Endophytic fungi: isolated from Cosmos caudatus Kunth and Cosmos sulphureus Cav.: a histologic observation, identification, and secondary metabolites chemical analysis

Cosmos caudatus and Cosmos sulphureus are sorts of plant commonly use for side dish vegetables. Some plant live in mutualism symbiotic interaction with some endophytic fungi that protect the host plant. This research is done to: 1) determine the endophytic fungi colonization in the Cosmos plant tissues by histologic observation, 2) isolation and identify the endophytic fungi species from Cosmos leaf, branch bark and flower petal tissues, 3) analyze alkaloid, flavonoid, tannin, saponin, and phenolic contents produced by the each endophytic fungi species. The endophytic fungi culture on PDA medium were cut into 5×1 cm in size and inoculated in PDB medium, then shake in the rate of 120 rpm for 7×24 hours. Afterwards the liquid centrifugated. The secondary metabolites contents were analyzed by using spectrophotometric method. This research result showed that: 1) the endophytic fungi position was found on the leaf palisade cell wall, epidermal cell wall, sponsa cell wall, neighbour cell wall; on the branch bark parenchyme cell wall; on the flower petal epidermis cell wall; 2) eleven endophytic fungi have been identified; 3) some variation in the secondary metabolites contents produced by each endophytic fungi.


Introduction
Cosmos spp.plant usually uses as a fence plant, because the flower has a beautiful colour, i.e: yellow, orange, pink.Some people eat the leaves as a side dish.This plant usually stay healthy and rarely attacked by microorganisms.Cosmos caudatus flower have a yellow colour and Cosmos sulphureus flower colour is pink.
These plants are ralatively resistance to diseases, so it was assumed that there is some endophytic fungi live in the plant tissues and have a mutualism symbiotic interaction with these plants.Some previous researches proved that several endophytic fungi species have a mutualism symbiotic interaction with the host plant and the host plant relatively resistant towards some pathogenic microorganisms [1,2].Some endophytic fungi species isolated from a medicinal plant, Hedycium acuminatum, potencially produce some antimicrobial compounds that can inhibite the growth of pathogenic bacteria that attacked host plant [3].The precious research have been reported that some species of endophytic fungi that lived in Cananga odorata leaf, branch bark and flower petal tissues have a mutuliasm symbiotic interaction with the endophytic fungi, i.e: Colletotrichum alienum, C. kahawae, C. aotearoa, C. alatae, C. queenslandicum, Nigrospora sphaerica, Mycelia sterile 1 and Mycelia sterile 2. The endophytic fungi could protect the host plant from bacteria infection by produce some antimicrobial secondary metabolites.Besides that, the endophytic fungi also get some advantages from the host plant, i.e: protection from environmental stress such as low humadity, high air temperature, drought, and also nutrition [1,2].The endophytic fungi live on the epidermis cell wall and parenchyma cell wall of Switenia mahagoni twigs, on the epidermis, vascular tissues and stoma guard cell wall of this plant leaves.The hyphae position can be determinated by microscopic observation.
This research is aimed to: 1) determine the position of endophytic fungi in the C. caudatus and C. sulphureus leaf, branch bark and flower petal; 2) Identify the endophytic fungi species from C. caudatus and C. sulphureus leaf, branch bark and flower petal; 3) Analyze several secondary metabolites compounds contents produced by each endophytic fungi species.

The microscopically observation on endophytic fungi position in C. caudatus and C. sulphureus plant tissues preparation
The leaves, branch bark and flower petal of the C. caudatus and C. sulphureus plant were cut in longitudinally and paradermally, then observe histologically the endophytic fungi hyphae position in the M. liliifera tissues in microscopic observation.

The endophytic fungi identification
C. caudatus and C. sulphureus leaf, branch bark and flower petal was rinsed and soaked in 1% NaOCl during one min., then washed in sterile distilled water.Afterwards the plant parts were dipped within 70% alcohol during a minute, then rinsed with sterile distilled water [4].The leaves and flower petals part cut in 1x1 cm 2 in size, the branch bark part cut with the branch bark.

The liquid culture preparation
The endophytic fungi isolates were inoculated on PDA medium, 5×1 cm 2 in size, and afterward put PDB medium, incubated in 27℃, shook in 120 rpm.Then, the liquid culture filtered and centrifugated for 10 minutes in 3000 rpm.The secondary metabolites, i.e. flavonoid, alkaloid, tannin, saponin, and Phenolic content were taked from the supernatant.

The preparation in sample and standard solution.
The 1 gram sample was crushed and dissolved in methanol to 10 mL, homogenized for 30 min.Then filtered and centrifugated for 10 minutes in 3000 rpm.Afterwards the supernatant was taken.The liquid sample in 1 mL volume were add methanol in the volume 5 mL and homogenized for 5 min.Then filtered with a vacuum filter.

2.4.1.3.
The determination of flavonoid content.Take 0.1 mL sample solution or standard solution, add with 0.1 mL 2% AL2CL3, afterwards homogenized for 60 minute.Then, add with aquadest 1 mL in volume.If color becomes red, it was proved that the sample contains flavonoid.The absorbance is λ = 420 nm.The determination of flavonoid concentration were done based on the regression standard equation.[6] 2.4.2.1.Preparation of standard solution.The standard solution is atrophine solution (100 mg/L), weigh 10 mg atropine, solute in 100 mL chloroform solution.Then the solutions were prepared in concentrations: 0; 1; 5; 10; 25; and 50 mg/L.

The preparation in sample and standard solution.
The 0,1 g sample was crushed and dissolved in 10 mL DMSO, added with 5 L bromocresol green solution and 1 mL 2 N HCL and 5 L phosphate buffer, homogenized for 60 min.Afterwards poured into a separatory funnel and mixed with 10 mL chloroform.Then, the solution were shaken until the layer were seen.Preparation of the standart solution were done by taking 5 mL atropine solution, add with 5 mL brom cresol green solution, 1 mL 2N HCL, and 5 mL buffer phosphate and homogenized.

Determination quantity of alkaloid.
Upper phase of the standard solution were taken in 11 mL.If yellowish-orange colour was seen, it was proved that the solution contained alkaloid.Afterwards the solution was added with chloroform to 5 mL in volume.The absorbance is λ = 470 nm.The determination of alkaloid concentration were done based on the regression standard equation.[6] 2.4.3.1.Preparation of standard solution.The standard solution is tannic acid solution (50 mg/L).Then dissolve 5 mg tannic acid in 20% ethanol to 10 mL.Then the solutions in concentrations: 0; 1; 5; 10; 25; and 50 mg/L was prepared.

The preparation in sample and standard solution.
The 0.1 g sample was crushed and dissolved in 10 mL methanol, homogenized for 30 minute.Then filtered and centrifugated for 10 min in 3000 rpm.Afterwards the supernatant was taken for next process.

The determination of tannin content.
The 5 mL standard solution and added with 0.5 mL 0.008 M K3Fe(CN)6 and 0.5 mL 0.1 M FeCl3, then homogenized for 30 minute.The solution dilluted with aquadest to 10 mL in volume.The absorbance is λ = 620 nm.The determination of tannin concentration were done based on regression standard equation.[7] 2.4.4.1.Preparation of standard solution.The standard solution is saponin solution (100 mg/L).Afterwards dissolve 10 mg saponin in 20% ethanol to 100 mL.Then, the solutions were prepared in concentrations: 0; 1; 5; 10; 25; and 50 mg/L.

The preparation sample and standard solution.
The 0,1 g sample was crushed and dissolved in 10 mL 90% ethanol, afterwards homogenized and heated in a water bath for 90 minute in 55℃.Then, the solution was filtered and then reextracted with 10 mL 90% ethanol.The mixed of both extracts heated at 90℃ until half of the solution remained.Afterwards the solution was poured in the separating funnel, add with 40 mL diethyl ether, shook and let stand until the solution was separated.Then, take the bottom phase, add 10 mL 5% NaCl and 60 mL butanol and filtered.Aferwards the filtrate was dried up at 60℃ to get dry saponin and then added with 5 mL 20% ethanol.

The preparation sample and standart solution.
The solid sample were crushed and measured for 0,5 -2 g, then diluted in methanol pa to a volume of 25 mL, homogenized and left to stand for 30 min., afterwards filtered and centrifugated in 3000 rpm for 10 min, the supernatant were taken.The solution were taken.The solution were uses for the next process.The liquid sample were taken for 1 mL in volume, then added with 5 mL methanol pa, pour in test tube, afterwards homogenized uses vortex for 5 min.The solution filtered with vacum filter, the filtrat were uses for the next proces.

Data analysis
The position of endophytic fungi hyphae in C. caudatus and C. sulphureus shown in Table 1 and Figure 1.Each endophytic fungi microscopical characteristics shown in Figure 2. Description data of ten endophytic fungi species from C. caudatus and C. sulphureus shown in Table 2.The endophytic fungi species secondary metabolite content shown in Table 3.

The observation of endophytic fungi position histologically in C. caudatus and C. sulphureus tissue
The observation result of the endophytic fungi position on the host plant tissue was determined by microscopic observation on leaf, branch bark, and flower petal of C. caudatus and C. sulphureus The endophytic fungi hiphae position shown in Table 1.The illustration of endophytic fungi hyphae on the plant part tissues shown on Figure 1.The histologic observation result of the hyphae position on leaf, branch bark and flower petal proved that the fungal hyphae attached on the pallisade cell wall, sponsa cell wall, neighbour cell wall of the leaves; on the parenchyma cell wall, and on the epidermis cell wall.There is no hyphae found insert into the host plant cell it is proved that there some endophytic fungi that live in C. caudatus and C. sulphureus as a host plant and does not make any damaged.

The identification result of endophytic fungi
The identification result showed that there were found 11 species, consist of: Mycelia sterile, Nigrospora oryzae, Sclerotium sp.  2).The macroscopic and microscopic character description of each fungi shown on Table 2. Some of these fungi species were found not specific in one of the plant part specifically, it could be found in the leaf and branch as well as in the flower petal plant tissue.It was found 11 endophytic fungi species isolated from C. caudatus and C. sulphureus leaves, branch bark as well as flower petal tissues.The endophytic fungi hyphae attached on the leaves epidermis cell, pallisade cell, and sponsa cell; on the branch bark epidermis cell and parenchyme cell; and on flower petal epidermis cell and parenchyme cell.There are no hyphae insert into host plant cell especially in the leaf, branch bark as well as in the flower petal.It is proved that the fungi does not make any damage to the host plant tissues, so the host plant always stay healthy.This fact also proved that there is a mutualism symbiotic interaction between the endophytic fungi and the host plant.The plant protected by the endophytic fungi and the host plant.The plant protected by the endophytic fungi from the pathogenic bacteria that attacked the plant.On the other way, the fungi protected from harmfull abiotic factors i.e: low humadity, high air temperature, lack of water, etc. [9]; the fungi also receive nutrition in intercellular space that cell not used by the host plant [10].and C. sulphureus plant could be isolated from another plant.Nigrospora oryzae also found as an endophytic fungi species in Avicennia marina leaf [11].C. aenigma have been isolated from Persea americana [12].C. alienum could be found as one of some endophytic fungi species in Michelia champaca L. [13]; in Physalis angulata L. leaf [14]; in Cananga odorata leaf and flower petal [15]; and Malus domestica plant [12].C. aotearoa also found as an endophytic fungi species ini Cananga odorata [15].Mycelia sterile also could be found in Chinese medicinal plant [16].

3
Fungi on The Leaf, Branch Bark, and Flower Petal Tissues of C. caudatus and C. sulphureus No

Table 2 .
The Description of Macroscopic and Microscopic Characteristic of Each Endophytic Fungi Species Isolated from C. caudatus and C. sulphureus

Table 2 .
(continued)Based on the analysis results it was proved that each fungi can produce flavonoid, alkaloid, tannin, saponin, and phenolic with different contents (Table3).Colletotrichum alienum has the ability to produce those secondary metabolites in the highest contents compared with the other fungi.The analysis results of each secondary metabolites content produced by the eleven fungi species shown on Table3.Eleven endophytic fungi species have been identified.Mycelia sterile, Nigrospora oryzae, Sclerotium sp. 1, Streptothrix sp.Corda, Verticillium sp., Sclerotium sp.2., Colletotrichum aenigma B. Weir & P. R. Johnst, Colletotrichum alienum B. Weir & P. R. Johnst, Rhizoctonia sp. 1, Rhizoctonia sp. 2, Colletotrichum aotearoa B. Weir & P. R. Johnst.These endophytic fungi species isolated from C. caudatus

Table 3 .
The Analysis Result of Secondary Metabolites Content Produced by The Eleven Fungi Species