Auricularia auricula is one of the main edible fungi widely cultivated in China. Aspergillus flavus that is the most common class of pathogenic bacteria fungi produced the high toxicity of the aflatoxins, which is one of the fungal diseases of A. auricula. Morphological observation of A. flavus mycelium and A. auricula mycelium in plate confrontation, mutual inhibition of growth at different germination times, and the interaction of A. flavus liquid culture solution and A. flavus volatiles with A. auricula mycelium were used to investigate the mechanism of the interaction between A. flavus and A. auricula mycelium. Mycelium of A. auricula and A. flavus had a mutual inhibitory effect, but A. auricula mycelia had stronger inhibitory effect on the growth of A. flavus mycelia, besides that the results of the interaction between A. flavus volatiles and A. auricula volatiles were also the same and the inhibition of A. flavus by volatiles from A. auricula mycelium about 11%. After 240 h, the inhibition rate of A. flavus liquid culture solution on A. auricula mycelium reached up to about 20%. It was analyzed that some antimicrobial substances such as small peptides and organic acids produced in the metabolites of A. flavus liquid culture solution were the main reasons of the growth inhibition of A. auricula mycelium. The main inhibitory substances were 2-Butanone, 2-Butanone dimer, etc. Further study of AFT B1 and AFT B2 revealed that aflatoxins could migrate from the medium to the mycelia and the fruiting body of A. auricula, but the migration rate was basicly lower than 10‱. The mycelia of A. auricula had strong degradation of aflatoxins in the growth process. So the mechanism of antifungal activity of these substances was studied to provide a theoretical basis for future chemical synthesis.
Research Article
Mutual inhibition of Aspergillus flavus and Auricularia auricula mycelium for the prevention of competing diseases during growth of fungi
https://doi.org/10.21203/rs.3.rs-3864001/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 10 May, 2024
Read the published version in Advanced Composites and Hybrid Materials →
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Aspergillus flavus
Auricularia auricula
Mutual inhibition
Aflatoxins migration
Auricularia auricula is a valuable medicinal and edible gelatinous mushroom, one of the major edible mushrooms widely grown in China, rich in protein, polysaccharides, minerals, vitamins and other nutrients. Although Aspergillus spp. (e.g. A. auricula, Aspergillus fumigatus and Aspergillus flavus) are not the main invasive pathogens of A. auricula, causing contamination of the culture substrate in strain cultivation and bag cultivation[1]. In particular, A. flavus is easily transmitted by air currents and various insects in a favorable environment spores to begin to grow and produce aflatoxins that are a class of mycotoxins produced mainly by A. flavus and Aspergillus parasiticus, which are highly toxic, carcinogenic and mutagenic to the human liver[2]. At present, 18 types of aflatoxins had been discovered and isolated and identified, common contamination of agricultural products were mainly aflatoxins B1, B2, G1, G2, M1, M2, of which AFT B1 was the most toxic[3, 4]. There was a research showing that edible fungal mycelial diseases were divided into invasive and competitive diseases, the two types of disease in the pathogenic species, performance characteristics, pathogenic mechanisms, epidemiological conditions[5]. As one of the competitive pathogens of edible mushrooms, A. flavus mainly causes contamination of the culture medium system during the growth of edible mushrooms.
Trichoderma is the most serious contamination of A. auricula pathogens. It could be directly entangled in A. auricula mycelium to inhibit its growth, but also through the production of metabolites to play an inhibitory role[6, 7]. It has been demonstrated that T. pleuroticola could completely inhibit the growth of A. auricula mycelium meanwhile produce antimicrobially active metabolites. T. pleuroticola could produce volatile and nonvolatile metabolites to have different degrees of inhibition on A. auricula mycelium, and fermentation production of T. pleuroticola produced active antifungal compounds to carry out their chemical synthesis production for further studying the biosynthetic pathway of these active antifungal compounds in T. pleuroticola. Aspergillus hartmannii produced active substances, including chitinase, protease and glucose endonuclease, which were able to degrade the fungal cell wall and destroy the fungal hyphae and spores, and Aspergillus pelta produced antifungal substances at low pH to inhibit the growth of edible fungi[8, 9]. While studies on the effect of A. flavus on A. auricula had only been able to demonstrate that it contaminated the A. auricula culture medium, it was not yet known whether A. flavus has an effect on A. auricula mycelium. There were few studies on the way the two interact and the main substances that produced this effect.
The interaction between A. flavus and A. auricula mycelium was studied by morphological observation of plate standoff test and scanning electron microscopy to determine the specific competition and to study the main components of the competition so as to provide a scientific basis for the prevention and control of A. flavus in the process of growth of A. auricula. Some studies have shown that A. auricula was the species with the highest microbial load, but studies on aflatoxins migration in food have been scarce since they were carried out long ago in the late 60s ~ 80s. Due to the complexity of the matrix, the migration pattern of metabolite production by pathogenic bacteria varies in different food groups, depending on the structure of the type of mycotoxin involved[10]. In addition, we studied the toxicity production pattern of A. flavus, the suspension of A. flavus spores was separately accessed to different culture substrates, and LC-MS determined the aflatoxins content, and the investigation of the accumulation of pathogenic fungi and their metabolites in edible mushrooms with the migration of aflatoxins during growth of A. auricula. The present study was of great significance in the evaluation of the safety of edible mushrooms[11–13].
2.1 Strains and materials
Hei 29 (H 29) and Heiwei 15 (HW 15) of A. auricula strains were purchased from the Institute of Microbiology (Heilongjiang, China). A. flavus was isolated and characterized from the A. auricula packages suspected to be contaminated by A. flavus. Aflatoxin (B1, B2, G1, G2) Standards were obtained from Sigma-Aldrich (St. Louis, MO, USA).
2.2 Instrumentation
Morphological was made using an environmental scanning electron microscope (SEM, Quanta 200, FEI Company, USA). A. flavus liquid culture solution was identified with liquid chromatography-mass spectrometry (LC-MS, UltiMate3000, Thermo Fisher, USA). Aflatoxin content was determined with high-performance liquid chromatograph-fluorescence (HPLC, UltiMate3000, Thermo Fisher, USA). The volatile organic compounds (VOCs) of the mycelium were identified with gas chromatography-ion mobility spectroscopy (GC-IMS, FlavourSpec® Gas Phase Ion Mobility Spectrometer, GAS Company, Germany).
2.3 Preparation of culture mediaA. auricula PDA growth medium: 1000 mL water and 20 g bran were added into 200 g peeled potato, boiling at 100 ℃ for 20min and filtering and removing dregs. 20 g glucose, 1 g vitamin B, 3 g peptone, 2 g KH2PO4 and 1 g MgSO4·7H2O were added into the filtrate. If it was a solid medium, the 20 g agar and water were added into the medium after rehydrating.
A. auricula bag cultivation medium: 90% cottonseed husk, 5% bran, 3% cornstarch, 1% gypsum powder, and 1% lime powder was mixed with an appropriate amount of water so that the water content of 60% ~ 70%. The 30 g bag material was put into a glass dish (d = 9 cm) and pressed into a 1.5 cm-thick cake.
A. auricula viscous slurry medium: the soaked dried A. auricula was pulped in a wall-breaking machine to a sticky consistency, and 30 g slurry was loaded into a glass Petri dish (d = 9 cm), flattened to make a 1.5 cm-thick cake.
Preparation of aflatoxin-containing medium: 100 µL spore suspension with a concentration of 106 spores/mL was inoculated into sterile PDB medium, mixing homogeneously and then sealing, incubating for 168 h at 150 r/min and 28 ℃. The filtered mycelium was collected after centrifuging at 8000 r/min for 10 min, filtering again. The concentrations of AFT B1 and AFT B2 in the resulting A. flavus filtrate were determined to be 474.21 ng/g and 11.36 ng/g, respectively[4].
2.4 Morphological examination
Plate standoff and scanning electron microscopy observed mycelial interactions. A. flavus inoculated on the PDA plate was cultured in the dark at 28 ℃ for 72 h. H 29 and HW 15 inoculated on the PDA plate were cultured in the dark at 28 ℃ for 240 h. The inoculated A. auricula discs were punched with a hole punch (d = 7 mm) and placed on one side of the PDA plate of a Petri dish (d = 9 cm). After the mycelium grew to half of the Petri dish, the other side was inoculated with A. flavus discs (d = 7 mm) so that the two discs were 7 cm apart to incubate at 28 ℃ in a constant temperature incubator protected from light[14]. The inoculated A. auricula discs and A. flavus discs were used as the control group. The growth radius of each colony was measured every day, and the growth inhibition of the two kinds of mycelium was calculated to study the interaction between A. flavus mycelium and A. auricula mycelium. Mycelial growth inhibition can be calculated by the following formula[14]:
2.5 Effect of Interaction between A. flavus and A. auricula mycelium during the developmental culture of A. auricula packets
A comparison study of the differences in the interaction between A. flavus and A. auricula mycelium in simultaneous and delayed inoculation mycorrhizal cultures has been conducted. The sterilized A. auricula packets were inoculated in the incubator, whose temperature was 25 ℃. Relative humidity (RH) was 40%. The experimental group was inoculated with both HW 15 and A. flavus strains. In contrast, the control group was inoculated with only HW 15 strains or A. flavus strains, and the growth of the two types of mycelium was observed and recorded[16]. The sterilized A. auricula bag was inoculated with HW 15 strains and then cultured in the incubator (25 ℃, RH = 40%). Some of the bag was taken out when the A. auricula mycelium covered half of the bag, and the bottom end of the bag was punched to inoculate with activated A. flavus spores to continue the culture[17]. The growth of the mycelium on the bag was observed and recorded. The inoculation of A. flavus was the experimental group, and the non-inoculation of A. flavus was the control group to study the interaction between A. flavus and A. auricula mycelium during the germination culture of A. auricula mycelium packages.
2.6 Effect of A. flavus liquid culture solution on the growth of A. auricula mycelium
After the effect of A. flavus liquid culture solution on A. auricula mycelium by Morphologica, LC-MS analyzed the culture solution for metabolic composition, and the substances that played a major role in the inhibitory effect of A. flavus liquid culture solution on A. auricula mycelium were investigated.
100 µL A. flavus spore suspension with a concentration of 106 spores/mL was inoculated into a sterile PDB medium, mixing homogeneously and sealing. The medium was incubated at 150 r/min and 28 ℃ for 48, 96, 144, 192 and 240 h, and the culture solution was filtered and centrifuged at 8,000 r/min for 10 min before being filtered again. The filtrate, which was 20% of the volume of the A. auricula growth medium, was added to the A. auricula solid growth medium. The activated strains (H 29 and HW 15) were inserted into the plate and incubated in darkness at 28 ℃ for 240 h, and the A. auricula discs were punched out with perforators (d = 7 mm) and placed in the center of the plate of A. auricula solid growth medium with the addition of A. flavus liquid culture medium. The solid-state growth medium of A. auricula without A. flavus liquid culture solution was used as the control and incubated at 28 ℃. The colony diameters of each group were measured every 96 h to calculate the inhibition rate of A. flavus liquid culture solution on the growth of A. auricula mycelium[18, 19]. Qualitative and quantitative analysis of liquid cultures of A. flavus incubated for 240 h by LC-MS has been investigated[20, 21].
2.7 Effect of A. flavus volatile substances on A. auricula mycelium
The interaction between A. auricula mycelium and A. flavus volatiles was observed morphologically, and the volatiles of A. auricula mycelium and A. flavus mycelium were determined to further study the main substances that played an inhibitory role by Gas Chromatography-Ion Mobility Spectrometry (GC-IMS)[22].
The activated A. flavus and A. auricula strains were separately extracted and simultaneously accessed to the ends of the same freshly prepared dichotomous PDA Petri dishes, which were sealed with sterile airtight parafilm (100 mm × 2 mm) sealing film and incubated at 28 ℃ at constant temperature in darkness[23]. The PDA dishes inoculated with A. auricula species alone and A. flavus species alone were used as the control, and the mycelial growth of each group was observed at 24 h intervals while measuring the radial growth radius of the colonies. The bacterial inhibition rate (P, %) was calculated according to the following formula to study the interaction of volatiles from A. auricula and A. flavus[24]:
2.8 Analysis of volatile organic compounds (VOCs) of A. auricula mycelium and A. flavus mycelium
1 mL sterile A. auricula solid medium was placed in a 20 mL headspace bottle. After the medium solidified, the activated A. flavus and A. auricula discs were punched with a punch (d = 2 mm) and inoculated in the center of the medium in different headspace flasks to incubate at 28°C for 72 h. VOCs were identified with GC-IMS. The test conditions were as follows: the headspace bath temperature was performed at 45°C for 20 min; the temperature of headspace injection was performed at 85°C; the injection volume was set at 500 µL, and the incubation speed was 500 rpm. The GC-IMS system was equipped with an MXT-5 (15 m × 0.53 mm and 1.0 µm) chromatographic column (RESTEK, USA). The chromatographic column was programmed to 60°C and worked for 60 min. Nitrogen (99.999%) was used as the carrier gas and drift gas, and the flow rate of drift gas was 150 mL/min.
2.9 Migration of aflatoxins during the growth of A. auricula
2.9.1 migration of aflatoxins in the liquid medium culture of A. auricula
A. auricula liquid growth medium was mixed with aflatoxin-containing medium in equal proportions (the initial concentration of aflatoxins in the medium was 237.11 ng/g for AFT B1 and 5.68 ng/g for AFT B2), and accessed to 5 mL A. auricula liquid strains, which were cultured at 150 r/min and 28 ℃ until the 96, 192, 288, 384, and 480 h. The filtered A. auricula mycelium was measured to determine the liquid culture and the aflatoxin content in the fungus mycelium.
2.9.2 migration of aflatoxins during solid-state medium culture of A. auricula
The aflatoxins migration under solid-state culture was monitored by a cellophane incubation method. The cellophane was cut into round pieces, moistened in sterile water and covered with Petri dishes (d = 9 cm) containing a solid medium of A. auricula. The disks (d = 7mm) were punched from the edge of 168-h-old A. auricula colonies and inoculated to 1 cm from the edge of the Petri dishes. The disks (d = 7 mm) were punched from the edge of 120-h-old A. flavus colonies and placed on the other side of the Petri dish, which was then sealed and incubated at 28°C. The inoculation was carried out on the incubation 192, 288, 384, and 408 h [25]. At 192, 288, 384 and 576 h, the mycelium of A. auricula was scraped off with a spatula, washed with distilled water and then put into a 2 mL centrifuge tube for grinding, and then the aflatoxins were extracted and detected. At the same time, the aflatoxins' content was extracted and detected by striking the aflatoxins' colony agar blocks in the corresponding culture medium.
2.9.3 migration of aflatoxins during the growth of A. auricula fruiting bodies
A. flavus spore suspension was mixed with sterilized A. auricula packet culture material at 100:1 (ml/kg) and then sealed to protect at a constant temperature of 28 ℃ in darkness for 168 h, so that A. flavus could fully grow in A. auricula packet medium and produce toxins, which was A. flavus material. The A. flavus material mixed with A. auricula culture material, so that the proportion of A. flavus material in A. auricula mixed culture medium were 0%, 25%, 50% and 100%, stirred evenly after the four different packages of medium were loaded into the polypropylene packet bag, and the tube was put the middle of a 10 mL centrifuge, sealing, and autoclaving at 121 ℃ for four h. The packets cooled down and then accessed to the 10 mL of A. auricula, sealed in a 25 ℃ and RH = 60% incubator. After 1080 ~ 1440 h, the A. auricula mycelium grew all over the mycelium bag. After the mycelium bag was perforated, it was protected from light in the mushroom room at 22 ℃ and RH = 80%, and could be sprayed with water when the black ear base was formed after 240 ~ 288 h. After 168 ~ 336 h, the earpieces were fully opened and the color changed from black to brown, and then they could be harvested. After harvesting, each group of A. auricula was dried naturally in the sun in the open air, and the aflatoxins content was measured. The aflatoxin content in the culture material of each group was also measured after the first crop of fungus was harvested.
2.10 Statistical Analysis
Excel software was used to present the experimental data, and all data analyses were performed by analysis of variance (ANOVA) using the SPSS statistical analysis system software (SPSS 22, Chicago, USA) and presented as the mean ± standard deviation (SD). Significant differences were identified using Duncan's new multiple range test at the 𝑃 = 0.05 level. Three replicates for each experimental treatment were considered and each measurement was performed in triplicate.
3.1 Morphological examination
The results of the plate standoff test of H 29 and HW 15 with A. flavus, respectively, are shown in Fig. 1. On 72nd h after the accession of A. flavus, the mycelium on the edge of H 29 was sparse but increased in thickness, and the edge of A. auricula HW 15 appeared obvious protruding velvet mycelium. The growth of A. auricula mycelium was inhibited, and the influence of A. flavus on the morphology of A. auricula mycelium was larger; on 120th h, there was a clear antagonistic line between the two bacteria; on 168th h the demarcation line between the two bacteria appeared, and the mycelium of the two bacteria in the contact position was no longer growing, while the mycelium of HW 15 grew almost all over the plate. The mycelium of HW 15 almost grew all over the plate, and the colony diameter of A. flavus in the confrontation plate was significantly smaller than that of the control group; at 216 h, obvious yellow coils appeared in the middle of the colony of A. auricula, which might be the aging of the strain. The colony diameter of A. flavus in the confrontation process was significantly smaller than that of the control group. From the picture of 72 h, the spore formation rate of the standoff group was also significantly lower than that of the control group, and at 216 h, there were obviously scattered protrusions of A. flavus on the back of the medium. Therefore, there was an interaction of morphology and mycelial growth between A. flavus, H 29, and HW 15.
The mutual inhibition rates of A. flavus and A. auricula (H 29 and HW 15) mycelia during plate standoff are shown in Fig. 2. The two mycelia had mutual inhibition during the confrontation, and the inhibition rate of A. auricula mycelia on A. flavus mycelia was significantly higher than that of A. flavus mycelia on A. auricula mycelia (P < 0.05). The mutual inhibition rate of the two mycelia increased significantly with time (P < 0.05), and the inhibition rate of A. flavus on HW 15 reached 18.40% at 144 h. The mycelial growth of A. flavus against HW 15 was always inhibited during the confrontation culture, and the inhibition rate decreased with 48 h of culture and then increased significantly (P < 0.05). At 144 h, the inhibition rate reached 29.10%, which was significantly higher than the inhibition rate against HW 15. The inhibition rate of A. flavus to H 29 in the 216 h of the confrontation culture process reached 31.10%, and the inhibition rate of H 29 mycelium to A. flavus reached 59.60%, which seriously affected the normal growth of mycelium.
Therefore, the growth of A. flavus filaments and A. auricula mycelia were mutually inhibited, and the inhibition rate of A. auricula mycelia on A. flavus filaments was significantly higher than that of A. flavus filaments on A. auricula mycelia. There was a significant difference in the inhibitory effect of A. flavus filaments on the growth of the two types of A. auricula mycelia, with a higher inhibition rate for HW 15 mycelia; the difference in the inhibitory effect of the two types of A. auricula mycelia on the growth of A. flavus filaments was not significant.
In order to further investigate the interaction between the mycelium of A. auricula and A. flavus, SEM was used to observe the mycelium, and Fig. 3 (a, b) were the electron microscope images of the mycelium of H 29 and HW 15. There was little difference between the two in the diagrams. The mycelium of A. auricula was smooth and thin, with obvious reticulation and small branches. Figure 3 (c, d) was the electron microscope diagrams of A. flavus filaments, which were thicker, had particulate matter attached, and had obvious spore heads. Figure 3 (e-h) shows the electron microscope diagrams of the interaction of the two mycelia. A. auricula filaments had the effects of wrapping, attaching, and entangling to the filaments of A. auricula, which could inhibit the growth of A. flavus mycelium from Fig. 3 (e-g). As shown in Fig. 3 (f), the spores of A. flavus were seen to attach to A. auricula mycelium, thus inhibiting the growth of A. auricula mycelium. A. auricula mycelium and A. flavus mycelium inhibited each other, which was consistent with the characteristics of competitive diseases.
3.2 Effect of Interaction between A. flavus and A. auricula mycelium during the developmental culture of A. auricula packets
The interaction between A. flavus and A. auricula mycelium during the germination culture of A. auricula mycelium packages was illustrated by the comparison of the results of simultaneous inoculation germination culture and delayed inoculation germination culture tests (Fig. 4). In the early stage of the simultaneous inoculation culture, the growth rate of A. flavus mycelium was significantly faster than that of A. auricula mycelium, and it was the dominant species. At about 288 h, A. flavus mycelium grew all over the whole packet from the top to the bottom, while the delayed inoculation culture inoculated with A. flavus, the two kinds of mycelium grew in opposite directions. After 120 h, the A. auricula mycelium covered the A. flavus colonies and grew downward, but the growth of A. flavus mycelia could not be observed. With the increase of time, the white mycelium of A. auricula inoculated at the same time gradually covered A. flavus mycelium. After 432 h, A. auricula mycelium was obviously in a dominant position, and after 720 h, A. auricula mycelium covered the whole mycelium bag, and the color of the bag gradually became white. Because of the early germination stage, there was still part of the oxygen in the mycelium, A. flavus, as aerobic bacteria grew rapidly. With the increase of time, the oxygen in the mycelium was gradually consumed. The growth of A. flavus slowed down, and the growth of A. auricula mycelium was accelerated. Gradually, the A. auricular mycelium in the contaminated A. flavus mycelium on the mycelium could also continue to grow. After the delayed inoculation culture inoculated for 480 h, A. flavus mycelium was completely covered by A. auricula mycelium, and the whole mycelium turned white.
As shown in Table 1, the growth rates of A. auricula mycelium and A. flavus mycelium in the experimental packages inoculated at the same time with germination culture were smaller than those in the control packages, and the growth of A. flavus mycelium and A. auricula mycelium in the packages had a reciprocal inhibition. The inhibition rate of A. auricula mycelium on the growth of A. flavus mycelium (43.52%) was significantly higher than that of A. flavus mycelium on A. auricula mycelium (7.95%). The results of this experiment were consistent with those of the plate standoff test, which indicated that the growth of A. flavus filaments and A. auricula filaments had a mutual inhibitory effect.
|
Strains |
Growth Rate (cm/d) |
Inhibition Rate (%) |
|
|---|---|---|---|
|
Control Bacterial Packages |
Test Group Bacterial Packages |
||
|
HW 15 |
0.447 ± 0.006 |
0.411 ± 0.008 |
7.95 ± 0.45 |
|
A. flavus |
0.873 ± 0.015 |
0.493 ± 0.011 |
43.52 ± 1.17 |
Delayed inoculation germination culture when compared with the control group, the growth of the test group of bacterial packets of A. auricula mycelium and A. flavus mycelium were inhibited to different degrees, as shown in Table 2. At the early stage of inoculation with A. flavus, the growth rates of A. flavus and A. auricula mycelium were not close to each other, and the growth rate of A. auricula mycelium was 7.87%. With the increase of time, the two kinds of mycelium were close to each other and covered each other, and growth was significantly inhibited. The growth rate of A. auricula mycelium to A. flavus was 42.61%, and the growth rate of A. flavus to A. auricula mycelium was 28.40%. Therefore, both inoculation methods showed that the growth inhibition rate of A. auricula mycelium to A. flavus mycelium was significantly higher than that of A. flavus mycelium to A. auricula mycelium.
|
Strains |
Growth Rate (cm/d) |
Inhibition Rate (%) |
|
|---|---|---|---|
|
Control Bacterial Packages |
Test Group Bacterial Packages |
||
|
HW 15 |
0.447 ± 0.006 |
Near A. flavus 0.482 ± 0.002 |
-7.87 ± 1.01 |
|
A. flavus coverage 0.257 ± 0.009 |
42.51 ± 2.85 |
||
|
A. flavus |
0.873 ± 0.015 |
Close to the A. auricula 0.625 ± 0.010 |
28.40 ± 1.05 |
After the A. auricula packet was contaminated by A. flavus, although A. flavus mycelium had a significant inhibitory effect on the growth of A. auricula mycelium, the A. auricula mycelium was still able to grow slowly and gradually prevailed. This was different from the ability of A. flavus to infest the mycelium. Some showed that when some of the A. flavus in the mycelium contacted the edible fungal mycelium, the edible fungal mycelium deepened in color, and the mycelium gradually withered[26–28]. Eventually, the mycelium was occupied by A. flavus.
3.4 Effect of liquid culture solution of A. flavus on the mycelium of A. auricula
The effects of A. flavus liquid culture solution on the mycelium of H 29 and HW 15 were shown in Fig. 5 and Fig. 6 (both figures showed the results of the experiment on the 192 h after inoculation with A. auricula strains). After adding A. flavus liquid culture solution, the diameter of the mycelium of the two strains of A. auricula gradually decreased with the increase of time, and the diameter of A. auricula colony was the smallest on the 240 h. However, the addition of A. flavus liquid culture solution had no obvious effect on the morphology of A. auricula mycelium. As shown in Fig. 6, it could be seen that different liquid culture times of A. flavus liquid culture solution inhibited the growth of both kinds of A. auricula mycelium, and there was a significant difference in the inhibition rate (P < 0.05). The highest inhibition rate of A. flavus liquid culture solution for 240 h was 15.85% and 22.92% for H 29 and HW 15 mycelium, respectively, and the lowest inhibition rate of A. flavus liquid culture solution for 48 h was 8.09% and 5.23% for H 29 and HW 15 mycelium respectively. Therefore, the liquid culture solution of A. flavus had a significant inhibitory effect on the mycelium A. auricula strains (H 29 and HW 15) and the inhibitory rate of HW 15 was higher.
The metabolites of A. flavus liquid culture solution was identified by the LC-MS method (Table 3), and the identified metabolites included small peptides, sugars, acids, ketones, alcohols and other compounds. Some small peptides in the results of this experiment might make the decomposition products of extracellular enzymes, and the specific components had to be further extracted and analyzed. The presence of some organic acids in the liquid culture solution of A. flavus might reduce the pH of the culture solution to inhibit the growth of A. auricula mycelium. On the other hand, some small peptide products and organic acids in the liquid culture solution of A. flavus could be used for the synthesis of compounds that inhibit the growth of fungal mycelium after further research.
|
Compounds |
Class I |
Formula |
|---|---|---|
|
Pantetheine |
CoEnzyme and vitamins |
C11H22N2O4S |
|
Cyclo (Pro-Phe) |
Amino acid and Its metabolomics |
C14H16N2O2 |
|
Phytosphingosine |
SL |
C18H39NO3 |
|
N4-Acetylcytidine triphosphate |
Nucleotide And Its metabolomics |
C11H18N3O15P3 |
|
Eicosanoyl-EA |
Organic acid And Its derivatives |
C22H45NO2 |
|
2,4-diacetamino-2,4,6-triphenoxy-D-mannopyranose |
Carboxylic acids and derivatives |
C10H18N2O5 |
|
Carnitine isoC4:0 |
FA |
C11H21NO4 |
|
N-Acetyl-5-aminosalicylic acid |
Benzene and substituted derivatives |
C9H9NO4 |
|
L-Dihydroorotic Acid |
Organic acid And Its derivatives |
C5H6N2O4 |
|
Nicotinic Acid |
CoEnzyme and vitamins |
C6H5NO2 |
|
N, N′-dicyclohexylcarbodiimide |
Alcohol and amines |
C13H22N2 |
|
Phe-Ala |
Amino acid and Its metabolomics |
C12H16N2O3 |
|
D-Erythronolactone |
Aldehyde, Ketones, Esters |
C4H6O4 |
|
(Rs)-Mevalonic Acid |
Organic acid And Its derivatives |
C6H12O4 |
3.5 Effect of A. flavus volatile substances on A. auricula mycelium
The volatile substances of A. flavus were detected, and the results of studying the interaction of volatile substances of A. flavus with the mycelium of A. auricula were shown in Table 4. The inhibitory effect of volatile substances of A. flavus on the growth of A. auricula mycelium was not significant, and the inhibition rates of A. auricula H 29 and HW 15 were 0.24% and 0.28%, respectively However, the inhibitory effect of volatile substances of A. auricula on the growth of A. flavus was significant, and the inhibitory rates of HW 15 and H 29 on A. flavus were 11.32% and 12.19%. Given the more significant inhibition of A. flavus by volatile substances of A. auricula mycelium, volatile substances in A. auricula mycelium were collected by fermentation extraction, isolated, and purified to produce active antifungal compounds might provide a new idea in the field of organic synthesis.
The results of GC-IMS presented the information on each sample's VOCs fingerprinting. The spectra of one of the blank control groups (KB) were selected as a reference. The volatile substances with a higher concentration of A. flavus (LXY) mainly included Ethanol, Isopropanol, Ethyl acetate, 3-Butyronitrile, Ethyl propionate, Isopentanol and Ethyl 2-methylpropionate; the volatile substances with a higher concentration of A. auricula (ME) mainly included Ketones and Alcohols, such as acetone, 2-Butanone, Isopropanol, Isopentanol, and Cyclohexanone, and the content of ethanol in ME was higher than that in the blank substrate. ME was compared with LXY, the volatile substances with higher concentrations in LXY mainly included Ethyl 2-methylpropionate, Ethyl propionate, 3-Butyronitrile, Ethyl acetate, Isopropanol, Ethanol, Furfural, and Ethyl butyrate, which could be used as a reference for the early prevention of A. flavus in the growth of A. auricula. The volatile substances with higher concentrations in ME mainly included 2-Butanone, 2-Butanone dimer, Isoamyl alcohol and acetone, which were mainly used to prevent A. flavus in A. auricula[29, 30]. According to previous studies, it was presumed that these substances had the potential to inhibit the growth of A. flavus filaments ( Fig. 7)[31].
3.6 Migration of aflatoxins during culture of A. auricula
3.6.1 migration of aflatoxins in liquid culture of A. auricula
As shown in Table 5, the migration accumulation as well as degradation of aflatoxins under liquid culture conditions of A. auricula mycelium and changes of aflatoxins content.
|
Time (h) |
AFT B1 Content (ng) |
Mobility (‱) |
Degradation (%) |
AFT B2 Content (ng) |
Mobility (‱) |
Degradation (%) |
||
|---|---|---|---|---|---|---|---|---|
|
Culture Medium |
A. auricula Mycelium |
Culture Medium |
A. auricula Mycelium |
|||||
|
0 |
11855.50 ± 98.46a |
ND |
-- |
-- |
284.00 ± 7.43a |
ND |
-- |
-- |
|
96 |
9463.65 ± 88.07b |
3.86 ± 0.18e |
3.26 ± 0.12e |
20.14 ± 1.12e |
227.70 ± 5.90b |
0.08 ± 0.006e |
2.64 ± 0.13e |
19.80 ± 1.13e |
|
192 |
8052.85 ± 46.81c |
11.94 ± 0.41a |
10.07 ± 0.22a |
31.97 ± 2.32d |
192.65 ± 7.72c |
0.19 ± 0.012a |
6.55 ± 0.24a |
32.10 ± 2.24d |
|
288 |
7276.85 ± 67.33d |
9.51 ± 0.38b |
10.00 ± 0.14b |
38.54 ± 1.84c |
175.60 ± 6.93d |
0.17 ± 0.004b |
6.06 ± 0.18b |
38.11 ± 2.18c |
|
384 |
6989.70 ± 40.65e |
7.53 ± 0.52c |
6.35 ± 0.25c |
40.98 ± 2.15b |
167.60 ± 4.51e |
0.15 ± 0.005c |
5.18 ± 0.16c |
40.93 ± 2.26b |
|
480 |
6121.40 ± 71.83f |
6.82 ± 0.36d |
5.75 ± 0.21d |
48.31 ± 2.61a |
145.55 ± 4.72f |
0.13 ± 0.007d |
4.58 ± 0.17d |
48.70 ± 2.37a |
| Note: The data represented the mean ± standard error of three replicates; different lowercase letters in the same column indicate significant differences between groups (P < 0.05); "ND" means not detected. | ||||||||
As seen from Table 5, A. auricula growing in the liquid environment containing aflatoxins would result in a part of aflatoxins migration and accumulation into A. auricula mycelium and the migration rate of aflatoxins tended to increase first and then decrease. At the early stage of cultivation, the aflatoxins content in A. auricula mycelium increased gradually with time. At 192 h of cultivation, the contents of aflatoxins AFT B1 and AFT B2 in A. auricula mycelium reached the highest values of 11.94 ng and 0.19 ng, respectively, and the aflatoxins mobility also reached the maximum values of 10.07‱ and 6.55 ‱. Again, the aflatoxins content in A. auricula mycelium decreased over time, as did the mobility. After 480 h, the content of AFT B1 and AFT B2 in A. auricula mycelium decreased to 6.82 ng and 0.13 ng, and the mobility of AFT B1 and AFT B2 decreased to 5.75‱ and 4.58‱. The adsorption of mycotoxins by microorganisms such as fungi and yeast was reversible, which might have led to fluctuations in mycotoxin mobility.
The aflatoxins content in the culture medium gradually decreased with time during the liquid culture of A. auricula mycelium with a large decrease (P < 0.05). Although partial migration of aflatoxins to the fungus mycelium occurred, the migration amount was low, and the mobility rates of AFT B1 and AFT B2 were only 5.75‱ and 4.58‱. So the A. auricula mycelium could not only adsorb a small amount of aflatoxins in the medium but also had a strong degradation of aflatoxins in the medium. The results showed that the degradation rate of AFT B1 and AFT B2 in the fungus mycelium reached 48.31% and 48.70% at 480 h[32].
3.6.2 migration of aflatoxins in the solid culture medium of A. auricula
The aflatoxins were detected in both the culture medium and agaric mycelium during the co-culture of agaric mycelium and agaric mycelium on a solid medium. The content of aflatoxins showed a trend of increasing and then decreasing, indicating that aflatoxins produced by A. flavus would also migrate to agaric mycelium during co-culture. However, the migration volume was less than liquid culture (Table 6). The maximum migration volume of AFT B1 and AFT B2 was only 0.1673 ng and 0.0046 ng.
3.7 The migration of aflatoxins during the growth of A. auricula fruit body
As could be seen from Table 7, the concentration of AFT B1 and AFT B2 of A. flavus spore suspension mixed with A. auricula culture medium at 100:1 (mL/kg) for 168 h were 4.69 ng/g and 1.48 ng/g, indicating that aflatoxins would be produced after A. flavus contaminated A. auricula culture medium. If there were aflatoxins in the culture medium, the aflatoxins would migrate from the culture medium to the fruiting body of A. auricula during the growth of A. auricula. The higher the content of aflatoxins in the culture medium, the greater the migration, but the migration rate was not high. The maximum migration rates of the two aflatoxins were below 6‱.
The A. auricula was cultivated with different content of aflatoxins (the material contaminated by A. flavus) and migrated to the fruiting body of A. auricula in different degrees (Table 8). The aflatoxins migration was low, and the aflatoxins content in the inoculum had decreased significantly (P < 0.05). After the first harvest of A. auricula, the content of AFT B1 in three kinds of fungus bags containing aflatoxins decreased by 52.01%, 51.54% and 49.87%, respectively compared with that before cultivation, and the decrease of AFT B2 was 53.24%, 50.99% and 49.19%, which indicated again that A. auricula mycelium could not only absorb a few aflatoxins in the medium, but also had a strong degradation effect on aflatoxins in the medium[33–35]. Therefore, it was speculated that some enzymes produced during the growth of A. auricula could also inhibit or degrade aflatoxins[36].
|
Time (h) |
AFT B1 content (ng) |
AFT B2 content (ng) |
||
|---|---|---|---|---|
|
Culture Medium |
A. auricula Mycelium |
Culture Medium |
A. auricula Mycelium |
|
|
192 |
1286.91 ± 22.61b |
0.0570 ± 0.0027d |
56.28 ± 1.63b |
ND |
|
288 |
1688.50 ± 23.79a |
0.1673 ± 0.0019a |
73.04 ± 1.92a |
0.0046 ± 0.0002a |
|
384 |
1120.88 ± 24.48c |
0.1468 ± 0.0026c |
52.34 ± 1.85c |
0.0039 ± 0.0002b |
|
480 |
917.65 ± 11.32d |
0.1460 ± 0.0018b |
48.38 ± 1.74d |
ND |
| Note: The data represented the mean ± standard error of three replicates; different lowercase letters in the same column indicate significant differences between groups (P < 0.05); "ND" means not detected. | ||||
|
Proportion of A. flavus (%) |
AFT B1 Content (ng) |
AFT B1 Mobility (‱) |
AFT B2 Content (ng) |
AFT B2 Mobility (‱) |
||
|---|---|---|---|---|---|---|
|
Culture Medium |
A. auricula |
Culture Medium |
A. auricula |
|||
|
0 |
ND |
ND |
-- |
ND |
ND |
-- |
|
25 |
586.25 ± 9.85c |
0.21 ± 0.01c |
3.58 ± 0.12c |
185.01 ± 5.68c |
0.08 ± 0.001c |
4.32 ± 0.03c |
|
50 |
1172.50 ± 10.46b |
0.46 ± 0.01b |
3.92 ± 0.13b |
370.04 ± 4.76b |
0.18 ± 0.003b |
4.86 ± 0.10b |
|
100 |
2345.00 ± 15.93a |
1.02 ± 0.02a |
4.35 ± 0.09a |
740.10 ± 6.58a |
0.38 ± 0.005a |
5.13 ± 0.09a |
| Note: The data represented the mean ± standard error of three replicates; different lowercase letters in the same column indicate significant differences between groups (P < 0.05); "ND" means not detected. | ||||||
A. flavus mycelium and A. auricula mycelium had mutual inhibition, and A. auricula mycelium had stronger inhibition on the growth of A. flavus mycelium. The A. auricula mycelium inhibited the growth of A. flavus mycelium by winding, attaching and fusing to the A. flavus mycelium. The spores of A. flavus attached to the A. auricula mycelium inhibit the growth of A. auricula mycelium. The degree of mutual inhibition of A. flavus and A. auricula mycelium was different at different stages of growth. A. flavus liquid fermentation broth had an obvious inhibitory effect on A. auricula mycelium. The longer the cultivation time, the more metabolites small peptides and acids produced, and the stronger the inhibitory effect on A. auricula mycelium. The volatile substances of A. flavus did not have a significant inhibitory effect on A. auricula mycelium, but the volatile substances of A. auricula mycelium had on the growth of A. flavus mycelium, in which the main volatile substances that played an inhibitory role were 2-Butanone, 2-Butanone dimer, Isoamyl alcohol, acetone and so on. Whether A. auricula was cultured in an aflatoxin-containing medium or co-cultivated with A. flavus, aflatoxins migration occurred from the medium to A. auricula mycelium and substrates, but the migration rate was low, basically below 10‱.
Some small peptides in the liquid culture medium of A. flavus could make the decomposition products of extracellular enzymes. The specific components had to be further extracted and analyzed. The presence of some organic acids would also reduce the pH of the culture medium, thus inhibiting the growth of A. auricula mycelium. The specific competition between A. flavus and A. auricula and the main components of the competition, as well as the migration of aflatoxins in the growth process of A. auricula were determined to provide a scientific basis for the safety of consumption of A. auricula and a scientific basis for the prevention and control of aflatoxins in the growth process of A. auricula. In the future, the isolation and purification of volatiles from A. auricula mycelium, which played a major role in inhibiting aflatoxins, will be used to produce these active antifungal compounds. At the same time, it will be possible to apply this discovery to the synthesis of materials, which will provide ideas for the preservation of fruits and vegetables. These active antifungal compounds might also be further investigated based on the biosynthetic pathway of these active antifungal compounds in A. auricula mycelium for chemical synthesis.
Author contribution: Haisheng Zhang, Hui Dang, and Zhe Wang designed this project and contributed to the main manuscript text. Mengjie Yang conducted experiments and wrote the main manuscript text. Baoshan Zhang, Ethan Burcar, Erin Witherspoon, William Winchester, ZME, MHH, MAA, Yu Zhao and Xianpan Bu have contributed to conducting the experiments, preparing figures, and writing. All authors reviewed the manuscript.
Funding This research was funded by the National Key Research and Development Projects during the 13th Five Year Plan (2019YFC1606702) and by the Key Laboratory of Selenium-rich Product Development and Quality Control of the Ministry of Agriculture and Rural Affairs opening project, China (Se-2021C06). The authors extend their appreciation to the Deanship of Scientific Research at Northern Border University, Arar, KSA, for funding this research "work through the project number "NBU-FPEJ-2024-ID-XXX". ZW would like to acknowledge the support from Oakland University.
Data availability The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request.
Competing Interests: The authors declare no competing interests.
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No competing interests reported.
- TableofContent.png
Mutual inhibition between Aspergillus flavus and Auricularia auricula, liquid metabolites of A. flavus inhibited 22.9% of A. auricula mycelium, while volatiles of A. auricula inhibited A. flavus by 12%, and migration and degradation of A. flavus-produced AFT B1/B2 in A. auricula mycelium belong to the competitive diseases.
