The Abundance of Arbuscular Mycorrhiza Infective Propagules Under Galam Stand at Shallow Peat of South Kalimantan

. Colonization of arbuscular mycorrhiza on plants has been reported to give benefit to plants, especially at extreme sites such as degraded peatland. Galam ( Melaleuca cajuputi ) is an indigenous peatland species which grows on acidic condition. The number of arbuscular mycorrhiza infective propagules is important to be determined concerning the galam regeneration due to its offered benefits that support colonization. This research aims to determine the abundance of arbuscular mycorrhiza infective propagules under galam stand and to describe symbiotic forms of AMF colonization on the roots of galam. The Most Probable Number (MPN) method, wet sieving, and root staining from the modification of Vierherlig et al ., 1996, and the calculation of root’s mycorrhizal colonization by grid line technique were the methods that were used in this research. The research used Complete Randomized Design (CRD) with a 5-fold factorial pattern. The results of this study indicated a significant difference between the abundance of AMF under galam stands at the depth of 0-15 cm and 15-30 cm respectively. The results of spores identification showed 4 genera of spores, namely: Glomus, Gigaspora, Scutellospora , and Acaulospora . The structure of root colonizations were hyphae, spores, vesicles, and arbuscular.


Introduction
Galam (Melaleuca cajuputi) is a woody plant species grows on shallow to moderately deep peat (1-2 m), which can adapt to acidic conditions with the pH range fro m 3-7. This plant has been recorded on shallow to moderately deep peat (1-2 m) in South Kalimantan [1]. Moreover, Galam can grow under flooded conditions due to the adaptation of its metabolis ms [2]. It is called paperbark in Eng lish due to its thin and papery white bark [3]. Galam poles are used for construction purposes, sawn timber, h igh-quality fuelwood, and charcoal. Due to the high demand for t imber, the availability of Galam has been decreasing due to overexp loitation. Galam is not cultivated. It has relied on natural regeneration and no y ield management in the field. Thus, the sustainability of Galam trees is threatened.
Most plants, including Galam, associates with arbuscular mycorrhiza fungi (AMF). AMF has the main three functions including increasing plant growth, enhancing the absorption of soil nutrients , and protecting plants from the pest and disease attack and critical site condition [4]. To conserve Galam, we need to know the benefit of AMF colonization on Galam. One of the ways to determine the benefit of AMF colonizat ion is by providing the nu mber of infective propagules under the Galam trees. Infect ive propagules including spores and sporocarps, extraradical myceliu m, and colonized roots; establish associations when they contact potential host-roots [5]. The object ive of this research is to determine the abundance of arbuscular mycorrhiza infective propagules under Galam stand and to describe symbiotic forms of AMF colonization on the roots of Galam.

Soil and root sample collection
Soil samples and root samples were collected under the galam trees of Liang Anggang shallow peat at South Kalimantan. Root sample co llect ion was carried out unproportionally fro m five galam trees; put in the envelopes and labeled. Soil samp les were collected fro m 5 galam trees and within each tree, samples were replicated fro m 3 points. The nu mber o f soil samples was approximately 1 kg for each point at two different depth: 0-15 cm and 15-30 cm. The soil samp les were put in plastic bags and labeled.

The Most Probable Number (MPN) method
To obtain the number of infective propagules, a series of 4-fo ld d ilutions were p repared. The soil samp les of 1 kg were air-dried and sieved at 0.5 cm x 0.5 cm sieve. This sample was used as a base for the next dilution and labeled substrate A. Then, 4 kg of soil sample was autoclave-sterilized, sieved, and dried. This sample is labeled as substrate B. Furthermo re, 11 kg of soil was autoclave-sterilized, un-sieved. This samp le then labeled as substrate C. The MPN method was then started with putting 100 g of substrate C in each pot. Then, the dilution of substrate A and B was applied 50 g in each pot. The top layer of the pots was put 50 g of substrate C. Three maize seeds were then sown in each pot, watered daily, and gro wn fo r 4 weeks. After 4 weeks, the plant's roots were then harvested, cleaned, and stained with the mod ification of the Vierheilig method [6]. In this method, the root samples were washed with tap water, soaked them in KOH 10% for 24 hours, soaked in HCl 0.1 N for 24 hours , and ink + vinegar 5% solution for 24 hours. The stained root samples were then checked for mycorrhizal colonizat ion, counted, and reported as infected (+) or not infected (-).

The wet sieving method for isolating the AMF spores
One hundred grams of soil sample was mixed with 1000 ml of tap water and mixed well. Then, it was poured onto a series of sieve 250, 125, 75, and 63 µm. The particle that was trapped in the sieve was then moved onto a Petri dish, checked, and counted under a stereomicroscope. The spores were then distinguished based on color and size.

Data analysis
The infective propagules were counted using a formula Log Ω = x . log a -K Ω: the number of infective propagules X: average number of infected pots a: dilution factors K: taken from Sieverding [8]. The number of spores and the arbuscular mycorrhiza colonizat ion in plant's roots was presented descriptively.

Number of infectives propagules
The number of infective propagules under the Galam stand is presented in Table 1. The number of infective propagules under the Galam stand varied with the lowest of ± 55667,28 and the highest of ± 85113,8. The average number of arbuscular mycorrhiza infect ive propagules under the Galam stand was ± 75298,29.

The arbuscular mycorrhiza spores
The arbuscular mycorrhiza spores that were isolated under the Galam trees were varied. There were six species namely Glo mus sp. 1, Glomus sp. 2, Glomus sp.3, Gigaspora sp., Acaulospora sp., and Scutellospora sp. The spores were presented in Figure 1.

Figure 1. Arbuscular mycorrhiza spores under Galam stand at shallow peat of South Kalimantan
The average number of spores per 100-gram soil is presented in Table 2. The average number of spores under galam trees at the soil depth of 0-15 cm and 15-30cm were 508.4 and 337.2, respectively.

Colonization of arbuscular mycorrhiza on roots
The percentage of roots colonization is presented in Table 3.  [10].
The symbiotic forms of AMF colonization on the Galam roots were found namely arbuscular and vesicular ( Figure 2). The AMF arbuscular structures look like a tree; consists of intraradical hyphae. Arbuscules functions as a place for the exchange of nutrients and carbon between AMF and host plant. The molecular and structural organizations of arbuscular facilitate and regulate the processes of nutrient exchange [9]. The vesicles are terminal swellings of hyphae formed inter and intracellularly having a storage function [7].

Discussion
The average number of infective propagules under the galam tree at shallow peat of South Kalimantan was ± 75298,29 per 50-gram soil; or 1505 per gram soil. According to The Indonesian Ministry of Agriculture Decree No. 70/ 2011 on Biofert ilizer Standard, the number of arbuscular mycorrh iza p ropagules shall higher than 50 per g ram soil. Moreover, if we are looking at the root colonization at 0-15 cm and 15-30 cm, both exceeded mo re than 50%. According to O'Connor [10], it was categorized as high colonization. Thus, according to The Indonesian Ministry of Agriculture Decree No. 70/ 2011 on Bio fertilizer Standard, the minimu m percentage of colonizat ion for the biofertilizer is more than 50%. It means, fro m the number of infect ive propagules and arbuscular mycorrhiza co lonization, we can determine that Galam can get the benefit from arbuscular mycorrhiza.
Glomus was the species that most found under the Galam trees at shallow peat of South Kalimantan, Glomus is known to be adaptable in various soil and has the widest habitat [11]. Glomus has a high tolerance to extreme conditions and can form symb iosis [12]. Ramadhani [13] added that Gigaspora is also able to survive in the extreme condition such as acidic or drought. Yuwat i et al [14] also found that Glomus sp was dominantly found at deep after burnt peatland at Central Kalimantan. The number of spores was higher at 0-15 cm than 15-30 cm. The deeper the peat soil, the mo isture is h igher. Mo isture could accelerate the spores to germinate [15]. It means, the deeper the soil, the arbuscular mycorrhiza could be in the form of hyphae than spore. Moreover, Delv ian [15] stated that high soil mo isture content could accelerate the spore to germinate and form co lonization on the host plant's roots. On the contrary, the formulat ion of new spores will increase the dry condition and roots colonizat ion will be lower. It is a natural response of arbuscular mycorrhiza to keep its existence in nature.
One of the ways to enhance land productivity is by silvicultural techniques; one of them is the utilizat ion of biofert ilizer. Considering the number of infective propagules, some spores, and colonization rates, the arbuscular mycorrh iza spores under the galam trees at shallow peat of South Kalimantan is promising to be utilized as biofertilizer.

Conclusion
The average number of arbuscular mycorrh iza infective propagules under the Galam stand of shallow peat South Kalimantan were 55667-85113 p ropagules per 50-gram soil. The number of spores at 0-15 cm and 15-30c m soil depth were 508 and 337, respectively. There were four genera of arbuscular mycorrh iza spores found namely Glomus, Gigaspora, Acaulospora, and Scutellospora. Among the four genera, Glomus was dominantly found. The colonization of arbuscular mycorrhiza on the roots was ranging fro m 57 -70%; categorized as high. These findings showed that Galam was able to get the benefit of AMF co lonizat ion and the soil under galam trees was the potential to be developed as biofertilizer.