Isolation and antagonistic test of fungi associated with pummelo citrus against Botryodiplodia theobromae in vitro

Botryodiplodia theobromae is one of the causal agents of diplodia stem-end rot in pummelo citrus. Currently reported, indigenous microbes which are associated with plants can improve plant health by controlling diseases and promoting plant growth. This study aims to determine the potential of fungi associated with pummelo citrus in inhibiting B. theobromae in vitro. Identification of the fungus which associates with pummelo citrus is determined by observing the cultural morphological and microscopic characteristics. The antagonist test was performed using dual culture method. The collected data was analyzed with a completely randomized design with 13 treatments and 3 replications. The results showed that there were isolates of fungi associated with pummelo citrus which were identified as Trichoderma, Aspergillus, Mucor and Fusarium genera. There were 7 isolates of fungi which have the potential to inhibit B. theobromae which were from genera Trichoderma and Aspergillus. The antagonist mechanisms that occurred in this study were competition and parasitism.


Introduction
Pummelo citrus is an iconic commodity in Eastern Indonesia, but currently the farmers less interested with this commodity because the productivity is very low due to a problem related to plant diseases, including diplodia stem-end rot caused by Botryodiplodia theobromae (Patouillard) Griffon and Maublanc.Losses caused by diplodia disease can reach 50-85% of the total production during the harvest season [1].
The type of control carried out by farmers in dealing with diplodia disease currently still relies on chemical control which can disrupt the balance of the ecosystem.Therefore, we need a method of controlling plant diseases that is environmentally friendly and able to support sustainable agriculture.
Control of plant diseases that support sustainable agriculture can be achieved by utilizing natural enemies such as antagonistic fungus that are cheaper and more environmentally friendly [2].Antagonistic fungus can suppress the growth of pathogens through several mechanisms including competition, antibiosis, and hyperparasites.In addition, several species of fungi are also able to produce growth hormones for plants [3].
The use of antagonistic fungus in controlling B. theobromae has been reported [4][5][6] that some antagonistic fungus associated with plants, 80-90% are able to inhibit the growth of B. theobromae in vitro.Therefore, research is required into antagonistic tests of fungus associated with pummelo citrus to know its potential in controlling B. theobromae causal agent of diplodia stem-end rot.

Sampling
Sampling of pummelo citrus stem infected with B. theobromae and sample for antagonist fungi was acquired in Padang Lampe Village, Ma'rang District, Pangkep Regency.Samples were obtained by taking mature leaves of pummelo citrus, samples of branches selected were from the second branch of the main stem and the samples of roots taken were at a depth of 15-20 cm from the soil surface with a length of 15 cm from the base of the stem and the diameter of the roots ranged from 1-1.5 cm [7].The plant parts used for the isolation of the fungus were obtained from healthy pummelo citrus, namely plants that did not show any disease symptoms such as yellowing, wilted, or overgrown fungal mycelium.

Fungi Isolation
Fungi isolation was carried out using the modified method by [8].Samples were rinsed with tap water and then cut into 1 cm.Root and branch samples were surface sterilized by immersing in 2.5% NaOCl solution for 2 minutes, then 70% alcohol for 1 minute twice, then rinsing with sterile distilled water for 1 minute twice, while leaf samples were surface sterilized by soaking in 2.5% NaOCl solution for 1 minute, then 70% alcohol for 30 seconds twice, then rinsing with sterile distilled water for 30 seconds twice and then dried on sterile filter paper.Isolation of pathogenic fungi was carried out following the method of [9] modified.The sample is rinsed with tap water then the part that borders the healthy tissue and infected tissue is cut approximately 1x1 cm.The plant parts were surface sterilized by immersing in 2.5% NaOCl solution for 1 minute, then rinsed 3 times with sterile distilled water and then dried on sterile filter paper.
Isolation of the fungus was carried out using the direct plating technique, namely by placing the sample pieces on PDA media.Each petri dish contains 3 samples.The growing fungus is then isolated into the new PDA as a pure culture and identified based on macroscopic and microscopic morphology using the handbook of fungus determination key [10].

Antagonistic Test
Fungal cultures from healthy plants and B. theobromae culture were taken using a cork borer with a diameter of 7 mm and then grown side by side at a distance of 4 cm, while the distance between each isolate from the edge of the petri dish was 2.5 cm [11].All the cultures were sealed and incubated at room temperature for 5 days.R1 = the measurement of the pathogen colonies in the opposite direction from the isolate of fungus associated with pummelo citrus, R2 = the measurement of the pathogen colonies that grow towards isolate of fungus associated with pummelo citrus, A = fungus which was isolated from healthy pummelo citrus and P = pathogen B. theobromae.Observation of antagonistic mechanisms was performed macroscopically through direct observation in dual cultures.The interaction mechanism that occurs between pathogen and antagonistic fungi is based on the criteria by [9,15,16]: a. Competition; the antagonistic fungus colonies cover the pathogen colonies and the growth of the antagonistic fungi is faster to fill a 9 cm diameter petri dish, at the contact area, hyphae of pathogen lysis.b.Antibiosis; between the pathogen and the tested fungus there is a clear zone, the shape of the pathogen hyphae changed and there is a pigment on the bottom surface of the tested fungus colony.c.Parasitism; the tested fungus hyphae grow on top of the pathogen.The tested fungus hyphae are found coiled the pathogen hyphae in the contact area and cause lysis.The fungus treatment that shows the best potential inhibition against B. theobromae will be observed again under microscope by taking a piece of mycellium in the contact area of the two fungi around 1 cm to observe hyphal interactions.The data obtained were analyzed using Analysis of Variance and further test using Duncan Multiple Range Test at the 5% probability level if significant differences are detected.

Result and discussion
The isolation results of the fungus from leaf, branch and root tissues found a total of 13 isolates with different characteristics.The fungi identified were from the genera Trichoderma (4 isolates), Aspergillus (3 isolates), Mucor, Fusarium and 4 other isolates could not be identified because they were only mycelia sterilia which did not produce conidia [10].The percentage inhibition of the fungus shows a variable effect on several treatments.Observations were still made even though the control had filled the petri dishes because some isolates were still active in inhibiting until the 5th day.Note: Numbers that followed with the same letter in the same column are not significantly different in Duncan's test at the 5% level.
Table 1 shows that in 1 dpi observation the control treatment and the fungus treatment not significant different, but in 2 to 5 dpi there was a difference.The inhibition test was carried out by calculating the growth diameter of the fungus mycelium.The growth rate of B. theobromae in the control was seen to be faster than in the fungus treatment, so that it can be seen that the isolated fungus from pummelo citrus was able to inhibit the growth of B. theobromae.The growth rate of antagonistic fungi is an indicator that fungi are able to compete with pathogenic fungi, the faster the growth of antagonistic fungi, the more effective it is in suppressing the pathogens growth [17].The ability to inhibit the development of pathogens is a requirement for an organism to be a biocontrol agent [11].
Based on the results of the inhibition test, it showed that there were variations in the effectiveness of all isolates.Trichoderma sp. 2 is a treatment that has the best average percentage of inhibition on day 5,

Asp
Tr P P (3) Asp P Tr P namely 77.04% and is included in the fungus which has very high effectiveness.This is in accordance with the category of inhibition percentage by [11,18] which states that the percentage of inhibition of the antagonist agent is grouped into; low (1-25%), moderate (26-50%), high(51-75%) and very high (76-100%).
Treatment of Aspergillus sp. has growth that spreads and faster, so it is known that the mechanism that occurs is competition [9,15,16].Another isolate that also exhibits a competition mechanism for nutrients and space are the isolate Trichoderma sp. 1, Trichoderma sp. 2, Trichoderma sp. 3 and Trichoderma sp. 4 because on 3 to 5 dpi it showed faster growth than B. theobromae [4,19].Microscopic observation of the contact area of B. theobromae and antagonistic fungi in figure 4 showed that cell lysis occurred in B. theobromae hyphae.The competition mechanism occurs when antagonistic fungal colonies cover pathogenic colonies and the growth of antagonistic fungi is faster to fill the petri dish and in the contact area, pathogenic hyphae underwent lysis [12].Aspergillus sp.produces hydrolytic enzymes which are able to inhibit the growth of pathogens.Those enzymes are protease, lipase and cellulase as well as pectinase and also extracellular enzymes such as glucoamylase, chitinase, lactase, catalase, invertase, α-amylase and ß-amylase that support the mechanism of inhibition of Aspergillus sp. to break the components of pathogens cell wall [20].
The diameter of the B. theobromae colonies decreased in the treatment of Trichoderma sp. 1, Trichoderma sp. 2, Trichoderma sp. 3 and Trichoderma sp. 4 because they were covered by antagonistic fungal mycelium.The antagonistic fungi that are able to cover pathogenic fungal colonies is referred to as parasitism [17].Observations on the contact area of the hyphae showed that the antagonist fungal hyphae entwined the pathogenic hyphae and lysis in figure 4. The antagonistic mechanism between Trichoderma sp. and pathogenic fungi are mycoparasitistic interactions that begin after the parasitic hyphae make physical contact with the host hyphae.The initial growth of the Trichoderma sp.mycelia elongates, then entangles and penetrates the host fungal hyphae untill vacuolization, lysis and finally disintegrates [12].Furthermore, these antagonists grow and coil in the pathogenic hyphae to inhibit the pathogen's spread [9,21].Trichoderma mode of action by producing cellulase and chitinase which are able to destroy pathogens hyphae.Besides producing enzymes, species Trichoderma also produces VOCs (Volatile organic compounds).Although it is just a tiny part of the fungus, but it can activate resistance mechanisms and protect plants from pathogens [22].

Conclusion
There were seven potential fungal isolates from healthy pummelo citrus with inhibition percentages above 50 % namely Aspergillus sp.

Figure 1 .
Figure 1.Schematic of isolate placement for in vitro antagonist test with dual culture method, fungi associated with pummelo citrus (A) and pathogens (P)

Figure 2 .
Figure 2. Matrix of interactions between antagonist fungi (A) and pathogens (P) Aspergillus sp. 1, Trichoderma sp. 1, Aspergillus sp. 2, Trichoderma sp.2, Aspergillus sp. 3, Trichoderma sp. 3 and Trichoderma sp. 4 are the isolate that has the potential to inhibit B. theobromae.The interaction between the tested fungi and B. theobromae shows competition and parasitism mechanisms, which can be observed in Figure 3.