaulospora sp. 13 isolated from post-tin mining area, Acaulospora sp. 5 and Acaulospora sp.18 isolated from natural forest significantly resulted on higher shoot dry weight than control plants.The symbiotic effectiveness of AMF may varies according to their preference for specific soils or host plant specificity [23], direct ability to stimulate plant growth [24], rate of infection [25], and competitive ability [26].The variation ability of AMF was also previously reported [27].
From the result of screening AMF, it showed that screening AMF is crucial in the context of ecological restoration efforts.By identifying and selecting appropriate AMF strains, it becomes possible to enhance the establishment and growth of native plant species in degraded ecosystems.AMF can play a significant role in improving soil health, nutrient cycling, and ecosystem functioning, thereby contributing to the restoration of biodiversity and ecosystem services.This knowledge can inform the development of targeted and sustainable approaches for utilizing AMF in agriculture and ecosystem management.For testing the screened AMF into tin tailing, seven AMF that showed good colonization, shoot P uptake, and biomass yield from post-tin mining and natural forest, and one AMF isolated from post-coal mining were used.The introduced selected AMF from post-coal mining was used as effectiveness comparison of AMF.Those seven AMF used were three isolated from natural forest area (Acaulospora sp. 5 (M1), Acaulospora sp. 15 (M2), Acaulospora sp.18 (M3)), and four isolated from post-tin mining (Acaulospora sp. 1 (M4), Acaulospora sp.7 (M5), Acaulospora sp. 8 (M6), Acaulospora sp. 13 (M7).
While AMF from post-coal mining was inoculated as M8.All inoculation of AMF successfully colonized the P.There was no AMF colonization found in control plants.Inoculation of M7 resulted in the highest AMF colonization among treatments, followed by M1, M4, M5, and M8.Plants inoculated with M1, M4, M5, M7, and M8 tended to have higher shoot P uptake than control (Figure 5A).production than control plants (Figure 5B).Inoculation of M4, M5, and M7 triggered the plants flowering emergence (Figure 5B).Generally, AMF isolated from post-tin mining area resulted on better plant growth performance than AMF from natural forest and introduced AMF isolated from post-coal mining area.The influence of AMF in flowering plants was previously\y reported [28].AM fungi can indeed stimulate new leaf formation and flowering in plants by providing various growth-promoting benefits, such as nutrient availability [29], hormonal regulation [30], stress mitigation, and photosynthetic efficiency [31].AMF enhance the uptake and transport of essential nutrients, including nitrogen, phosphorus, and micronutrients, from the soil to the plant roots.By improving nutrient availability and uptake efficiency, AMF can support the plant's metabolic processes and provide the necessary resources for plant growth development such as leaf growth and flowering [32].By enhancing nutrient uptake efficiency, AMF can provide the necessary nutrients that support the transition from vegetative growth to reproductive stages, including flowering.
AMF can influence plant hormone dynamics, particularly auxins, cytokinins, and gibberellins, which are involved in regulating plant growth and development [33].These hormones play a critical role in leaf initiation and expansion, including flowering.By modulating hormone levels and signalling pathways, AMF can promote the formation of new leaves and contribute to leaf expansion and differentiation.AMF-mediated hormonal changes may contribute to the regulation of flowering processes in P. nigrum.However, further research is needed to determine the specific mechanisms involved.AMF can enhance photosynthetic efficiency in plants, which is crucial for energy production and supporting reproductive processes [34].Efficient photosynthesis is vital for energy production which required for flower bud formation, development, and subsequent flowering, including fuels for new leaf formation.By improving nutrient availability, optimizing water uptake, and modulating physiological processes, AMF can increase photosynthetic rates and carbon assimilation, leading to improved leaf growth and the production of new leaves and promotion of flowering P. nigrum.
Among treatment, AMF isolated from post-tin mining showed superior growth.Research has shown that indigenous AMF often have a better symbiotic relationship with native plant species compared to introduce AMF strains [35].Indigenous AMF have coevolved with native plant species over long periods, leading to a higher degree of genetic compatibility and specificity.This coevolutionary history results in a better match between the plant's signalling molecules and the indigenous AMF strains, allowing for more efficient nutrient exchange and symbiotic interactions.Furthermore, Indigenous AMF strains have adapted to the specific environmental conditions of the region where they naturally occur.
were 9 Acaulospora sp.collected from natural forest.Post-tin mining resulted 16 Acaulospora Start Soil sampling from natural forest and post-tin mining area Isolation of AMF by wet sieving and decanting method Screening of AMF for their nutrient uptake and yield production AMF had not good result Morphological identification and propagation of AMF spore AMF had good result Inoculation of AMF into Piper nigrum growing in tin tailing Analysis of AMF colonization, shoot P concentration, new leaf number, and flowering Statistical analysis

Figure 3 .
Figure 3. Shoot P concentration of Sorghum bicolor with or without inoculation of arbuscular mycorrhizal fungi growing in sterilized sand for screening purpose.

Figure 4 .
Figure 4. Shoot dry weight of Sorghum bicolor with or without inoculation of arbuscular mycorrhizal fungi growing in sterilized sand for screening purpose.

Figure 5 .
Figure 5. Shoot P concentration, AM colonization, number of new leaf, and flowering number of Piper nigrum with or without inoculation of arbuscular mycorrhizal fungi growing in tin tailing.

Table 1 .
Spore density and type of arbuscular mycorrhizal fungi (AMF) collected from natural and post-tin mining area.
Generally, plants inoculated with AMF stimulate on the new leaf number