Endophytic microbe in stem tissue of oil palm colonized by Ganoderma sp.

Infection with Ganoderma sp. results in pseudoschlerotium, a distinctive feature of oil palm tissue trunks. The goal of this study was to study the microbial communities in Ganoderma sp. colonized oil palm trunk tissue. Endophytic bacteria are isolated from oil palm trunk tissue using selective media such as Ashby, Pikovskaya, King’s B, and a half-NA media to isolate N2 fixing bacteria, P solubilizing bacteria, Pseudomonas sp., and endophytic microbes, respectively. To prepare the sample, two methods were tested: 1) sterilizing the surface of the sample and then directly planting the stem tissue in a medium, and 2) sterilizing the surface of the tissue, crushing the tissue with a sterilizer mortar, and centrifuge for further seeding the supernatant in the selected medium. The first procedure was found to be effective in isolating N2-fixing bacteria from the top, middle, and bottom of the stem. We also recovered P solubilizing bacterium, which was yellow in colour and only located in the centre part of the stem, whereas the Pseudomonas sp. isolate was white in colour and found throughout the stem except at the bottom in the left side of the pseudosclerotium. We were able to isolate endophytic bacteria with a white hue using a half-NA medium. Method 2 did not succeed in isolating Azotobacter sp., but it can still isolate P solubilizing, Pseudomonas sp., and endophytic bacteria in all sections of the stem. The number of bacteria successfully isolated using the culture approach was low, particularly in the pseudosclerotium area. However, the larger quantity of Pseudomonas bacteria found in infected Ganoderma sp. tissue should be investigated further to establish the role of these bacteria, particularly in plant defence against Ganoderma sp.


Introduction
Ganoderma sp. is a major pathogen in oil palm plantations caused by basal stem rot (BSR).The habitat of this fungal is soil and infects oil palm trees through plant roots.Controlling of BSR caused by Ganoderma sp using antagonist microbe has been developing through the application of Trichoderma sp, Gliocladium sp.Paecillomyces, Hendersonia sp. in a single isolate or in a mixed culture.The problem in controlling stem rot disease is the ability of Ganoderma sp. to form necrosis or pseudoscklerotium in oil palm trunk tissue.This ability causes Ganoderma to be able to survive in oil palm trees and even causes trees to fall.Some microbes as active ingredients of bio fungicides are generally microbes that grow outside the tissue.The evidence of Ganoderma infection is detected in oneof oil palm field, Deli Serdang, North Sumatera in April 2020.The observation was performed in unproductive age group in the second and third year (TBM II and TBM III) of oil palm.The data showedthe average evidence in 1243 (2023) 012019 IOP Publishing doi:10.1088/1755-1315/1243/1/012019 2 TBM II is 0.3-0.6%,while in TBM III 0.4-0.6%[1].More detailed observationby Paterson (2019), for North Sumatra, Sumatra, South Sumatra, and inland Sumatra, respectively, the percent of current disease levels in 2019 were 37, 39, 51, and 52% [2].
However, exploration of endophytic microbe in oil palm stem infected with Ganoderma has not been studied deeply.Research on endophytic in relation to the status of plant survival, as well as its rolein increasing plant resistance to disease, has begun to be carried out.Endophytes are defined as microorganisms including bacteria, fungi, and actinomycetes that inhabit intra-and intercellular plant tissues for all or part of their life cycle.Fungal endophytes have the ability to produce numerous extracellular enzymes; such as pectinases, cellulases, lipases, amylases, laccases, and proteinases.These fungal enzymes play a key role in biodegradation and hydrolysis processes which are significantly important mechanisms against pathogenic infection and to obtain their nutritional need from the host plants [3].The ability of fungal endophytes to produce different enzymes has been reported by [3,4], however further quantitative assays for fungal endophytic enzymes and their metabolic potential are required to understand the ecological and biological role of these fungal endophytes.
Some research evidence shows that there are differences between healthy plant tissue and diseased plant tissue.Exploration and understanding of the role of endophytic in oil palm plants attacked by Ganoderma have not been done much as well as interactions between endophytic and Ganoderma.The results of research on sweet potato plants show that some endophytes are able to play a role in plant growth, whereas in tomato plants, endophytes can produce plant growth hormones.Endophytic microbial communities in the Prunus ovium L genotype that are easy and difficult to propagate are very significantly different.The dominant bacteria found are included in Mycobacterium sp.In the genotype that is easily propagated, there are Rhodopseudomonas sp. and Microbacterium sp. that have the ability to produce growth hormones [5].The results of exploration on sweet potato plants obtained 11 different strains belonging to the genus Enterobacter, Rahnella, Rhodanobacter, Pseudomonas, Stenotrophomonas, Xanthomonas and Phyllobacterium, these microbes play a role in increasing plant growth for example because of their ability to produce IAA and are able to fix N2 from atmosphere [6].This study was aim to explore the selected cultivated endophytic microorganism in the oil palm stem colonized with Ganoderma sp.Selected isolation methods will be used to capture as many endophytic microbes present in the stem tissue, the next activities can be carried out to screen the microbes that are antagonistic to Ganoderma and study the distribution of endophytic microbes that are beneficial in improving plant health, especially throughincrease nutrient availability.

Plant sampling and study area
A 15-year-old diseased oil palm tree, as a source of the sample, was growing in the area of the Indonesian Research Institute for Biotechnology and Bioindustry in Bogor at an altitude of 150 asl.The diseased tree showed typical symptoms of spear leave, fruiting body formation, and there are leaves that form skirts.A 30 cm long stem part was cut off transversally and longitudinally from three regions: the top (A), the middle (T), and the bottom (B) parts of the tree.The inner piece of the stems was collected and placed in sterile polyethene bags and then brought to the laboratory.

Endophytes isolation
The reagent used for endophytes isolation were 70% alcohol, 5.25% NaOCl, chloramphenicol, nutrient agar (NA), Ashby medium, Pikovskaya medium, Kings'B medium, and sterilized distilled water.The sample preparation was done using two different methods, which are explained below.
For the first method, the stems are cut into cubes (5x5 mm) and washed with running water to remove the debris and epiphytic microorganisms attached to the outer surface and then subjected to sequential 1 minutes of sterile distilled water, immersed in 75% ethanol for 1 min, and allowed to standfor 1 minute.In the next stage immersed in 5.25% NaOCl for 0.5 minutes, immersed again in 75% ethanol for 30 s, and let stand for 1 minute.The stem tissue was immersed in 5.25% NaOCl for 0.5 minutes, then in 75% ethanol for 30 seconds, and finally washed in sterile distilled water three times.The plant tissue was taken using a flammed-sterilized scalpel and then placed on Ashby medium agar plates, Pikovskaya medium agar added with 0.05 mg/litre chloramphenicol, Kings' B medium agar, and half-strength nutrient Agar.After incubation at 28 ± 2°C, all microbial growth that appeared onthe plates was then subcultured on the same agar medium to obtain a single isolate.The isolates were then subcultured again to confirm their purity.The isolates were preserved on agar slants and then stored at 4°C for further study.The second method is as follows, five grams of the stem samples were surface sterilized by washing in sterilized distilled water, soaked in 70% ethanol for 1 min, 5.25% sodium hypochlorite for 5 min, 70% ethanol for 1 min, and finally washed in sterilized distilled water three times.The surfacesterilized samples were crushed in a sterilized mortar, and the plant extract was centrifuged at 3000 g for 10 min.One ml of the supernatant was serially diluted up to 10 −3 and 0.1 ml of the suspension was then spread onto the agar medium as described before [7].The microbial isolates obtained were then purified and stored as described before.The endophytic microbes isolated were then characterized based on their morphological characteristics.

Cross-sectional observation of trunk tissues
Based on the observation result, the transverse section of the oil palm stems, showing internal symptoms of G. boninense.Ganoderma formed irregular growth, and the boundary between the colonized or decayed stem tissue and fresh stem tissue is a contrasting dark coloured area, dry rot lesions of both the stem and bole, occasionally with visible mycelial growth and narrow, dark bands of tissue between the light-brown lesions (Figure 2).The rotten part emits turbid-coloured liquid and smells.A study performed the analysis of mycelium morphology and somatic incompatibility study [8].The research showed that every single individual morphological characteristic varied in the colony's shape.The shape could be irregular with concentric rings, sub-ungulate, or flabellate.The color ranged from white to yellowish brown.

Isolation of endophytes
In the first methods of sample isolation, the Azotobacter was successfully isolated from the samples.Isolates were only found on the middle and top of the part stems, and decreased in number in areas near the occurrence of colonization of Ganoderma (pseudosclerotium).The production of extracellular enzymes by endophytes may imply a resistance strategy of the host plant against pathogenic microorganisms, and in another side to improve plant nutritional status [4].The characteristics colony of Azotobacter shows white-mild yellow in colour with the slime on the colony surface.It also shows the clear zone surrounding the isolate.Moreover, the pH of culture media from oil palm stem revealed lower pH which are 5.77 (sample of the top area of the stem), 5.96 (sample of the middle area of the stem), and 5,81 (sample of the bottom area of the stem), comparing to the control isolate which is the culture media without bacteria has pH 6.88.This data leads to the hypothesis that the Azotobacter sp.activity affects the culture media acidity.Also, for the temperature, ideally, the Azotobacter spp were grown in a range of 30-34 0 C. Azotobacter sp is well known a bacteria that could produce hormones such as IAA.[5].The results of exploration on sweet potato plants obtained 11 different strains belonging to the genus Enterobacter, Rahnella, Rhodanobacter, Pseudomonas, Stenotrophomonas, Xanthomonas and Phyllobacterium, these microbes play a role in increasing plant growth for example because of their ability to produce IAA and are able to fix N2 from the atmosphere [6].Although the obtained results also revealed the ability of these endophytes to produce IAA in the absence of tryptophan i.e. the rate ofIAA production was related to the increase of tryptophan concentration.Further analysis for the antagonism test between Azotobacter sp. and Ganoderma sp. was done.The data shows that the Azotobacter isolate does not inhibit the Ganoderma's growth.Thus, Azotobacter sp.only fixing N2, but not inhibit Ganoderma sp.growth.[9].Bacteria that are able to dissolve phosphate could be isolated from both preparation methods.However, the ability to grow and solubilized the phosphate is generally very slow.In addition, the ability of the bacteria to solubilise P in media were disappeared after subculture.Pseudomonas can be collected from both methods in a high population.The higher number of Pseudomonas bacteriain the infected tissue of Ganoderma sp.possibly needs further analysis to determine the function of these bacteria, especially in plant defence against Ganoderma sp.
Though these fungal endophytes have various biological and biochemical properties potentially useful.The bacterial endophytic microbiome promotes plant growth and health and beneficial effects are in many cases mediated and characterized by metabolic interactions.These fungal isolates might behad a considerable impact on plant growth parameters including root elongation as a result of ammonia and IAA production.[10,11] showed that a nonsporulating strain of fungal endophyte which failed to sporulate was described as sterile hyphae.This is a common problem concerning the identification of fungal endophytes.We have just started to explore the endophytic microbe in the oil palm trunk infected by Ganoderma sp.However, further research is needed to more efficiently make use of beneficial plantmicrobe interactions and to reduce pathogen infestation as well as antimicrobial activity of endophytes against Ganoderma sp. as the results of plant-microbe interactions, imply a resistance strategy of the host plant against pathogenic microorganisms and improve plant nutritional status.On the other side, it could be revealed the novel bioactive substances produced by the microbes for commercial interest.

Conclusion
P-solubilizing bacteria, Pseudomonas can be isolated using methods 1 and 2 while Azotobacter only by method 1.The population of Azotobacter and phosphate solubilizing bacteria decreased in the area near pseudosclerotium while Pseudomonas was found in all parts of the stem even in the area near to the pseudosclerotium.

Figure 1 .
Figure 1.Sample collection of oil palm stem in the PPBBI area.Stem position in the field (A), activities of oil palm sample collection (B, C, and D)

Figure 2 .
Figure 2. The oil palm section stem which are used for the research: A) the bottom, B) the middle, C) the top part