The potential of dark septate endhophyte fungus from white jabon (Neolamarckia cadamba (Roxb.) Bosser)

Endophytic fungi are one of the forest microbes that can be solutions for increasing the resistance of forest plants to environmental stress. Exploration and collection of endophytic fungi from the forest is important because the condition of the forest as a natural habitat for microbes has been disturbed due to degradation and deforestation. A potential plant species for forest and land rehabilitation is white jabon (Neolamarckia cadamba (Roxb.) Bosser). The research objective was to isolate and characterize endophytic fungi and to obtain the dark septate endhophyte from the roots of the white jabon which have the potential to increase plant growth. The fungus was isolated by surface sterilization method and then characterized and identified morphologically. Pathogenicity test was carried out to determine the pathogenicity of the fungus. The results of the study found ten isolates. Most of the isolated fungi showed sterile mycelia with septate hyphae, so they were not identified morphologically. The isolate of CPGS 3 isolates probably belonged to the genus Aureobasidium sp. CPGS 3, CPKR 6, and CPPS 11 isolates are candidates for dark septate endophyte fungi which have the potential to promote plant growth and not become pathogens in chinese cabbage (Brassica rapa).


Introduction
White jabon (Neolamarckia cadamba (Roxb.)Bosser), synonymous with Anthocephalus cadamba (Miq.) is one of the potential trees species widely cultivated in various sites, both on a large scale in regions like North Sumatra, Riau, and Central Kalimantan, and on a smaller scale by farmers, especially in Java and South Kalimantan [1] [2].White jabon shows promising prospects for industrial plantation forestry, reforestation, and as a revegetation option for former mining areas in Indonesia Genotype and environmental factors such as nutrients, soil, and association with microorganisms influence the growth of white jabon.Indonesia possesses vast biodiversity, including a diverse array of microorganisms found in forests.Nowadays, forest microorganisms offer potential solutions to various global challenges, particularly in environmental and forest restoration.One of the microorganisms that influence plant growth is endophytic fungi.Endophytic fungi, such as Dark Septate Endophyte (DSE), provide numerous benefits to host plants by enhancing their growth and productivity without causing pathologies [3][4] [5].DSE fungi also enhance host plant compliance to biotic and abiotic environmental pressure [6] [7].Given the continuous degradation and deforestation of natural habitats, the exploration and collection of endophytic fungi from forests are essential.The readiness of plant seedlings to withstand stress, such as drought, is crucial, especially for root systems that require specific conditions.Plant roots suitable for extreme land conditions should have a well-developed root system and can form symbiotic relationships with specific fungi.
Endophytic fungi can produce secondary metabolite compounds according to their host plants.The enhancement of host plant growth occurs through mechanisms like improved nutrient absorption or the production of growth-regulating hormones [8].However, there is limited research on endophytic fungi in white jabon.Therefore, the purpose of this research was to isolate and characterize endophytic fungi and to obtain the dark septate endhopyte fungi from the roots of white jabon trees which have the potential to increase plant growth.

Method 2.1. Time and Location of Research
The DSE fungi exploration activity was conducted at the provenance-progeny test tree stand of white jabon at Parungpanjang forest areas with special purposes (PFRS), Bogor (06°20'42" S, 106°06'15" E, 52 m asl altitude).The provenance-progeny test was established in 2013 with a spacing of 3 m x 3 m.[9].The PFRS area is a specific zone designated for research, and weed growth is rapid after land clearing.It has a podzolic soil type with low soil nutritional (low grade of C-organic and N, P, K with pH of 4.8) (Table 1).The average temperature and annual rainfall are 27.8°C and 2,440 mm, respectively.The process of isolating, purifying, and testing the fungi at the Laboratory of Forest Plant Pests and Diseases, Bogor Forestry and Environmental Standard and Instrument Implementation Center (BPSILHK Bogor).Macroscopic and microscopic characterization of the fungi at the Pathology Laboratory, IPB University.
2.1.Isolation and purification.Root samples were collected from white jabon trees selected through purposive sampling, with the criteria of strong and healthy parent trees.The root samples were thoroughly washed and sterilized using the surface sterilization technique in pure culture [5], then isolated in a 50% Corn Meal Agar (CMA) medium and incubated at room temperature.Pure cultures were prepared by extracting fungal mycelium from each fungal colony that appeared morphologically distinct based on macroscopic features such as color, shape, edge, and colony distribution pattern.Purification of the fungi was performed on 50% Corn Meal Malt Yeast Agar (CMMYA) medium.

Potential as an endophytic fungus.
Research on non-pathogenic fungi that promote growth is determined by the response of seeds after being treated with endophytic fungi.Growth-promoting fungi are fungi that can increase plant height and root length, resulting in better nutrient absorption and increased vigour in plants.If the planted seeds fail to grow, it means that the endophytic fungal suspension does not affect the growth of chinese cabbage (Brassica rapa) plants.A total of 10 sterilized chinese cabbage seeds were planted in PDB medium in jam jar containers containing endophytic fungal cultures.As a control, seeds were placed in a PDB medium without the fungal culture.Observations were made by measuring shoot height, root length, and biomass after 14 days of planting.The nonpathogenic nature of the endophytic fungi is determined based on pathogenicity tests, specifically, the influence of endophytic fungi on the rate of development of chinese cabbage seeds in the laboratory.

Morphological characterization of fungi (macroscopic and microscopic).
Identification was carried out on all pure isolates.The observed macroscopic morphology included colony color, colony texture (smooth like cotton, hairy, or velvety), colony diameter on CMMYA media after three weeks, colony surface (granular, powdery, raised, or smooth), and reverse colony color.Fungal morphological identification was conducted by referring to the identification book by [11] Microscopic structures observed included mycelium, conidiophores, conidia, appressoria, and chlamydospores.

Results and Discussion
In this study, each root sample was found to harbor endophytic fungi, indicating a symbiotic relationship between the white jabon plant and endophytic fungi.The capability of endophytic fungi to colonize the roots of white jabon is believed to be connected to the environmental conditions of white jabon plants in PFRS Bogor with low soil fertility and acidity levels.It is known that the symbiosis of endophytic fungi with host plants serves as a defense mechanism against environmental stress, including extreme environmental conditions.The mechanism of environmental defense occurs through the formation of bioactive compounds and secondary metabolites [12].Changes in metabolite production are caused by environmental stress, causing the endophytic fungi to activate dormant genes as an adaptation to extreme environmental changes [13].
Colonies of endophytic fungi on white jabon root tissue began to appear on days 5 to 7 after the start of incubation.The diameter of the CPKR 8.2 colony reached 8-9 cm within 22 days after incubation, while the other isolates only reached 3.3 -4.5 cm in diameter.The color of the endophytic fungus colonies produced varies, which are generally dark white, gray to dark in color.Several isolates form concentric lines in their colonies.The various fungal colonies that were successfully isolated are shown in Table 2.The endophytic fungal isolates produced in this study are difficult to describe in terms of their characteristics because they exhibit sterile mycelium.Microscopic observations of the fungal somatic structures show that the hyphae are generally septate.These septate hyphae are a characteristic identification feature for fungi belonging to the Ascomycetes.According to [14] endophytic fungi are divided into four classes.Endophytic fungi classified in the second class are of the Ascomycete type, with only a few belonging to the Basidiomycete type.The fungi in this second group of fungi usually covers plant tissue such as the roots, stems, and leaves of plants and can thrive in environments with high-stress conditions.
The fungal isolates CPGS 1, CPGS 2, CPKR 6, and CPPS 8.2 exhibit sterile mycelium with dark hyphae and septate.Meanwhile, the isolate CPGS 3 and CPPS 11, the possibility is that they belong to the Aureobasidium sp.The characteristic features of Aureobasidium sp.include dark conidia arranged in chains or solitary forms, and having various shapes such as round, cylindrical, and irregular.The hyphae of Aureobasidium sp. are dark and septate.Endophytic fungi infect and complete almost their entire life cycle within the tissues of healthy plants, being associated with healthy plants and showing no symptoms.The inability of endophytic fungi to cause disease symptoms is suspected to be due to the absence or loss of pathogenicity-related genes.Therefore, pathogenicity tests are conducted to select potential endophytic fungi.
The results of the pathogenicity test on six candidate DSE (Dark Septate Endophyte) isolates showed varying abilities in inducing plant resistance (Table 3).Meanwhile, isolates CPGS 1, CPGS 2 and CPKR 8.2 tend to inhibit the growth of chicory seeds, so they are suspected to be pathogens.CPGS 3, CPKR 6 and CPPS 11 isolates showed better root and biomass growth compared to controls.Meanwhile, isolates CPGS 1, CPGS 2 and CPKR 8.2 tend to inhibit the growth of chicory seeds, so they are suspected to be pathogens.[15] further added that culture extracts of endophytes can increase the development of host plants.In their research, Ernst et al. (2003) stated that isolates of endophytic fungi from the genus Stagonospora can increase the biomass of wheatgrass.Similarly, [16] showed in their study that endophytic treatment can increase the biomass weight of the host plants.
The endophytic isolates DSE CPGS 3, CPKR 6 and CPPS 11 from the white jabon plant are expected to not only be able to induce plant resistance in vitro but also to demonstrate their ability in vivo.The further research is still needed at the nursery level to strengthen the in vitro test results.This is because not all endophytic isolates may show synchronization between in laboratory and in the greenhouse test results.Nevertheless, in laboratory tests can serve as an initial selection to narrow down the scope of in vivo testing, ultimately identifying endophytic fungal isolates with multifunctional and genuine development potential.Endophytic fungi have the ability to produce several compounds that can function as anti-bacterial, anti-fungal, growth-promoting hormones, and insecticides.

Conclusion
The results of this study showed that six isolates fungi, namely CPGS 1, CPGS 2, CPGS 3, CPKR 6 and CPKR 8.2, and CPPS 11.The isolates fungi of CPGS 3, CPKR 6, and CPPS 11 had the potential to become DSE fungi due to their ability to increase plant growth.

Table 2 .
The character of endophytic fungus candidate isolate.

Table 3 .
Growth of chinese cabbage (Brassica rapa) sprouts on pure culture media of DSE candidate fungus isolates from roots of white jabon.