Endomycorrhizae and Soil Ameliorant Applications on Growth Performance of Red Jabon (Anthocephalus macrophyllus) in Lime Stone Post Mining Land of PT. Holcim Indonesia Tbk

2.1 Research time and location

The planting was carried out at the site of the post-mining limestone land of PT. Holcim Indonesia Tbk. The research was carried out from September 2016-March 2018. Then the data will be presented in 2023.

2.2 Research materials and equipment

The research materials used were aluminum foil, label paper, tissue rolls, H₂SO₄, 70% alcohol, 10% KOH, alkaline H₂O₂, 1% HCL, 0.05% trypan blue solution, and red Jabon plant seeds (A. macrophyllus) from the nursery from previous research, then planted on limestone post-mining land. Plants grown in the field are adjusted to each existing treatment. The tools used in the research were plant watering tools, Petri dishes, permanent markers, rulers, caliper, meter tape, tape measure, knife, hoe, shovel, scissors, object glass, cover glass, camera, microtome, microscope, and Chlorophyll Meter MC -100.

2.3 Cultivation of red Jabon plants on limestone post-mining land

Mycorrhizal and non-mycorrhizal red Jabon seedlings six months old in the nursery were then transferred to the field. The average height of mycorrhizal seedlings planted was 35 cm, and the average height of non-mycorrhizal seedlings was 25 cm. Before planting, the land is cleared, the distance between the plants is measured, and the planting holes are made with 40 cm × 40 cm × 40 cm. Plants are planted in holes, with a distance between planting holes of 3 m × 3 m. The total combination of treatments was 12; each treatment was repeated 20 times, so the total number was 240 plants. The experimental research methods were used multi-level and multi-factor. Furthermore, maintenance activities include weeding, watering, and controlling pests and diseases. Observations and data collection were carried out once a month for 18 months.

2.4 Observed plant parameter

2.4.1 Increase in plant height

The plant height data were measured from the base of the stem to the highest growing point of the plant; measurements are made once a month for six months.

2.4.2 Increase in plant diameter

Plant diameter data were obtained by measuring the diameter of the plant at a distance of 1 cm from the root neck using calipers. Seedling diameter data were measured once a month for six months.

2.4.3 Percentage of root colonization by AMF

The percentage of mycorrhizal colonization was calculated using the Slide Method. Ten pieces of colored roots are taken randomly and then arranged on a glass object, as many as ten pieces. The colonization percentage is calculated based on the formula:

$\%$ Colonization $=\frac{\text { Number of infected root pieces }}{\text { The total number of root pieces observed }} \quad\quad\times 100 \%$

2.4.4 Proportion analysis of xylem, phloem, cambium, and pith

(1) Preparation of samples

The samples were taken from three seeds at each treatment and carried out the observation in the nursery. The samples were prepared by taking cross-sections or slices on the tiller stems. Slices were made at a height of 1 cm from the base of the root. Furthermore, the stem pieces were used as preparations; then, the preparations were photographed using a micro capture microscope regarding the Sass method (1958).

(2) Measurement

Microscopic observation of the test sample incisions was carried out by measuring the proportion of cells that make up the xylem, phloem, cambium, and pith tissues. The photos taken are then processed using the ImageJ software to calculate the proportion of each constituent cell in the seedling.

(3) The amount of leaves chlorophyll

The amount of leaves chlorophyll was measured using a Chlorophyll Meter MC 100. This tool is clipped to a leaves so that the leaves chlorophyll value will automatically appear. These measurements were made on the lower, middle, and upper leaves blades. Then the values are averaged. The mean value indicates the amount of leaves chlorophyll in each 1 cm² area. Furthermore, each plant's area and the number of leaves are calculated to analyze the amount of chlorophyll. The area of each leaves can be measured using millimeter block paper so that the total amount of chlorophyll for each plant can be determined.

(4) Root anchor index (IJA) and root grip index (ICA)

Each sample plant was excavated and exposed to its roots to be observed, and its dimensions were measured according to the variables used. There are several variables used in observing the development of plant roots, namely: 1) the position of root penetration in the soil layer, 2) root architecture, 3) Root Anchor Index (IJA), and 4) Root Grip Index (ICA).

The position of root penetration in the soil layer for each observed plant sample can refer to the classification of the type of root penetration proposed by Gray and Sotir [9].

Root architecture can also be observed based on the branching pattern when observing the position of root penetration in the soil layer. Architectural observations can refer to the theory [10] put forward, namely VH-type, H-type, V-type, R-type, and M-type.

In this study, the rooting variables observed were Root Anchor Index (IJA) and Root Grip Index (ICA). IJA is the ratio between the diameter of the vertical roots and the diameter of the stem. At the same time, ICA is the ratio between the diameter of the horizontal roots and the diameter of the stem [11]. Each plant sample was measured for horizontal root diameter, vertical root diameter, and stem diameter. A root is classified as a horizontal root if the angle between the root and the vertical plane is more than or equal to 45°, while if it is less than 45°, it is classified as a vertical root. Root diameter, horizontal and vertical, was measured at a distance of 1 cm from the base of the root. The stem diameter of the sample plants was measured at the height of 130 cm for the tree and pole level, 25 cm from the root base for the sapling level, and 10 cm from the root base for the seedling level. The classification (Table 1) and formula for calculating IJA and ICA is as follows:

$\mathrm{IJA}=\sum \frac{d v^2}{d b^2} \mathrm{ICA}=\sum \frac{d h^2}{d b^2}$

Information:

IJA = Root Anchor Index

ICA = Root Grip Index

dv = Vertical Root Diameter

dh = Horizontal Diameter of Lateral Root

db = Stem Diameter

Table 1. Classification of IJA and ICA scores [10]

2.5 Data analysis

Several stages of analysis were carried out in this study to improve the accuracy of the research results, namely the data normality test using the Shapiro-Wilk test; after the data was known to be expected, the homogeneity test of variance was carried out. The homogeneity test of variance was calculated using the Bartlett test. After that, an Analysis of Variance (ANOVA) was carried out, and further tests were carried out with the Duncans New Multiple Range Test (DMRT) methods at 95% [12] to see the effect of each treatment being tested.

4.1 Leaves chlorophyll content

Table 2 shows that the interaction of 2 factors or three treatment factors shows that the interaction only significantly affects the amount of leaves chlorophyll parameter. In contrast, the other parameters are not significantly different. In comparison, the single-factor treatment of AMF on A. macrophyllus plants planted on limestone post-mining land significantly affected the parameters of plant height increase. The percentage of AMF colonization and the amount of leaves chlorophyll were not significantly different from the increase in plant diameter. The effect of a single phosphate factor showed a significant effect on the height gain parameter. It was highly significant on the leaves chlorophyll amount parameter for the limestone planting area. In contrast, the influence of a single factor CLW showed no significant effect on all plant parameters.

Duncan's test results of the interaction of 3 treatment factors that gave the highest amount of leaves chlorophyll in the limestone area were the M2P1L1 treatment with a percentage increase of 283.63% compared to the control.The chlorophyll content of leaves of A. macrophyllus at the age of 6 months varied between AMF treatments. The highest amount of leaves chlorophyll of A. macrophyllus was found in the AMF treatment compared to the control. Leaves chlorophyll plays an essential role in the process of photosynthesis. The results of this study indicate that AMF symbiosis can support plant photosynthesis through the absorption of Mg as an essential component of leaves chlorophyll [13]. According to Widiastuti and Tahardi [14], AMF inoculation can increase magnesium uptake. Magnesium is the center of the chlorophyll molecule needed for photosynthesis and an activator of enzymes in photosynthesis and respiration [15, 16]. Thus, when magnesium levels increase, the essential processes it influences will also increase so plants can grow better than plants without AMF.

The amount of leaves chlorophyll of A. macrophyllus was significantly affected by AMF treatment (Table 2). The amount of leaves chlorophyll of A. macrophyllus inoculated with AMF was more than without AMF. The presence of chlorophyll in the leaves can take advantage of low light intensity. The mechanism of photosynthesis of individual plants and leaves is genetically controlled and can adapt to specific environments [17]. Furthermore, the exceptionally high light intensity can also cause excessive transpiration, causing plant stems to shorten, leaves to become thick with small sizes, increase the number of water-carrying tissues and decrease growth. The high light intensity can also increase leaves temperature so that it can cause stomata to close and decrease photosynthesis. Photosynthesis can produce enough carbohydrates to be sent to the root area and soil microbes. Plants with sufficient chlorophyll content can carry out the process of photosynthesis, which produces assimilation for further plant growth [18].

4.2 Plant height increase

Based on Duncan's test results, the single AMF treatment that increased the height of A. macrophyllus plants in the limestone area was the M2 treatment, with a percentage increase of 60.95% compared to the control (Figure 5). A single phosphate treatment with the highest increase in the height of A. macrophyllus plants in the limestone area was the P1 treatment, with a percentage increase of 17.70% compared to the control (Figure 7).

The presence of AMF in plants will increase P uptake [19, 20] due to external hyphae spreading widely into the soil [21]. In addition to P, the presence of AMF can also help increase calcium absorption [14], which plays a role in cell division and elongation [16]. The increase in plant height is also influenced by the activity of several hormones, such as gibberellin and cytokinins [22]. According to Anas and Santosa [23], AMF on plant roots can produce several growth regulatory hormones, such as gibberellin and cytokinins, so the presence of AMF on plant roots can play a role in the process of stem elongation. Thus the presence of AMF can increase the average increase in plant height better compared to the control; this is in line with the results of this study. Besides that, the role of AMF can increase the ability of plant life both in suitable habitats and habitats on marginal lands. Various types of AMF are also capable of symbiotic and play a role in stabilizing and absorbing heavy metals in polluted land [24]. Other studies have also shown that Kayu Kuku plants with local AMF applications are suitable for post-mining nickel land, planted in the shade and the open [25]. The growth performance of the red Jabon plant on the location of post-mining limestone land is quite good (Figure 13).

13.jpg

Figure 13. The performance of A. macrophyllus plants aged 18 months after planting on limestone post-mining land of PT. Holcim Indonesia Tbk

4.3 Plant diameter increase

The increase in plant diameter is a secondary growth strongly influenced by nitrogen [22]; with the presence of AMF, the N content also increases and ultimately plays a role in increasing the diameter. N uptake was more significant in mycorrhizal plants than in plants without mycorrhizal. It is evident from the tissue analysis results conducted in previous research on the A. macrophyllus test plant [2]. The increase in plant diameter is strongly influenced by photosynthesis and the availability of H₂O supply [26]. The presence of AMF can help increase the supply of H₂O with the help of external hyphae, which can enter into soil cavities smaller than the root diameter [27].

The presence of AMF is significant for the availability of nutrients such as P, Mg, K, Fe, and Mn for plant growth. It occurs through the formation of hyphae on the root surface, which function as root extensions, especially in areas with poor nutrient and water conditions. Mycorrhizal plants' roots increase nutrient absorption from the soil faster than non-mycorrhizal plants [28, 29]. The benefits of these mycorrhizal fungi are seen if the soil conditions are poor in nutrients or dry conditions, whereas in fertile soil conditions, the role of these fungi is insignificant [30, 31].

However, in field studies, the results were statistically different from the greenhouse scale, where the treatment effect showed no significant difference in the increase in plant diameter (Table 2). This study suggests that physiologically young plants are more likely to optimize vertical growth first rather than diameter growth.

4.4 AMF colonization percentage

The root system of a 6-month-old A. macrophyllus plant grown on limestone post-mining land has been colonized by AMF (Figure 10). In post-mining limestone land, AMF treatment was able to colonize plant roots ranging from 69.66-100%, while plant roots treated without AMF were also colonized by AMF, which was relatively high, ranging from 67.79-96.66%.

The presence of AMF structures on the roots of plants that were not treated with AMF is thought to be colonized by AMF, which naturally occurs in the post-mining land. AMF structures, such as internal and external hyphae and vesicles, can contribute to the growth of A. macrophyllus plants post-mining.

AMF treatment can increase plant growth compared to control (Table 2). The inoculated AMF formed a symbiosis with the root system of A. macrophyllusaged six months. The statement is evidenced by the discovery of AMF structures in plant roots, such as internal and external hyphae and vesicles. These AMF structures have different roles. External hyphae absorb nutrients and water needed by plants [32]. The existence of the AMF structure can help improve water and nutrient status and increase plant growth.

The increase in plant growth of mycorrhizal A. macrophyllus was strongly associated with the role of AMF in improving the nutrient status and water absorption (Figures 11 and 12). The host of AMF infection on the plant root system will produce hyphae intensively so that mycorrhizal plants can increase their capacity to absorb nutrients and water [32, 33]. Referring to the results of several studies conducted by other researchers, it was reported that AMF was significantly able to help the absorption of macronutrients (N, P, and K) and micro (Mn, Zn, Cu, and Fe) under conditions of metal contamination [3-6]. In addition, AMF also reduces the entry of heavy metals into plant tissues [5, 6]. The ability of AMF to absorb nutrients and suppress metal uptake into plant tissues can have a positive impact on plant growth and biomass [34].

However, what is no less critical in transferring nutrients from AMF to the roots is the efficiency of AMF [32]. With a high percentage of colonization but low efficiency, the presence of AMF will only help the plants a little. The statement follows [35] which shows that the significant value of colonization in the roots is only sometimes followed by better plant growth. Plants associated with AMF are determined by the size of the percentage of AMF colonization in the roots and the efficiency and effectiveness of AMF in transferring nutrients to the root cells.

The size of the percentage of AMF colonization in the roots only partially reflects its ability to assist the absorption of nutrients for plants [36]. Barber [19] stated that the internal hyphae of AMF in the roots transfer nutrients from the external hyphae into the root cells of the host plant. However, the structure of AMF, which is no less critical in this process, is the external hyphae responsible for absorbing nutrients from the soil. Calculating the percentage of AMF colonization in the roots only determines whether or not AMF colonizes the roots and passes external hyphae scattered in the soil [36].

There is a considerable difference between AMF in the colonization ratio of internal hyphae (in the roots) to external hyphae in the soil. A large percentage of AMF colonization in roots does not necessarily have a lot of external hyphae and vice versa [36]. Thus, a close relationship between the percentage of AMF colonization and plant growth parameters can be obtained if an overall calculation is carried out on AMF colonization in the roots and those widely distributed in the soil. In addition, AMF efficiency and effectiveness tests involving compatibility at the molecular level with host plants need to be carried out. With the association of AMF with A. macrophyllus test plants, this study shows that AMF can be used as an alternative to increasing the growth of forestry plants on post-mining land and marginal land, which has so far been less popular.

4.5 Anthocepalus macrophyllus Wood Anatomy Tissue Proportions

Observation of stem tissue development of A. macrophyllus was carried out at the age of 6 months after planting in the field. Observations were made by cutting across the stem of the plant. Generally, the stem consists of two parts, the outside and the middle. The outer part of the stem consists of a protective layer, namely the epidermis and cortex. The middle part (stele) consists of the inner xylem and outer phloem; there is cambium tissue between the two tissues, and the stele's innermost part is called the pith [37].

The results of observations of stem tissue of A. macrophyllus aged six months after planting on limestone post-mining land showed that AMF, phosphate, and CLW treatment affected the formation of the proportions of a wood anatomical tissue (Table 3). The effect of AMF treatment on the proportion of xylem tissue formation showed that the average proportion of xylem and pith formed was more significant in the presence of AMF treatment compared to the control. Conversely, the average proportion of phloem and cambium formed was smaller. The effect of phosphate treatment on the formation of xylem and pith tissue showed that the average proportion of xylem and pith formed was more significant with phosphate treatment compared to the control, and vice versa, the average proportion of phloem and cambium formed decreased. The effect of the CLW treatment on the proportion of xylem tissue formation and the proportion of pith formed showed that the mean proportion of xylem and pith formed was smaller with the CLW treatment compared to the control. Conversely, the average proportion of phloem and cambium formed was more significant.

The anatomical structure of wood includes shape, size, nature, function, proportion, and arrangement of the cells that make up the wood. In contrast, wood properties are nothing but a measure of the quality or description of the wood itself. The properties of wood are determined and depend on its anatomical structure. In other words, the nature of wood is inherent in the structure of the cells that make up the wood. The relationship between the anatomical structure of wood and the nature, use, and processing in the context of optimal and proper utilization of wood is very close. The properties of wood depend heavily on its anatomical structure and are inherent in the structure of the cells that make up the wood. The overall properties of wood together will determine its use, and the processing that will be applied must be adapted to the anatomical structure of the wood [38].

4.6 Root anchor index (IJA) and root grip index (ICA) values

IJA (Index of Root Anchoring) is the ratio between the diameter of vertical roots and stem diameter. At the same time, ICA (Index of Root Binding) is the ratio between the diameter of horizontal roots and stem diameter [11]. IJA and ICA values can be used to describe the root distribution of a plant.

Table 4 shows that the A. macrophyllus plants planted on limestone post-mining land have IJA values in the medium category, while ICA values are in a low category. It indicates that the A. macrophyllus plant can support the plant upright, but its grip on the soil is not good. The A. macrophyllus plant does not yet have strong roots, so the plant is prone to toppling when disturbed. One of the essential root characteristics of the growth and control of landslides, such as the condition of the limestone post-mining land, is that it can be seen from the ability of roots to penetrate the soil layer. Vertical and horizontal roots that can penetrate deeper soil layers will provide stability to plant growth. The role of the roots will be more effective if the roots cut through the landslide area on unstable soils [39]. Reubens et al. [40] also stated that roots penetrating deep soil layers would provide good shear strength and bending effects. The result will support the soil layer above it and is quite effective in controlling landslides so that the roots' grip on the soil is firmer than before. Horizontal roots that spread in the surface layer of the soil will grip the soil, and vertical roots as anchors will support the tree upright so that the movement of the soil mass does not easily topple it. Plant vigor is one factor that affects a plant species's survival. The level of plant sturdiness can be determined by dividing the plant height by the plant diameter [41]. A high value of toughness will indicate a low survivability due to an unequal ratio between height and diameter. At the location of the limestone post-mining land, it can be seen that the ICA value is relatively low, so the grip of the roots of the A. macrophyllus plant on the soil looks not good; this is suspected because the soil layer/soil solum on the limestone post-mining land tends to be very thin so that the roots do not grip the ground less.