Abstract: Ectomycorrhizal fungi, Scleroderma spp., is potential to promote the growth of seedlings for forestry plants. This research explored the Scleroderma spp., from rhizosphere of Fagaceae in School of Biology forest, investigated the compatibility of Scleroderma spp., with Lithocarpus urceolaris seedlings and studied its effectiveness. The result showed that there were three species of Scleroderma: Scleroderma sinnamariense, Scleroderma columnare and Scleroderma citrinum. Lithocarpus urceolaris inoculated with Scleroderma sinnamariense, resulted in the highest growth of plants (56.55 cm) compared to S. columnare, S. citrinum and control. Diameters of seedlings inoculated with the three species of Scleroderma did not show significant different but they were significant different from control. The three species of Scleroderma had the same growth of colonizations (30%) classified as middle colonizations. There were changes in morphology and anatomy of roots from the infection of three species of Scleroderma. Mantle was clearly observed to cover the root surface and the mycelia formed the Hartig net. There was compatibility between L. urceolaris and three species of Scleroderma. It is suggested that inoculating these Scleroderma to L. urceolaris is necessary to increase the quality of growth seedling.
INTRODUCTION
The School of Biology Forest (SBF) of Andalas University is one of the protected lowland forests in West Sumatera as a natural water resource for Padang City. Besides its economic value and environmental service as town lungs, SBF is also used as a research and education areas. SBF has approximately 150 ha width with hilly topography, 10-30% slope, located in 260-465 m above sea level with temperature ranges from 24-27°C and relative humidity 68-90%.
Actual condition of SBF has been disturbed, for instance there are areas of farming, illegal logging and land clearing by fire. As a protected forest, these conditions have disturbed the ecosystem and endangered the existent populations that leads to extinction. However, SBF is an unique area since it is dominated by Fagaceae plants. This circumstances gives great chances for scientists to study about Fageceae especially about its association with ectomycorrhizal fungi in West Sumatera. Fagaceae had high growth rates of diameter and physically hard wood of a stem (Yoneda et al., 1997). It has economy value since it could be used for floor, furniture and construction. Its fruits are usually used for medicinal and food.
High domination of Fagaceae in SBF gives a significant contribution to the structure and function of this lowland forest ecosystem. So, a good quality of Fagaceae seedlings are really needed for the conservation of SBF. Breeding this plant using seeds take a long time period and small percentage of seed germinations. One of the methods to increase the growth of seedlings is by utilizing the ectomycorrhizal technique. Introducing the ectomycorrhizal is a solution to recover the quality of forest which has been already damaged. Ectomycorrhiza used could be obtained from the same species of plants or others.
Ectomycorrhiza is one of mycorrhiza which usually infects the forest plants. The fungi of ectomycorrhiza obtain the source of carbon from their host plants, on other hand, mycelium of the fungi help the plants to absorb water and nutrient from soil. The ectomycorrhizal fungi not only accelerate the shoot growth but also rehabilitate degraded land. According to Bougher et al. (1996) ectomycorrhizal fungi have great benefits to ecological function and have role in food chains in rhizospheres. Smits (1992) mentioned that Dipterocarpaceae seedlings from seeds and vegetative multiplication, need the ectomycorrhizal fungi for their growth.
Some of the forest trees such as Pinaceae, Betulaceae, Fagaceae, Dipterocarpaceae and Myrtaceae associated with the ectomycorrhizal fungi (Wilcox, 1983). Most of the fungi forming ectomycorrhiza, are Basidiomycetes such as Scleroderma sp., Laccaria sp., Amanita sp., Pisolithus tinctorius, Boletus sp., Telephora sp., Russula sp., Suillus sp., (De la Cruz, 1983; Bougher et al., 1996).
Scleroderma forms ectomycorrhizal associations with a wide range of woody plants, including members of the Pinaceae, Myrtaceae, Fagaceae, Mimosaceae, Dipterocarpaceae and Cistaceae (Sims et al., 1997; Jeffries, 1999). Some beneficial isolates can vigorously compete with other ectomycorrhizal fungi in field (Jeffries, 1999; Martin et al., 2003).
No studies have addressed the diversity or distribution of ectomycorrhizal fungi on Garry oak (Quercus garryana Hook). In spite of interest in habitat, systematic, tree health, natural regeneration and restoration of Garry oaks, no studies have gone below ground to see which fungal associates are present (Valentine et al., 2002).
The SBF as a part of the lowland tropical forests has significant biological resources, therefore the existence of ectomycorrhiza is expected to give contribution for forest trees. The loss of various types of plants as a result of forest destruction will be followed by the loss of ectomycorrhizal fungi associated with plants of the forest. So far there has not been a study to investigate the potential of indigenous ectomycorrhiza in West Sumatera forests.
The existence and diversity of ectomycorrhizal fungi in SBF in colonization of Fagaceae is very important to find indigenous Scleroderma. Utilizing indigenous Scleroderma is expected to give an effective method with a low cost to promote initial growth of Lithocarpus urceolaris (Fagaceae). Objectives of this study were to explore Scleroderma spp., from rhizosphere of Fagaceae plant in SBF and study their compatibility to observe their effectiveness to Lithocarpus urceolaris seedlings.
MATERIALS AND METHODS
Collection of ectomycorrhiza fungi, Scleroderma spp.: Fruit bodies of Scleroderma spp., found in rhizosphere of Fagaceae were collected and identified. The fruit bodies found were taken carefully by taking some mycelia in soil attached to roots of host plants by using small shovel. The fruit bodies were cleaned from dirts and then put in brown paper bags separately then placed in plastic bags. Then they were labeled and identified. In laboratory the fruit bodies were collected in formalin (4%) after being washed with water then with sterile water and alcohol. Identification was based on basidiocarp characteristics referred to Sims et al. (1995) and Bougher et al. (1996).
Preparation of Scleroderma spp., inoculum: The inoculum was obtained from the fruit body tissues. The young fruit bodies were chosen to be isolated because their tissue were actively growing. There fore the mycelia grew rapidly. Isolation was done by cutting fruit bodies aseptically then hyphae were taken from center of gleba with inoculation needle and shifted to the Petri dishes filled with MMN medium then incubated. The mycelium suspension was made by blending the mycelia in water by adding one drop Tween 20 for 500 mL water. The ratio between mycelium and water was 1:10 (m/v). Every seedling was inoculated with 1 mL mycelium suspension.
Seed germination: The seeds of Lithocarpus urceolaris were collected from SBF. The surfaces were sterilized by sodium hypochlorite solution (NaOCl) and rinsed by sterile water. Germination phase was done in seedling trays which were filled by the mixture of soil and sand with 1:1 volume ratio. This medium was sterilized by autoclave for 30 min at 1.5 atm and 121°C. The medium was not compact, so it had enough porosities to prevent damage to the roots at the time of weaning. The seedlings were watered to keep the humidity.
Preparation of seedling media: Media used for the growth of Lithocarpus urceolaris seedlings was a mixture ultisol and sand with ratio 1:1. The medium was sterilized by autoclave for 30 min at 1.5 atm and 121°C. The sterilized medium was put into polybags.
Compatibility test: The prepared medium in the polybag was watered until saturated. The two weeks of L. urceolaris seedlings which had uniform growth were selected and moved into the polybags. Then, the roots of seedlings were inoculated by indigenous Scleroderma spp. As a control, seedlings were not inoculated. The seedlings were not watered for the first two days after inoculated to prevent inoculum from being leached. After that, watering was done once in two days. This research used randomized block design. Data were analyzed by ANOVA and Duncan’s New Multiple Range Test (DNMRT) with 95% confidence interval.
Ten months after inoculation all seedlings were harvested. Root systems of the seedlings were photographed and root samples were taken for further analysis and examination under a microscope. The seedlings were taken out from the polybags and carefully washed without damaging the roots. The clean roots were observed to find out the progress of mycorrhizal based on the colonization percentage of ectomycorrhiza. Colonization percentage of ectomycorrhiza were observed visually.
Growth parameters observed were height and diameter of seedling measured after 10 months. Height of seedling was measured from the base of seedling to the top. Stem diameter measured was the part at the ground surface using caliper.
Root preparation for microscope observation: To make sure that the roots were infected by mycorrhiza, root histology was observed. Roots specimens were prepared following the method of Sass (1958) which covered the process of fixation, dehydration, parafinas, cutting and staining. Ectomycorrhizal roots of L. urceolaris were soaked in Formalin Alcohol Acetic Acid (FAA) for 24 h and sliced using a rotary microtome for 5-10 μm thick, then stained with tryphan blue. The existence of mantle and Hartig net indicated that the seedling had been infected by ectomycorrhiza Scleroderma spp.
Morphology of ectomycorrhiza from L. urceolaris: The characteristics of root morphology like branch patterns and colors of sheath were also observed using loop. Roots with mycorrhiza symbiont were marked by the existence of mantle or hyphae covering the roots.
RESULTS
Exploration of Scleroderma spp.: The result showed that the isolates found in rhizosphere of Fagaceae in School of Biology Forest, Andalas University showed variation in the shape, size, color and the stipe of their basidiocarp, also the shape and color of their spores. Morphologically, the result showed that there were three species of Scleroderma found in rhizosphere of Fagaceae in School of Biology Forest, Andalas University, i.e., S. citrinum, S. columnare and S. Sinnamariense.
According to Hawksworth et al. (1995) cited in Bougher et al. (1996) Scleroderma ectomycorrhizal fungi could be classified into:
| Division: | Basidiomycotina |
| Class: | Holobasidiomycetes |
| Sub-class: | Gasteromycetes |
| Order: | Sclerodermatales |
| Family: | Sclerodermataceae |
| Genus: | Scleroderma |
| Species: | Scleroderma citrinum, Scleroderma columnare and Scleroderma sinnamariense |
Microscopically external hyphae surrounding the ectomycorrhiza were septate and formed clamp connection which is one of characteristics of fungi in Basidiomycetes (Fig. 1).
Growth of L. urceolaris seedling: Effects of Scleroderma spp., inoculation on L. urceolaris seedlings were presented in Table 1 and Fig. 2.
Inoculation of three species of Scleroderma on L. urceolaris showed significant effect on seedling height and diameter.
| Table 1: | Effects of Scleroderma spp., inoculation on growth of Lithocarpus urceolaris seedlings |
| Numbers in the column followed by the same lower case letter are not significantly different according to DNMRT at 5% significance level | |
| Fig. 1(a-c): | Hyphae Scleroderma spp., with clamp connection (white arrow), (a) Scleroderma citrinum, (b) Scleroderma columnare and (c) Scleroderma sinnamariense |
| Fig. 2: | Lithocarpus urceolaris seedlings inoculated with three species of Scleroderma, a: Scleroderma citrinum, b-c: Scleroderma columnare, d: Scleroderma sinnamariense and e: Control |
| Fig. 3(a-c): | Cross section of L. urceolaris roots infected by Scleroderma spp. (a) Scleroderma sinnamariense, (b) Scleroderma columnare and (c) Scleroderma citrinum. M: Mantle, HN: Hartig net, CC: Cortex cell |
Height and diameter of seedlings inoculated with three species of Scleroderma were higher compared to those of control. Among the treatments, seedling height showed significant effect but not in diameter. The highest seedlings were obtained from inoculation with S. sinnamariense (56.55 cm) followed by S. columnare (40.25 cm) and S. citrinum (39.45 cm). Figure 2 showed the seedlings of L. urceolaris grew much better than control.
Percentage of ectomycorrhizal colonization: Inoculation of Scleroderma spp., on L. urceolaris affected the colonization percentage of ectomycorrhiza. The control seedlings were clear from the ectomycorrhizal since they were not inoculated. Based on the morphology analysis, the ectomycorrhiza development occurred on roots of L. urceolaris seedlings inoculated with Scleroderma spp. Three species of Scleroderma in this research showed the same colonization growth (30%).
Mantle and Hartig net thickness: At cross section of roots, mantle and the Hartig net were not observed on seedlings of L. urceolaris uninoculated with Scleroderma. On the other hand, the inoculated seedlings by S. sinnamariense, S. columnare and S. citrinum showed a complete structure of mantle and Hartig net. The mantle was clearly shown to cover the surface of the roots and the mycelia of Scleroderma formed the weaving Hartig net (Fig. 3). The three types of Scleroderma formed a single layer mantle. The thickness of the mantle was 5-20 μ and the one of the Hartig net was 15-20 μ (Table 1).
Morphology of ectomycorrhiza on L. urceolaris seedlings: Ten months after inoculation, L. urceolaris seedlings were well colonized by S. sinnamariense, S. columnare and S. citrinum. Macroscopically, ectomycorrhiza formed on the roots of L. urceolaris seedlings associating with three species of indigenous Scleroderma in SBF, had the same morphological characters resulting in monopodial branches. Most of the surface sheaths were white.
DISCUSSION
The higher height and diameter of seedlings indicated that there was association between L. urceolaris with Scleroderma spp. The positive effect of the association was also shown on Scleroderma. This indicated that association gave benefits for both seedlings and ectomycorrhizal Scleroderma.
All three species of Scleroderma found had the same colonization growth (30%), classified into medium colonization. Marx et al. (1991) divided the percentage of colonization into four groups, (1) 75-100% (very good/high), (2) 50-74% (good), (3) 24-49% (medium) and (4) 1-24% (bad/low). Infection of Scleroderma ectomycorrhiza caused the change in root structures. In infected root there was found hyphae covering roots of L. urceolaris seedlings. Mantels were formed to cover roots and Hartig net was formed among cortex cells. This depended on the compatibility between host plants and ectomycorrhizal fungi penetrating roots, age of root seedling inoculated and environment that support growth of fungi mycelia. Every species of plant should have compatibility with a fungi to form mycorrhiza. The compatibility is necessary to form symbiosis between fungi and plants. Every species of ectomycorrhizal fungi has a very high compatibility with its host plant. It is marked by higher percentage of ectomycorrhizal colonization, high growth rate and formation of mantle and Hartig net. According to Wolf and Wolf (1947), anatomically and morphologically roots infected by mycorrhiza are generally marked by formation of mantle covering roots and formation of Hartig net consisting of hypha penetrating among cortex tissue. If both structures can be observed in root colonized, it means that ectomycorrhizal fungi inoculated compatible with species of seedling inoculated. Peterson and Farquhar (1994) said that roots colonized by ectomycorrhizal fungi mostly undergo changes in morphology and anatomy. Infected roots usually undergo alteration of their diameter, length and branches. According to Zak (1971, 1973), different host plant species showed different branch forms and sheath color of ectomycorrhiza.
There was compatibility between L. urceolaris seedling with indigenous Scleroderma in SBF. Therefore, colonization level and compatibility of three species of Scleroderma on L. urceolaris would influence seedling growth. Host plant species and different level of association are important in relation to introduction of exotic trees species.
In ectomycorrhiza, sometimes it took longer time for fungi to infect host plants. Lu et al. (1998) reported that colonization percentage of ectomycorrhiza Scleroderma on Eucalyptus urophylla was very low after nine months inoculations. The success of root colonization depends on spore germination, mycelia growth in soil and susceptibility of roots against mycorrhizal infection (Bowen, 1994; Reddy and Natarajan, 1997). Physiological and ecological difference between the same and different species of ectomycorrhiza influence its success in colonizing roots (Bonfante et al., 1998). Selection of compatible ectomycorrhiza species is very important for the success of developing inoculation program (Dell et al., 1994). Generally, the existence of association of ectomycorrhiza in plant roots could increase the plant growth. The increase in plant growth means the L. urceolaris seedling can be moved to field earlier so that it could decrease the cost of seedling rearing.
Scleroderma is one of groups of ectomycorrhizal fungi growing in Indonesia forests. Hyphae Scleroderma has a major role in absorbing nutrients and water for host plants. According to Chen et al. (2006), Scleroderma inoculation could increase seedling growth of Pinus and Eucalyptus up to 105% after colonization.
Inoculation has a role to form mycorrhizal colony because spores is a reproductive organ of ectomycorrhizal fungi that in turn would develop to form hypha in suitable environment. On the following stage hypha will grow to cover roots in infection area to form colony and finally hypha will penetrate roots (Alexopulos and Mims, 1979). Inoculation of indigenous ectomycorrhizal Scleroderma in SBF on L. urceolaris seedling was meant to study the compatibility between the two.
Shemakhanova in Julich (1988) indicated that infecting fungi inoculums to seedlings on one of Fagaceae like oak (Quercus sp.) could increase leaves size up to 70% or two fold bigger than control. The existence of mycorrhiza indicated that there was mutualism functional interaction between certain plants with one or more mycobion strain and vice versa in space and time.
Successful formation of ectomycorrhiza showed the compatibility between S. sinnamariense, S. columnare and S. citrinum with L. urceolaris. This means that L. urceolaris could become the host plant for S. sinnamariense, S. columnare and S. citrinum. Most Scleroderma sp., have a wide range of host plants (Jeffries, 1999) but they need a specific host for producing fruiting bodies. For example, S. columnare needs Pinus merkusii and S. dictyosporum needs dipterocarps to produce fruiting bodies. During the field observation, fruiting bodies have never been found. Potential ectomycorrhizal association often indicated by the formation of fruiting bodies. According to Massicotte et al. (1994), for Scleroderma the percentage of ectomycorrhizal colonization on the secondary host was lower than that in the primary host. The low colonization on the secondary host has also been found for other ectomycorrhiza fungi.
CONCLUSION
There were three species of Scleroderma identified associated with L. urceolaris in School of Biology Forests at Andalas University: S. sinnamariense, S. columnare and S. citrinum. Symbiosys of the three species could increase the growth of L. urceolaris seedlings. There was changes in morphology and anatomy of roots infected by the three species of Scleroderma. There was also compatibility between L. urceolaris with the three species of Scleroderma. It is suggested to inoculate L. urceolaris seedlings with the three species of Scleroderma to increase seedling growth.
ACKNOWLEDGMENT
This study was funded by Indonesian Directorate General of Higher Education with grant number 486/SP2H/PP/DP2M/VI/2010.