Styrax sumatranus J.J.Sm endophytes fungi and its potential as phosphate solubilizing and cellulose decomposition

Styrax sumatranus J.J.Sm is a plant from North Sumatra, which is widely used by community for religious rituals, medicines and it has economic value. Endophytic fungi are fungi that harbour inside the plant tissue without causing any negative effects to their host plant. Endophytic fungi isolated from the leaves and bark of Styrax sumatranus J.J.Sm. The aim of this research was to obtain endophytic fungi that have potential as phosphate solubilizing and cellulose decomposition. The bark and leaves of Styrax sumatranus J.J.Sm as a source of endophytic fungi were taken from Pandumaan village, Pollung District, Humbang Hasundutan Regency, North Sumatra. Test the potential of endophytic fungal as phosphate solubilizers using selective Pikovskaya media and as cellulose decomposition using carboxyl methyl cellulose (CMC) media plus congo red 0.1%. Then calculate the phosphate solubility index and cellulolytic index. The result show that, of 20 isolates tested, there were 12 isolates that had potential as phosphate solubilizers and 12 isolates had potential as cellulose decomposition. Phosphate solubility index dan cellulolytic index obtained range from 2.10-2.44 and 0.22-0.55 respectively.


Introduction
An endophyte are microorganisms that have the ability to colonies plant tissues during the life cycles without causing damage to the plant [1,2].This microorganism are known to have beneficial for plants through some mechanisms, namely spurring growth, increasing resistance against several pathogens and attacks pest and increase resistance plants to environmental conditions unfavorable [3].Endophytic fungi are one of the microorganisms that live in plant tissues and play an important role in ecosystems and plant growth [4].This fungus is able to produce bioactive substance [5], phytohormones [6] and act as phosphate solubilizer [7].Endophytic fungi can be found in all parts of plant such as roots, stem, barks, flowers, fruits and leaves [8].
Phosphate solubilizing fungi are able to assist in the supply of phosphorus nutrients for plants [9].Fungi have long been known for their potency to chelate and incorporate phosphate from insoluble form into available form to plants through the involvement of organic acids which released the ion (PO 4 ) from adsorption complex [10][11][12][13].The Aspergillus, Penicillium and Fusarium genera are types of fungi that have a good ability to help increase the availability of P nutrients [14,15].Fungi are known as good decomposer of organic matter.In general, the ability of fungi to decompose cellulose and lignin as the main constituents of organic matter is higher than that of actinomycetes and bacteria [16].Cellulolytic fungi are able to break down cellulose into compounds that can be used by plants.Cellulolytic fungi have the ability to produce cellulase enzymes which can decompose organic matter [17,18].The Acremonium, Aspergillus, Chaetomium, Curvularia Penicillium, Trichoderma and Rhizopus, are capable of producing cellulose enzymes [19,20].
Each higher plant have can contain several endophytic microorganisms.Styrax sumatranus J.J.Sm is a typical plant from North Sumatra [21], which contain endophytic fungi.On the leaves and bark of the S. sumatranus J.J.Sm plant, endophytic fungi have been identified [22][23][24].However, the potential endophytic fungi as phosphate solubilizers and cellulose decomposition is not yet known.So the research objective was to test the potential of endophytic fungi as phosphate solubilizer and cellulose decomposition.

Collection of leaves and bark of Styrax sumatranus J.J.Sm
The leaves and bark of Styrax sumatranus J.J.Sm were taken from Pandumaan village, Pollung District, Humbang Hasundutan Regency, North Sumatra.Leaves and bark were taken from healthy and disease-free trees.Furthermore, the leaves and bark are put into plastic bag and taken to the laboratory.

Isolation of endophytic fungi
The procedure for isolating endophytic fungi follows [25] in the following way: leaf sample and bark were cleaned and washed under running tap water.Surface sterilized was carried out by immersing the leaves and bark samples with 70% alcohol (2 minutes), then immersed in 5% sodium hypochlorite solution (2 minutes).After sterilization step, the plant parts were rinsed using sterile distilled water three times.Leaf and bark that have been sterile and dry, cut into pieces with a size of size of 2cm x 2cm using a sterile knife.The sterile piece of leave and bark were transferred to potato dextrose agar (PDA) medium in a petri dish.To suppress bacterial growth, the antibiotic chloramphenicol was added.The third water rinse was used as a control.If here is no contamination in the petri dish, the endophytic fungi will grow.Petri dish and pieces of leaves and bark were incubated for 7 days at 25 °C.The growth of fungal colony was observed every day.Colonies different characteristic were purified and then stored for potency testing and identification.

Testing the potential of endophytic fungi as phosphate solubilizers and cellulose decomposition
All endophytic fungi that were successfully isolated were tested for their potency qualitatively.Endophytic fungi are grown on different selective media.To determine the potential of endophytic fungi as phosphate solubilizer, isolate were grown on Pikovskaya media.To determine the potential of endophytic fungi as cellulose decomposition, Carboxyl Methyl Cellulose (CMC) media plus 1% congo red was used.The potential of fungus as a phosphate solubilizer and cellulose decomposition is indicated by the formation of clear zones around the growing colonies.The phosphate solubility index and cellulolytic index were determined for each isolate by measuring the colony diameter and clear zone diameter.To calculate the phosphate solubility index, the sum of the colony diameter and clear zone diameter was divided by the colony diameter [26].Similarly, the cellulolytic index was calculated by subtracting the colony diameter from the clear zone diameter and then dividing the result by the colony diameter [27].

Morphological identification of fungal isolates
Endophytic fungal isolates were characterized and determined morphologically down to the genus level.All purified fungal isolates were grown back on potato dextrose agar (PDA) medium and allowed to grow for 3 days.Observations were made macroscopically (colony diameter, colony color, and reserve color of the colony), and microscopically (namely colony shape, conidial shape, conidiophore shape and conidia color).All the characteristics obtained were then adjusted to the fungal identification book [28,29].

Isolation of endophytic fungi
A total of 20 isolates were obtained from the isolation, 10 isolates came from leaves and 10 isolates came from the bark of Styrax sumatranus J.J.Sm.Each higher plant has one or more endophytic microbes in its body, both of fungi and bacteria.According to Fouda et al. [8] endophytic microbes can be found in all parts of the plant, be it stems, leaves, bark, flowers or roots.Endophytic microbes enter the plant tissues through stomata, roots or injured plant parts.Once inside in the plant tissues, endophytes will be settle and be in the spaces between cells.According to Khiralla et al. [30] environmental factors affect the existence of endophytes, in addition the species of plant and the type of host plant tissue.Each part of the plant will have different types and numbers of endophytic microbes.Endophytes residing within plant tissues contribute to plant adaptation in adverse environments, enhance nutrient availability, secrete enzymes for cell wall degradation, serve as biocontrol agents, and possess the capability to synthesize phytohormones.

Testing the potential of endophytic fungi as phosphate solubilizers and cellulose decomposition
In Table 1 and Table 2, the result of calculating the phosphate dissolution index and cellulolytic index are presented.Dissolving index and cellulolytic index describe the ability of each isolate to carry out its respective function.The greater the index value, more effective the isolates are in dissolving phosphate or decompose cellulose according to their respective function.The clear zone formed (Figure 1a) on each isolate an indicator that Ca 3 (PO 4 ) 2 dissolution has occurred by the isolate.If no clear zone is formed, it means that phosphate dissolution does not occur and the the endophytic fungus isolate has no potential as a phosphate solubilizers (Tabel 1).The dissolution of phosphate by each isolate is due to the presence of organic acids secreted by the fungus in its metabolic process.This organic acid will form bonds with calcium so that phosphate can be free into solution.The phosphate present in solution is the available phosphate.Table 1 shows that are 12 isolates that have potential as phosphate solubilizers (8 isolates from leaves and 4 isolates from bark).The phosphate dissolving index formed was not the same between one isolate and another.Dissolution index values range between 2.10 to 2.60 (Table 1).KS3 is the isolate with the highest dissolution index value.The occurrence in the effectiveness of each isolate is caused by differences in isolates in producing organic acids, both of types and amounts [13,31].Research result found that the endophytic fungi isolated from Shorea leprosula and Shorea selanica plants were able to dissolve phosphate.Endophytic fungi identified as Trichoderma spirale and Melanconiella elisii were able to dissolve phosphate in Pikovskaya media with dissolution index of 0.12 and 0.64 respectively [32].While found 38 isolates endophytic fungi isolated from mangrove plants had potential as phosphate solubilizers with varying dissolution index (ranging from 1.2-13.7)[33].The results of research [34] obtained 6 isolates of endophytic fungi from seaweed that were able to dissolve phosphate with a phosphate dissolution index ranging from 1.3 to 3.5.
Cellulolytic fungi on CMC media also produces clear zones (Figure 1b) as an indicator of cellulose decomposition.Meanwhile, the cellulolytic describe the ability of an isolate to decompose cellulose qualitatively.The greater the cellulolytic index value, the more effective the isolate.The cellulolytic index produced by each isolate varied between 0.22 to 0.55 (Table 2).There were 12 isolates formed clear zone (8 isolates from leaves and 4 isolates from bark).DS1 is the isolate with highest cellulolytic index.Differences in cellulolytic index values indicate that there are differences in the effectiveness of each isolate.Even though the cellulolytic index values of all isolates were less than 1.The value of the cellulolytic which is less than 1 indicates that all isolates fall under the criteria of being low in decompose cellulose.According Sutari [27] the criteria for the cellulolytic index values are as follows: less than 1 (including low criteria), between 1and 2 (moderate), greater than 2 (high), if does not form a clear zone, it means there is no cellulolytic activity.In addition to measuring the cellulolytic index, the potential of endophytic fungi as cellulose decomposer can be done by calculating the cellulase enzyme produced.The results of research [35] found that endophytic fungi isolated from Taxus cupidata can decompose cellulose by producing cellulase enzyme of 5.27 U/mL.

Table 1 .
Potential of endophytic fungi as phosphate solubilizer and phosphate dissolving index.

Table 2 .
Potential of endophytic fungi as celllose decomposition and cellulolytic index.Of the 20 isolates, 12 isolates have the potential as phosphate solubilizing and cellulose decomposition.