Characterization of endophytic bacteria of Mindi (Melia azedarach) and Balsa (Ochroma pyramidale) plants and potential inhibition in plant diseases

Plant diseases attack all parts of the plant, such as leaves, stems, and roots. The cause of pest attacks is due to several factors, namely environmental conditions that support pathogens, selected plant conditions that are susceptible to disease, and poor maintenance. Disease attacks on the roots include attacks that are harmful to plants, so it is necessary to have effective control carried out by considering the ecology and economy. One of the techniques for controlling plant-disturbing organisms (OPT) can be carried out by utilizing endophytic microbes. This study aimed to explore the types of endophytic bacteria found in balsa and mindi, which have the potential as biological control agents, as well as the effect of using endophytic bacteria in suppressing the growth of root diseases in vitro. The study found that as many as 20 endophytic bacteria were purified from balsa, and 25 individuals were purified from mindi. The hemolysis test and plant hypersensitivity test results showed seven pure isolates with endophytic potential, namely three isolates from balsa and four from mindi.


Introduction
Plant diseases attack all parts of the plant, such as leaves, stems, and roots.The cause of pest attacks is due to several factors, namely environmental conditions that support pathogens, selected plant conditions that are susceptible to disease, and poor maintenance.Among the disease attacks, a disease that attacks the roots is dangerous because it can cause death.Many root diseases, such as soil-borne diseases, are caused by Fusarium oxysporum, Rhizoctonia solani, Sclerotium rofsii, Phytophthora, Rigidoporus sp.[1,2], as well as root and stem rot by Ganoderma sp.[3].Disease attacks on the roots include attacks that are harmful to plants, so it is necessary to have effective control carried out by considering the ecology and economy.One of the techniques for controlling plant-disturbing organisms (OPT) can be carried out by utilizing endophytic microbes (fungi, bacteria, and actinomycetes).Endophytic bacteria are one of the most widely developed biological control agents because they produce antibiotics and degrading enzymes that can inhibit pathogens in vitro and increase plant resistance to pathogens by inducing plant resistance reactions.
Balsa (Ochroma pyramidale) is a fast-growing species that is often used as an indicator of forest regeneration, has a dependence on arbuscular mycorrhizal fungi (FMA) and endophytic microbes, and can increase nutrient uptake [4,5].In addition, the mindi plant (Melia azedarach Linn.) is also a fastgrowing species, and its leaves can be used as a natural biopesticide.The benefits of mindi leaves are such as being able to inhibit attacks by Aspergillus sp. and cabbage liver caterpillar pests and can inhibit gall rust disease in sengon seedlings [6,7].The content of balsa and mindi can be utilized by isolating endophytic bacteria from plant parts, especially roots, which have a high diversity of microbes.Endophytic bacteria in balsa and mindi roots can be tested on Fusarium sp. and Ganoderma sp.Therefore, this study aimed to explore the types of endophytic bacteria found in balsa and mindi, which 1315 (2024) 012033 IOP Publishing doi:10.1088/1755-1315/1315/1/012033 2 have the potential as biological control agents, as well as the effect of using endophytic bacteria in suppressing the growth of root diseases in vitro.

Method
The research was conducted at the Plant Nematology Laboratory, Department of Plant Protection, and Pathology Laboratory, Department of Silviculture, IPB University.The research was conducted from May to December 2023.The tools used for this research were laminar air flow, autoclave, pan, knife, stirrer, measuring cup, Erlenmeyer flask, toothpick, spatula, tweezers, beaker glass, petri dish, micropipette, spreader, mortar, tip, bunsen, analytical balance, tube rack, test tube, cutter, loop needle, syringe, black cloth, cryogenic vial, container, plastic wrap, aluminum foil, microscope, and slide glass.The materials used in this study were potato, dextrose, agar, distilled water, chloramphenicol, powdered NA media, cotton, alcohol, agar, mindi root, balsa root, hypochlorite solution, sterile water, tissue, KOH fluid, blood medium, media planting material, filter paper, and endophytic bacteria isolates.
2.1 Research Procedure 2.1.1.Preparation of media and disease isolates.Making 1 liter of tryptic soy agar (TSA) media requires 30 grams of instant powder, 20 grams of nutrient agar (NA), and 39 grams of potato dextrose agar (PDA) media for each 1 liter of distilled water in the three media.The media was put into an Erlenmeyer flask and covered with cotton.Sterilization was continued on all three medium (TSA, NA, and PDA) in an autoclave at 121°C at 1 atm pressure for 15 minutes.Fusarium sp. and Ganoderma sp.isolates were obtained from the collection of the Forest Pathology Laboratory of IPB and PT.Nusantara Plantation Research (RPN) Bogor City.

Endophytic bacteria isolation.
Endophytic bacteria isolation was obtained from the roots of mindi and balsa forestry plants.As much as 1 gram of mindi and balsa roots were washed and sterilized with 70% alcohol, 1% NaOCl, and sterile water three times.The sterile roots were macerated until smooth with the addition of 1:10 water.0.1 mL of the suspension was isolated on NA medium and observed at room temperature for 24 hours.Bacteria that grow with different colors, shapes, and surface characteristics are purified on new media in each dish.

Data Analysis 2.2.1. Biosafety test.
Hemolysis Activity Test.This test was carried out using isolates of endophytic bacteria that were streaked on the blood of mammals and incubated for 24 hours at room temperature.The isolation results obtained if they do not show any changes in the color of the media or hemolysis zone will be used in the next test because they are safe for mammals [8].
Hypersensitivity Reaction Test.This test was carried out to detect the pathogenicity of endophytic bacteria.The method used was the Klement and Goodman (1967) method by growing endophytic bacteria in 3 ml of tobacco leaves for 48 hours.Plants that do not produce spotting symptoms are used for testing [8].

In vitro root disease inhibition test with endophytic bacteria.
This test aims to determine endophytic bacteria capable of inhibiting the growth of root diseases, namely Fusarium sp. and Ganoderma sp.Testing with the double culture method with a total of 63 isolates.The design used was 2-factor RAL, with the factors being types of endophytic bacteria and types of pathogens.The endophytic bacteria to be tested were three isolates on balsa roots (A2, B1, F1) and four isolates on mindi roots (02, 07, 09, & 14).Each treatment was repeated three times to reduce the bias in the resulting value.
The growth of pathogens towards and against the bacteria was observed every day until the seventh day.The formula calculates the inhibition percentage [9].In vitro growth test of root disease colony diameter on PDA media and TSA media tested for inhibition with endophytic bacteria with a factorial RAL pattern according to [10], the model used is as follows: Information: Yij : The response to inhibition of root disease that received a combination of treatments given the ith endophytic bacteria, the j-th spacing, and the k-replication µ : General mean value ai : Effect of endophytic bacteria from level i ßj : The influence of the factors giving the disease level j (aß)ij : Effect of interaction between administration of endophytic bacteria from level I and disease administration from level j Eij : The effect of the ith and jth treatment errors on the kth experimental unit

Characterization endophyte bactery
Isolation of endophytic bacteria from both plants, namely balsa (O.pyramidale) and mindi (M.azedarch), was carried out on the roots, which are the tissues that become a source of absorption of nutrients in the soil.Roots are the main route for the entry of endophytic bacteria into plant tissues apart from stems, leaves, flowers, and cotyledons.Endophytic bacteria in plants are not found with certainty but can be seen from isolation using agar media [13].The media used to isolate bacteria is nutrient agar (NA) media rich in yeast extract, peptone, NaCl, and agar.NA media is good to use because it has a similar composition to plant conditions.Pure cultures resulting from biosafety tests were cultured on tryptic soy agar (TSA) media.TSA media is a nutrient-rich medium containing peptones derived from casein, soy meal, agar, and sodium chloride [11].TSA media can isolate many plant endophytic bacteria [12].
The isolated balsa and mindi roots produced 45 colonies which were successfully purified.Twenty balsa root isolates and 25 mindi colonies were grown on NA media.In general, more isolates were obtained from the mindi root than the balsa root.Each colony that is the same in one root is grouped based on the shape of the bacterial colony, the surface, and the color of the colony.Bacterial colonies in balsa were 11 out of 20, and mindi had 12 out of 25 different colonies.Bacterial colonies have different shape conditions, while mindi bacterial colonies have a variety of quite different colors.The isolation results are purified and grouped in one cup based on the same characteristics to facilitate selection.Bacterial isolates in balsa were grouped based on shape because they were more diverse, while bacterial isolates in mindi were grouped based on color because they differed more significantly.The characteristics of bacterial isolates seen from the shape and size of the bacterial colonies, color, surface, edges, and condition of the endophytic bacterial colonies [12].
A total of 11 colonies obtained on balsa root were tested for biosafety by dialysis tests on blood media and hypersensitivity tests on tobacco leaves.The results of the biosafety test showed that three colonies (A2, B1, and F1) were safe for mammals and plants.Bacterial colonies on mindi roots were tested for biosafety 12 colonies resulting in 4 colonies (02, 07, 09, 14) safe for plants and mammals.Endophytic bacteria found in balsa and mindi roots were seven colonies with different characteristics, as shown in Table 1.

In vitro disease inhibition
The results of the Ganoderma sp. and Fusarium sp.disease inhibition test by the seven obtained endophytic bacteria can be seen in Figure 1.
The endophytic bacteria obtained were tested against the pathogen Fusarium sp. and Ganoderma sp., showing a significant natural effect.Endophytic bacteria show inhibition of pathogens which can be seen in Figure 2. The mechanism of suppressing pathogen growth includes antibiosis.The mechanism of antibiosis is the inhibition of pathogens by metabolic compounds produced by biological agents such as enzymes, volatile compounds, and other antibiotic compounds [13].The results of the observations can be seen in Figure 2.There is a clear zone between the pathogenic and endophytic bacterial colonies that are visible (GB1, GF1, FA2, and FF1).This inhibition is also evidenced in the inhibition graph (Figure 1), which shows inhibited pathogen colonies on the first day compared to controls.Pathogen inhibition produced significant values on the first day and remained stagnant until the end of the observation [14].Based on the morphological inhibition test results, it can be seen in Figure 1 that all the isolates obtained inhibited the pathogen from the first day and remained stagnant until the seventh day.However, isolates FB1 and F02 showed significantly lower values from the third day.Pathogen inhibition test calculated with SPSS 27 showed that bacteria inhibition has a significant effect on pathogens.This can be seen in Table 2. Based on the data analysis, it was found that endophytic bacteria could inhibit the pathogen Fusarium sp. and Ganoderma sp.Pathogen Fusarium sp. is the cause of fusarium wilt in some plants.Fusarium sp. is a genus of fungi that cause disease in many plants.Fusarium wilt disease usually enters through root wounds and develops in plant tissues [15].Endophytic bacteria testing on F07, F09, and F14 resulted in high inhibition of Fusarium sp., so it can be a recommendation to reduce higher attacks.In addition, in GA2, the pathogen inhibition test of Ganoderma sp.produces the highest inhibition and can be used as a recommendation for disease inhibition.Ganoderma sp. is a pathogen that causes root and stem rot [16][17].This disease is dangerous because it can damage and even cause death to the host.Endophytic bacteria A2, which can inhibit well in vitro, can be a potential for controlling root rot and stem rot diseases.

Conclusion
Endophytic bacteria were produced on seven isolates of balsa and mindi roots.Characterization of bacteria can be determined based on the shape and size of the bacterial colonies, color, surface, edges, and conditions of the endophytic bacterial colonies.The endophytic bacteria inhibition test produced a value that had a significant effect.

Figure 2 .
Figure 2. Disease inhibition test of Fusarium sp.(F), Ganoderma sp.(G), and Control (K) on the seventh day

Table 1
Characteristics of endophytic bacterial colonies on balsa and mindi

Table 2
Inhibition of 7 selected endophytic bacterial isolates from balsa and mindi plants against the pathogen Fusarium sp. and Ganoderma sp.Numbers in the same column followed by the same letter show no significant difference based on Duncan's test at the 5% level.