MATERIALS AND METHODS

Time and study site: The study was conducted from May-July 2018. Soil samples were collected from mining contaminated sites Bukit Ngalau, PT Semen Padang, Indarung, Lubuk Kilangan district, Padang, West Sumatera, Indonesia (Fig. 1).

Isolation of arbuscular mycorrhizal fungi (AMF): Isolation of indigenous AMF was carried out by taking a sample of the soil in the field according to the methods of the ICRAF⁸. The AMF isolate taken from roots of vegetation area with deep of soil about 5-20 cm (rhizosphere area). On each post sample will taken ±500 g of soil (composite). Soil samples were taken put in plastic and labeled in accordance with the type of plant as well as the date of sampling soil (Fig. 2).

Propagation of arbuscular mycorrhizal fungi (AMF)/ trapping: The AMF trapping was done using the "Open pot culture method" according to Brundrett et al.⁹. Cultures are grown in a greenhouse at least 1 months. Trapping is necessary step to obtain many healthy spores of colonizing fungi for identification and as inoculum to establish monospecific cultures.

Extraction and identification of arbuscular mycorrhizal fungi (AMF): Extraction and identification of spores from soil samples was carried out by referring to the pour and wet filter method¹⁰ with centrifugation modifications⁹. This pouring and filtering technique procedure, first is mixing the soil sample as much as 50 g with 500 mL of water, then stirring until the granules of the soil are destroyed. Then filtered in a set of filters with a size of 500, 125 and 63 μm sequentially from large to small size of filters set. The top filter is sprayed with water to make it easier for the spores to escape. Then the top filter was removed and a number of residual soil left on the 125 and 63 μm sieve was transferred to the centrifuge tube and centrifugation technique was carried out. Filter results in centrifugal tubes plus 60% glucose using pipettes. The centrifuge tube is closed and centrifuged at 3000 rpm for 5 min.

Fig. 1:	Study sites Bukit Ngalau PT Semen Padang, West Sumatera, Indonesian
	(Source : www.google.com/maps)

Fig. 2:	Isolation of AMF with plot 20×20 m2 at Bukit Ngalau, PT semen Padang, West Sumatera, Indonesia

Then the supernatant solution was poured into 45 μm filter paper or Petri dish and observed under a binocular microscope for spore density (abundance) calculation. Observation of the characterization of FMA type is based on morphological characteristics of spores. The FMA was identified to the genus level with reference to morphological descriptions of AMF genus.

RESULTS

In this study found 3 genus of the AMF in the rhizosphere of 14 plants species at Bukit Ngalau, PT Semen Padang (Table 1) consists of Glomus, Gigaspora and Acaulospora.

Table 1:	Genus of AMF were found associate with vegetation in mining contaminated sites Bukit Ngalau, PT Semen Padang

Fig. 3:	Genus of spores found associated with plants rhizosphere in Bukit Ngalau PT Semen Padang

These types of AMF are associated with different types of plants. The greatest dominant genus found was from Glomus taxa which is associate with 8 species of vegetation with different habits and has the highest abundance of spore 38/50 g of soil. The Glomus population was found as the highest compare to other mycorrhizal genus Acaulospora and Gigaspora. On the other hand, Acaulospora and Gigaspora genus only found associate with 3 types of vegetation and the lowest value for spores abundance was from Acaulospora (9/50 g of soil) (Fig. 3).

DISCUSSION

This study found the AMF Indigenous from post mining land Bukit Ngalau that can be beneficial for rehabilitation of post mining land especially through revegetation. The result showed that Glomus was the dominant taxa with the highest spore abundance and form the association with most of plants at study site. Glomus is considered as a generalist species at contaminated areas. Glomus is also adapted to the stress caused by different concentrations of heavy metals¹¹. Saidi et al.¹² mentioned that some of Glomus species produces higher glomalin than the other species. Glomalin serve to protect the hyphae of drought and destruction attack by microbes. According to Husin et al.¹³, Glomus spores are also found dominant in various rhizosphere in the critical land of Sumatra.

All 14 plants found at Bukit Ngalau, PT Semen Padang showed a strong symbiotic relationship between plants species and AMF but significant differences were observed in the different of host plant species and the spore density varied among plant species. There be selectivity between host plants and AM fungi. The number of AM fungi detected was influenced by host plants under certain conditions¹⁴. Holste et al.¹⁵ also mentioned that the diversity and density of AMF spores would determine by the type of plants on the ground. Both of mycorrhiza and plant diversity can stimulate or retard each other¹⁴. The exudates that was produced by the roots of the host plant also can affect the germination of spores and the growth of hyphae. The root exudates produce a signal that can be responded by the AMF, if there is a match then the spores will germinate and grow to form hyphae^16-18.

The highest abundance of Glomus spore may also have been influenced by trapping process. Trap cultures is an important factor influencing mycorrhizal development, especially spore formation¹⁹. Glomaceae isolates generally are the fastest genus to colonize plant roots. Meanwhile, Acaulosporaceae isolates generally colonized roots more slowly, at a rate more similar to spore-regenerating Gigasporaceae. Glomaceae isolates showed more extensive colonization than others isolate²⁰. The extensive colonization from Glomus may be one of the reason why Glomus has higher abundance of spore compared to Acaulospora and Gigaspora. On trap culture, there are also some species present in the field soil that were lost after trap culture and the frequency and relative abundance of some species were quite different before and after trap culture¹⁹. This may due to differences in the ability of AMF to sporulate. According to Wang and Jiang²¹, Glomus are thought to require less time to produce spores than others species. Glomus spores germitate faster where it only take 4-6 days of germination^9,22. The AMF indigenous found in this study is beneficial for the revegetation on post-mining soil. AMF indigenous can guarantee and help plants become established in harsh ecosystems such as post mining soil. In Indonesia, the government recommend to use native plants in land rehabilitation, this study will help the success of using native plants as rehabilitation strategy that associated with AMF.

CONCLUSION

There is three genera of AMF were found in the rhizosphere of vegetation at Bukit Ngalau, PT Semen Padang. The AMF identified fell within Glomus, Gigaspora and Acaulospora. Glomus was the dominant genus with the highest spore abundance (38/50 g soil) compared to both of Gigaspora and Acaulospora.

ACKNOWLEDGMENT

This study was supported by Andalas University who has given financial support for this study with the grant number 4917.01 $and PT Semen Padang for facility and access to study sites.