Characterization of rhizosphere fungi of the ebony plant (Diospyros celebica Bakh.) which has the potential to produce Indole-3-Acetic Acid and Gibberelic Acid Phytohormones

Ebony (Diospyros celebica Bakh.) is a slow-growing species that is an impacting factor in the extinction of this species. Efforts to reduce the ebony population can be performed by employing microbes to stimulate ebony growth. This study aimed to obtain bacterial isolates with the potential of producing IAA and GA3 that can be further utilized as biofertilizer agents. Research stages encompassed soil sample isolation, isolation of bacteria, and analysis of IAA and GA3. The research was in complete randomized design (CRD). Thirteen isolates were successfully purified and analyzed with their IAA and GA3 hormones. The highest IAA concentrations were observed in EP1b at 48 incubation hours (44.39 ppm), EP3d at 72 incubation hours (34.93 ppm), and EP3c at 72 incubation hours (34.48 ppm). EP2c, EP2e, and EP2a produced the highest GA3 concentration at 24 incubation hours which were 3.88 ppm, 3.88 ppm, and 3.81 ppm, respectively.


Introduction
IAA (Indole acetic acid) is the principal hormone that regulates several plant physiological processes, including cell development and division, differentiation of plant tissues, and light and gravity responses.One of the plant organs that are extremely sensitive to IAA concentration is the root.Plants respond to IAA through the mechanisms of primary root elongation, lateral root development, and adventitious root formation [1].There are two groups of IAA hormones: endogenous IAA and exogenous IAA.Gibberellin is one of the growth regulators that affect plant expansion, so it is said that gibberellin's ability to stimulate plant growth is greater than that of auxin when given alone.Gibberellin also plays a role in germination, specifically in the breakdown of dormancy.Although gibberellin is present in seeds, Characterization of rhizosphere fungi of the ebony plant (Diospyros celebica Bakh.) which has the potential to produce Indole-3-Acetic Acid and Gibberelic Acid Phytohormones 1230 (2023) 012078 IOP Publishing doi:10.1088/1755-1315/1230/1/012078 2 exogenous gibberellin must be applied for germination to compensate for the lack of gibberellin in the seeds to be germinated [1].
Eboni (Diospyros celebica Bakh.) is an Indonesian species that grows abundantly on the island of Sulawesi, making it an endemic species with distribution in Central Sulawesi, North Sulawesi, and South Sulawesi [2].Ebony is a slow-growing species with a lengthy growth period.Because the seeds of ebony are recalcitrant, they cannot be stored for an extended period of time [3].In addition, environmental factors are an ecosystem condition for the growth of a plant type, so it can be said that the development of a plant type is strongly influenced by the environment in which it grows, including soil.
It has never been reported that rhizosphere fungi that are capable of producing plant growthpromoting compounds such as IAA and GA from ebony plants have been investigated.Consequently, this research must be conducted to investigate the rhizosphere fungi of ebony plants that produce IAA and GA hormones as biofertilizer agents, which can be utilized for growth enhancement and efforts to produce high-quality seeds.

Research methods
This study will be conducted between January and June of 2022 at a specific time and location.This study was conducted in two stages, the first of which involved the collection of ebony plant soil samples from the Hasanuddin University Educational Forest in the Maros Regency.The second phase, Isolation and Identification Activities, was conducted at the Biotechnology and Tree Breeding Laboratory and the Integrated Laboratory of the Biology Unit of the Faculty of Forestry at Hasanuddin University in Makassar.

Sampling
In the educational forest of Unhas, Cenrana District, Maros Regency, ebony stands were sampled.Three tests were conducted in the shade of an ebony tree.25 cm deep soil samples are taken from rhizosphere areas.Each sample was collected in all four cardinal directions.Each test was sampled at four locations, which were then combined into a single soil sample.

Making media of Rhizosphere fungi culture
Making PDA (Potato Dextrose Agar) media by weighing up to 39 grams of PDA, 10 grams of glucose, and 10 grams of gelatin.Put all the ingredients in an Erlenmeyer and add 1,000 ml of aquades.The Erlenmeyer is then sealed with aluminum foil and homogenized with a magnetic stirrer hot plate.At a temperature of 121°C, the substrate was placed in the autoclave for 15 minutes.Transferring sterile media to Laminar Air Flow that has been UV-sterilized 90 minutes prior.Turning on the Bunsen, heating the petri dish on top of the Bunsen, pouring warm media into the dish, and then closing it to prevent contamination, after the solidifying media has been glued together with plastic wrap.

Recurrence and rejuvenation of Rhizosphere fungi
In order to revitalize fungal isolates, instruments and materials must be prepared.Spray your hands with alcohol to sterilize them, then open the petri dish containing the isolates and heat the Petri dish's lip.Utilize a cork borer that has been treated with a burn-dying technique to puree a colony of fungi.The media agar contained within the fungus should be scraped into the PDA media.Reheat the Petri dish's lip, then close and glue it with plastic wrap to prevent the occurrence of breast milk-containing bacteria.The fungus was observed for 7 days.

IAA concentration measurement
IAA concentration was determined by qualitatively testing IAA-producing fungi with Salkowski reagents using the colorimetric method.Isolates that pass qualitative tests are then subjected to quantitative analysis using the spectrophotometry technique.Three pieces of cork borer were added to 45 mL of PDB medium to which L-tryptophan had been added, and the cells were then incubated.The culture was incubated for seven days and shaken at 150 revolutions per minute.A total of 4 mL of culture was removed aseptically and placed in an Eppendorf tube before being centrifuged at 10,000 rpm for 15 minutes to separate the filtrate from the supernatant.Take a total of 0.5 mL of the g supernatant, add 1 mL of Salkowski reagent, and incubate in a dark room for 30 minutes.After incubation, the absorbance of the supernatant was measured using a UV-Vis Spectrophotometer with a 520 nm wavelength, and the IAA concentration was calculated using the IAA standard curve equation.The standard curve of IAA concentration in the PDB medium is derived from the correlation between variations in IAA concentration and absorbances measured with a spectrophotometer at 520 nm.There were three repetitions of sampling at 0, 24, 48, and 72 hours of incubation [4,5].

GA3 concentration measurement
On GA3 hormone-producing hormones, Gibberellic Acid (GA) measurements were conducted.Using a cork borer, up to four pieces of fungus isolates grown on PDA media were transferred to PDB media, incubated for seven days, and shaken.The culture is then harvested, centrifuged at 8000 rpm for 10 minutes, transferred 15 ml into a test tube, and 2 ml of Zinc acetate solution is added.After 2 minutes, 2 ml of a solution of potassium ferrocyanide centrifuged for 10 minutes at 10,000 rpm is added.The supernatant is diluted with 5 ml of hydrochloric acid at a concentration of 30%.The absorbance was measured using a spectrophotometer at a wavelength of 254 nm, with blanks containing 5% hydrochloric acid.The absorbance was measured [6].Samples were collected at 0, 24, 48, and 72 hours of incubation with three repetitions of incubation [4,5].

Variable research
This study employs qualitative and quantitative approaches as variables.A qualitative approach is characterized by observing the color of the fungus colony, the diameter of the fungus colony, the texture of the fungus colony, the surface of the fungus colony, and the change in the color of the sample to pink, which indicates that the isolates produce IAA and GA, whereas a quantitative approach measures the absorbance of isolates using a spectrophotometer to determine the concentration of IAA and GA produced.

Data analysis
Using Microsoft Excel and regressive equations, the identification of rhizosphere fungi is determined quantitatively by examining the absorbance value of IAA and GA3 concentrations.Upon comparison with the absorbance of the IAA standard solution, the IAA concentration is calculated.With the equation Y=0.064x+0.009[4], the Concentration of GA3 is measured by using the regression equation made from the serial dilution of the stock solution GA Y= 0.888x+0.441[7] to obtain the concentration value, where Y= Absorbance Value (nm) and X= Concentration (ppm).After obtaining the IAA and GA3 absorbance values in the RAL (Complete Randomized Design) analysis, the Duncan test was conducted to determine if the treatment had a real effect [8].

Isolation of Rhizosphere fungi
13 bacterial isolates were obtained by isolating bacteria from ebony rhizosphere soil samples in the educational forest of Unhas, Maros Regency, Cenrana District, South Sulawesi Province.The activities of tillage, fertilization, and liming can cause alterations in the rhizosphere environment of plants, particularly differences in microbial populations.In addition, it affects not only the number of microorganisms in the soil but also their diversity [9].The IAA production test employs a spectrophotometer for quantitative analysis (Figure 1.).Based on quantitative test results, EP1b, EP3d, and EP3c isolates from ebony produced the highest IAA successively at 44.39 ppm, with the highest results obtained at incubation times of 48 hours, 34.93 ppm and 34.48 ppm at 72-hour incubation time as shown in the following chart: Based on the results of isolation and purification research conducted on soil samples taken from around the roots of ebony plants in the Hasanuddin University Education Forest, the Maros Regency obtained ten isolates, which were then tested for IAA and GA hormones, and the five isolates with the highest concentrations of IAA and GA were obtained with distinct morphological features.According to Wulandari et al (2013), biotic and abiotic environmental factors influence microbial growth [10].The availability of nutrients necessary for their survival is one of the environmental factors that affect the growth of microorganisms (particularly fungi).The number of microbial populations residing in the soil can be used to predict its burial.Because fungi function as an ionizer of organic compounds into nutrients available to plants, a high number of fungi indicates a high level of soil fertility and indicates that the soil contains sufficient organic matter and other compounds for the growth of fungi.Soil moisture influences soil aeration, temperature, and chemical reaction.

Morphological characteristics of fungi
The results of macroscopic observations, such as colony color, texture, and diameter of the fungus colony, can be seen in table 1.The morphological characteristics of all fungal isolates differ from one isolate to the next.This is evident from the predominantly white, yellow, and brown hues of the upper and lower colonies.The ten observed fungal isolates had a velvety, cotton-like texture.This is consistent with the findings of Astuti (2017),who successfully identified microbial characteristics in fungal isolates from ebony plants in Barru Regency with velvet and fine cotton-dominated texture [12].
3.3.IAA and GA production capability test 3.3.1.IAA production.The results of testing the production capability of IAA on 10 isolates of fungi isolated from ebony revealed discoloration, although not statistically significant, in each isolate.This indicates that the IAA hormone is produced at relatively low levels.The majority of isolates did not experience a significant change in concentration during the incubation periods of 0, 24, 48, and 72 hours, as determined by absorbance measurements utilizing a spectrophotometer for all tested isolates.At the IAA phytohormone production stage, incubation is conducted in the dark to prevent the occurrence of IAA degradation by fungi caused by high light intensity [13].
The discoloration of fungal isolates is grown on liquid PDB media, and then Salkowski reagents are added.Indicative of an indole reaction by Salkowski's solution, the concentration of IAA formed is indicated by a change from yellow to pink in the color of the supernatant solution following the reaction with the Salkowski reagent.According to Restu et al (2019), the reddish color change in isolates after dripping the Salkowski reagent is caused by a reaction between the Salkowski reagent and IAA or IAAforming compounds [14].Indole-3-acetic acid (IAA) binds to FeCl3 and HClO4, the constituent compounds of the Salkoswski reagent, to form a tris-(indole-3-Aceto) iron (III) complex, which gives the reagent its red color.Obtaining isolates of the IAA-producing rhizosphere fungi benefits plant development and makes the soil fertile, which has a positive effect on plant development.4.17  5.40  Description: Numbers followed by different letters show a significant difference based on the mrtD test at a confidence level of 5%.
Based on the ANOVA test, two incubation periods with a significant effect were identified, and a subsequent test was conducted using the DMRT (Duncan Multiple Range Test) tests in table 3.According to additional tests, DMRT 5% isolated three fungi with the capacity to produce IAA hormones, namely EB 3, EB 4, and EB 10.IAA concentrations averaged 6.44 ppm, indicating that EB 3 isolates with a 24-hour incubation period had the greatest impact on IAA levels.EB 10 has the lowest value, with an average IAA production of 4.17 ppm over the same 24-hour incubation period.The production of the IAA hormone is significantly affected by the fungus isolates, L-tryptophan concentration, growth rate, and length of incubation.In addition to the factors above the IAA concentration, environmental factors or the conditions of each sampling location, such as carbon source, pH, and dissolved oxygen concentration, also influence the IAA concentration [15].In general, the concentration of IAA in fungal cultures grown on media supplemented with L-tryptophan is higher than in cultures grown on media without L-tryptophan [16].
The measurements of IAA hormone results by ebony rhizosphere fungi revealed that the average yield capacity increased, albeit not significantly, in all isolates.After 72 hours of incubation, more IAA was produced by each isolate than after 24 hours.This research is consistent with that of [17], who found that 72 hours of incubation produced the highest concentration of h-acyl, which was significantly higher than 24 hours.According to Retnowati et al. (2002),IAA production is directly proportional to the length of the incubation period; that is, the longer the incubation period, the greater the increase in IAA production [18].This is influenced by nutrient-sufficiency factors during the growth phase of the fungus and the presence of pure L-tryptophan as a precursor for IAA synthesis.
The IAA growth hormone results test revealed a phenomenon in the EB 3 isolate in which a fungus isolate was able to produce the highest hormone among other isolates after 24 hours of incubation, but its production decreased after 72 hours because the isolate used the IAA hormone it produced to metabolize.There are EB 8 isolates early on in the incubation of high nutrient sources so that IAA production is high and continues to rise until the end of the incubation, although not significantly.The highest IAA production by rhizosphere ebony fungi occurred after 48 hours of incubation and decreased after 72 hours.This is consistent with the research Retnowati et al (2002) indicating that the highest IAA production is achieved after 48 hours of incubation [18].During the 48-hour incubation period, the fungus typically enters its final stationary phase, resulting in a very high IAA concentration.This is influenced by the abundance and activity of enzymes used in the bioconversion of L-tryptophan to IAA, such as L-tryptophan monooxygenase, IAM hydrolase, indol-pyruvate decarboxylase, and IAA1d dehydrogenase.Almost all isolates of IAA production are still low after 24 hours of incubation.This is because, during the incubation period, the fungus is still in the lag phase (adaptation), which is the phase of adjusting the activity of fungi to a new environment and can last from a few minutes to several hours, and also because the enzymes that convert L-tryptophan into IAA are still low.
There is a phenomenon in EB isolates 1, 2, and 5 that, after 48 and 72 hours of incubation, demonstrates a stationary phase, i.e., the growth of the fungus is relatively stable.This is due to the equilibrium between cell division and cell death [19].In EB isolates 3, 6, 7, 9, and 10 with 72-hour incubation periods, the fungus enters the final phase of its growth, the death phase, and IAA production decreases.IAA production decreases at 72 hours due to the release of IAA-degrading enzymes, including oxidase and peroxidase [18].

GA productions.
Compared to the control, the color of the test results for each fungus isolate changed.The change in color indicates that the rhizosphere fungus isolates of the ebony plant are able to produce the GA hormone.In tests for GA hormones, three isolates with the potential to produce GA hormones were identified: EB 2, EB 8, and EB 10.Based on the ANOVA test, four incubation periods with a significant effect were identified: 0 hours, 24 hours, 48 hours, and 72 hours.Additional tests were conducted using the DMRT (Duncan Multiple Range Test) tests in table 4. According to additional tests of DMRT 5%, there are two isolates with a very good effect, namely EB 2 with a concentration of 4.54 ppm in the incubation period of 0 hours and EB 8 with a concentration of 5.19 ppm in the incubation period of 48 hours, while EB 8 isolates have the lowest concentration value with a concentration of 3.98 ppm in the incubation period of 0 hours.Abbas (2022) tested 16 rhizosphere fungal isolates from red jabon stands with GA concentrations ranging from 2.48 to 4.19 ppm [20].The range of 3.98 to 5.19 ppm for the GA concentration of rhizosphere fungus isolates is somewhat higher than the range of 2.48 to 4.19 ppm.The concentration results were significantly higher than those of Nurdiyanti (2019) study, which examined 27 fungal isolates from the rhizosphere of mahogany stands in two locations [21].Takalar's location of sixteen rhizosphere isolates with GA concentrations between 2.41 ppm and 3.08 ppm.Isolates of Maros 11 with an average GA concentration between 3.29 and 3.93 ppm.According to Imawan (2019) research, the concentrations produced by 14 rhizosphere fungal isolates of pecan stands ranged from 2.88 to 5.08 ppm, indicating that the concentrations produced were comparable to the results of this study [22].This demonstrates that the obtained fungus isolates are classified as being able to produce high levels of GA hormones, making them highly beneficial for plant growth and physiological processes.
Gibberellin is one of the essential plant growth hormones.The isolates of fungi discovered on ebony plants in the county of Maros produced relatively high concentrations.Therefore, it is possible to propagate isolates with the highest GA concentration, which can be utilized as biofertilizers in the future.Biofertilizers, which are formulations of living microorganisms and may be applied to plant nurseries, plant surfaces, or soil, form colonies in the rhizosphere, promote plant growth, and increase the availability of soil nutrients.Thus, if successful, the use of chemical fertilizers can be reduced and replaced with environmentally friendly and sustainable fertilization technology in order to improve plant growth.

Identification of Rhizosphere fungi isolates
Isolate identification is performed microscopically by observing vegetative structures (hyphae) and generative structures (spores), which are then matched with the key book of fungal determination "Pictorial Atlas of Soil and Seed Fungi" [23] and other literature such as journals and theses that discuss the identification of fungi, so that the genus can be determined from the results of the identification.The outcomes of the observations are shown in Table 4.These 5 isolates have the potential to produce IAA and GA hormones, and they have been successfully identified to the level of genera, including Aspergillus, Trichoderma, and Penicillium, with Penicillium being the dominant genus based on the results of the identification.According to Nurdiyanti (2019)research, the results of rhizosphere fungus identification in mahogany stands in each provenance are distinct [21].In Maros Regency, the genus Trichoderma and Rhizopus were identified in as many as 4 isolates, whereas in Takalar Regency, Aspergillus was identified in as many as 7 isolates and Penicillium in as many as 5 isolates.
Aspergillus is cosmopolitan, its spores are very small and light, and they are easily dispersed through the air; therefore, it plays a significant role in polluting organic materials [24].The isolates of this fungus, according to macroscopic observations, are white with yellow edges and have a cotton-like texture.Observations under a microscope reveal that this fungus has a large conidia head that is round and dark brown in color, as well as an upright, long, unbranched conidiophore.This is consistent with the findings of Syaifuddin (2017) research, which indicates that the upper conidia of Aspergillus appear black or brownish-black under the microscope [25].The top is augmented.The smooth conidiophores are either colorless or yellow-brown in hue.The vesicles on the upper portion are enlarged and rod-like at the end.Conidia are round and dense.Table 4 presents the results of macroscopic observations of the Genus Aspergillus.
Trichoderma is a beneficial saprophytic soil microorganism that naturally attacks pathogenic fungi.Trichoderma fungus is a type of fungus that can be used as a biological agent to control soil pathogens.Trichoderma fungus is widespread in almost all soil types and habitats [26].This fungus colony has a green surface and a cotton-like texture, as observed macroscopically in the Trichoderma genus (Figure 4).These isolates belong to a genus with erect, branched conidiophores and thin-walled conidia.The obtained identification results are consistent with those discovered by Gusnawaty e al. (2018), in which the conidiophore branches, specifically the lower portion of the lateral branch, repeat, whereas the more distal branches, become shorter.After reaching adulthood, the mycelium of Koloni is yellowish-green or dark green, especially in the portion that indicates a high concentration of conidia [26].Table 4 displays the results of macroscopic observations of Trichoderma species.
Penicillium is a soil-dwelling fungus that grows rapidly and is antagonistic to other fungi [27].This fungus can grow between 25 and 45 degrees Celsius.These fungus isolates were white and had a velvety texture and fine cotton, while microscopic examination revealed conceptual hyphae and branched diophore coni.The results of this observation are consistent with those of Sari (2017), who states that the conidiophores of Penicillium have a distinctive microscopical shape [28].Conidiophores are upright and branched near their ends.Single and double conidiophores of Penicillium are circularly branched and resemble the branching of a bush.Table 4 displays the results of macroscopic observations of the Genus Penicillium.

Conclusion
As many as five isolates of ebony rhizosphere fungi with the ability to produce IAA and GA were obtained from three genera, including Aspergillus, Trichoderma, and Penicillium.The three best IAAproducing isolates were obtained from EB 3, EB 4, and EB 10 after 24 and 48 hours of incubation, while the best GA-producing isolates were obtained from EB 2, EB 8, and EB 10 after 0, 24, 48, and 72 hours of incubation.

Figure 2 .
Figure 2. IAA concentrations produced by isolates of rhizosphere ebony fungi.

7 Figure 3 .
Figure 3.The concentration of GA produced by isolates of rhizosphere fungi ebony.

Table 1 .
Macroscopic characteristics of the growth of isolates of rhizosphere ebony fungi on PDA media during the seven-day incubation period.

Table 4 .
Microscopic characteristics of growth of Ebony Rhizosphere Fungi isolates.