Antifungal activity of volatile organic compounds from Streptomyces alboflavus TD-1

Authors

  • Changlu Wang,

    Corresponding author
    • Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, China
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  • Zhifang Wang,

    1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, China
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  • Xi Qiao,

    1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, China
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  • Zhenjing Li,

    1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, China
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  • Fengjuan Li,

    1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, China
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  • Mianhua Chen,

    1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, China
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  • Yurong Wang,

    1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, China
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  • Yufang Huang,

    1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, China
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  • Haiyan Cui

    1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, China
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Correspondence: Changlu Wang, Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology No. 29, the 13th Avenue, TEDA, Tianjin 300457, China. Tel.: +86 22 6027 2219; fax: +86 22 6027 2219; e-mail: changluwang2009@gmail.com

Abstract

Streptomyces sp. TD-1 was identified as Streptomyces alboflavus based on its morphological characteristics, physiological properties, and 16S rDNA gene sequence analysis. The antifungal activity of the volatile-producing S. alboflavus TD-1 was investigated. Results showed that volatiles generated by S. alboflavus TD-1 inhibited storage fungi Fusarium moniliforme Sheldon, Aspergillus flavus, Aspergillus ochraceus, Aspergillus niger, and Penicillum citrinum in vitro. GC/MS analysis revealed that 27 kinds of volatile organic compounds were identified from the volatiles of S. alboflavus TD-1 mycelia, among which the most abundant compound was 2-methylisoborneol. Dimethyl disulfide was proved to have antifungal activity against F. moniliforme by fumigation in vitro.

Introduction

Mold spoilage and mycotoxin contamination are some of the major concerns in agriculture. Mycotoxin contamination of feed is mainly caused by three genera of molds: Aspergillus, Penicillium, and Fusarium (Magan & Aldred, 2007). Molds directly affect the feed palatability, reduce the feed nutrition value, and cause livestock and poultry poisoning. Moreover, the residue of mycotoxins in animal food can be passed through the food chain, which affects human health by inducing cancer, deformity, renal toxicity, liver toxicity, genetic toxicity, and suppression, and other adverse effects (Kolosova & Stroka, 2011). The economic consequences of mycotoxin contamination are also profound in agriculture.

Prevention is essential because only a few methods can completely overcome the problems once mycotoxins are present. A number of approaches have been used to inhibit the growth of molds, including physical, chemical, and biological control strategies (Kolosova & Stroka, 2011). The addition of effective preservatives is widely used in feed mildew. Commercial products are mainly based on mixtures of salts of aliphatic acids such as propionic, sorbic, and benzoic acids (Kolosova & Stroka, 2011). However, the use of chemicals is becoming increasingly restricted because of environmental and health concerns, as well as drug resistance. Therefore, natural antimicrobials are extensively investigated, and considered safe and reliable.

Volatile organic compounds (VOCs) are low-molecular-weight carbon-containing compounds that easily evaporate at normal temperature and pressure. Given fact that VOCs can diffuse through the atmosphere and soil, they are ideal ‘infochemicals’ (Morath et al., 2012). The biocontrol activity of VOCs excreted by microorganisms is gaining attention, and specific methods for fungicidal treatments have been investigated in many diverse areas. VOCs are biodegradable and do not leave toxic residues on the surface. They are also effective in disease control as conventional fungicides (Mercier & Smilanick, 2005). Using antifungal VOCs from microorganisms to control molds under airtight conditions has potential use in the feed industry.

Previous studies have reported that the conidiophores and hyphae of five fungi (Aspergillus giganteus, Fusarium oxysporum, Penicillium viridicatum, Trichoderma viride, and Zygorhynchus vuilleminii) exhibit abnormal morphology when treated with the VOCs of nine bacteria and an actinomycete (Moore-Landecker & Stotzky, 1973). Methyl vinyl ketone emitted by Streptomyces griseoruber can reportedly inhibit the spore germination of Cladosporium cladosporioides (Herrington et al., 1985). The antifungal activity of volatiles produced by Muscodor albus has been extensively reported, indicating that volatile antibiotics can inhibit or kill not only plant pathogens (e.g. Botrytis cinerea, Colletotrichum acutatum, Geotrichum spp., Monilinia fructicola, Penicllium spp., Rhizopus spp., Rhizoctonia solani, Pythium ultimum, Aphanomyces cochlioides, Verticillium dahliae, F. oxysporum f. sp. betae, Sclerotinia sclerotiorum, and Phytophthora capsici), but also building pollution molds (Strobel et al., 2001; Stinson et al., 2003; Mercier & Jiménez, 2004, 2007; Mercier & Manker, 2005; Mercier & Smilanick, 2005; Gabler et al., 2006; Schnabel & Mercier, 2006). Given the unusual antimicrobial properties of M. albus, its use in agriculture has been patented and is being developed as a commercial antimicrobial biofumigant product (Mercier & Smilanick, 2005).

VOCs produced by the Irpex lacteus isolate Kyu-W63 has been found to lighten the powdery mildew caused by Oidium sp. under greenhouse conditions (Koitabashi, 2005). Bacillus subtilis and Bacillus amyloliquefaciens can also reportedly produce antifungal volatiles (Arrebola et al., 2010). Thus, numerous microorganism species can produce antifungal VOCs.

In addition, the volatiles of wild-type antagonistic F. oxysporum negatively influence the mycelial growth of different formae speciales, and repress the gene expression of two putative virulence genes in F. oxysporum lactucae strain Fuslat10. These VOCs also promote lettuce growth and expansion through A5 gene expression (Minerdi et al., 2009, 2011). These studies indicate that the VOCs of microorganisms may directly or indirectly act to influence the growth, morphogenesis, and gene expression of microorganisms or plant. Recently, the VOCs of Streptomyces platensis have been proven to show antifungal activity against R. solani, S. sclerotiorum, and B. cinerea (Wan et al., 2008). The volatiles of Streptomyces globisporus have also been found to exhibit antifungal activity against Penicillium italicum in Citrus (Li et al., 2010). However, the VOCs of Streptomyces can reportedly stimulate the spore germination of Gigaspora margarita (Carpenter-Boggs et al., 1995).

Little information is currently available on the activity of VOCs from Streptomyces against storage fungi. Streptomyces sp. TD-1 isolated from soil surrounding a granary has been proven to have antagonism action for controlling nine storage fungi simultaneously (Liu et al., 2012). The present study investigated the taxonomy of Streptomyces sp. TD-1, the effects of VOCs generated by Streptomyces sp. TD-1 on storage fungi, the VOCs released from the mycelium of Streptomyces sp. TD-1, and the anti-Fusarium moniliforme activity of an individual compound (dimethyl disulfide) from the mixture of volatiles.

Materials and methods

Antagonist Streptomyces sp. TD-1

Streptomyces sp. TD-1 was isolated from soil surrounding a granary (Tianjin, China), and deposited in the China General Microbiological Culture Collection Center and CGMCC No. 4666. Studies on the cultural and physiological characteristics of the producing strain were carried out following the methods recommended by the ISP (Shirling & Gottlieb, 1966), and the utilization of carbon sources was tested according to the growth condition on plates containing different sugar sources. The morphological properties were observed with a scanning electron microscope (SU-1510 Hitachi). The 16S rRNA gene fragment was sequenced by China General Microbiological Culture Collection Center.

Preparation for VOC production

Streptomyces sp. TD-1 was cultured on test tube slants of Gause's synthetic agar. Spore suspensions were prepared by washing the agar slant surface with sterile distilled water. Gause's synthetic medium in an Erlenmeyer flask was inoculated with the prepared spore suspension. The flasks were incubated at 28 °C for 5 days. The fermentation broth was filtered, and the mycelium of Streptomyces sp. TD-1 was used for measurement.

Fungi strain

Fusarium moniliforme, Aspergillus flavus, Aspergillus ochraceus, Aspergillus niger, and Penicillum citrinum were obtained from the Tianjin Institute of Animal Husbandry and Veterinary Science. For conidial production, they were grown on potato dextrose agar (PDA).

Effect of VOCs on the mycelial growth of storage fungi in vitro

Two bottom dishes of a sterilized Petri dish (9 cm in diameter) were used. The dish containing mycelium of the fermentation broth of strain TD-1 was prepared and covered with the other dish containing PDA inoculated with a 6 mm-diameter plug of storage fungi. The two dishes were then sealed with Parafilm™ to obtain a double-dish chamber (Arrebola et al., 2010).

Collection and analysis of VOCs by GC/MS

The VOCs of strain TD-1 were collected by the headspace solid-phase microextraction (HS-SPME) technique (Gu et al., 2007). The HS-SPME syringe contained 50 : 30 divinylbenzene : carburen on polydimethylsiloxane (DVB/CAR/PDMS) on a stable fiber (65 μm). The gas chromatograph was equipped with an ion-trap detector (Varian-4000 GC/MS; Varian Co.). A VF-5 ms fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm-thick film; Varian Co.) was used to separate the volatiles. Helium was used as the carrier gas at 1 mL min−1. The injector temperature was maintained at 250 °C. The working temperature for the volatile-separation column was programmed as follows: set at 40 °C for 3 min at the beginning, increased to 150 °C at 4 °C min−1 held at 150 °C for 1 min, and further increased to 250 °C at 8 °C min−1 held at 250 °C for 2 min. The temperature was set at 220 °C for the electronic bombard ion source (70 eV) and 260 °C for the transfer line.

Effect of dimethyl disulfide against F. moniliforme

Two covered bottom dishes were used as described above. The top dish contained a fungal plug, and the bottom dish contained a piece of autoclaved filter paper, to which the selected individual compound was added.

Results

Taxonomy of the produced Streptomyces sp. TD-1

Streptomyces sp. TD-1 formed well-developed and branching substrate mycelium, but rare aerial mycelium. It grew well on most of the media and produced no distinctive soluble pigment. The mature spore chained as straight or slightly curved, the spores were cylindrical to oval shaped with sizes of 0.5–2 μm, and surface was smooth as observed under a scanning electron photomicroscope.

The culture characteristics of the strain are summarized in Table 1. The carbon utilization and physiological characteristics of the strain are described in Table 2.

Table 1. Culture characteristics of Streptomyces sp. TD-1
MediumAerial mediumSub-mediumPigment
Czapek's agarCream whiteBrownish redNo
Glucose asparagineWhiteBrownish redNo
Glycerol asparagine (ISP5)WhiteLight redNo
Inorganic salts-starch (ISP4)WhiteBrownish redNo
Yeast-mall extract agar (ISP2)WhiteBrownish redNo
Oatmeal agar (ISP3)WhiteCream whiteNo
Gause's no. 1Cream whiteOrange redNo
Yeast peptone agar (ISP6)Cream whiteOrange redNo
Table 2. Carbon utilization and physiological characteristics of Streptomyces sp. TD-1
Carbon Carbon Carbon Carbon Physiological characteristics 
Glucose+Raffinose+Ribose+Sodium acetateCoagulation of milk+
Mannose+Trehalose+InulinSodium gluconate+Liquefaction of gelatin+
Mannitol+Fructose+GlycogenSodium malate+Use of cellulose
LactoseXylose+ErythritolSodium succinate+Peptonization of milk
Galactose+Melibiose+RhamnoseSodium malonateTyrosinase
SorboseMelezitoseStarch+Sodium tartrate+Amylase+
SorbitolRhamnoseCellobiose+Sodium citrate+  
Maltose+Glycerin+l-arabinose+Sodium propionate  

The 16S rRNA gene sequence of Streptomyces sp. TD-1 (GenBank accession number JX915780) contained 1401 nucleotides. The blast results indicated that Streptomyces sp. TD-1 deeply branched within a member of genus Streptomyces and had the highest similarity to Streptomyces alboflavus NBRC 3438T (Fig. 1). The sequence similarity was 99.7%, and most morphological and physiological characteristics were highly similar, except for the carbon utilization for sucrose, synanthrin, arabinose, fructose, and rhamnose. Thus, Streptomyces sp. TD-1 was identified as S. alboflavus TD-1.

Figure 1.

Phylogenetic tree showing the position of Streptomyces sp. TD-1 on the basis of 16S rDNA gene sequence analysis. mega version 5.1 software was used to align 16S rRNA gene sequence. The tree was constructed using the neighbor-joining algorithm. Boot strapvalues based on 1000 replications are shown at the nodes of the tree. Streptomyces lydicus strain ATCC 25470 was used as the outgroup.

Suppression of the mycelial growth of storage fungi by the VOCs of S. alboflavus TD-1

Figure 2 shows the effects of the VOCs produced by the mycelium of the fermentation broth of S. alboflavus TD-1 on the mycelial growth of storage fungi. For all five filamentous fungi tested, > 24.8% inhibitory rate of VOCs on fungi was achieved.

Figure 2.

The Inhibitory rate of storge fungi developed in the presence of mycelium of Streptomyces alboflavus TD-1. 5 days of incubation at 28 °C. Inhibitory rate of fungal radial mycelial growth was calculated according to the formula: (R1 − R2/R1) × 100, where R1 is a control value, R2 is the measurement of the fungus in antagonist-fungal (Arrebola et al., 2010). Values are the means of inhibitory rate ± SD, means based on 6 replicates, Error bars represent the SD.

GC/MS analysis of VOCs produced by S. alboflavus TD-1

GC/MS analysis showed that the most frequent 27 VOCs from the mycelium of the fermentation broth of S. alboflavus TD-1 (Supporting Information, Fig. S1 and Tables 3). These compounds were categorized into several classes: alcohols, alkenes, aromatic hydrocarbons, aldehydes, and sulfide. Among them, 2-methylisoborneol (2-MIB) was the most abundant VOC and its antifungal activity has been studied in our previous paper. Dimethyl disulfide reportedly has a significant effect on the mycelial growth, sporulation, or conidial germination of P. italicum (Li et al., 2010). Thus, this volatile compound was selected for further individual testing of antifungal activity against F. moniliforme.

Table 3. VOCs from the mycelium of Streptomyces alboflavus TD-1 fermentation broth detected by GC/MS analysis
Possible compound, Relative peak area (%)Possible compound, Relative peak area (%)
  1. The 27 compounds were identified by comparing the mass spectra and retention times from those of available standards in the Library of the National Institute of Standards and Technology (NIST05). Minor compounds were detected are not included in this table.

Dimethy disulfide, 0.393-Cyclohexen-1-ol,3-ethyl, 0.13
6,7-Dimethyl-1,2,3,5,8,8a-hexahydronaphthalene,0.13Cyclohexane,1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, 0.27
Benzene,1,2,3-trimethyl, 3.44Trans-1,10-Dimethyl-trans-9-decalinol, 4.28
4-Isopropenyl-4,7-dimthyl-1-oxaspiro[2,5]octane,0.211,3-Cycloexadiene-1-carboxadehyde,2,6,6-trimethyl, 2.76
Bicyclo[3.1.1]hept-2-ene-2-carboxaldehyde,6,6-dimethyl, 10.06Bicyclo[5.3.0]decane,2-methylene-5-(1-methylvinyl)-8-methyl-, 0.27
3-Caren-10-al, 0.28Cedran-diol,8S, 14-, 0.63
2-Methylisoborneol, 51.34Humulen-(v1), 0.21
Camphenol,6-, 0.22Oleyl Alcohol, 0.23
1,7,7-Trimethylbicyclo[2.2.1]hept-5-en-2-ol, 0.10Isoledene, 1.16
Cis-1,4,Dimethyladamantane, 12.13(Z)6-Pentadecen-1-ol, 0.11
1-H-Indene,1-ethylideneoctahydro-7a-methyl-,(1Z,3a,alpha,7a,beta)-, 2.92Naphthalene,1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-, 0.12
1,4-Dimethyladamantane,[1.alpha.,3.beta.,4.beta.,,5.alpha.,7.beta]-, 0.101H-Cycloprop[e]azulen-4-ol,decahydro-1,1,4,7-tetramethyl-, 0.50
Bicyclo[4.3.0]non-3-ene,3,4,7-trimethyl, 0.73Cyclohexanemethanol, 0.11
Cycloehexane,1,1,4,4,-tetramethyl-2,6-bis(methylene)-, 2.64

Effect of dimethyl disulfide on the mycelial growth and sporulation F. moniliforme

Dimethyl disulfide had inhibitory activity on the mycelial growth of F. moniliforme at the tested concentrations, as shown in Table 4. Mycelial growth and sporulation were completely inhibited when the dimethyl disulfide was 10 μL plate−1.

Table 4. Effect of dimethyl disulfide on Fusarium moniliforme
Volume of dimethyl disulfide (μL plate−1)0110100
  1. a

    Mycelial growth and sporulation were measured after incubation for 5 day at 28 °C.

  2. b

    Statistical results are expressed as means ± SD.

  3. c

    Means based on six replicates followed by the same letters within the column were not significantly different (P < 0.05) according to the LSD test.

Mycelial growth (cm)68 ± 4a59 ± 8b0 ± 0c0 ± 0c
Sporulationa (× 105 sporesplate−1)20 167 ± 2639a5750 ± 689b0 ± 0c0 ± 0c

Discussion

In this paper, Streptomyces sp. TD-1 was identified as S. alboflavus according to its morphological characteristics, physiological properties, and 16S rRNA gene sequence. Recently, a series of novel cyclic hexadepsipeptide with antifungal activity has been isolated from the fermentation broth of S. alboflavus (Guo et al., 2009; Ji et al., 2012). In our previous study, the antifungal secondary metabolite of S. alboflavus species includes not only non-volatile antifungal substances, but also volatile antifungal substances that inhibited the molds (Liu et al., 2012). All these studies suggest that strain S. alboflavus TD-1 has potential use for storage fungi biocontrol.

Research on volatile metabolites from Streptomyces has revealed considerable biosynthetic diversity and antifungal activity in culture plates or in planta (Schöler et al., 2002; Li et al., 2010). In the current work, we showed that the growths of mold colonies of F. moniliforme, A. flavus, A. ochraceus, A. niger, and P. citrinum were simultaneously inhibited by the VOCs of S. alboflavus TD-1 in vitro, especially F. moniliforme and A. flavus. No direct contact was observed between the fungus and mycelium of S. alboflavus TD-1 in a double-dish chamber. The antifungal effect was also nullified when activated charcoal, a well-known volatile absorbent, was added. (data not published, Qiao Xi). So, the VOCs from the strain S. alboflavus TD-1 have the antifungal activity by fumigation. VOCs are convenient to use under an airtight condition because they are small molecules that can easily diffuse through the porous structure and over great distances in the atmosphere. This result may provide a new method of biocontrolling molds or plant diseases caused by these organisms.

A total of 27 VOCs from S. alboflavus TD-1 were identified by GC/MS analysis. Two earthy-smelling substances, 2-MIB and geosmin, were found at the same time. These volatile compounds were simultaneously produced by other microorganisms, including Streptomyces spp. AMI 240, S. aureofaciens ETH 13387, S. aureofaciens ETH 28832, S. griseus ATCC 23345c, S. murinus NRRL 8171, S. murinus DSM 40091c, S. olivaceus ETH 7437, and S. griseus ATCC 10137 (Wilkins & Schöller, 2009). 2-MIB and geosmin are tertiary alcohols with low water solubility, weak polarity, fat solubility, and semi-volatility at room temperature. They are usually used in the drinking water industry as control targets (Jüttner & Watson, 2007). Seven of the VOCs detected in this study, including dimethy disulfide, 1-H-indene, 1-ethylideneociahydro-7a-methyl (1Z,3a,alpha,7a,beta), cycloehexane,1,1,4,4,-tetramethyl-2,6-bis(methylene)-, trans-1,10-dimethyl-trans-9-decalinol, isoledene, and naphthalene,1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-, are reportedly components of volatiles from S. globisporus JK-1 (Li et al., 2010), but had different relative peak areas. Among them, dimethyl disulfide produced by numerous microorganisms and found in garlic oil has been found to have antifungal activity (Schöler et al., 2002; Li et al., 2010). A previous study has shown that dimethyl disulfide has antifungal activity against P. italicum in vitro and in planta through fumigant action (Li et al., 2010). In this study, dimethyl disulfide was also proven as able to inhibit mycelial growth from plugs of F. moniliforme by fumigation. The mycelial plugs of F. moniliforme fumigated by dimethyl disulfide for 7 day were unable to produce hyphae when transplanted onto fresh PDA plates, showing fungicidal effects on spores. A previous study has shown that B. subtilis and its secondary metabolite, fengycins, can inhibit the growth of F. moniliforme (Hu et al., 2007). Information on antifungal activity against F. moniliforme as a fumigant is limited. Given that dimethyl disulfide has an undesirable odor, it is unsuitable for use in large concentrations but can be considered as a candidate for one active ingredient of a blend of fumigation. Three of the volatile compounds detected in this study, including 3-caren-10-al, humulen-(v1), and isoledene, are reportedly components of antimicrobial volatiles in essential oils of Satureja montana or Magnolia liliflora Desr., ether extracts of Panax ginseng, hexane extracts Abutilon Indicum, or leaf essential oil of Gossypium barbadense (Bajpai et al., 2008; Xu et al., 2009; Essien et al., 2011; Serrano et al., 2011; Shanthi et al., 2011). Other volatiles detected in this study, including cedran-diol, 8S, and 14-,(Z)6-pentadecen-1-ol have been found as components of Hybanthus enneaspermus and Momordica charantia (Anand & Gokulakrishnan, 2012). Other individual volatile compounds that can suppress pathogens possibly exist and warrant further investigations.

The production of VOCs by microorganisms is both complex and dynamic. The characterization of VOCs is also difficult because they are produced in small quantities. In our study, the antifungal activity varied with the age of the fungal colony, culture conditions, temperature, and other environmental parameters. Most of the VOCs were the same morphologically but differed in quantity. No volatile compound has been identified by analysis of the relationship between the antifungal activity and production of an individual volatile compound. To our knowledge, information on the biosynthesis of VOCs from microorganisms is limited. The only pertinent report is that of Gianoulis et al. (2012), who used genomics, transcriptomics, and metabolomics to correlate the production of eight-carbon volatiles of Ascocoryne sarcoides with the expression of lipoxygenase pathway genes. Their purpose was to illustrate the mechanism of VOC production. These methods provide a promising research design to study the biosynthesis of VOCs from microorganisms.

In conclusion, the present study showed that VOCs from the mycelium of S. alboflavus TD-1 fermentation broth exhibited efficacy against some storage fungi such as F. moniliforme, A. flavus, A. ochraceus, A. niger, and P. citrinum in vitro. The antifungal substance dimethyl disulfide also presented an ability to inhibit the growth of F. moniliforme by fumigation. The effect of the VOCs on fruits or feeds contaminated with mold was not examined in this research, and further studies with regard to the safety of these compounds to the environment and human health are needed.

Acknowledgements

The authors wish to thank the Research Center for Modern Analysis Techniques, Tianjin University of Science & Technology for the GC/MS analysis. There is none financial/commercial conflicts of interest in this work by all authors.

Authors' contribution

Z.W. and C.W. are joined first authors of this publication.

Ancillary