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.