Qian Li,Xiaoman Zhu,Yanli Xie*,Jingmeng Liang

Henan Key Laboratory of Cereal and Oil Food Safety Inspection and Control,College of Food Science and Engineering,Henan University of Technology,Zhengzhou 450001,China

ABSTRACT The harm of Aspergillus flavus(A.flavus)and aflatoxin is a severe food safety problem worldwide,which causes huge economic losses every year.Therefore,it is urgent to control the growth of A.flavus and the biosynthesis of aflatoxin.Plant-derived natural compounds are superior to synthetic fungicide in inhibiting the growth of A.flavus benefiting from their high safety to the environment,humans and stock,and low cost.This study aimed to evaluate the antifungal effects and potential antifungal mechanisms of three plant-derived compounds(octanal,nonanal and decanal)against A.flavus.We determined the minimum inhibitory concentrations (MICs) and action mechanism of the three volatile aldehydes on A.flavus and also performed calcofluor white(CW)staining for visualizing the distribution of septa.Cell respiration metabolism and the pathogenicity on maize kernels were also carried out to evaluate the efficacy of the three volatile aldehydes on the growth of A.flavus.The results showed that the three volatile aldehydes could inhibit the germination of spores and mycelial growth of A.flavus,the MICs on spores and mycelia were:octanal(1.0 and 0.5 μL/mL),nonanal(0.5 and 2.0 μL/mL),and decanal(1 and 5 μL/mL).The three volatile aldehydes could strongly damage the integrity of both the cell wall and the cell membrane of A.flavus.Meanwhile,they could decrease the content of total lipid and inhibit respiration metabolism of A.flavus cell.Results of in vitro antifungal test showed that all the three volatile aldehydes could effectively prevent the growth of A.flavus on maize kernels.The study revealed that octanal,nonanal and decanal could effectively inhibit the growth of A.flavus both in culture medium and on maize kernels to different extent.The results confirmed that the plant-derived compounds could be developed into promising antifungal agents applied in the preservation of grains.This study provides a theoretical basis for the research and application of potential antifungal agents.

Keywords:Aspergillus flavus Octanal Nonanal Decanal Antifungal mechanism

1.Introduction

Aspergillus flavus(A.flavus)is an opportunistic pathogen of agricultural crops,such as maize,peanut and cottonseed[1].In addition,A.flavus also produces highly toxic secondary metabolites known as aflatoxins(AFB1,B2,G1,G2)[2].Therefore,it is urgent to prevent contamination by A.flavus with safe and effective methods.

With the ever-increasing numbers of fungicide-resistant pathogens,plant essential oils have been widely studied as antifungal compounds,as well as to determine the mechanism underlying their antifungal effects.In recent years,quite a few natural products have been authorized by the U.S.Food and Drug Administration(FDA)as food additives,such as cinnamaldehyde,citral,thymol,etc.[3].

Octanal,nonanal and decanal are three common aldehydes present in essential oils extracted from many plants,e.g.,green tea and soybean[4],broom bush[5],fish-mint[6],and camphor bush[7].They have already been authorized as safe food additives by the China National Center for Food Safety Risk Assessment(CFSA).Octanal,nonanal and decanal are also GRAS compounds [3].Besides, octanal,nonanal and decanal have been reported to inhibit Penicillium spp.and some other fungi[8–10].Zhou et al.[11,12]have studied the inhibitory mechanism of decanal,the alteration of the proteome and genome of Penicillium expansum (P.expansum).However, the antifungal effects and the mechanisms of the three volatile aldehydes on A.flavus are still unknown.

Octanal, nonanal and decanal share a similar carbon backbone,where the only difference in the structure is the respective length of their carbon chains(C8-C10)(Chemical formulas are shown in Fig.1).Knowledge of whether this structural difference correlates with antifungal abilities is still lacking.Therefore,the susceptibility of A.flavus in response to the three volatile aldehydes(minimum inhibitory concentration,growth of A.flavus on maize kernels)and the underlying antifungal modes of action were investigated from the following four aspects: (1) the integrity of cell walls; (2) the permeability of cell membranes; (3) total lipid content;and(4)cell respiration metabolism.

2.Methods

2.1.Antifungal activity determination of the three volatile aldehydes

2.1.1.Germination inhibition rate

The inhibitory effects of octanal(97%,CAS:124–13-0,Shanghai Aladdin Reagent Co., Ltd., Shanghai, China),nonanal(97%;CAS:124–19-6,Macklin Biochemical Co.,Ltd.,Shanghai,China)and decanal(97%;CAS:112–31-2,Shanghai Aladdin Reagent Co.,Ltd.,Shanghai,China)on A.flavus spore germination were tested in a 96-well plate.In detail, spores (5 × 105spores/mL) were added into a 96-well plate.The final concentrations of octanal,nonanal and decanal were 0(set as control),0.125,0.250,0.500,1.000,and 2.000 μL/mL,respectively.The plate was incubated at(28±2)°C for 48 h,and then was placed under an inverted optical microscope(BM-37XBC,Shanghai BM Optical Instruments Manufacture Company Ltd.,Shanghai,China)for observing the spores.The minimum inhibitory concentration (MIC) is represented as the minimum concentration at which the germination inhibition rate was 100%at 48 h.The germination inhibition rate was calculated as follows:

Fig.1.Chemical formulas of octanal,nonanal and decanal.

where S1and S0represent the numbers of un-germinated spores and the spores at the seeding time point, respectively.Three images were included for the counting.

2.1.2.Mycelial growth inhibition rate

The three volatile aldehydes were mixed with 10 mL Sabouraud's Dextrose Agar(SDA,AR,Kermel Chemical Reagent Co.,Ltd.,Tianjin,China)and poured into sterilized Petri dishes (60 mm diameter).We have found from the preliminary experiments that decanal failed to inhibit the mycelial growth at a concentration lower than 2 μL/mL.Therefore,we selected the final concentrations of 0,0.25,0.50,1.00,and 2.00 μL/mL for octanal and nonanal,and 0,2,3,4,and 5 μL/mL for decanal.A 6-mm A.flavus mycelium plug was placed onto the amended SDA plate,which was incubated at (28 ± 2) °C for 48 h.Colony diameter was measured at 24 h and 48 h, respectively.The MIC is represented as the minimum concentration at which the 48-hour mycelial growth inhibition rate was 100%.The mycelial growth inhibition rate (MGI, %) was calculated as follows:

where dc(cm)is the mean colony diameter for the control sets and dt(cm)is the mean colony diameter for the treatment sets.Each experiment was repeated for 3 times.

2.2.Cell wall integrity

To investigate the influence of the three volatile aldehydes on the cell wall,Calcofluor White(CW,CAS:4404-43-7,Maokang Biotechnology Co.,Ltd.,Shanghai,China)was used for assessment of any changes to cell wall integrity[13],as it can specifically bind with chitin[14].Septa are formed by the concave deformation of the outer cell wall,which are special intracellular structures of mycelia to separate mycelia into small cells.Chitin is the main component in septa.Therefore,the number of septa could reflect the cell wall integrity.A.flavus suspensions (50 μL, 5 × 105spores/mL) were inoculated into 10 mL of Sabouraud's Dextrose Broth(SDB;4%glucose&1%peptone)and incubated at(28±2)°C for 24 h.Then,each volatile aldehyde was added into SDB to reach the concentrations of 0,1/2MIC and MIC,respectively.After incubated for another 24 h, mycelia were placed onto a glass slide and stained with CW and 10% KOH (AR, Kermel Chemical Reagent Co.,Ltd.,Tianjin,China)as described previously[14],and mycelia septa were observed with a fluorescence microscope(DFC 7000 T,Leica,Germany)at an excitation wavelength of 440 nm and an emission wavelength of 520 nm.The number of septa was counted in eight images for each group.The septa loss rate was calculated as follows:

where S1represents the septa number of A.flavus after the addition of each of the three volatile aldehydes,S0represents the septa number of A.flavus in the control group.Each experiment was repeated for 3 times.

2.3.Cell membrane integrity

2.3.1.PI staining

Cell membrane integrity is important in maintaining cell homeostasis and normal life activities.To assess the membrane permeability,fresh mycelia were harvested from the respective SDB flasks and placed onto a glass slide.After washing with phosphate buffered saline(PBS,AR,Kermel Chemical Reagent Co., Ltd., Tianjin, China), the mycelia were incubated with 10 μL propidium iodide(PI)(1 μL/mL,Solarbio Science&Technology Co.,Ltd.,Beijing,China)for 20 min at room temperature in darkness.After washing several times with PBS, samples were visualized with the fluorescence microscope at an excitation wavelength of 480 nm and an emission wavelength of 630 nm, and the fluorescence intensity was quantified by Image J (1.52v,National Institutes of Health(NIH),Bethesda,USA).

2.3.2.Release of cell constituents

Once the cell wall and membrane are damaged, there could be a leakage of cellular constituents (e.g., nucleic acids,proteins,etc.).The content of cell constituents that released into the culture medium was measured according to the method described previously [15].Briefly, 50 μL of spore suspension were inoculated into SDB (10 mL) and shaken(150 r/min)for 24 h at(28±2)°C.Then,mycelia were treated with the three volatile aldehydes at their respective 1/2MICs and MICs.After incubation for another 24 h,10 mL of each culture filtrate was collected.The absorbance of the filtrate was measured at a wavelength of 260 nm under a UV/Vis Spectrophotometer (UV-6100S,Shanghai Mapada Co., Ltd., Shanghai, China).SDB was used as the blank control.

2.3.3.Determination of relative conductivity

Relative conductivity was assessed using a method described previously[13]for representing the leakage of electrolytes from cells.Briefly,two mycelium plugs(6 mm in diameter) from the margins of one-week-old SDA plates were added to 50 mL SDB.After 24-hour shaking(150 r/min) at (28 ± 2) °C, the broth was treated with one of the three volatile aldehydes at their respective 1/2MICs and MICs,and shaken for another 24 h.The untreated A.flavus was used as the control.Mycelia were then collected and washed several times with pure water.After vacuum filtering,0.2 g of mycelia from each group was suspended in 10 mL pure water.The electrical conductivity of each mycelium sample in pure water was measured at 0,5,10,20,40,60,80,100,120,140,160 and 180 min using a conductivity meter(DZS-706-C,INESA Scientific Instrument Co.,Ltd.,Shanghai,China).Afterwards,the mycelium suspensions were boiled for 10 min to measure the final conductivity.Relative conductivity of pure water without mycelia was also measured before and after boiling as the background.The relative conductivity was calculated as follows:

where C1represents the conductivity of A.flavus after adding the three volatile aldehydes,CW1and CW2represent the conductivities of pure water before and after boiling,respectively,C2represents the final conductivity.

2.3.4.Determination of total lipid content

As a main component of cell membrane,lipid is essential in regulating fluidity and in improving the stability of cell membranes.The content of total lipid was investigated following instructions described previously[16,17].Briefly,two mycelium plugs(6 mm in diameter)from the margins of one-week-old SDA plates were added to 50 mL SDB.After 24 h of shaking (150 r/min) at (28 ± 2) °C, the broth was treated with one of the three volatile aldehydes at their respective 1/2MICs and MICs,and shaken for another 24 h.Mycelia were then collected and washed.Then,the samples were dried with a vacuum freeze drier(LJC-10C,Beijing Sihuan Co.,Ltd.,Beijing,China).Approximately 0.01 g of lyophilized mycelium powder was suspended in 1 mL distilled water,and then 200 μL of the sample suspension was extracted with a methanolchloroform mixture (1:1) and vigorous shaking for 30 min.After centrifuging at 4000 g for 10 min,the lower phase containing lipids was mixed with 0.2 mL saline solution and then centrifuged at 4000 g for another 10 min.Then, 200 μL of the lower phase containing lipids was mixed with 0.5 mL H2SO4and boiled for 10 min.Subsequently,3 mL of phosphovanillin was added to react with lipid and form a colorimetric product.After incubation for 10 min at room temperature,the absorbance of the samples was measured at 525 nm.As the standard,cholesterol was dissolved in 70%chloroform to reach the final concentrations of 0.125,0.250,0.500,1.000,2.000,4.000 mg/mL.The measurement for cholesterol was conducted the same as described above from the step of mixing with 0.5 mL H2SO4.

2.4.Determination of respiratory inhibition rate

To assess the influences of the three volatile aldehydes on the respiration of A.flavus when exposed to aldehyde volatiles, dissolved oxygen was measured by the method described previously[13].In brief,7.2 mL phosphate buffer(pH 7.0)was added to the spore suspension(2 mL,5×105spores/mL).After equilibration for 5 min,0.8 mL 1.0%glucose was added to the mixture and mixed thoroughly.The dissolved oxygen was measured with a dissolved oxygen meter (DZS-706-C; Leici, Shanghai, China).Following a 10 min incubation, each volatile aldehyde was added to the tube at concentrations relating to their respective 1/2MICs and MICs, and the content of dissolved oxygen was measured at 25 min.The time point when glucose was added was set as 0 min.Respiratory rate(R)was calculated as follows:

where O25(μmoL)and O0(μmoL)represent the oxygen content at 25 min and 0 min,respectively.According to the respiratory rate of A.flavus before and after the addition of octanal, nonanal and decanal, the respiratory inhibition rate for A.flavus was calculated using the following equations:

where IRrepresents the respiratory inhibition rate of A.flavus after the addition of the three volatile aldehydes,and R0and RIrepresent the respiratory rate of A.flavus before and after the addition of the three volatile aldehydes,respectively.

2.5.Assessments of in vitro antifungal effects on A.flavus

Fresh maize kernels were washed with sterile water and dried briefly, and then exposed to ultraviolet light for 20 min under laminar flow.The sterilized maize kernels were put into Petri dishes(3 maize kernels for each Petri dish) and inoculated with A.flavus spore suspension (10 μL/maize kernel,5×105spores/mL).Those maize kernels with only spore suspension were set as the control.The experimental kernels were also coated with one of the three aldehydes at their respective 1/2MICs and MICs against spore germination.The maize kernels were then incubated at(28±2)°C,and growth of A.flavus(as colony diameter,mm)was monitored and recorded over 72 h.Each Petri dish was a replicate,and for each aldehyde treatment there were three replicates.

2.6.Statistical analysis

All assays were performed in triplicate and the results were presented as mean±SD.The significant differences between mean values were determined by One-way ANOVA using Duncan's multiple range test(P<0.05).The statistical analyses were performed by SPSS software(SPSS 20.0,IBM,Armonk,NY,USA).

3.Results

3.1.Antifungal activity of the three volatile aldehydes

The inhibition effects of octanal,nonanal and decanal on the spore germination and mycelial growth of A.flavus were shown in Tables 1 and 2.The results showed that the MICs against spore germination were 1.000, 1.000 and 0.500 μL/mL for octanal,decanal and nonanal respectively (Table 1), and the MICs against mycelial growth were respectively 0.50, 2.00 and 5.00 μL/mL for octanal,nonanal and decanal(Table 2).The results demonstratedthat nonanal and octanal respectively possessed the highest effect in inhibiting spore germination and mycelial growth.

Table 1 Germination inhibition rate of octanal,nonanal and decanal for A.flavus at 48 h.

Table 2 Mycelial growth inhibition rate of octanal, nonanal and decanal for A.flavus at 48 h.

3.2.Effects of octanal, nonanal and decanal on cell wall integrity

Chitin is one of the main structural components in the cell wall of A.flavus,and it is often the target of antifungal drugs[18].In Fig.2,septa in the cell wall for the control was bright,those in the 1/2MIC groups of octanal,nonanal and decanal were also intact.The number of septa in the mycelia treated with octanal,nonanal and decanal at their MICs decreased by 70.04%,67.51%and 52.74%,respectively (Table 3).Therefore, the three volatile aldehydes have potential to greatly damage the integrity of cell walls in A.flavus at their MICs.

Fig.2.Fluorescence micrographs of septa of A.flavus before and after treatment with octanal,nonanal and decanal at various concentrations.Scale bar:20 μm.

Table 3 Changes in A.flavus septa number upon treatment with octanal,nonanal or decanal at their 1/2MICs and MICs.

3.3.Effects of octanal,nonanal and decanal on cell membrane integrity

3.3.1.Influence on fluorescence intensity of A.flavus mycelia after PI staining

The results of PI staining showed that the fluorescence intensity of A.flavus mycelia increased in a concentrationdependent manner(Table 4).For example,fluorescence intensities of mycelia treated with octanal, nonanal or decanal at their respective 1/2MICs were almost four,fiveand two times that of the control,while those at their respective MICs were almost eight, seven and four times that of the control.The results demonstrated that the three volatile aldehydes could markedly destroy the cell membrane of A.flavus.As same as their effects on the cell wall,decanal had the weakest effect on the cell membrane in comparison with the other two aldehydes.

Table 4 Changes in fluorescence intensity and OD260 of A.flavus under treatment with octanal,nonanal and decanal at different concentrations.

3.3.2.Influence on the release of cell constituents

After treatment with octanal,nonanal and decanal,we measured the absorbance of mycelium filtrate at 260 nm,which directly reflects the release of nucleic acids.As shown in Table 4, in comparison with control, samples treated with the three volatile aldehydes exhibited a significant increase in absorbance value(OD260)at their 1/2MICs or MICs,indicating that there was a release of cell constituents.The results confirmed,from the other side,that all the three volatile aldehydes could damage the cell membrane of A.flavus.

3.3.3.Influence on relative conductivity

To further examine the effects of the three volatile aldehydes on cell membrane permeability(i.e.,leakage of electrolytes) of A.flavus mycelia, we tested the relative conductivity of samples treated with the three volatile aldehydes.The results showed that the relative conductivity increased significantly in comparison with that of the control(39.23%±3.22%)and was in positive correlation with the concentration of the volatile aldehydes(Fig.3).The 180-min relative conductivities at their respective 1/2MICs and MICs were (78.96% ± 3.63%) and (92.63% ±0.95%) for octanal (Fig.3A), (70.78% ± 2.66%) and(84.94%±4.65%)for nonanal(Fig.3B),and(79.88%±4.74%)and(87.29%±6.10%)for decanal(Fig.3C).The results also proved that all the three volatile aldehydes indeed increased the permeability of mycelia.

3.3.4.Influence on total lipid content

As the cell membrane integrity was destroyed by the three volatile aldehydes,the total lipid content of A.flavus was further quantified.As shown in Table 5, treatment with the three volatile aldehydes could result in decrease of the total lipid content,and this effect was dose-dependent.In detail,the total lipid content was(182.22±1.93)μg/g for control.The total lipid content of A.flavus treated with octanal,nonanal and decanal at their respective 1/2MICs and MICs were(91.25±2.08)and(25.37±1.02)μg/g,(121.05±1.09) and (31.63 ± 1.02) μg/g, and (146.21 ± 2.28),(57.20 ± 1.04) μg/g.Distinctly, the highest loss rate of total lipid content was found in the octanal treated groups(1/2MIC: 50%; MIC: 86%) compared with the other two groups.Herein,octanal showed the strongest destroying effect on the total lipid after 24 h treatment.

Table 5 Effects of octanal,nonanal and decanal treatment on total lipid content of A.flavus mycelia.

3.4.Effects of octanal,nonanal and decanal on respiration of A.flavus

A glucose utilization experiment was conducted to investigate the influence of octanal, nonanal and decanal on A.flavus respiratory metabolism.The respiration inhibition rate of octanal,nonanal and decanal at MICs were 38.83%,61.10%and 58.94%respectively(Fig.4)at 25 min.

Fig.4.Respiration inhibition rates for A.flavus treated with octanal,nonanal and decanal.

3.5.In vitro antifungal effects of octanal,nonanal and decanal

Based on the promising inhibitory effects of octanal,nonanal and decanal on A.flavus,their antifungal capabilities were assessed on maize kernels in vitro.In the control group,mycelia began to grow at 24 h.Except for a low relative inhibition effect of octanal at 1/2 MIC(10%,72 h),the other volatile aldehydes could inhibit the growth of A.flavus by more than 50% at their 1/2MICs (Table 6).No mycelial growth was observed under 72-hour treatment of the three volatile aldehydes at their MICs(Table 6 and Fig.5).Therefore, in vitro test results demonstrated that the three volatile aldehydes exhibited higher antifungal capabilities against A.flavus on maize kernels.

Table 6 A.flavus colony diameter on maize kernels treated with octanal,nonanal and decanal at 1/2MICs and MICs for 72 h.

4.Discussions

Benefiting from their beneficial characteristics,such as high safety to human health and environment,quite a few of plant-derived natural compounds have been authorized to be applied in grains and foods as antifungals[19].In recent years,many natural compounds have been found to exert promising antifungal effects on A.flavus, such as cinnamaldehyde, citral, thymol, etc.[20–22].Therefore,plant-derived natural products are predicted to serve as potential fungicides when applied in edible commodities in the near future.

In this study,the inhibitory effects of three volatile aldehydes (octanal, nonanal and decanal) on A.flavus were evaluated.First of all,all the three aldehydes could effectively inhibit the spore germination and mycelial growth in a dose-dependent manner,wherein octanal and nonanal exerted stronger inhibitory effects than decanal, as indicated by their respective MICs.Although an earlier study has reported that octanal exerted stronger inhibition effect on mycelial growth of A.flavus than nonanal[4],our results showed that the inhibitory efficacies of octanal and nonanal varied on spore germination and mycelial growth.Interestingly,not consistent with our previous assumption,the inhibitory effect was not correlated with the number of carbon atoms(C)in the linear aldehyde structure.In our recent study,we have found out that the relative positions of some functional groups(e.g.,hydroxyl group and aldehyde group)on the benzene ring were considered as the key factors in antifungal effects[23].Another report proposed an increase in the antifungal effects of aldehydes occurring with the presence of an α,β-unsaturated bond adjacent to the carbonyl moiety [24].Since the three aldehydes do not fall into the above two situations,we propose that the modes of action of them are varied(e.g.,antifungal target(s)),the converting of aldehydes getting inside cells,etc.).

Fig.5.Photographs of A.flavus on maize kernels after 72-hour treatment with octanal,nonanal and decanal.

More and more studies have addressed that some aldehydes target the cell wall integrity, such as cinnamaldehyde-treated Geotrichum citri-aurantii [14] and HMB-treated A.flavus [13].As the outermost barrier of fungi,cell wall plays an important role in maintaining the intrinsic morphology and integrity of cells,as well as resisting environmental stresses[25].Besides the dose-dependent effects of octanal,nonanal and decanal on the cell wall integrity of A.flavus, the lowest loss rate of speta number suggested that decanal exerted the weakest strength.The fact that septa are composed of chitin directed to the conclusion that the three aldehydes,especially nonanal and octanal,target chitin.However,the mode of action of them on the metabolism of chitin requires further demonstration.

Cell membrane is located adjacent to cell wall,serves as the inner barrier and plays an important role in maintaining cell viability[26].And lipids are the main components of cell membranes and have many essential functions,including reducing the permeability of water-soluble materials and increasing membrane stability[27].In this study,we conducted PI staining and determination of the released cell constituents as well as the relative conductivity to comprehensively evaluate the changes of cell membrane permeability,as PI is not able to bind with intact cell membranes and as well in this case,cell components would not be released out of cell membranes.All these three aldehydes are capable to destroy cell membranes in a dosedependent manner, and again, among them, decanal treated mycelia showed the lowest PI fluorescence intensities and the highest content of total lipids,indicating that cell membrane damage capacity of decanal was the lowest.Whether the present lipid is broken down or the synthesis of lipid is suppressed by the three volatile aldehydes requires further elucidation.Therefore,octanal and nonanal could target cell membranes of A.flavus by damaging cell membrane integrity,as well as by reducing the total lipid content.Besides A.flavus, octanal [8], nonanal [10] and decanal could also damage cell membranes of G.citriauranti,Penicillium cyclopium,and P.expansum[12],respectively.In addition,other aldehydes have also been reported to act on cell membranes,such as citral-treated Penicillium italicum[28],citronellal-treated Penicillium digitatum[29]and cuminaldehyde-treated A.flavus[30].All these investigations suggest that cell membranes of pathogenic fungi can be considered as a common target for the antifungal actions of plant-derived aldehydes.

Apart from cell walls and cell membranes, in recent years,mitochondria-associated respiratory is addressed as another antifungal target of plant-derived aldehydes.Besides supplying and recycling energy,respiratory metabolism could also improve the elasticity of cells and provide intermediate metabolites[31].In this study,the addition of octanal,nonanal and decanal significantly inhibited the rate of oxygen consumption.The strongest inhibition occurred for nonanal and decanal treated spores.In combination with the previous results, it can be concluded that octanal and nonanal are more prone to act on cell walls and cell membranes,nonanal and decanal are more prone to act on the respiration of A.flavus.Therefore, nonanal seems a promising antifungal agent with versatile antifungal targets.These results direct to the priorities for our further research.

Besides the above assays in culture medium, we also evaluated the inhibition of the three volatile aldehydes on maize grains.Maize is extremely susceptible to A.flavus[32], therefore inhibiting growth of the fungus can help mitigate infection.We found that all three volatile aldehydes could completely inhibit the growth of A.flavus on maize kernels for at least 72 h.In combination with our previous studies,the isomers of vanillin proved to be a promising natural product by inhibiting the growth of A.flavus[13,23].All these results facilitated the application of these plant-derived natural compounds into practice.

5.Conclusions

In conclusion, based on the susceptibility test against A.flavus,we found out that the inhibition effects of octanal,nonanal and decanal were achieved by their destruction on chitin that serves as scaffold for the integrity of cell walls.In addition,they could also destroy cell membranes by inducing the release of cell components.Cell respiration was also found to be prohibited as A.flavus spores could not effectively consume dissolved oxygen.Therefore, cell walls,cell membranes,and respiration serve as antifungal targets during the inhibition of all three volatile aldehydes with varied extent.And in comparison with all these phenotypic results,nonanal is regarded as a versatile antifungal compound.Importantly,all three volatile aldehydes effectively controlled the infection of A.flavus on maize kernels.As all the three volatile aldehydes are recognized as safe,they are prospected to be promising in future applications in the preservation of grains and agricultural products either in single or complex form.

Author Contributions

Qian Li: Conceptualization, project administration;Xiaoman Zhu:Data curation,formal analysis,investigation,original draft writing;Jingmeng Liang:Investigation;Yanli Xie:Supervision.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgement

This study was funded by the Doctor Research Fund of Henan University of Technology (grant number 2019BS019)and the Natural Science Research Projects of Education Department of Henan Province(21A550005).