Introduction

Maize or corn is a food ingredient with the second-highest carbohydrate content after rice in Indonesia and comes third in the world after wheat. Maize or corn is used as a raw ingredient for the animal feed manufacturing industry. The need for corn in Indonesia was increased by 1.11% per year in 2010‒2014. The official statistics for the province of Bali reported that corn production in 2015 was 40.603 tons, a decrease compared to 2013. The decline was caused by several factors, such as the decreasing of land areas, the farming transitions into horticultural crops, and lack of water supply (CBS 2014).

Corn as a raw and feed ingredient must have good quality. The most frequent problem in public and among corn farmers is the contamination of aflatoxin compounds produced by A. flavus (Montalbano et al. 2021). This fungus grows easily in tropical areas. In Indonesia, the fungus A. flavus has the potential to grow well and produce aflatoxins because supported by optimal levels of humidity, rainfall, and temperature for the growth of A. flavus. Frequent contamination of aflatoxins can easily be found in whole grains such as corns and peanuts. Several causes of aflatoxins contamination are the post-harvest handling, harvest storage, and processing of products made from corn. These factors are common in corn farmers and companies that use corn as raw material (Rahayu et al. 2010). The contamination of aflatoxin compounds in corn kernels results in losses to corn farmers and health problems in animals and humans, so the control and prevention of A. flavus are needed.

The prevention of aflatoxins contamination in raw and feed ingredients can be conducted more effectively by inhibiting the growth of A. flavus. It can be physically conducted through reduction of water content in the materials, lowering of temperature, and modification of storage conditions. It can be chemically conducted by giving disinfectants acidic and alkaline substances. Chemical control requires a lot of money regarding the high cost of the chemicals needed. In addition, at the farmers’ level, it is difficult to do so; therefore it is necessary to do the control biologically. The biological control of fungal pathogens can be achieved by using antagonistic bacteria such as PGPR (Sharf et al. 2021), and various antagonistic fungi such as species of Trichoderma (Ali et al. 2020), Aspergillus (Khan and Javaid 2022a, b) and Penicillium species (Khan and Javaid 2022c). According to Mauro et al. (2018), biological prevention is more effective than physical and chemical prevention. Given the relatively fast microbial growth with a short generation time, it can be produced on a large scale.

Rhizopus oligosporus is a nonpathogenic fungus. This fungus is likely used for the fermentation process of raw ingredients becoming products that have high nutritional value, such as the fermentation of soybeans using the Rhizopus spp. yeast becoming into tempeh. Nursadin and Supriyanto (2012) reported that Rhizopus spp. has a high ability to compete and able to inhibit the growth of pathogenic fungi. The results of previous studies showed that Rhizopus sp. could inhibit Fusarium oxysporum by 60%. Therefore, in the present study, R. oligosporus was evaluated for its potential to control the growth of A. flavus FNCC6109.

Materials and Methods

Research location

The research of the potential of R. oligosporus filtrate in controlling the growth of A. flavus FNCC6109 was conducted in the Microbiology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences Udayana University, Indonesia.

Isolation of R. oligosporus in tempeh

The isolation of R. oligosporus was conducted by aseptically taking as much as one loop of the part of the colony suspected of being R. oligosporus which grew on the surface of the tempeh. This part of the colony was then placed right in the middle of Petri dishes which already contained PDA media. The Petri dishes were incubated at 28°C temperature for four days. The growth of fungal colonies on Petri dishes was observed macroscopically and microscopically by referring to the Pitt and Hocking (1997) identification book. The fungi that showed the characteristics of R. oligosporus were then being re-isolated until getting a pure culture.

Regeneration of A. flavus FNCC6109

The A. flavus FNCC6109 isolate was obtained from the stock culture of the Microbiology Laboratory, Department of Biology FMIPA, Udayana University. The stock culture was rejuvenated by taking the hyphal flakes using a needle and then implanted right in the middle of the Petri dish that contained PDA media, then being incubated at 28^oC for four days. The growing colonies of A. flavus FNCC6109 were used for the next testing stage.

Inhibition test of R. oligosporus against the growth of A. flavus FNCC6109

The inhibition of R. oligosporus was conducted in vitro using the dual culture method. The procedures started with taking the culture of R. oligosporus and A. flavus FNCC 6109 with a 5 mm diameter cork borer. Both colonies were grown side by side with a distance of 3 cm in Petri dishes containing PDA media, then being incubated at 28^oC and measured the diameter for four days. Subsequently, the same control was conducted; however, only one type of fungi was grown. The antagonism effect of R. oligosporus against A. flavus FNCC6109 could be calculated with the PIRG (percentage inhibition of radial growth) (Singh and Vijay 2011):

PIRG: Percentage Inhibition of Radial Growth

R1: Colony area of A. flavus FNCC6109 without the antagonist (control)

R2: Colony area of A. flavus FNCC6109 with the antagonist (dual culture)

Inhibition test of R. oligosporus culture filtrate against the growth of A. flavus FNCC 6109

A bottle of 100 mL PDB (potato dextrose broth) was prepared and then inoculated with the R. oligosporus that had the potential of inhibiting A. flavus FNCC 6109. Then, it was incubated for 3, 4 and 5 days at 28°C. Once the incubation period was over, the inhibition of the R. oligosporus culture filtrate was tested in vitro against the growth of A. flavus FNCC6109. This test started off with preparing the culture filtrate of R. oligosporus and took as much as 1 mL of it and placed on the Petri dish, which would be poured with PDA media afterward, then left to solidify. Next, the A. flavus FNCC6109 colony was taken using a 5 mm diameter cork borer and incubated for four days. After that, control was made by growing colonies of A. flavus FNCC6109 on PDA media without being given any culture filtrates. The effect of culture filtrate could be determined by measuring the colony area of A. flavus FNCC6109 using the formula:

L1: Colony area of A. flavus FNCC6109 without the antagonist (control)

L2: Colony area of A. flavus FNCC6109 with control

Inhibition of R. oligosporus culture filtrate against A. flavus FNCC 6109 in corn kernels

A sterile Petri dish was prepared and contained with solid PDA media; colonies of A. flavus fungi were planted right in the middle of the Petri dish with a diameter of 5 mm and then incubated at a temperature of 28°C for four days. The grown colonies dripped with sterile water as much as 5 mL, then rubbed it on the colonies' surface using a spatula. The liquid which contained the spores was then taken using a Pasteur pipette and then collected into a sterile bottle. To determine the density of the spores in the suspension, calculations were made using a hemacytometer.

The design used to determine the inhibition of the R. oligosporus culture filtrate on the growth of A. favus FNCC 6109 on corn seeds was a Completely Randomized Design (CRD). Types of treatment of R. oligosporus filtrate on the growth of A. flavus FNCC6109 on corn seeds were as follows: a) Corn kernels without treatment (control), b) Corn kernels + 5 mL suspension of A flavus, c) Corn kernels + A. flavus + 10% R. oligosporus filtrate, d) Corn kernels + A. flavus + 20% R. oligosporus filtrate, e) Corn kernels + A. flavus + 30% R. oligosporus filtrate, f) Corn kernels + A. flavus + 40% R. oligosporus filtrate, and g) Corn kernels + A. flavus + 50% (v/v) R. oligosporus filtrate.

The giving treatments were conducted by preparing as much as 100 g of corn kernels for each treatment which was placed in 8 sterile containers. The corn kernels were sprayed with as much as 15 mL of R. oligosporus filtrate and then adding 5 mL of A. flavus FNCC6109 spores were. After being given the treatment, they were stored for 15 days at 28^oC. After the incubation period ended, the total population of A. flavus FNCC6109 was calculated using the plating method. To obtain the representative data, all treatments were repeated four times.

Data analysis

The quantitative data were analyzed with ANOVA. If the data obtained have a significant difference at the test level of 5% (P ≤ 0.05), it would be followed by the Duncan test to determine the differences in each treatment.

Results

The isolation and identification of R. oligosporus

The macroscopic characteristics of the isolated R. oligosporus colonies in tempeh showed the color of white to the grayish, diameter of 4 cm in the four days incubation period, cottony texture, no zoning, no radial and growing zone. Whereas on the microscopic observation through 40 × 10 magnification on the R. oligosporus showed round or elliptical sporangium which had no insulation on the hyphae, there was a stolon that connected two sporangiophores and had rhizoid (Fig. 1). Macroscopic observations showed that R. oligosporus had a white to grayish colony color, textured like cotton, had no radial lines, had a growing zone, and had no zonation.

In vitro inhibition of A. flavus FNCC6109 by R. oligosporus

Inhibition of R. oligosporus against A. flavus FNCC6109 using the dual culture method has obtained the average colony area of A. flavus FNCC6109 as much as 5.77 ± 0.773 cm². In comparison, it had as much as 13.00 ± 1,154 cm² of colony area in 4 days incubation under control. In Table 1, it can be seen that the average percentage of inhibition power of R. oligosporus on the growth of A. flavus FNCC6109 was 56.08 ± 10.103% on PDA media with four days incubation period. The colony area of A. flavus FNCC6109 on treatments seemed smaller than those with no treatments (Fig. 2). There were allegations on the treatment of the R. oligosporus to be able to suppress the growth of A. flavus FNCC6109.

In vitro culture filtrate test of R. oligosporus against A. flavus FNCC6109

The inhibition of the culture filtrate of R. oligosporus, which tested against the growth of A. flavus FNCC6109 in vitro showed that the culture filtrate had the ability to inhibit A. flavus FNCC6109. In Fig. 3, it can be seen that the colonies of A. flavus FNCC6109 could not grow well in the presence of the culture filtrate of R. oligosporus, while on the control, colonies of A. flavus FNCC6109 seemed bigger.

In numbers, the inhibition test of the R. oligosporus culture filtrate against A. flavus FNCC6109 can be seen in Table 1. Table 1: Average and percentage of R. oligosporus inhibition against A. flavus FNCC6109 on PDA media at a certain incubation period at 28^oC

Treatment	Inhibition (cm² or %)
Control (cm²)	13.00 ± 1.154
R. oligosporus (cm²)	5.77 ± 0.77
Not incubated (%)	56.08 ± 10.103
Three days incubation (%)	61.27 ± 5.13
Four days incubation (%)	64.07 ± 7.05
Five days incubation (%)	67.27 ± 2.70

Fig. 1: A. Colony morphology of R. oligosporus on PDA media with an incubation period of 3 days at a temperature of 28°C

B. Microscopic structure of R. oligosporus under a binocular microscope at 400× magnification (arrow S = Sporangium; SP = Sprongiophore; R = Rhizoid; Sn = Stolon)

The five-day incubation period of the culture filtrate of R. oligosporus on PDA media had the highest percentage of inhibitory, which was 67.27 ± 2.70%, while the three days and four days incubation percentages were 61.26 ± 5.13% and 64.07 ± 7.04%. The culture filtrate of R. oligosporus showed positive results in inhibiting A. flavus FNCC6109 in vitro, presumably due to the presence of an active compound or enzyme capable of suppressing the growth of A. flavus FNCC6109. Meanwhile, A. flavus FNCC6109 grown on PDA media without culture filtrate suspension showed good growth of A. flavus FNCC6109 with a larger colony area.

The population of A. flavus FNCC6109 in corn kernels added with R. oligosporus

The corn kernels that gave the treatment of adding R. oligosporus culture filtrate had less population of A. flavus FNCC6109 colonies than the corn kernels that had not been given the culture filtrate. Table 2 represented several treatments of the culture filtrate concentration added to the corn kernels, which showed the varied population numbers of A. flavus FNCC6109. The calculation showed that at a concentration of 50%, the culture filtrate of R. oligosporus had the highest inhibitory ability with the average number of A. flavus FNCC6109 colonies 3 × 10⁴ CFU g^-1, whereas in treatment B, which was only given a suspension of A. flavus FNCC6109 spores had a larger average population, namely 42 × 10⁴ CFU g^-1 after 15 days of the incubation period. This showed that the culture filtrate of R. oligosporus was able to suppress the population of A. flavus FNCC6109 in corn kernels.

Table 2 showed the lower the concentration of R. oligosporus culture filtrate added into the corn kernels, the higher the population of A. flavus FNCC6109 after an incubation period of 15 days. Whereas in treatment A (corn kernels without culture filtrate of R. oligosporus and A. flavus FNCC 6109), there were neither R. oligosporus nor A. flavus FNCC 6109 found. This proved that the corn kernels used in this study were free of contaminants from the two fungi.

Discussion

Microscopically it forms non-septate hyphae, has stolons and rhizoids, and the shape of the sporangium is spherical. These characteristics were in accordance with the description stated by Yuliansih (2007) and Dolatabadi et al. (2014) in which R. oligosporus macroscopically and microscopically have white to grayish colonies, have rhizoid like roots, the hyphae are not insulated, single sporangiophores, the sporangium is round or elliptical and have stolons. The difference between the genus of Rhizopus and other fungi is that the non-insulated hyphae have rhizoids and a distinctive sporangium shape. Firmansyah (2007) had also isolated and identified R. oligosporu spp. in tempeh. The results obtained three species those were R.oligosporus, R. stolonifer and R. oryzae. The same research had been conducted by Virgianti (2015), which showed that the isolated R. oligosporus colonies in tempeh macroscopically had the characteristics of having white to grayish color and grew like cotton. Rahmawati et al. (2013) reported the results of the isolation and identification of molds in corns and found the presence of R. oryzae and R. stolonifer. Furthermore, McKelvey and Murphy (2017) stated the existence of R. oligosporus in cornflour had a role in producing cellulose, xylanase, and protease enzyme activities.

The results of research by Nursadin and Supriyanto (2012) showed that R. oligosporus was able to inhibit Fusarium oxysporum by 60%. Furthermore, it was conveyed that R. oligosporus could be used as a competitor because it has a very high competitive ability and very fast growth. The same thing was also conveyed by Anuragi and Sharma (2016) that R. oligosporus could be used as a biocontrol agent because of its ability to inhibit Fusarium oxysporum with an inhibitory percentage of 56.76%. Moreover, Adebola and Amadi (2010) reported that R. oligosporus could inhibit the growth of the pathogen Phytophthora palmivora with a 76% inhibition rate in 7 days incubation period. The inhibition mechanism of R. oligosporus against the growth of A. flavus FNCC6109 other than by suppressing it was suspected that R. oligosporus produced metabolites that could inhibit A. flavus FNCC6109. R. oligosporus was also able to produce enzymes and bioactive compounds that are antimicrobial and antifungal. Virgianti (2015) reported that isolated R. oligosporus from local tempeh was capable of producing antimicrobial bioactive compounds. The bioactive compounds produced were able to inhibit enteric pathogenic bacteria and have different inhibition zones.

Table 2: The population of A. flavus FNCC6109 colonies in corn kernels added with culture filtrate of R. oligosporus before and after the incubation period

Treatment	Total Population of A. flavus FNCC6109 (CFU g^-1)		Enhancement of A. flavus FNCC6109 (%)
Treatment	Population Before the Incubation (T₀)	Population After the Incubation (T₁₅)
A	0.00	0.00^a ±0.00	0
B	21 × 10⁴	44.7 × 10^4b ±0.068×10⁴	53
C	14 × 10⁴	18.0 × 10^4c ± 0.062×10⁴	22.2
D	11 × 10⁴	13.7 × 10^4cd ± 0.051×10⁴	19.7
E	9 × 10⁴	10.3 × 10^4de ± 0.134×10⁴	14.4
F	6 × 10⁴	6.7 × 10^4df ± 0.127×10⁴	10.4
G	5 × 10⁴	5.3 × 10^4f ± 0.196×10⁴	5.7

T₁₅is the average value with three replications and with different letter notations in the same column indicating a significantly different average value (P ≤ 0.05) based on the Duncan test after conducting the analysis of variance

Fig. 2: Inhibition of R. oligosporus against A. flavus FNCC6109 on PDA media with an incubation period of 4 days and at a temperature of 28°C (K = Control; P = Treatment; A = A. flavus FNCC6109; R = R. oligosporus)

Fig. 3: Inhibition of R. oligosporus culture filtrate against A. flavus FNCC6109 in vitro on PDA media with an incubation period of 4 days and at a temperature of 28°C (Information on the figure KF = filtrate culture R. oligosporus.; A = A. flavus FNCC6109)

The culture filtrate of R. oligosporus at five days incubation period had the highest inhibitory ability in inhibiting A. flavus FNCC6109. During the five days incubation period, the enzymes produced by R. oligosporus were thought to have optimum enzyme activity. This is supported by the statement of Pujiati et al. (2017) that the levels of crude protein cellulase enzymes from R. oligosporus in sugarcane bagasse substrate incubated for 3 to 12 days had an increase in the amount of protein content. The increase in protein content also indicated the activity of the cellulase enzyme has increased. This happened due to the increasing length of fermentation time. R. oligosporus was able to produce β-glucanase enzymes. The β-glucanase enzyme is an extracellular enzyme capable of hydrolyzing carbohydrates of the glucan group. This meets the statement of Ravindran et al. (2018) that R. oligosporus is capable of producing β-glucanase enzymes. Glucans are one important component in making the cell walls of fungi in general. Glucans can be hydrolyzed by the β-glucanase enzyme produced by several types of fungi, namely R. oligosporus and Trichoderma spp. The β-glucanase enzyme produced also has an important role in the self-defense mechanism against pathogenic fungal attacks. Research conducted by Lorito et al. (1994) stated that the β-glucanase enzyme showed antifungal activity by hydrolyzing the glycan structures present in the cell walls of pathogenic fungi. The structure of glucans is known to be mostly found at the tip of the hyphae, so the pathogenic fungal hyphae are not able to grow properly in the presence of the β-glucanase enzyme. Furthermore, it was also confirmed by Budiarti and Widyastuti (2011) that the β-glucanase enzyme was able to influence the growth of hyphae, where the hyphae experienced swelling and necrosis.

Based on the statistical test, it showed the effect of the concentration treatment of R. oligosporus culture filtrate, which was given into the corn kernels during an incubation period of 15 days against A. flavus FNCC6109 in corn kernels. The average population of flavus FNCC6109 in treatment B (without culture filtrate) was 44.710 CFU g^-1, which was significantly different (P ≤ 0.05) with treatment G (corn kernels with A. flavus and culture filtrate of R. oligosporus with a concentration of 50% (v/v). The concentration treatment of R. oligosporus culture filtrate given in corn kernels was able to decrease the population of A. flavus FNCC6109. It was proven by the decreasing tendency in the population of A. flavus FNCC6109; the higher the concentration of the culture filtrates given, the lower the population of A. flavus FNCC6109. This is thought to be R. oligosporus experiencing very fast growth and ability to compete in obtaining nutrients. In addition, it is also suspected that the performance of enzymes and other metabolites played a role in damaging the spore wall components of A. flavus FNCC6109 so that the spores of A. flavus FNCC6109 were not able to grow properly. This statement is supported by Calestino et al. (2006), who stated that R. oligosporus is able to produce β-glucanase enzyme, which is thought to be able to damage the spore walls of A. flavus FNCC6109. Furthermore, R. oligosporus is able to produce volatile compounds, which are thought to affect in inhibiting the growth of A. flavus FNCC6109. This is supported by the statement of Huang et al. (2019) that R. oligosporus is able to produce volatile compounds such as ethanol, isobutyl alcohol, and three methyl butanol, where these compounds are thought to be able to inhibit the growth of A. flavus. Similar results were stated by Guneser et al. (2017) that R. oligosporus produces volatile compounds. The isolation results of the volatile compounds by R. oligosporus obtained in fermented soybeans and barley, volatile compounds, namely ethanol, acetone ethyl acetate, 2-butanone, 2-methyl-1-butanol.

Based on the presumable performance of the enzymes and bioactive compounds produced by R. oligosporus, it can be attempted that R. oligosporus could be used as a biocontrol agent in inhibiting the growth of pathogenic fungi. This statement is supported by Monk et al. (2020) that the inhibition mechanism of R. oligosporu spp. against A. flavus and A. parasiticus by binding to element C (Carbon) contained in beans, causing A. flavus and A. parasiticus to lose. The utilization of R. oligosporus culture filtrate has been conducted in many industries and can be used as a probiotic agent. Besides its ability to be a biocontrol in inhibiting the growth of pathogenic fungi and degrading mycotoxin compounds, R. oligosporus is thought to increase the quality of food quality as reported by Maryana et al. (2016), which stated that R. oryzae culture could increase the protein content in liquid tofu waste. The results obtained during fermentation for 48 h could increase the protein content by 0, 47%. Suarti and Budijanto (2021) stated that the use of R. oligosporus in concentrate feed with fermentation process was able to increase weight and reduce phytic acid levels. Phytic acid is a phosphorus compound that can bind mineral components such as iron, calcium, and zinc so that it cannot be absorbed directly by the body. According to Bhavsar and Khire (2014) that giving R. oligosporus culture filtrate in concentrate, feed is thought to be able to produce phytase enzymes that affect in breaking down the phytic acid.

Based on the ability of the culture filtrate tested on corn kernels, it generally had a positive correlation, both in vitro and in vivo, in inhibiting the growth of A. flavus FNCC6109. So it can be seen that the culture filtrate of R. oligosporus has the potential in inhibiting the growth of A. flavus FNCC6109 in corn kernels with a decrease in the population of A. flavus FNCC6109 after an incubation period of 15 days.

Conclusion

The R. oligosporus filtrate inhibited A. flavus FNCC6109 with the highest percentage of inhibition was 61.92, and the lowest was 44.42. The highest inhibition rate was 67.27±2.70 with an incubation period of 5 days, and the lowest was 61.27±5.13 with an incubation period of 3 days. The filtrate concentration of 50% suppresses A. flavus FNCC6109 with the lowest population average of 55.3 × 10⁴± 0.196 CFU g^-1 after an incubation period of 15 days. Further research needs to be conducted on the enzymes and active compounds produced by R. oligosporus, as well as testing the decrease in aflatoxin content produced by A. flavus FNCC6109 in corn kernels.

Acknowledgments

The authors would like to thank the Head of the Microbiology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, and the Joint Laboratory of the Faculty of Mathematics and Natural Sciences, Udayana University.

Author Contributions

IBGD, as the head of the research, was in charge of designing research and isolation of R. oligosporus and A. flavus FNCC6109; AAKD and Arofi were in charge of preparing tools, media, and conducting dual culture tests; IWS performed fungal growth measurements and tabulated data; and IKW performs data analysis, translation, and publication.

Conflicts of Interest

The authors declare no conflict of interest

Data Availability

Data presented in this study will be available on a fair request to the corresponding author.

Ethics Approval

Not applicable in this paper

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