Fractional inhibitory concentration index of the allicin and flavonoids combinations against Ganoderma boninense

Allicin and several flavonoids were known to have antifungal activity against various fungi. The antifungal capability of allicin and flavonoids varies depending on the target fungi; the combination of both compounds was expected to increase its antifungal activity. As a major disease of oil palm, basal stem rot (BSR) caused by Ganoderma boninense always be a top concern of oil palm farmers. This study aimed to evaluate the effect of combining allicin with hesperidin and rutin on antifungal activity against G. boninense through the fractional inhibitory concentration (FIC) index. The FIC index was determined using a checkerboard assay with media poisoning methods. The allicin concentration tested was 0.1%, 0.2%, 0.4%, and 0.8%, while the hesperidin and rutin concentrations were 1.25%, 2.5%, 5%, and 10%. All combinations were done in triplicate and incubated for twelve days. The result showed that a combination of allicin with hesperidin or rutin was the additive interaction that lowers the concentration of each compound with similar inhibition rate of G. boninense in vitro. Combining allicin with rutin was considered better than hesperidin due to the higher inhibition rate of G. boninense. The allicin-rutin combination has a lower concentration with a 100% inhibition rate of G. boninense in vitro.


Introduction
Allicin, the organosulfur compound found in crude garlic extract, was known for its wide-ranged spectrum antimicrobial activity that inhibits the growth of membrane cells of fungi and bacteria [1][2][3][4].Against fungi, inhibition activity causes damage to the cytoplasm, destroys the membrane, and leads to the malformation of mycelium [3,5].The flavonoids had various benefits, from antioxidant to fungicidal activities.Even though the use of flavonoids was quite popular in pharmacological fields [6][7][8][9], the use of these compounds as fungicides in the agricultural field was less studied.The flavonoids used in this study were hesperidin and rutin.Hesperidin was found in the orange peel extract, while rutin was abundant in the leaf extract.Hesperidin can reduce the growth rate of Fusarium semitectum, Aspergillus parasiticus, and A. flavus up to 33-39% at 0.25 mM [10].Rutin also has antifungal activity against several fungi, such as Fusarium oxysporum, Diplodia oryzae, Rhizoctonia solani, Aspergillus carneus, and Helminthosporium turcicum [11].
Ganoderma boninense is the pathogen that causes basal stem rot (BSR) disease in oil palms [12,13].This disease has been a major problem for years, causing losses of up to 43% [13,14].The management of BSR disease in oil palm was focused on preventive action.Until recently, no effective methods have been proven to control this disease curatively [15,16].
Based on our previous study, allicin, hesperidin, and rutin has antimicrobial activity against G. boninense in vitro (publication in press).The inhibition rate of these compounds varied depending on their concentration.For efficiency and safety, the pesticide formulation should contain the lowest concentration of active ingredients with an effective result [17].To increase the inhibition rate and lower the concentration, adding another active ingredient with a synergistic effect was a good solution [18][19][20].Combining two active ingredients would result in several interactions: synergism, additive, no response, and antagonistic [21].A combination that resulted in the synergism and additive interaction was the one that can increase effectivity.In contrast, the no response and antagonistic interaction were avoided because the combination has lower effectiveness than a single active ingredient.Determining the effect of the combination of two active ingredients was essential to evaluate the concentration of each compound.This process can be done with in vitro bioassay against target fungi.The in vitro assessment was crucial in discovering and screening new active compounds [22].This study evaluated the effect of combining allicin with hesperidin and rutin as an active ingredient against G. boninense through a fractional inhibitory concentration index.

Allicin, hesperidin, and rutin preparation
Allicin was extracted from crude garlic (Honan garlic) following the method of Nguyen et al. [33].The alicin extract was then mixed with water as a solvent.The study used Hesperidin extract (product of Quanao Biotech, CAS 520-33-2), extracted from the orange peel with 98% purity.Rutin used in this study was also a product of Quanao Biotech extracted from Sophora japonica with 95% purity.

Compatibility test
The compatibility test between both active ingredients was done using a checkerboard assay adapted from Aleixo et al. [23] and tested using the poisoned food technique [24][25][26] with dilution methods in agar media [27].Each potato dextrose agar (PDA) containing the active ingredient with the same ratio of multiple concentration were combined to evaluate the antifungal activity against G. boninense inoculum (Figure 1).The tested allicin concentration was 0.05, 0.1, 0.2, 0.4, and 0.8%.Conversely, the tested concentration of hesperidin and rutin were 1.25, 2.5, 5, and 10%.The highest tested concentration for each active ingredient was the minimum inhibitory concentration (MIC) from the previous study.
The G. boninense was isolated from the infected oil palm, has been tested as high-virulence isolates and has been molecularly characterized as Ganoderma boninense isolate UPMLD1806.The G. boninense inoculum was two weeks old and 10mm in diameter, inoculated to PDA plate containing the combination of tested compounds.The Petri dish used in this study was 10cm in diameter.All Petri dish sealed and incubated at room temperature.The diameter of colonies was recorded after 1, 3, 6, 9, and 12 days.This study was conducted in a completely randomized design (CRD) with three repetitions.The inhibition rate was calculated using the following formula: (diameter of control colonidiameter of treatment coloni)/ diameter of control coloni x 100%.The fractional inhibitory concentration (FIC) index was determined by dividing each active ingredient's MIC when combined with each active ingredient's MIC when used alone.The FIC index indicates the interaction of combination.FIC index ≤0.5 means synergistic, 0.51 -1.0 mean additive, 1.1 -4 mean indifferent, and FIC index > 4 mean antagonistic interaction [28].

Allicin and flavonoids compatibility test
The observation duration of this study was limited to 12 days after inoculation (DAI) because the colony of G. boninense would fully grow generally on a 10 cm Petri dish in 10 days.The study was focused on determining FIC index of allicin combined with hesperidin or rutin.MIC of each compound was required to calculate the FIC index.MIC is the lowest concentration of antimicrobial compound that totally inhibits the visible growth of the tested organism [27,29].Based on a previous study, the MIC of allicin with a 100% of inhibition rate of G. boninense colonies was 0.8%, while 10% was the MIC for both hesperidin and rutin.

Table 1. The effect of various concentrations of allicin and hesperidin combination on colony diameter, inhibition rate of the G. boninense colonies, and fractional inhibitory concentration index
The observation result showed that the diameter colony of G. boninense was inhibited by the combination of allicin and hesperidin, with the inhibition rate varying depending on concentration (Table 1).The treatment with 0% allicin and hesperidin was counted as a control.Nine days after inoculation, the control colony of G. boninense was fully grown on a 10cm Petry dish.However, we continue the observation until 12 DAI to evaluate the growth of G. boninense on other treatments.The inhibition rates were calculated by comparing the diameter of control and treatment colonies at 12 DAI.The minimum concentration of allicin that affect the growth rate of G. boninense was 0.4%, with a 17.1% inhibition rate.This rate was similar to the result of a previous study conducted by WallockRichards [30] that reported the minimum concentration of garlic extract that inhibits Burkholderia cepacian was 0.5%.The concentration of allicin, temperature, and cultivation condition of the garlic source will affect the antimicrobial capability [31].In this study, the lowest concentration of allicin required to inhibit the growth of G. boninense entirely was 0.8% (Figure 2).After 12 days, no growth of G. boninense was observed.
Hesperidin was reported to inhibit the growth rate of several microbes ranging from 11-39% at 0.25 mM [26], but against G. boninense there is no previous study reported.The lowest concentration of hesperidin that inhibited 14% of the growth rate of G. boninense was 2.5%, which is not statistically different (P<0.05) from the control.The minimum hesperidin concentration with an inhibition rate of G. boninense up to 92% was 5%.Although this result was similar (P<0.05) with 100% inhibition, this inhibition rate will likely decrease along incubation time.However, the 5% hesperidin could inhibit the growth of G. boninense in the first nine days after inoculation.This result also indicates that 5% of hesperidin concentration was insufficient to kill the G. boninense.
Combining allicin and hesperidin would reduce the concentration required for both compounds to inhibit G. boninense growth.Based on the FIC index, the interaction between allicin and hesperidin as an antifungal against G. boninense was additive.All the FIC index with synergistic category has an inhibition rate that is not different (P>0.05) from the control.The additive interaction indicates that the combination of both compounds is the sum of the antifungal activity of each compound [32].One compound does not disturb the antifungal activity of another.Although the additive interaction has less effectiveness than synergistic interaction, it is still favourable due to the reduction of each concentration.The combination of 0.05% allicin and 5% hesperidin gave the best inhibition result at 12 DAI, even though the inhibition rate was not 100% (Figure 2).Like hesperidin, the minimum concentration of rutin to fully inhibit the growth of G. boninense in vitro was 10%.Although after 12 DAI, 5% rutin has zero growth of G. boninense, it starts to grow at 14 DAI (data not shown).This result indicates that both hesperidin and rutin have the same MIC against G. boninese.The interaction of allicin and rutin against G. boninense was additive.In contrast with the allicin-hesperidin combination, the additive interaction of allicin and rutin inhibited G. boninense growth in vitro by up to 100% (Figure 2).The combination of 0.1% allicin and 5% rutin resulted in a 100% inhibition rate of G. boninense (Table 2).The lower concentration of allicin (0.05%) and 5% rutin resulted in a statistically indifferent (P<0.05)inhibition rate, but this combination has additive interaction and a lower inhibition rate.

Conclusion
The fractional inhibitory concentration index of allicin combined with hesperidin or rutin has an additive interaction.Combining allicin with hesperidin and rutin can reduce the concentration of both compounds to produce a similar inhibition rate against G. boninense.The allicin-rutin combination was considered superior because of the capability to reduce the concentration and result in total inhibition against G. boninense.

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.Illustration of checkerboard assay to evaluate the compatibility between active ingredients.

Table 2 .
The effect of various concentrations of allicin and rutin combination on colony diameter, inhibition rate of the G. boninense colonies, and fractional inhibitory concentration index