CHARACTERIZATION OF SOIL ACTINOMYCETES FROM MALINO PINE FOREST RHIZOSPHERE OF SOUTH SULAWESI

Actinomycetes are a group of Gram-positive bacteria that produce active compounds with a wide distribution range in nature especially in soil. The purpose of this study was to isolate actinomycetes from the rhizosphere soil of the Malino pine forest, South Sulawesi. This research is a descriptive qualitative study of 15 actinomycetes isolated from the rhizosphere soil of the Pine Forest. These 15 isolates have been successfully identified to the genus level. Actinomycetes were isolated by direct dilution method and further morphological identification was carried out. Actinomycetes were isolated on yeast malt agar (YMA) medium. The growing isolates showed colony colours brown, grey, and white. Produces yellow, brown, and beige colour pigments. Based on the characterization carried out, all isolates were identified into the genus Streptomyces.

study was conducted to determine the characteristics of actinomycetes from rhizosphere soil from the Malino Pine Forest in Gowa Regency, South Sulawesi, which provides information on the diversity of biological resources in the pine forests particularly Malino pine forests.
Samples were taken from the ground near the roots of pine trees by digging at a depth of 10-15 cm from the ground. It is estimated that 10 grams of soil are taken and placed in sterile plastic clips, closed tightly transported to the laboratory, and stored at 4° C before isolating actinomycetes. Measurement of environmental parameters such as temperature, humidity, and soil pH was carried out using a soil thermometer and soil tester. The sample was dried for 7 days (Nurkanto, 2010) and filtered to separate large dirt and homogenized sample (Susilowati et al., 2007).

Morphological Identification
The growing actinomycetes colonies were taken and transferred one by one to yeast malt agar (YMA) media. Incubation was carried out at room temperature for 1-14 days to give the isolates a chance to grow completely. Identification up to the genus level was carried out by macroscopic and microscopic observation. Macroscopic identification was carried out by observing the colony colour and the resulting pigment. Microscopic identification was carried out by observing the arrangement of the spore chains under a microscope (Miyadoh, 1997;Holt et al. 1994;Nurkanto, 2007).

Results and Discussion Environmental Parameters of Malino Pine Forest
Samples were taken from five locations of Malino Pine Forest, South Sulawesi. The sampling process is carried out aseptically at a depth of 10-15 cm because it greatly affects the presence of microorganisms. Soil depth affects the population of microorganisms. The population of microorganisms including actinomycetes is mostly found at soil depths of 10-15 cm (Bhattarai, 2015). Algafari (2014) added that at the depth of the soil 10 cm is recommended for the isolation of actinomycetes because in its oxygen and nutrients for the growth of actinomycetes are available in abundant quantities. Other factors that affect the presence of microorganisms are chemical and physical include moisture, pH, and soil temperature (Table 1). The diversity and types of actinomycetes are greatly dependent on chemical, physical, and biological factors in the environment. To determine the effect of these factors, environmental parameters are measured. The results showed that the average humidity at the five sampling locations was 1 %, while the average pH in Patappang (Lembanna), Malino (Pendidikan Street), Lombosang, and Gantarang was 6.9. the average pH at the Taman Wisata Resort. Alam (TWA) Malino which is 6.8. Furthermore, the temperature obtained at five locations ranged from 24.6 °C -26 °C. The pH, temperature, and humidity values in the malino pine forest showed the optimum for the growth of actinomycetes.
Determination of soil type and environmental conditions is veritably important in the isolation of actinomycetes because their growth is influenced by soil conditions such as geographical location and biotic factors (temperature, soil type, depth, soil pH, the content of organic compounds, aeration and humidity) (Samar et al., 2018). The measurement of pH range at the sampling location (pH 6.8-6.9) supports the optimal growth of the genus Streptomyces. This is correlated with the characterization result which showed that all genera found at the sampling location refer to the genus Streptomyces. As mentioned by Zanane et al., (2018) that the pH of Streptomyces growth is range from 6.5 to 8.0.
The abundance of actinomycetes populations is various infertile soils with humidity ranging from 0.67 % to 0.89 %. However, actinomycetes can survive and grow at low humidity (0.67 %) and grow optimally at 0.89 % humidity (Zenova et al., 2007). As for the physical factors, the suitable temperature for the growth of actinomycetes is 25 °C to 30 °C (Adriani & Tulak, 2013). Actinomycetes usually grow at a soil depth of 11-15 cm above the soil surface. At this depth, there is a combination of pH and water content that optimal for actinomycetes growth (Miyadoh & Otoguro 2004).
According to Kanti (2005), waterlogged soils are not suitable for the growth of actinomycetes. However, soils in dry or semi-dry areas can maintain a fairly large population of actinomycetes. This is due to the resistance of spores to drought. Actinomycetes can grow with a large population in dry areas. The drier the soil sample and the less water content, the more live actinomycetes particularly the genus Streptomyces ( Zenova et al., 2007). Soil sampling in this study was conducted in Malino Pine Forest, South Sulawesi. The Perhutani Forest Resources Development Center stated that the loss of water from rainfall due to the interception process from pine forests was the highest, namely 15.7 % compared to other forests, agathis (14.7 %) and puspa (13.7 %) (Indrajaya & Handayani, 2008). This reduces the amount of water content in the soil causes the soil to become drier and allowing for the growth of actinomycetes.

Isolation and Morphological Characterization of Actinomycetes
It was found that 15 actinomycetes colonies with different characteristics on YMA media were shown in table 2. The observed macroscopic characteristics were colony colour and pigment. Based on these characteristics, all isolates were identified in the genus Streptomyces. Based on the result, actinomycetes isolates that grew on Humic Acid Vitamine Agar (HVA) media were presumed. The isolation method used was the spread method. The suspected isolates obtained were purified on Yeast Malt Agar (YMA) media. Actinomycetes are a type of soil bacteria with a resistant spore to heat, dryness, and chemicals. In nutrient-poor media such as HVA, actinomycetes grow for a long time, which is about 1-2 weeks due to their complex life cycle. The introduction of colonies to the media should be relatively easy and simple. Actinomycetes can be distinguished from other bacteria easily based on their colony shape. The selection results based on the diversity of colony, 15 pure isolates of actinomycetes were obtained with various colony morphologies, air mycelium, and are able to sporulate at the age of 7-14 days. Four isolates were obtained from the Gantarang, 4 isolates from Lombosang, 4 isolates from Resort TWA Malino, while the other 2 isolates were obtained from Malino (Pendidikan Street), while the remaining 1isolate was isolated from Patappang (Lembanna). Based on the observation of colony characteristics, all the isolates obtained showed characteristics refer to the genus Streptomyces.
In different with other bacteria, actinomycetes colonies are not formed from a uniform collection of single cells but in branched filamentous masses form. Actinomycetes colonies that grew in YMA media in this study were vegetative mycelium with powdery air mycelium, grew widely attached to the media and the whole unit was easy to take with loop wire. Initially, the surface of the Streptomyces colony was relatively smooth. Gradually with the development of aerial hyphae floccose, granular, powdery, or velvety characteristics was formed. Streptomyces filaments and spores are slight and no more than 1 µm in diameter. Spores are formed from filament fragmentation and arranged in straight, wavy, or helical chains (Hasani et al., 2014). Filaments allow efficient use of nutrients and allow Streptomyces to colonize substrates better than other unicellular microorganisms. When the colony becomes old and its nutrients are depleted, filamentous branches develop from the surface of the colony and produce aerial mycelium that grows vertically. (Olanrewaju & Babalola, 2019). All the fifteen actinomycetes isolates are showed the genus Streptomyces. This indicates that the Streptomyces group is the dominant actinomycetes group in the Malino pine forest. According to (Hasani et al., 2014), Streptomyces are the most abundant microorganism in the soil and makeup 40 % of the total bacteria in the soil. This is due to the filamentous structure of streptomyces cause condensation of soil texture, and protect them from wind and eradication of rain.
Another distinctive characteristic of the 15 isolates from this study is the distinctive earthy odour that only belongs to the genus Streptomyces. Akbar et al., (2017) stated that this is because the main habitat of Streptomyces is on the ground. Streptomyces compose approximately 70 % of microorganisms in the soil and can be isolated in various environments, even from an unusual environment. According to Zhi et al., (2016) this soil odour is caused by Streptomyces producing a metabolite called Geosmin. This compound is a sesquiterpenoid component consisting of carbon, oxygen, and hydrogen. Acetic acid, acetaldehyde, ethanol, isobutanol, and isobutyl acetate have been identified as odour compounds also produced by Streptomyces.

Macroscopic Observation
Macroscopic observations on the surface of the medium showed that 15 actinomycetes isolates had morphological characteristics powdery air mycelium, flat and convex colony surfaces, and vegetative mycelium which was firmly attached to the media surface as shown in Figure 1. According to Ambarwati et al., (2011), the characteristics of the genus Streptomycetes are involved vegetative hyphae and air mycelium. Vegetative hyphae will produce broad branched mycelium which rarely has fragments. The air mycelium in the adult phase form three too many spores (more than 50 spores). Meanwhile, Korn Wendisch & Kutzner (1992) stated that some species of Streptomyces have short spore chains in the vegetative mycelium. Spores are nonmotile. The colonies that appear are relatively smooth, but then there is a development that reveals the air mycelium in the form of floccose, powder grains, or velvet.
The colours of the vegetative mycelium and air mycelium of the 15 isolates obtained can be grouped into 3 different groups. they are brown, white, and grey and turn to black over time. The colours in the vegetative and air mycelium are caused by the formation of special metabolites called pigments. The pigmentation of actinomycetes varies according to the type of actinomycetes obtained. Many Streptomyces produce pigments in their vegetative mycelia or around agar media (Bennett et al., 2018). Pigmentation can range from white or grey to yellow, orange, lavender, blue and green, so it is often referred to as a colour wheel. Some pigments consist of 2 to 3 compounds, while others can consist of 7 to 10 or even 15 compounds (Abdulla et al., 2008).
Approximately 200 actinomycetes pigments have been identified with available data regarding chemical properties and then divided into several products such as acyclic compounds, aromatic compounds, quinones, oxygencontaining heterocyclic compounds, nitrogen-containing heterocyclic compounds, cidromycin. Each type of pigment can provide a specific colour and can be used to classify Streptomyces. A greenish pigment which may indicate viridomycin is produced by Streptomyces with air mycelia grey, pink, and yellow-greenish. Pigments associated with antibiotics of the type rhodomycin, griseorodinrubromycin, and lithmosidin produced by Streptomyces with air mycelia grey and pink (Salvameenal et al., 2009). Sathi et al., (2002) identified 4hydroxynitrobenzene in brown and cream-coloured Streptomyces pigments, Streptomyces with brownish pigments reported to have an antimicrobial activity that inhibits the growth of pathogenic bacteria (Azimi et al, 2014). Prashanti et al (2015) also reported that the yellow pigment produced by Streptomyces griseoaurantiacus JUACT 01 contains carotenoids with anticancer activity.
Morphological characters shown in the formation of pigments in the media by actinomycetes are used to identify genera or species. According to the pigment, formation is influenced by media pH, aeration, growth temperature, carbon, and nitrogen sources. Pigments produced by actinomycetes in the media are usually various, such as blue, purple, red, yellow, brown, green, and black. these pigments may dissolve in the media or remain on the mycelia. (Anandan et al., 2016). The same Actinomycetes species can produce different pigment colours depending on the growth medium, possibly due to the result of hydrogen ion concentration (Akilandeswari & Pradeep, 2017).

Microscopic Observation
This study obtained 5 types of spores arrangement of Streptomyces isolates based on microscopic observation. They are flexuous arrangement as seen in Figure 2, loops primitive spirals hooks as shown in Figure 3, open spirals, straight, and monoverticillate with spirals as shown in Figure 4. Bergeys Manual of Determinative Bacteriology noted that the genus Streptomyces has 10 types of spore chain arrangement is straight, flexuous, fascicled, monoverticillate no spiral, open loops primitive spirals hooks, open spiral, closed spiral, monoverticillate with spiral, biverticillate no spiral, and biverticillate with spiral (Bergey & Holt 1994).  Observations using a 400x microscope magnification showed that 15 actinomycetes isolate obtained from the rhizosphere soil of the Malino Pine Forest of South Sulawesi generally had spores in the form of streptococci, include long elliptical chains in aerial hyphae, tight and dense mycelium. The results are in accordance with Armaida's (2016) statement that Streptomyces is a Gram-positive bacteria and streptococci arranged in a spiral, measuring about 0.8 -2 μm. The spore chains form the air mycelium are rarely undergo fragmentation but the spores are formed from the segmentation of hyphae. Krieg and Holt (1994) explained that Streptomyces contains L-DAP and glycine as the characteristics of