The Influence of Patchouli (Pogostemon cablin Benth.) and Turmeric (Curcuma domestica Val.) essential oils on the growth of Rhizoctonia sp. in vitro

Rhizoctonia sp. is a soil-borne pathogen that causes damping-off in various forestry plant seedlings. Using plant-based pesticides is a disease control solution that is more environmentally friendly than chemical pesticides. Essential oil is a component that can be used as a biopesticide because it contains antifungal properties, such as the compounds in patchouli and turmeric essential oils. This study examines the inhibition of patchouli and turmeric essential oils and their combinations with various concentrations on the growth of the pathogenic fungus Rhizoctonia sp. in vitro. Observations were made using patchouli, turmeric, and a combination of patchouli and turmeric essential oil with concentrations of 1%, 5%, and 10% on PDA (Potato Dextrose Agar) and PDB (Potato Dextrose Broth). The in vitro pathogen inhibition test results showed that the type and concentration of oil in PDA significantly inhibited the growth of Rhizoctonia sp.. In contrast with PDB, only oil concentration significantly inhibited the growth of Rhizoctonia sp.. The combination of patchouli and turmeric essential oils with a concentration of 10% on PDA and turmeric essential oil with a concentration of 10% on PDB showed the highest inhibitory response.


Introduction
Rhizoctonia sp. is a pathogenic fungus that infects various cultivated and forestry plants.It can attack plant roots, prevent root growth, and disrupt nutrient absorption, potentially leading to losses in the forestry and agricultural plantation sectors.According to Istikorini and Sari's [1] research, the severity of Rhizoctonia sp.attacks on 5-month-old sengon seedlings in open fields reaches up to 75%.
Dumping-off symptoms caused by Rhizoctonia sp. have been observed in several forestry plants at the seedling stage, such as calliandra [2], sengon [1], pine [3], acacia [3], teak [4], and spruce [5].Rhizoctonia is classified as a soilborne pathogen, making it capable of causing root rot.Research by Senjaya et al. [6] indicates that Rhizoctonia sp. can naturally persist and survive in the soil while rapidly attacking plants.
Nurseries play a crucial role in supporting the success of Industrial Plantation Forests by providing seedlings adapted to fields.The host range of Rhizoctonia includes commercial plant species frequently used in plantations for timber production.Therefore, controlling Rhizoctonia is essential to prevent losses.Intensive chemical pesticide use for control purposes can negatively impact humans and the environment.Using chemical pesticides may lead to leaching into the soil surface and water bodies, 1315 (2024) 012067 IOP Publishing doi:10.1088/1755-1315/1315/1/012067 2 causing residue.This poses significant dangers when humans consume water from these bodies or serve as a habitat for living [7].Therefore, it is essential to explore alternative approaches for minimizing the reliance on chemical pesticides in the management of Rhizoctonia sp., such as the utilization of botanical pesticides.
One of the botanical products suitable as a pesticide is essential oil.Indonesia, one of the main producers of essential oils, offers easy accessibility to these natural products.According to Negari and Saskara's research [8], Indonesia is a major producer of essential oils, with approximately 15 Indonesian native essential oils being commodities for export, including patchouli oil.Patchouli oil, known for its antifungal properties, can inhibit several types of fungi, including Sclerotium rolfsii [9].Another essential oil with antifungal properties is turmeric oil.Yuwono [10] shows that turmeric essential oil can inhibit the growth of Rhizoctonia sp. at a concentration of 5%.This research aims to examine the inhibitory effect of patchouli and turmeric essential oils, as well as their combinations at different concentrations, on the in vitro growth of Rhizoctonia sp.

Time and location of research
The research took place between March and June 2023 at the Forest Pathology Laboratory within the Department of Silviculture, Faculty of Forestry and Environment, Bogor Agricultural University.

Preparation of culture media.
The culture media used in this research are Potato Dextrose Agar (PDA) and Potato Dextrose Broth (PDB).PDA is prepared by using the following ingredients: 20 g of agar, 20 g of sugar, 200 g of potatoes, antibiotics (chloramphenicol), and 1 L of distilled water.PDB is prepared using the same ingredients and procedure as PDA but without adding agar [11] 2.2.2 Preparation of pathogen isolates.The Rhizoctonia sp.pathogenic strains utilized in this research were acquired from the Forest Pathology Laboratory within the Department of Silviculture, Faculty of Forestry, IPB (Bogor Agricultural University).Subsequently, the Rhizoctonia sp.isolates are propagated by rejuvenating them on PDA.

Preparation of essential oil concentrations.
The essential oils used in this research are patchouli and turmeric.These essential oils are sourced from the Forest Pathology Laboratory, Department of Silviculture, Faculty of Forestry and Environment, Bogor Agricultural University.Both essential oils are from Subang, West Java, with 100% pure important oil content obtained through steam distillation of patchouli leaves and dried turmeric rhizomes.The concentrations of the essential oils used are 1%, 5%, and 10%.The essential oils are used in single and combined compositions, mixed with PDA and PDB.The testing of essential oil mixtures with PDA is conducted to determine Rhizoctonia sp.'s growth and inhibition activity by measuring the diameter.In contrast, the combinations with PDB are used to assess inhibition activity by measuring the biomass of Rhizoctonia sp.The single and combined essential oils are dissolved with Tween 20 at a 1:1 ratio.The solution is then added to sterile water for 10 ml of essential oil.The composition used follows a modified research approach based on Firmansyah and Yuwono [12] and Pratiwi [13] (Table 1 and Table 2

Preparation of Growth Media and Inoculation of Isolates.
The essential oil solutions are adjusted according to their types and concentrations and added to the PDA and PDB growth media.For each petri dish, 9.9 ml of liquid PDA is combined with 1.1 ml of the concentration solution, while for each jar, 29.7 ml of PDB is mixed with 0.3 ml of the concentration solution [12].The essential oil and media mixture is carefully performed using a syringe within a controlled laminar airflow environment.Subsequently, the revitalized isolates are meticulously placed into the media containing the essential oil using a cork borer and spatula; all carried out within the laminar airflow setup.

Observations on PDA.
To monitor the growth of Rhizoctonia sp., daily observations are conducted on the PDA.The vertical and horizontal lengths of the Rhizoctonia sp.mycelium in the Petri dishes are measured at regular intervals of 24 hours.Additionally, the radial diameter growth of Rhizoctonia sp.mycelium is quantified using this formula [14]: Øx = Diameter of axis x Øy = Diameter of axis y

Figure 1. Calculation design of isolate diameter
In addition to observing the growth of diameter, the macroscopic morphology of the pathogen is also examined to determine the impact of essential oils on the morphology of the pathogen.Inhibition of pathogen growth by essential oils on PDA can be assessed by measuring the radial diameter.The formula for calculating the percentage of mycelium inhibition on PDA is as follows [15]: Explanation: dc = diameter of the control colony (cm) dt = diameter of the treatment colony (cm)

Observation of PDB.
Observation of Rhizoctonia sp.isolates on PDB is carried out by weighing the mycelium after a 7-day incubation period.Before consideration, the mycelium is separated using filter paper oven-dried at 60 °C for a period of 24 hours.Subsequently, the mycelium is oven-dried again for 24 hours at 60 °C.Once the oven-drying process is completed, the mycelium is transferred into a desiccator to create a dry and moisture-free environment.It is then weighed to determine its dry weight and the relative inhibition level is calculated accordingly.The formulas for calculating mycelium biomass [14] and relative inhibition [12] are as follows: Relative Inhibition (%) on PDB = B1 -B2 B1 x 100% Explanation: B1 = biomass of the control colony (g) B2 = biomass of the treatment colony (g)

Data Analysis
The data from this study underwent analysis utilizing a completely randomized factorial design with three replications, followed by the application of SAS software and subsequent analysis using analysis of variance (ANOVA).Significant effects observed in the results were subjected to further scrutiny through Duncan's test.The model for the design is formulated as follows [16]:

Inhibition Test of Rhizoctonia sp. on PDA
Observing the pathogen's diameter growth on PDA provides crucial information about the pathogen's response to the growth environment by measuring the isolate diameter on the media.Table 3. Result of the variance analysis for the inhibition of Rhizoctonia sp.

Source of Variation
P-value Essential oil type 0.0067 Concentration 0.0001 Essential oil type and concentration 0.1479 The results of the variance analysis for the inhibition of Rhizoctonia sp. on PDA (Table 3) indicate that the different types and concentrations of essential oils significantly affect the inhibition value.In contrast, their interaction does not have a significant impact.The Duncan test was conducted on the considerable analysis results.Values within a column that share the same letter are not significantly different at the 5% significance level.
The essential oil that provided the highest inhibition is the combination at 54.69%, while the highest inhibitory concentration is 10%, with a percentage of inhibition at 66.05%.The average increase in diameter of Rhizoctonia sp. is presented in Figure 2. The control isolate showed the highest daily growth.The isolate with a combination of 10% essential oil exhibited the lowest increase with a diameter of 1.77 cm, followed by isolates treated with 10% patchouli oil, 10% turmeric oil, 5% combination, 5% turmeric oil, 5% patchouli oil, 1% combination, 1% turmeric oil, and 1% patchouli oil with diameters of 3.32 cm, 3.82 cm, 4.07 cm, 4.18 cm, 5.5 cm, 6.13 cm, 6.42 cm, and 6.9 cm, respectively.The significant difference in growth observed between the control isolates and the treated samples indicates inhibition resulting from the antifungal properties of the essential oils.This finding is consistent with the research conducted by Setyaningrum et al. [17], which demonstrated the effectiveness of patchouli oil in inhibiting the growth of both C. albicans and T. mentagrophytes, highlighting its antifungal properties.Moreover, Darmawan and Anggraeni [18] also showed that turmeric extract could control the growth of Pythium sp.The restriction of fungal hyphal growth by essential oils signifies the inhibitory impact of these oils on fungal hyphae, suggesting potential antifungal properties.The hyphae of the control group showed a broader and longer increase, while the hyphae exposed to crucial oils experienced inhibition in their development (Figure 4).The percentage of growth inhibition rate (Figure 4) is a parameter used to evaluate the inhibitory effect of essential oils on pathogen growth.The result from this research reveals that the essential oil exhibits the highest daily inhibition rate is the 10% combination of essential oils, followed by 10% patchouli oil, 10% turmeric oil, 5% combination, 5% patchouli oil, 5% turmeric oil, 1% combination, 1% turmeric oil, and 1% patchouli oil.This suggests that the higher the essential oil concentration, the greater its inhibitory power on pathogen growth.Essential oils with lower concentrations tend to have less inhibitory effects because the amount of active compounds in the oil is smaller, limiting the available active compounds to interact with the inhibited target.

Inhibition test of Rhizoctonia sp. on PDB
Observing pathogen diameter growth on PDB provides crucial information about the pathogen's response to the growth environment by measuring isolated biomass on the media.The analysis results of the Rhizoctonia sp.inhibition (Table 5) indicate that different essential oils do not significantly affect.In contrast, the concentration of essential oils does influence the inhibition value.The significant results of the analysis were further tested using the Duncan test.The Duncan test result in Table 6 showed that the concentration with the highest inhibitory effect is 10%, followed by 5% and 1%.However, there was no significant difference between the 1% and 5%.Therefore, the recommended concentration of essential oil in the preparation of botanical pesticides based on essential oil is 10%.The higher the essential oil concentration, the greater its inhibitory power [11].Testing fungi on PDB can evaluate the inhibitory effects of essential oils on fungal growth, and biomass measurements can provide information about enzyme activity or metabolic processes occurring in fungi.Lower biomass observed in treated samples compared to the control suggests inhibiting specific enzyme activities or metabolic processes.(Figure 6).The type of essential oil did not significantly affect the inhibition.However, based on Figure 6, the essential oil with the highest inhibition is 10% turmeric, with an inhibition value of 53.40%.Turmeric can act as an antifungal agent.Turmeric's antifungal mechanism involves reducing the quantity of desaturase, leading to a decline in ergosterol and ultimately causing cell death through the generation of reactive oxygen species (ROS) [19].Turmeric comprises an α-phellandrene compound that induces inhibition of fungal mycelium by disrupting the integrity of the fungal cell membrane.Disruption results in the leakage of cellular constituents and potassium ions, an elevation in total lipid content, extracellular pH, and membrane permeability [21].The presence of α-guanine in patchouli oil is an antifungal agent and can damage fungal cell membranes [22].

Conclusion
Patchouli and turmeric essential oils have the potential to inhibit the growth and biomass of Rhizoctonia sp.The concentration level of the two essential oils significantly affects pathogen inhibition.The increase in oil concentration corresponds to a higher level of inhibition in both the growth and biomass of Rhizoctonia sp.In the PDA, the growth of isolates treated with the combination of essential oil at a concentration of 10% gave the highest inhibition response.Testing pathogenic biomass on PDB, 10% turmeric has the highest inhibitory response to pathogens.
(Dry Weight of Filter Paper + Dry Weight of Mycelium) -Dry Weight of Filter Paper εijk Explanation: Yijk = Response of Rhizoctonia sp.inhibition with the combination of the I type of essential oil treatment, the j concentration of essential oil treatment, and the k replication.µ = Overall mean value.αi = Effect of the i level of the essential oil type factor.βj = Effect of the j level of the essential oil concentration factor.(αβ)ij = Interaction effect between the I level of the essential oil type factor and the j level of the essential oil concentration factor.εijk = Effect of the error in the i and j treatment units in the k experiment.

Figure 2 .
Figure 2. The growth rate of Rhizoctonia sp.isolates

Figure 4 .
Figure 4.The inhibition rate of Rhizoctonia sp.isolates

Figure 6 .
Figure 6.Percentage of inhibition in Rhizoctonia sp.biomass

3. 3 .
Microscopic observation of Rhizoctonia sp.hyphae on PDA Inhibition by essential oils results in changes in the morphological form of Rhizoctonia sp..The hyphae of Rhizoctonia sp. in the control without added essential oils have a normal and organized format.The hyphae of Rhizoctonia sp.inhibited by essential oils show deformations with irregularly branched shapes.The changes in the hyphal form of Rhizoctonia sp. after treatment with essential oils indicate a negative effect on the structure and development of the hyphae.Essential oils can affect cell integrity and hyphal membrane, resulting in hyphal growth and form abnormalities.The damage to fungal cells is associated with changes during the cell adhesion process, causing disturbances in membrane integrity and permeability [20].

Table 1 .
). Composition of single essential oil concentrations

Table 2 .
Composition of combined essential oil solution

Table 4 .
The Duncan test results on the effect of various types and concentrations of essential oils on the inhibition of Rhizoctonia sp.

Table 5 .
Result of the variance analysis for the inhibition of Rhizoctonia sp.

Table 6 .
Duncan test results effect of essential oil concentration on the inhibition of Rhizoctonia sp.Values within a column that share the same letter are not significantly different at the 5% significance level.