Isolation and Identification of Endophytic Fungi from Leave and Stem of Calopogonium mucunoides

Thirty-seven isolates of endophytic fungi were isolated from leaves and stems of Calopogonium mucunoides collected from PTPN PTPN XII (Persero) Rubber Plantation, Klatakan, Kecamatan Tanggul, Kabupaten Jember, Jawa Timur. All isolates were identified based on morphological characteristics using the light microscope. The 37 isolates of endophytic fungi are members of Deuteromycota and Basidiomycota and classified to genera Phoma, Phomopsis, Corynespora, Rhizoctonia, Helicosporium, Curvularia, Torulomyces, Gliocladium, Gloeosporium, Acremonium, Tripospermum, Aureobasidium, Colletotrichum, Humicola, Fusarium, Sclerotium, and sterile hyphae. Article history: Received 22/01/2018 Received in revised form 04/04/2018 Accepted 04/04/2018

Healthy mature leaves and stems of C. mucunoides were washed thoroughly under running tap water, then the samples were sterilized by dipping them in 75% ethanol for 30 s, followed by immersing in 3 % sodium hypochlorite for several times, rinsed in sterile distilled water, and finally dried on sterile filter paper on petri dish. A piece of each leaves and stems were removed with a sterile scalpel then cut into small pieces about 1 to 1.5 cm, each piece was put on a Petri plate containing Potato Dextrose Agar (PDA) medium and incubated at room temperature (27-280C) to promote fungal growth and sporulation. After 7-8 days, individual hyphal tips of actively growing fungi were picked up for subculturing by Figure 1. Colony morphology variations of endophytic fungi from Calopogonium mucunoides on PDA at day seven. Number 1 to 19 from leave (isolat 1:ED1, isolat 2:ED2, isolat 3:ED3, isolat 4:ED4, isolat 5:ED5, isolat 6:ED6, isolat 7:ED7, isolat 8:ED8, isolat 9:ED9, isolat 10:ED10, inoculating it onto new PDA medium plate individually and incubated at room temperature (27ºC) for one week. The purified fungal isolates were labelled for further use.

Morphological identification
Identification of endophytic fungal isolates was done by observing trypan blue stained slides prepared from stock cultures using a bright-field and phase contrast microscope.
Identification was based on morphological characteristics such as growth pattern, hyphae, the colour of the colony and medium, surface texture, margin character, aerial mycelium, mechanism of spore production and conidial characteristics.
Obtained data were then compared with the descriptions of endophytic fungi and identified based on Selim et al. (2012) and Zuber (2011).

Results
A total of 25 fragments leaves and stem was used from C. mucunoides. In this study, a total of 37 endophytes were isolated (Figure 1). The endophytes were isolated using potato dextrose agar (PDA, Merck). The total numbers of colonies of endophytic fungi from the stem are lower than the ones from leaves.
Based on morphological characteristics identification ( Figure 2 and 3), almost all endophytic fungi which were isolated are members of Deuteromycota group (Table 1).

Discussion
Identification of fungi was done based on Selim et al.
(2012) and Zuber (2011) also by using standard protocol of Barnett and Hunter (1972). Identification is based on morphological characteristics of the fungi grown on the culture medium (PDA). The morphology including macroscopic and microscopic characteristics. The  Deuteromycota (White, 2009). Deuteromycota or fungi imperfecti is a group of fungi which sexual reproduction has not been identified. They are member of Ascomycota or some from Basidiomycota (Venkatesan and Suryanarayanan, 2013). The term of deuteromycota was not used anymore in present, due to the concept of DNA-based classification (Deoxyribose Nucleate Acid). However, some literature still uses this term to group all the fungi not belonging to other divisions (Taylor, 2011;Shenoy, 2007).
The result also discovered 12 fungal isolates that did not show the formation of conidia. These isolates were grouped as mycelia sterile. Isolates belonging to the mycelia sterile group because they did not show the form of anamorph or teleomorph during the observation process.  (Roza et al., 2011;Katoch et al., 2014;Ramesha & Srinivar, 2013;Powthong et al., 2012). Results showed that based on the morphological characteristics, the endophytic fungi isolated from legume species classified into Basidomycota, Ascomycota, and Deuteromycota (Dandu et al., 2014;Shankar & Shashikala, 2010;Phowtong et al., 2012;Senna & Sridhar, 2004 A more realistic approach is needed to characterize the endophyte species from a single host or group of hosts. The number of samples required for the isolation of the endophytic fungus related on the distribution and abundance of fungi in the host plant and the tissue types (foliage, stem, bark, xylem, root). More intensive sampling method will increase the recovery of rare species, which are likely also to occur on many hosts, but the most common species on a specific host will be widely distributed on that host (Stone et al., 2004).

Conclusion
A total of 37 endophytes were isolated and assigned to 16 genera based on the morphological characteristics , in which 33 (56,7%) isolates are Phoma sp., Phomopsis sp.,