In vitro antifungal activity of several organic compounds against Ganoderma boninense

Ganoderma boninense is the pathogenic fungi responsible for oil palm basal stem rot (BSR), one of the major diseases that can lead to plant death. Until recently, the most effective methods for controlling BSR disease were still in search. We tested several organic compounds that had the potential as fungicides for G. boninense, namely allicin, hesperidin, rutin, and polyphenol. The G. boninense used in this study was isolated from Bah Jambi, North Sumatra. The in vitro antifungal activity was tested using the media poisoning methods with 0.25%, 0.5%, 1%, 2%, and 4% concentrations with three replications. The growth capacity of treated G. boninense colonies was done using the Bavendamm test. The organic compounds showed different degrees of inhibition and increased with increasing concentration. Allicin was the most effective compound against G. boninense, with a 100% inhibition rate at the lowest concentration, followed by polyphenol. The G. boninense treated with allicin and polyphenol has neither grown nor produced a reddish-brown area underneath when reinoculated to a tannic acid-containing medium. This result indicates the treated inoculum has no growth capacity. Allicin and polyphenol showed high potential as active compounds of organic fungicide against G. boninense.


Introduction
Basal stem rot (BSR) disease caused by Ganoderma boninense has been the most critical disease in oil palm plantations for over 80 years [1].This major disease can cause economic losses of up to 43% [2].The incidence of BSR disease would increase along the plant age and reach 25% of the total area at 22 years after planting [3].Until recently, the management of BSR disease was focused on several actions, such as using resistant planting materials, biological control agents, mechanical (cultural) control, and chemical control using fungicides and fertilizer [4].
One of the curative actions that can be applied to control BSR disease is a fungicide.A previous study reported that the application of hexaconazole was claimed to reduce the number of dead oil palm plants caused by upper stem rot (USR) up to 48.76% in two different places [5].Even though that study showed the potential of hexaconazole to control Ganoderma spp., there is currently no effective method to combat G. boninense infection on oil palm [4].Besides, synthetic fungicides are harmful to humans and the environment because of their toxicity and high use.The solution we can seek for this matter is to use organic fungicides that contain active ingredients derived from organic sources.The organic compounds should possess the activity to suppress or even kill G. boninense.
The organic compounds tested in this study were Allicin, Polyphenol, Hesperidin, and Rutin.Allicin has long been known to have fungicidal activity against many fungi, such as Aspergillus, Candida, Dermatophytes, and more [6].Allicin is naturally found in garlic extract and has an antimicrobial activity inhibiting the growth of membrane cells of fungi [7][8].This inhibition leads to damaged cytoplasm, membrane destruction, and malformation of mycelium [8,9].Polyphenols found abundant in smoke-liquid possess antimicrobial activity against post-harvest fungi [10], Rigidiporus spp.[11], Ganoderma sp., and Curvularia sp.[12].Hesperidin and rutin were flavonoid compounds found in orange peel and leaf extract.Both compounds were selected through the molecular docking method with receptor target Erg11, which has a role in ergosterol biosynthesis.Hesperidin and rutin have the highest binding affinity with Erg11, next after hexaconazole as control.Ergosterol positively correlates with biomass and the inoculation period of G. boninense [13].
Before proceeding to the field test, in vitro bioassay was necessary to evaluate the antifungal activity against G. boninense.The in vitro assessment was crucial in discovering and screening new active compounds [14].This study aimed to evaluate the antifungal activity of allicin, polyphenol, hesperidin, and rutin against G. boninense in vitro.

Organic compound preparation
Allicin was extracted from crude garlic and mixed with a solvent.The polyphenol used in this study was a smoke liquid from the coconut shell through pyrolysis [15].In comparison, hesperidin and rutin used in this study were commercial products with 98% and 95% purity, respectively.

Antifungal assay
In vitro antifungal activity of organic compounds was tested using the poisoned food technique [16].Each compound was mixed with potato dextrose agar (PDA) to meet specific concentrations of 0.25, 0.50, 1, 2, and 4% and was tested in triplicate.Hexaconazole 1.35% was used as a positive control, and pure PDA plate as a negative control.The Petri dish used in this study was 10 cm in diameter.A mycelial disc with a 10mm diameter cut out using a cork borer from a 14-day-old isolate was aseptically inoculated onto the PDA plate containing the tested compound.The inoculated plates were incubated at room temperature, and colony diameter was measured and recorded after 2, 5, 8, and 10 days.Percent growth inhibition was calculated using formulas described by Vincent [17].This study was conducted in a completely randomized design (CRD) with 22 treatments.

Bavendamm test
The Bavendamm test confirmed the growth capacity of the treated G. boninense isolates with a 100% inhibition rate.The source of inoculum from the previous in vitro assay was sampled and inoculated to 3 PDA supplemented with 1% tannic acid and incubated for 2-7 days.The reddish-brown area around the inoculum was the indicator of its viability.

In vitro antifungal assay
Based on our observation result, the colony of G. boninense would fully grow on a 10 cm Petry dish in 10 days.Hence, we limited the observation duration to 10 days for all treatments.In vitro assay of antifungal activity of organic compound showed that allicin and polyphenol could inhibit the growth of G. boninense colonies up to 100% at minimum concentrations of 0.5% and 2%, respectively.While hesperidin and rutin also indicate an inhibition activity against G. boninense with a requirement of higher concentration than allicin and polyphenol (more than 4%) (Figure 1).Although in this study, the concentration of both compounds was not high enough to cause a 100% inhibition rate, but not statistically different (P>0.05)ten days after inoculation (Figure 2).All organic compounds tested possess antimicrobial activity against G. boninense that increases along with the increase in concentration.Hesperidin was reported to inhibit the growth rate of several microbes from 11-39% at 0.25 mM [16].Rutin extracted from natural sources was also known for its antimicrobial activity against several microbes, such as Pseudomonas aeruginosa, Klebsiella pneumoniae [18], and Mycobacterium smegmatis [19].These two compounds were commonly known in the pharmacological field as a supplement and anti microbes [20][21], but their use in the agricultural field was still not much studied.Although it requires a higher concentration to cause 100% inhibition than allicin and polyphenol, it is proven to have antimicrobial activity against G. boninense.The minimum inhibitory concentration (MIC) of hesperidin and rutin against G. boninense was 2%.Among the compounds tested, allicin was the most effective that could inhibit 100% of the growth of G. boninense colonies at the lowest MIC, followed by polyphenol.This result was equal to hexaconazole as a positive control.The MIC of allicin to 100% inhibit G. boninense was 0.05%.This result was in line with Wallock-Richards [22] study that reported the MIC of allicin-containing garlic extract against Burkholderia cepacian was 0.5%, and the MIC of pure allicin was 0.008-0.062%.Previous studies reported that the MIC of allicin from garlic extract varied depending on the target plant pathogen fungi.Allicin completely inhibited the growth of Verticillium dahliae at MIC 150 M, while V. longisporum was least inhibited by allicin at 200 M [7].
The colony of G. boninense inoculated on PDA treated with polyphenol and hesperidin 0.25-1% has significantly (P<0.05)higher growth rates compared to the control (Table 1).This result indicates that the use of a sub-lethal dose of polyphenol and hesperidin induces the growth rate of G. boninense colonies.Previous studies reported that the application of a sub-lethal dose of carbendazim induced the sclerotial production and conidial germination of Botrytis cinerea [23].Another study about the effect of sub-lethal concentrations of fungicides stimulated the mycelial growth of Pythium [24] and Sclerotinia sclerotiorum [25] was also reported.Although both hesperidin and rutin are flavonoids, rutin requires less concentration to inhibit G. boninense growth.The inhibition rate of allicin 0.5-4%, polyphenol 2-4%, hesperidin 4%, and rutin 4% were not significantly different (P<0.05) from hexaconazole.Even so, only allicin and polyphenol could 100% inhibit G. boninense growth.The growth capacity of the treated inoculum was confirmed through the Bavendamm test.The result showed that the treated inoculums were neither grown nor produced a reddish-brown area underneath (Figure 3).The reddish-brown area on tannic acid-containing media showed ligninolytic activity produced by Basidiomycota [26].G. boninense also produces a ligninolytic enzyme that can be examined through this procedure [27].We suspect this result indicates that all the treated inoculums with a 100% inhibition rate lost their growth capacity or died.

Conclusion
All tested organic compounds possess antifungal activity against G. boninense that varies depending on concentration.Allicin gives the most promising result with the lowest concentration that totally inhibits G. boninense growth, followed by polyphenol with a higher concentration.The treated G. boninense with a 100% of inhibition rate lost its growth capacity when reinoculated to a new medium.

Acknowledgment
The research was a part of the Palm Oil Grant Research 2022 project, funded by the Palm Oil Fund Management Agency.

Figure 1 .
Figure 1.Minimum concentration of each tested organic compound with the highest inhibition rate against G. boninense colonies ten days after inoculation.

Figure 2 .
Figure 2. The effect of several organic compounds at various concentrations on the diameter of the G. boninense colonies.Different letters within graphics indicate a statistically significant difference (Tukey Test at 95% confidence level).

Figure 3 .
Figure 3.The Bavendamm test result of treated G. boninense with a 100% inhibition rate.

Table 1 .
The effect of several organic compounds at various concentrations on the inhibition rate and growth rate of the G. boninense colonies.Numbers followed by different letters in the same column are significantly different according to Tukey Test at 95% confidence level. *