Potential Selection of Arbuscular Mycorrhizal Fungi (AMF) Indigenous Ultisols Through the Production of Glomalin

The arbuscular mycorrhizal fungi ( AMF ) with plants able to increase the capacity of plants to absorb nutrients and water from the soil. Recently, research was indicated that AMF hyphae containing glomalin as a glycoprotein that serves to unify the dispersed soil particles. The content of glomalin in soil is positively correlated with soil aggregate stability. The research potential of AMF species indigenous of Ultisol Darmasraya District of West Sumatra and glomalin production in experimental pots of sterile sand medium has been carried out. The purpose of this study was to determine the diversity of AMF species on Ultisol and to seeking indigenous AMF isolates that had the best glomalin production capability. AMF spores were isolated and identified from the rhizosphere soil of corn in Ultisol. AMF species that had been identified experimentally were tested in culture medium pot of sand and zeolite (w / w 1:1) using corn crops. The results found nine of the AMF species indigenous of Ultisol Darmasraya, namely Acaulospora scrobiculata, Glomus etunicatum, Glomus luteum, Glomus mosseae, Glomus verruculosum, Glomus versiforme, Scutellospora gregaria, Scutellospora heterogama and Gigaspora sp. AMF species that showed better colonization ability in corn was G. luteum, G. verruculosum and G. versiforme. All three species produced glomalin significantly higher than the other species, i.e. 1.29 mg g-1; 1.17 mg g-1; 1.15 mg g-1, respectively. [How to Cite: Eddiwal, A Saidi, I Lenin, EF Husin and A Rasyidin. 2014. Potential Selection of Arbuscular Mycorrhizal Fungi (AMF) Indigenous Ultisols through the Production of Glomalin. J Trop Soils 19: 181-189. Doi: 10.5400/jts.2014.19.3.181]  [Permalink/DOI: www.dx.doi.org/10.5400/jts.2014.19.3.181]  


INTRODUCTION
Arbuscular mycorrhizal fungi (AMF) can perform symbiosis with plants and are largely found in various ecosystems. The diversity of AMF in the soil may differ in environmental conditions from one another. AMF symbiosis with host plants can improve the absorption of nutrients and water for plants. Recent findings indicate that the role of the AMF to improve soil physical properties can be explained by increase in soil aggregation.
AMF activities has an important function in the ecosystem in the form of contributions to the improvement of soil structure that is highly correlated with soil aggregate stability and environmental quality (Wright and Upadhyaya 1998;Rillig and Mummey 2006;Treseder and Turner 2007;Borie et al. 2008). The role of AMF in soil aggregation is through AMF activity that produces a protein called glycoprotein as "glomalin" . Glomalin is first discovered in 1996 , in which this protein is abundant in the soil associated with the activity of AMF (Wright and Upadhyaya 1998). Rillig (2004) uses a new term to describe the soil protein which is called "glomalin-related soil protein (GRSP)" as the source of C (carbon) and N (nitrogen) soil and it is important in nutrient cycling and ecosystem (Treseder and Turner 2007;Hodge and Fitter 2010). Rillig et al. (2001) found that approximately 4 to 5% of the total soil C and N in Hawaii came from glomalin. Furthermore, Lovelock et al. (2004a) reported that levels of glomalin in the soil of La Selva at a depth of 0-10 cm was 3.94 mg g -1 , which contributed to soil C and N which were 3.2% and 5%, respectively. Glomalin (GRSP) instead secretion of AMF hyphae, but more than 80% making up the walls of hyphae and spores of AMF (Driver et al. 2005). Glomalin form of adhesive (glue) is produced by the AMF for the transport of nutrients and water activity. Glomalin serves to protect the hyphae from drought and destruction attack by microbes, which after activity stalled hyphae (in weeks) apart along the hyphae and glomalin fused with minerals (sand, dust and clay) and organic matter clump stable soil aggregates Rillig 2004;Borie et al. 2008;Coraqueo et al. 2010). Wright and Nichols (2002) explained that the blob structures formed by binding soil glomalin is quite stable, resistant to water and wind erosion, porous enough to pass air and water. It can hold more water, preventing scale formation (crusting) in the surface layer of the soil and supports the development of plant roots.
Glomalin production by AMF hyphae during colonization of the plant root activity can differ between species.  found that the amount of glomalin extracted per unit weight of hyphae differ between species Gigaspora gigantea, Glomus intraradices and Glomus etunicatum. Similar result was reported by Wright and Upadhyaya (1999), where the total production of glomalin was significantly different between the Gi. rosea, G. caledonium and G. intraradices. Furthermore Levelock et al. (2004b) observed between the result of different glomalin results of Acaulospora morrowiae, Gi. rosea, G. etunicatum and G. intraradices, where A. morroaiae produce the highest concentration of glomalin and G. intraradices the lowest.
Based on the above, the management of indigenous mycorrhizal fungi as biological resources of land, is a breakthrough technology for the development of sustainable agriculture. How large the potential diversity of AMF species indigenous to the Ultisol unknown. The research about the potential of AMF species to produce glomalin in corn is necessary needs to be done.
The purpose of this study was to determine the diversity of AMF species indigenous on Ultisol and seek isolates that have the best ability to produce glomalin.

Exploration and Identification of AMF
Sampling site is on the District Darmasraya West Sumatra wich soil order is Ultisol. Soil sampling was taken from two locations, sample taken from planting corn area at the Village of Pisang Berebus Sitiung and the Sungai Langkok Sitiung II. At each location 5 corn planting areas were taken for observation of root colonization and AMF spore, analysis of glomalin, aggregate stability, and soil chemical properties. Corn planting area measuring 0.5 to 1 hectare was used for random sampling. Soil sampling as much as 2.0 kg in the rhizosphere of plants and their root samples were taken from each planting areas as many as 10 plants. Soil samples were taken at a depth of 5-20 cm, air dried for one week and put it in a plastic bag labeled and stored at a temperature of 15-20° C prior to analysis.
Research activities began with the observation of AMF colonization on the roots of corn plants. The roots were cleaned with tap water and cut into 2 cm. Roots were immersed in potassium hydroxide (KOH) 10% for 4 days. KOH was removed and rinsed with distilled water. HCl solution 1% was added and let it stand for one night. HCl then was discarded, 0.05% trypan blue dye was added. Colonization was measured by the gridline intersect method (Giovannetti and Mosse 1980). Soil samples indicated the presence of infection in the roots of corn followed AMF spore isolation and identified. Every single type of spore in one group was placed on a glass preparations (glass slides) in a mixture of 1:1 (v/v ) of PVLG (Polyvinyl -Lacto -Glycerol) and Melzer 's reagent for identification. Spores were identified by observing the type and morphology of spores by publication INVAM (http: //invam.caf.wvu.edu/ fungi/taxonomy) as well as a variety of sources such as Wilson et al. (1983), Morton (1988), Morton and Benny (1990) and Brundret (2004).

Tests on Indigenous AMF Isolates
AMF species that have been identified was breeding on sterile sand medium with corn plants for 6 weeks. AMF inoculation treatment as much as 30 spores in suspension introduced on the roots of corn plants were grown on a culture medium. AMF inoculant material from the culture medium showed colonization or spores contained in the media was used as a treatment in pot culture experiments.

Research Parameter and Analyzed Method
Parameters observed on soil sampling were normal stability index aggregates (Six et al. 2000), total glomalin USDA_Glomalin Extraction), Diversity AMF (Dandan and Zhiwei, 2007) included spore density (SD), species richness (SR), Shannon-Wiener diversity index (H`), the isolation frequency (IF) and the relative abundance of spores (RA). Parameter observed on the planting medium pot experiment included a total glomalin and spore density (SD). Samples of plant roots were observed for the presence of AMF colonization.
Total glomalin was determined according to the USDA method (USDA_Bio-Rad Bradford Protein Assay). Briefly, 1 g of sample media / soil was added in 8 mL of 100 mM sodium pyrophosphate (Na 4 P 2 O 7 .10H 2 O), pH 9.0 and autoclaved at 121°C for 1 hour. Samples were centrifuged 5000 rpm for 15 min, the supernatant was removed and repeated up to 3 times until the extracts was strawcolored or colorless. All supernatants were combined and measured the volume of extract then centrifuged 10,000 rpm for 10 minutes. Move as much as 1 ml of the supernatant was poured into a micro tube for measuring the content of glomalin.
Glomalin concentration was measured by a colorimetric Bradford protein assay (USDA method) using BSA (bovine serum albumin) as a standard. Measurements were conducted in 200 µl (micro liter) of PBS (Phosphate buffered saline), 10 µl of Bio -Rad dye reagent Coomassie Brilliant Blue R -250 Staining Solution (produced by Bio -Rad Laboratories. Inc.) was added. Color reaction was read by microplate reader at a wavelength of 595 nm after 5 min. Optical density is measured and compared against a standard curve of known concentration (1.25 -5.0 µl) of the BSA. In the PBS standard solution, the addition of 100 mM sodium pyrophosphate equivalent to sample volumes (200 µl PBS -sample volume) was added.

Statistical Analysis
The AMF community structure indigenous on Ultisol was determined by measuring the density of spores, species richness, relative abundance, frequency of isolation and the Shannon -Wiener diversity index. Spore density is a replication of AMF biomass. The relative abundance as a percentage of the number of spores of a species that demonstrates the ability of sporulation of AMF species. Frequency of isolation describe the distribution of AMF species in an ecosystem. All data were analyzed statistically using the Excel Analysis ToolPak and CoStat program version 8.0.

The Diversity of Arbuscular Mycorrhizal Fungi on Ultisol of the Darmasraya District
The AMF species found in Ultisol Darmasraya District is as much as 9 species. Characteristics of the diversity of AMF in both locations can be seen in Table 1 and Figure 1. At the location of Pisang Berebus one species found in Acaulospora, 5 species of Glomus, 2 species of Scutellospora and 1 species of Gigaspora. In the Sungai Langkok found 7 species consists of 5 species of Glomus, 1 species of Scutellospora and 1 species of Gigaspora. Species of the genus Glomus was found predominantly in the Ultisol from both locations, while the other genera found little.
At the location of Pisang Berebus found as many as 2714 spores per 100 g of soil, which is the largest spores of G. luteum as many as 1009 spores and G. versiforme 999 spores which have relative abundance (RA) respectively 37.18% and 36.81%. In the Sungai Langkok found 367 spores per 100 g of soil with the highest number of spores of the species G. versiforme 134 spores (RA = 36.52%) and G. luteum 107 spores (RA = 29.16%). Fairly wide distribution of AMF species are at the location of Pisang Berebus (IF > 80%), but at the Sungai Langkok, AMF species have low prevalence. The diversity of AMF species at both locations showed that the location of Pisang Berebus there are species richness (SR) 7.8 and 4.8 in the Sungai Langkok. Value of the Shannon diversity index (H`) is equal to 1.575 at the Pisang Berebus and 1,566 in the Sungai Langkok.
We found that the AMF Glomus is the type that has spread is quite extensive and dominant (IF> 80%) in the Ultisol Darmasraya, and more than 90% spores at both locations is derived from Glomus. It is almost as was also reported by Prihastuti (2007), where in the acid dry land in Central Lampung found 8 species of AMF, including G. mosseae, G. versiforme and Gi. margarita is more dominant than others. In the hot dry valley areas in Southwestern China, Dandan and Zhiwei (2007) reported that there are six dominant species namely G. clariodeum, G. clarum, G. fasciculatum, G. verruculosum, Glomus sp 2 and Gigaspora sp1. They added that more than 80% of the total number of spores derived from Glomus. Lee and Eom (2009) was also reported that the AMF Glomus is dominant in soils from organic farms or in the conventional farms. The genus Glomus is often  Liu et al. 2009;Wu et al. 2010;Fokom et al. 2012).
Activity of AM fungi in the Pisang Berebus also better, which can be seen from the number of spores found 7 times more than in the Sungai Langkok. It is also supported by the content of total glomalin higher (3,604 mg g -1 ) at the location of Pisang Berebus than the Sungai Langkok (1.008 mg g -1 ). It can be understood that glomalin as building blocks in the walls of hyphae and spores (Driver et al. 2005), where an increase of activity AMF will generate high levels of glomalin. Differences of AFM morphology Characteristics Acaulospora scrobiculata Spores pale yellow color, form spores globose / 300 lm sieve rounded. Spore wall of three layers in which the outer layer is rather bright and pale yellow middle layer. Visible next to the separate components of the spore wall Glomus etunicatum Spores globose shaped reddish orange with a sieve size of 106 lm at 53 lm sieve. Spore wall of two layers, the outer arise turned a brownish red in Melzer's. Layer next to the colored bright yellow to arrange Glomus luteum Spores form globoses yellow orange sieve 106 lm to 53 lm. Four layers of the spore wall, outside light hyaline, pale yellow on the inside and a layer of rigid, no change of color in Melzer's Glomus mosseae Spores brownish orange, globus and slightly rounded shape. Sieve of 300 lm to 106 lm. There are 3 layers of the spore wall, the outer bright colors and turned into a pink reddish in Melzer's. The middle layer is rather light and separate the inner brownish yellow color.

Glomus verruculosum
Color yellow orange spore globose shape, sieve size of 300 lm to 106 lm. There are two layers of the spore wall, the outside such as light-colored peel and blend with the inner layer of yellow orange

Glomus versiforme
Spores brownish red, globose shaped with a size between 53 lm and 106 lm. Spore wall of two layers, the outer rather bright and not reacting in Melzer's. The inner layer of brownish yellow Scutellospora gregaria Spores brownish red color, globose shaped sieve size of 500 lm at 300 lm trapped. Spore wall of two layers, the outer rigid dark brown and light brown inner visible, reacting in Melzer's reddish Scutellospora heterogama Spores dark red slightly rounded shape / subglobose. Spore size sieve 300 lm to 106 lm. Outer spore wall light brown and reddish brown inner Gigaspora sp. Pale yellow spore color, shape rounded sieve size of 500 lm at 300 lm stuck. Spore wall seen two layers, the outer brown color and a reddish orange color inside. not react in Melzer's glomalin content for both sites also appear to be associated with soil aggregate stability. Soil aggregate stability was positively correlated with the content of glomalin (Wright et al. 2000;Rillig et al. 2001;Rillig 2004;Wright and Nichols 2002).

Soil Aggregate Stability
Observations on the aggregate stability of both sites showed that the location of Pisang Berebus had an average value of NSI higher than the Sungai Langkok. The relationship between the content of glomalin to soil aggregate stability of the two locations can be seen in Figure 2.
Aggregate stability for the location of Pisang Berebus was better than Sungai Langkok with NSI values > 0.8 (Figure 2). The NSI values at the location of Sungai Langkok ranged from 0.6 -0.74. Total glomalin at both location also showed the relationship with the NSI values and were positively correlated. Aumtung, (2010) have reported that the total glomalin was positively correlated with soil aggregate stability. He added that the cultivation of upland rice on dry land containing a total glomalin was higher than in lowland rice.
The existence of AMF in the soil plays an important role in soil aggregation through hyphae. Subowo (2010) has explained that the fungal hyphae is capable of uniting the soil aggregates bind to each other, so it is not easily damaged and resistant to physical stress or erosion. Hyphae and spores of the AMF is composed of 80% glomalin (Driver et al. 2005) that functions as an organic adhesive uniting stable soil aggregates . Hoorman et al. (2011) have explained that the three processes were simultaneously soil aggregation by AMF hyphae. At first, physically unite the hyphae of soil particles. Secondly, fungi physically protect the clay and organic particles that make up the microaggregate. Finally, the plant root and fungal hyphae form glomalin that glues micro-aggregates and smaller macro-aggregates together to form larger macro-aggregates. Although bacterial effects on soil aggregate formation are found primarily on microaggregates but mycorrhizal effects are more evident on macroaggregates (Rillig and Mummey 2006).
The density of spores from both locations are also different (Table 1) which showed a positive correlation with the content of soil glomalin (Figure 3). The highest total glomalin found in the Pisang Berebus locations i.e. from 2.14 to 4.54 mg g -1 with spore density between 1824-4199 spores per 100 g soil. The Sungai Langkok location found a total glomalin <1.5 mg g -1 and the number of spores between 155-637.
It is almost similar as was reported by Wu et al. (2010) where the density of AMF spores in the rhizosphere soil of wheat ranged from 350-1,380 per 50 g of soil. He added that the content of glomalin was negatively correlated with ratio C/N, but positively with the density of spores. Furthermore, Bai et al. (2009) reported a significant and positive relationship between the density of spores and glomalin. Thus, the evidence indicates that glomalin is contained in AMF spores and hyphae wall (Driver et al. 2005;Purine and Rillig 2007), so the greater spore production by AMF may indicate higher glomalin production. Fokom et al. (2013) has explained that the high positive correlation between total glomalin and soil water stable aggregate (WSA), carbon and total glomalin, organic matter and WSA as well as organic matter and total glomalin suggests that glomalin, C and N may contribute to the formation of WSA, and thereby promote the build up of soil structure. Guo et al. (2012) added that glomalin may be an appropriate index for evaluating soil fertility. Thus, that glomalin can be used as an indicator of soil quality (Fokom et al. 2012). Based on the above results, it can be stated that the activities of the AMF can be explained from the measured total glomalin. The higher total glomalin illustrated the number of hyphae and spores produced by AMF were too much. It can be understood that glomalin are the building blocks of hyphae and spores wall. This component is produced during symbiosis activities with plants, as described by  and Driver et al. (2005).

The Content of Glomalin in Cultur Media
Each species of AMF will have the ability to grow and develop differently during activity symbiosis with the host plant. One way to determine the activity of AMF symbiosis by measuring the levels of glomalin produced.
The results test of 9 species showed significantly different glomalin content, especially G.verruculosum, G. versiforme and G. luteum ( Table 2). The highest levels of glomalin was G. verruculosum (1.29 mg g -1 ), followed by G. versiforme was 1.17 mg.g -1 and G. luteum was 1.15 mg g -1 . Glomus mosseae (0.65 mg g -1 ) showed the same effect with S. gregaria and S. heterogama. Total glomalin extracted ranged of 0.18 to 1.29 mg g -1 soil, the lowest in the range reported by Wright and Upadhyaya (1998). Wright and Upadhyaya (1998) reported glomalin concentrations ranged from 1 to 21 mg g -1 soil in thirty-seven soil of five geographical locations. On the other hand, the concentration of glomalin in pot trial study by Antibus et al. (2006) ranged from 1-5.5 mg g -1 soil. This was also confirmed by Lovelock et al. (2004b) that the glomalin concentration was low for pot cultivation.
Interestingly, it turns out the concentration of glomalin or proteins were also measured on the treatment without AMF inoculation. The concentration of glomalin in the planting medium was the lowest in the treatment with no inoculation (0.0367 mg g -1 ), which showed no significant difference by treatment with A. scrobiculata inoculation. However, both of these treatments were statistically significantly different with other treatments. According to Rosier et al. (2008), a protein produced by organisms other than the AMF in the rhizosphere can be detected. He also found Glomus tends to produce higher concentrations of glomalin, where the results are more varied with different AMF species. Therefore, differences in glomalin production by nine of indigenous AMF species were statistically different. The same thing Nichols and Wright (2004) also reported that glomalin concentrations varied between five species of AMF, namely: G. etunicatum, G. viscosum, G. caledonium, Gi. rosea and Gi. gigantea. In fact, they reported differences between isolates of the same species. Lovelock et al. ( 2004b ) reported that glomalin production varies significantly across species of AMF in pot culture with maize as a host plant. They found that A. morrowiae produced glomalin higher than Gi. rosea, G. etunicatum and G. intraradices, and glomalin production by G. intraradices significantly lower than Gi. rosea and G. etunicatum. The differences in glomalin production may be due to differences in the activity of fungi (Bedini et al. 2007), and environmental stress conditions can affect the production of glomalin (Hammer and Rillig 2011).

CONCLUSIONS
This study is a pioneer to determine the mycorrhizal diversity in Ultisol and glomalin production of corn from farmland in the District Darmasraya West Sumatra. Our results indicated that the density of spores and AMF community were varied in the soil. We found there were nine of indigenous AMF species, where Glomus was relatively dominant in the rhizosphere soil of corn. In pot culture test, the results showed that the species of G. verruculosum, G. versiforme and G. luteum produces glomalin was significantly higher than the other species. Total glomalin of the three species were 1.29; 1.17 and 1.15 mg g -1 of soil respectively. experimental nitrogen addition in three forest communities in northern lower Michigan. Plant Soil 288: 173-187. Aumtung S, P Sirinikorn, P Susingsa and N Maungjai.
2010. Glomalin-related soil protein influence on soil aggregate stability in soil of cultivated areas and secondary forests from Northern Thailand. Papers  Spore density (SD)