Identification and effectiveness of biological agents against pathogens causes of white root fungus disease Rigidoporus sp. in vitro

Diseases affect the process of plant growth. One of the pathogens that causes white root fungal disease, Rigidoporus sp., can be transmitted through root contact. This study aims to determine the types of biological agents and their effectiveness in inhibiting Rigidoporus sp. in vitro. Identification is carried out macroscopically and microscopically. Inhibition testing was conducted on Potato Dextrose Agar (PDA) media, followed by a variance test with a One-Factor Complete Random Design. The results showed that Trichoderma sp. with a rejuvenation age of 8 days (T8) was the most effective in inhibiting the growth of Rigidoporus sp. and had the highest percentage at 71%.


Introduction
The problem often found in the forest is the attack of pathogens, which can affect the health condition of the woods.Pathogen have an important influence on a tree's growth and production process.Pathogens in forests usually attack plants, ranging from leaves and roots to all parts of the plant.Pathogens that attack plants are generally caused by fungi, one of which is Rigidoporus sp., often known as disease-causing pathogens white root fungal (WRF) disease.
Rigidoporus sp. is a polyphage fungus that can attack various plants.Rigidoporus sp.It is a soil infectious pathogen.That is, most of its life cycle is in the soil and can infect the roots or base of the stem so that it can cause infection and death for plants [1].So controlling it requires quite expensive costs.White root fungus can attack young plants that are already producing or productive.Cathrina [2] shows JAP disease pathogens can attack plants ranging from nurseries to adult plants.Symptoms of diseases caused by the pathogen Rigidoporus sp. are the formation of white rhizomorphs that spread along the roots.The mycelia of such rhizomorphs attach to the roots.Plant roots infected with JAP pathogens show the presence of fungal mycelia in the form of white threads.Affected plants show symptoms of dull leaves, yellowing, and falling off easily.Heavy attacks cause root rot so that the plant falls easily.Transmission of WRF is generally through contact of healthy plant roots with diseased plants [6].Some WRF control techniques are mechanical, technical culture, chemistry, biological or biological control.Automatic control is by removing dikes wood stamp or woody plant tissue to eliminate the source of infection.Technological and cultural management is carried out by planting legume-type cover crops.A common chemical control for WRF disease is to is using pesticide sprinkled on the soil around plant roots.Chemical control over a relatively long period can cause adverse side effects on the environment and human health.
Biological control of WRF has better prospects.It has advantages compared to chemical control, in addition to not having harmful side effects on the environment, and is also effective if used for a long time.This biological control uses biological agents antagonistic to disease-causing pathogens that are beneficial because of their strong negative properties in inhibiting the growth of pathogenic fungi.This study was conducted to determine the name of a biological agent and its effectiveness in inhibiting the growth of pathogens in vitro.

Time and location of research
This research was conducted from March to June 2023 at the Forest Pathology Laboratory, Department of Silviculture, Faculty of Forestry and Environment, Bogor Agricultural University.

Procedure 2.2.1 Identification of biological agents
This identification activity was carried out macroscopically and microscopically.Macroscopic Identification was carried out by observing the color and structure of the hyphae.The antagonistic fungus used in this study is the result of field exploration.The location for antagonistic fungi exploration is soil from guava tree roots in the Bogor Agricultural University Gymnasium.The technique used in investigating this antagonistic fungus is use the baiting method.The media used for bait is rice media.The soil taken from the roots is placed in a container, then rice is placed on it to cover all parts of the ground, and its growth is observed for seven days.Microscopic Identification was carried out using the Riddle method.Making Riddle preparations is done by placing isolates on the practices and covering them with a cover glass, previously soaked in alcohol.This immersion is done to prevent contamination from the outside, which can inhibit the growth of hyphae on the preparations.The preparations were placed in a petri dish and then given cotton moistened with distilled water, and the petri word was closed again.According to Istikhorini [4], the manufacture of Riddle preparations aims to observe the microscopic structure of the isolate as a whole.Identification activities using the help of a light microscope with a magnification of 10 x 40.

Culture Media Production
The medium used in this research is Potato Dextrose Agar (PDA).Making PDA media for 1 L of media uses 200 grams of potatoes.Peel the potato skin, wash it thoroughly, then cut it into small cubes.After the potatoes are diced, potatoes are boiled with 1 liter of distilled water until soft.The results of the boiled potatoes were filtered and then separated between the potatoes and the boiled water extract.Potato extract was measured to reach 1 L. The extraction that had been measured and added with distilled water was then added with 20 grams of agar powder and 20 grams of sugar, then mixed with a chloramphenicol capsule and stirred.Then, it was transferred into an Erlenmeyer and sterilized.

Isolation
The first step of this process is making a pure culture of Rigidoporus sp. by finding a location for sampling Rigidoporus sp.The sample that has been taken is then cut off with a knife or cutter that has been sterilized using 70% alcohol.Pieces of dried plant samples were transferred to a petri dish containing PDA and then incubated at room temperature until the fungus colonies filled the petri dish.The fungi on the media were observed and purified by transferring them to another Petri container containing PDA media until only one type of fungus was found in each petri dish.Isolates that had been pure were measured for growth in diameter every day until the diameter of the fungus-filled cup.

Test the inhibition of biological agents and disease-causing pathogens
A pathogen inhibition test was conducted to determine the interaction between Trichoderma sp. and Rigidoporus sp.Inhibition testing was carried out in a Laminar Air Flow so that the aseptic conditions were maintained and carried out using the dual culture method in vitro.Trichoderma sp.mushroom colonies.Furthermore the pathogenic fungus Rigidoporus sp. was transfer with a cork borer.The fungi on the media were observed and purified by transferring them to another petri dish containing PDA until only one type of fungus was found in each petri word.Isolates were measured for growth in diameter every day until the fungus filled four borers into culture plates with a diameter of 0.5 cm.Both colonies were taken and placed side by side at a distance of 3 cm on PDA in a petri dish.Control treatment was compared by isolating pathogenic fungi on PDA without antagonistic fungal treatment.All tests were carried out with three repetitions for each treatment, and growth was observed.The negative effect of Trichoderma sp.against the pathogenic fungus Rigidoporus sp. can be known by calculating PIRG (Percentage Inhibition of Radial Growth) based on the formula: PIRG = x 100% Explanation: PIRG = Percentage Inhibition of Radial Growth (Inhibition Power) R1 = The area of the pathogen colony approaching the petri dish R2 = Colony area of pathogen approaching antagonist fungi

Data Analysis
The experimental design used in this study was a one-factor, completely randomized design.The factor is the age of rejuvenation of Trichoderma sp., which consists of 4 levels.Each treatment used three replications.Data processing uses SAS 9.3.1 software and Microsoft Excel 2019.The analysis uses a variance or ANOVA test and Duncan's advanced test for factors significantly affecting parameters.

Isolation and Identification Pathogen
Disease isolation activities in fungi produce pure culture isolates that are pathogens that cause white root fungal disease.Macroscopic observations of pure cultures grown using potato dextrose agar (PDA) and incubated at room temperature 27°C showed colony growth with visible characteristics in white hyphae like cotton.Most saprophytic fungi initially have a white mycelium, and then the color can change as the mycelium matures.Growth of Rigidoporus sp.isolates, symmetrical and hyaline.Pathogenic fruit bodies Rigidoporus sp.spherical, slightly leathery, orange-brownish-red in color.Hyaline hyphae, thick hyphal walls with septa and hyphae, have many branches.Basidiospores are spherical with thin, smooth, hyaline walls.

Identification of Biological Agents
After seven days of observation, it showed spores growing on green rice's surface.In general, antagonistic fungi that have a green color are Trichoderma sp.Trichoderma sp.It is an aggressive fungus that easily grows in various habitats and environments.The green spores on the surface of the rice were then isolated on PDA to obtain a pure culture.
Types of Trichoderma sp.It is more commonly found around vegetated roots than in non-vegetated soil.Rhizosphere soils have a high C-organic value.The higher the C-organic content in the ground, the higher the total population of fungi, bacteria, and other microbes because the availability of organic matter is optimally met.
This mechanism can occur at one time.Types of endophytic fungi are known to have more than one role.This fungus influences plant health.Endophytic fungi provide additional nutrition to plants or stimulate plant growth while suppressing plant diseases.Conversely, plants also provide a habitat for fungi that suits their needs.
Macroscopic observation carried out by looking at morphological characteristics, including color, shape, direction of growth, and colony surface.Trichoderma sp. it has characteristics including the color of the colony, which varies from white, greenis-white, yellowish-green, and dark green.Syahputra show the colony's surface is rough, and some are smooth with white smooth edges.Some have concentric circles [5].Can be seen in Figure 2.

Growth of Rigidoporus sp.
Growth of Rigidoporus sp.fungi colonies.It occurs gradually to fill the petri dish on the fifth day of observation.Colony growth rate of the pathogenic fungus Rigidoporus sp. can be seen in Figure 3.The three isolates of Rigidoporus sp., grown on PDA on the first day, had relatively the same growth of 2.3 cm.On the second day of observation, the three isolates had similar growth diameters.Comments on the third day isolate RG1 had faster growth compared to the other two isolates, which was 7.1 cm.On the fourth day, the development of the RG3 isolate was more rapid than the other two isolates.On the fifth day, the three isolates managed to fill the cup.All environmental factors in the media can be controlled.Media containing high carbohydrate and nitrogen sources are required for fungal growth at a pH range of 5-6 and a temperature range of 15-37°C.The PDA medium is commonly used for culturing various saprophytic pathogenic fungi [6].Factors that influence the growth of fungal hyphae include temperature, carbon and nitrogen sources, and sources of lignocellulosic substrates [3].The high substrate content in the media, such as the content of carbohydrates, protein, glucose, and agar, also affects the growth of these pathogens.The high nutritional composition of PDA media can provide nutrients for colony growth so that within five days of incubation, the isolates have successfully filled a petri dish with a diameter of 9 cm.Colony growth of Trichoderma sp. is faster than the growth of colonies of Rigidoporus sp. because Trichoderma sp. is an antagonist agent that can grow fast so that it can compete with the pathogen Rigidoporus sp. and can inhibit the growth of pathogens.Rapid colony growth causes Trichoderma sp.It is easy to isolate even by using simple methods.The growth rate of antagonistic fungi is an indicator of the mechanism of competition for space and nutrients with pathogens.The faster growth of antagonistic fungi, the more effective it will be in suppressing the growth of pathogens.

In vitro Antagonism Test of Biological Agents
An antagonistic test conducted on PDA for both isolates showed that Trichoderma sp. can inhibit the growth of the fungus Rigidoporus sp.The interaction between the two colonies was shown on the first day of observation.The first day of word showed that the territories of Rigidoporus sp. have yet to experience growth while Trichoderma sp. is already experiencing growth.Observations also indicated the occurrence of inhibition growth of Rigidoporus sp. by Trichoderma sp.The Rigidoporus sp.colony radius, which approached the colony of Trichoderma sp., was smaller than the radius of the pathogen colony approaching the edge of the dish.The factor that causes the mechanism of antagonism using competition is the existence of competition between two or more microorganisms that require the same food or nutrition and limited space to grow.Each Trichoderma sp.isolated based on the age category of rejuvenation, T2, T4, T6, and T8 inhibited the growth of Rigidoporus sp.respectively by 30%, 49%, 69%, and 71%.These results show that each isolate that has been tested has a different ability to inhibit the growth of Rigidoporus sp.colonies.One of the factors causing the difference in inhibition or antagonism in each isolate is the relationship between the production of antibiotic compounds, including the enzymes produced, the age of the culture, and the incubation time.Illustrate the difference in the ability of each isolate to inhibit the growth of pathogens.Based on the results of research conducted, the highest percentage of inhibition by T8 isolates was 71%.The results also showed that the culture age factor also affected the ability of each isolate to inhibit the growth of pathogens.Based on the results of the Duncan test, isolate T8 had the highest average inhibition percentage of 71%, and isolate T2 had the lowest inhibition percentage.Duncan's test results also showed that isolate T2 significantly differed from isolates T6 and T8.This result indicates that isolate T8 is an isolate that has the most effective ability to inhibit the growth of Rigidoporus sp. because it has the highest inhibition percentage.Presumably because, at the age of the rejuvenation, Trichoderma sp. are in the final phase of growth, where these fungi produce high levels of antibiotic compounds and cellulase enzymes that can inhibit the growth of pathogenic fungi and have the most increased antagonism activity.

Conclusion and suggestion 4.1 Conclusion
The age of rejuvenation of Trichoderma sp.significantly affects the percentage of growth inhibition of Rigidoporus sp.The highest percentage of inhibition was produced by isolate T8, with a large rate of 71%.Proves that T8 isolate is the most effective in inhibiting the pathogen Rigidoporus sp.The factor that influences the difference in inhibition of each isolate is the age of the culture.Period 8 days is the effective age for Trichoderma sp.inhibited Rigidoporus sp.

Suggestion
Future research can directly apply Trichoderma sp. to control disease attacks caused by the pathogen Rigidoporus sp.It can perform disease control testing on other soil-borne diseases by using antagonistic fungi and to find out more about the factors that influence the ability of antagonistic fungi to inhibit pathogens.

Figure 3 .
Figure 3. Graph of growth of Rigidoporus sp.

Figure 4 .
Figure 4. Graph of growth of Trichoderma sp.

Figure 5 .
Figure 5. Antagonism test of Trichoderma sp.against Rigidoporus sp.The results of the percentage inhibition of Trichoderma sp. in the treatment based on the age category of rejuvenation of the pathogen Rigidoporus sp. are as follows.

Table 1 .
Duncan Test Results Effect of Isolate Type Trichoderma sp. to the Inhibition Percentage Based on Duncan advanced test result The numbers followed by different letters in the table show a significant effect at the 5% level.