Isolation And Characterization Of Endophytic Molds On Leaves And Stems Of Tea Mistletoe (

, purification and macroscopic and microscopic characterization. Endophytic fungi that have been isolated were characterized by macroscopic and microscopic characterization. The results of the macroscopic and microscopic characterization research showed that seven isolates of mold were successfully isolated and characterized. Microscopic characterization found five different genus among the molds Alternaria sp., Penicillium sp., Aspergillus sp., Cladosporium sp. and Fusarium sp..


INTRODUCTION
Mistletoe tea (Scurrula atropurpurea (Bl.)Dans) is a parasitic plant that lives on its host, but this plant has potential as an herbal plant.This plant has several metabolites in it.
Metabolite compounds are found in the leaves and stems of tea parasites such as alkaloids, flavonoids, terpenoids, glycosides, triterpenes, saponins, and tannins (Nasution, 2012).Pharmacologically metabolite compounds are not only produced from plants but are also produced by microorganisms that grow in plants.Based on research Simlai, et al (2014) proved that the isolation of antibioticproducing microorganism species from various plant organs such as leaves, roots, and stems.
Endophytic molds are molds that live in plant tissue and are able to form colonies in the tissue and do not harm the host itself.In plants there are one or more endophytic microorganisms consisting of molds or bacteria (Rante et al, 2013).Healthy plant tissue is found in endophytic fungi that live intracellularly and can induce the host and can produce secondary metabolites.
The metabolite compounds produced by endophytic fungi have potential as herbs.This is because endophytic molds are easy to grow and have a short cycle and can produce large amounts of bioactive compounds.Especially in the content of the mistletoe tea has active compounds that can be used as herbs.However, the existence of the mistletoe tea has a very limited stock and must be preserved.The mistletoe tea needs to be isolated and the endophytic mold taken to find out the genus name, so that the metabolite compounds from the endophytic mold can be used as herbs.
Based on the description that has been described, it is important for this research to isolate and characterize endophytic molds in mistletoe tea macroscopically and microscopically and to measure diameter growth.So that later the data obtained is able to provide information on the character of microorganisms, especially endophytic molds contained in the mistletoe tea and the discovery of the genus name of endophytic molds.

Sampling
Samples of mistletoe tea were obtained in Ketindan Village, Lawang District, Malang Regency, East Java.Samples of tea parasites were identified and determined at the Balai Materia Medica, Batu City, East Java.The parasitic mistletoe tea organs used as samples were leaves and stems stalks.Leaf and stems organs are taken from whole and healthy parts.Samples were stored in the refrigerator (freezer) at a temperature of ± 5°C

Preparation of Potatoes Dextrose Agar Media (PDA)
Media PDA used is the label Merck.PDA media were weighed as much as 10 grams and 0.13 grams of antibiotics were added.PDA media and antibiotics were dissolved with 250 mL of distilled water.All of these materials were heated to boiling using a hot plate at a temperature of 100°C and stirred using a magnetic stirrer until homogeneous.The media was sterilized using an autoclave at a temperature of 121°C, a pressure of 1 atm for 15 minutes (Fardiaz,1998).

Endophytic Mold Isolation
Samples from the leaves and stems organs of the mistletoe tea were selected and selected healthy ones to be used as research samples.The leaves and stems that have gone through the select process are washed under running water for 10 minutes.Furthermore, the sterilization process of planting materials such as samples, 70% alcohol, and distilled water was carried out in Laminar Air Flow (LAF) for 30 minutes.After the sterilization process in the LAF the sample was put into 70% alcohol solution for 1 minute, then put into 5% NaOCl (Sodium Hypochlorite) solution for 4 minutes, then put into 70% alcohol solution for 1 minute, then rinsed using sterile distilled water 3 times each for 30 seconds.Furthermore, the dried samples were cut into small pieces with a size of 1cm x 1cm using a sterile scalpel.
Samples of leaves and stems of mistletoe tea that have been cut are placed on the surface of PDA (Potato Dextrose Agar) media that has been added with antibiotics.Isolation of endophytic molds was carried out by direct seed planting technique.Inoculation was carried out on the media and each petri dish contained two sample pieces (duplo).The isolation process was carried out aseptically in the LAF.The isolation process was incubated for 2-14 days in an incubator at room temperature of 30°C.

Endophytic Mold Purification
Endophytic molds growing on PDA media were purified in stages.Each mold colony that had grown on isolation media was considered a different isolate based on macroscopic observations and purification was carried out.The purification process (Purification) is to regrow colonies on a petridish that already contains PDA media.Purification was carried out by cutting mold hyphae with a size of 1 cm x 1 cm using a round tip sterile ose needle, then implanted in a cup containing PDA media.The results of the purification of the mold were incubated in an incubator for 2-14 days at room temperature of 30°C.

Endophytic Mold Characterization
The characterization of endophytic mold isolates was carried out macroscopically.Characterization was carried out directly including the color of the upper surface of the colony (surface of colony), the color of the lower surface (reserve of colony), the surface texture of the colony, drops of exudate on the colony, growth zone (zone growth), zoning, radial lines from the center.colonies towards the edge of the colony (radial furrow) (Gandjar,1999).Microscopic characterization of endophytic fungi was carried out using the slide culture method.The slide culture method is a mold observation method by growing a mold culture which is considered better than the simple preparation method (Ramadhani,2019).The slide culture method can show the microscopic structure of the mold more fully and completely.The slide culture results were incubated in an incubator at room temperature 30°C for 3-7 days.Cultures of endophytic molds that had grown were observed microscopically using a binocular microscope.Microscopic observations using lactophenol cotton blue were dropped on a new object glass, then the cover glass used as a culture cover on the slide culture was placed on top of the droplets.Microscopic observations using a microscope were carried out using the smallest magnification of 40 times to 1000 times.Microscopic characterization includes hyphal septum (insulated or non-insulated), hyphal growth, presence or absence of conidia, and conidia shape (round, oval, or irregular) (Gandjar, 1999).

RESULTS and DISCUSSION
Isolation of endophytic molds was obtained from the leaves and stems of mistletoe tea taken from Ketindan, Lawang, Malang Regency.Each isolate had different macroscopic and microscopic characteristics.In this research, seven isolates were successfully isolated and characterized macroscopically and microscopically.While the genus found amounted to five genus (table 1) and refers to the Introduction to General Tropical Molds book Gandjar (1999) and Illustrated Marga Of Imperfect Fungi (Barnet,1998).

Isolate DBT
Macroscopic characteristics of endophytic mold colonies growing on PDA media (Potato Dextrose Agar) have a black upper Surface of the colony, black reverse side of the colony, the surface texture of the colony is similar to velvet, there is a growth zone.there is zoning, there are no radial forrows, and there are drops of colony exudate.
Microscopic characteristics of endophytic mold colonies growing on PDA using the slide culture method.There are conidia, and the shape of the conidia is oval with the tip resembling a septate beak of a duck, the conidiophores are bent.Microscopic Macroscopic characteristics of colonies of endophytic molds growing on PDA media have a black top surface (surface side), black reverse side of the colony, a velvety-like colony surface texture, and a growth zone.there is zonation, has a radial forrow, and has drops of colony exudate.
Microscopic characteristics of colonies growing on PDA media (Potato Dextrose Agar) using the slide culture method.This colony has a character with hyphae (insulated), hyphal growth (branching), hyphal color like hyaline.There are conidial heads that are round, Macroscopic characteristics of colonies of endophytic molds growing on PDA media have a black top surface (surface side), black reverse side of the colony, a velvety-like colony surface texture, and a growth zone., there is zonation, there are radial forrows, and has drops of colony exudate.
Microscopic characteristics of colonies growing on PDA media using the slide culture method.This colony has a character with hyphae (insulated), hyphal growth (branching), hyphal color like hyaline.There are conidial heads that are round, conidiophores are branched, sterigma is metula, conidia are round to semi-round and there are foot cells.Microscopic characterization refers to the Introduction to General Tropical Molds book, isolates DBT 2 belongs to the genus Penicillium sp.2.Macroscopic characteristics of endophytic mold colonies growing on PDA media have a white upper surface of the colony (surface side), the lower surface of the colony (reverse side) is yellow-black in the middle, the surface texture of the colony is similar to cotton, there is a growth zone, there is zonation, there are radial forrows, and there are drops of colony exudate.

Isolate TDBT 1
Microscopic characteristics of colonies growing on PDA media using the slide culture method.This colony has a character with hyphae (insulated), hyphal growth (branching), hyphal color like hyaline.There are round conidial heads, relatively long conidiophores, round vesicles, metula-shaped phyalids, round conidia and foot cells.Macroscopic characteristics of endophytic mold colonies growing on PDA media have a white colony surface side, a brownish yellow reverse side of the colony, the surface texture of the colony is similar to wool, there is a growth zone.There is zonation, there were no radial forrows, and there were no drops of colony exudate.
Microscopic characteristics of colonies growing on PDA media using the slide culture method.This colony has a character with hyphae (not insulated), hyphal growth (branching), hyphae color like hyaline.There are conidia, round conidia, and elongated conidiophores.Microscopiccharacterization refers to the Introduction to General Tropical Molds book, isolate TDBT 2 belongs to the genus Cladosporium sp.1.Macroscopic characteristics of endophytic mold colonies growing on PDA media have white colony surface side, white colony reverse side surface, colony surface texture similar to cotton, there is a growth zone.There is zonation, no radial forrows, and no drops of colony exudate.

Isolate TDBT 3
Microscopic characteristics of colonies growing on PDA media using the slide culture method.This colony has a character with hyphae (insulated), hyphal growth (branching), hyphal color like hyaline.There are conidia, and the shape of the conidia is oval, the conidiophores are elongated.Microscopic characterization refers to the Introduction to General Tropical Molds book, isolates of TDBT 3 belong to the genus Cladosporium sp.2.Macroscopic characteristics of endophytic mold colonies growing on PDA media have black colony surface side, black colony reverse side is orange in color, colony surface texture is similar to velvet, there is a growth zone, there is zonation, no radial forrows, and has exudate drops.
Microscopic characteristics of colonies growing on PDA media using the slide culture method.This colony has a character with hyphae (insulated), hyphal growth (branching), hyphal color like hyaline.There are conidia which include crescent-shaped microconidia with rounded ends and macroconidia with crescent-shaped, canoeshaped, or slightly turned and tapered ends, cano-shaped and oval conidia.Conidiophores are erect and insulated.Microscopic characterization refers to the Introduction to General Tropical Molds book, isolate TDBT 4 belongs to the genus Fusarium sp.

DISCUSSION
Based on the results of the research, the mold isolates from the leaves and stalks of the mango parasite had different macroscopic and microscopic characteristics.The endophytic mold isolates were characterized macroscopically and microscopically to the genus level as shown in Figures 1 to 7. Based on the macroscopic and microscopic characterization, it could be seen that the isolates obtained from the isolation of the leaves and stalks of the tea parasite consisted of the genera Altenaria, Penicillium, Aspergillus, Cladosporium and Fusarium.The Altenaria genus was found in DBT 1 isolates, the Penicilluium genus consisted of DBT 2 and DBT 3 isolates, the Aspergillus genus was found in TDBT 1 isolates, the Cladosporium genus consisted of TDBT 2 and TDBT 3 isolates, and the TDBT4 isolate was the Fusarium genus.
Endophytic microorganisms such as fungi and bacteria are found in more than one plant that lives in it (Rante, 2013).In plant tissues that live intracellularly are called endophytic fungi.Plant tissues such as roots, stems, leaves and fruit contain many endophytic microorganisms in them, but these endophytic molds do not have a negative impact on the host (Sofiyani, 2014).These endophytic molds live in healthy plant tissues and can induce their host to produce secondary metabolites.Recombinant genetics or coevolution lead to the induction of endophytic molds in plants (Sia, 2013).The metabolites produced by endophytic molds produce bioactive secondary metabolites, both known compounds and new compounds (Alvin et al, 2014).
In addition to secondary metabolites produced by endophytic molds, endophytic fungi are widely known for their biological activity benefits.Altenaria sp.including types of parasites or saprophytes on plants.In Furi's research (2018), Altenaria mold can cause leaf spots to appear on strawberry plants.Meanwhile, according to research by Arisanti, et al (2012) that penicillium is a type of mold  (Suciatmih, 2010).

CONCLUSION
Based on the results obtained in this research, the isolates of endophytic molds obtained from isolation and macroscopic and microscopic characterization of the leaves and stems of the mistletoe tea were obtained as many as 7 isolates.Of the 7 isolates can be characterized and produce 5 different genera.Isolat DBT 1 belongs to the genus Alternaria, DBT 2 and DBT 3 belongs to the genus Penicillium, TDBT 1 belongs to the genus Aspergillus, TDBT 2 and TDBT 3 belongs to the genus Cladosporium and TDBT 4 belong to the genus Fusarium.

Table 1 .
genus found on the leaves and steams of mistletoe tea.
Directorate General of Higher Education, Ministry of Education, Culture, Research and Technology of the Republic of Indonesia, with a Research Contract in 2021Number:567/G164/U.LPPM/K/B.07/IX/2021Who Has Provided Grants for Basic Research for Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT) with the title "Eksplorasi dan Domestikasi Benalu Teh Dan Benalu Mangga Sebagai Bahan Baku Fitofarmaka Jamu Antihipertensi" PUI-PT Research Center for Biological Molecular Engineering (BioMe) University Airlangga Surabaya, which has assisted in the process of identifying the genus of endophytic molds isolated from the leaves and stems of the mistletoe tea