Open Access Austin Journal of Biotechnology & Bioengineering Special Article - Fungal Biotechnology: Current and Future Perspective Mycofum igation for the Biological Control of PostHarvest Diseases in Fruits and Vegetables: A Review Go m e s AAM 1, Qu e iro z MV 1 an d Pe re ira OL2 * Departam ento de Microbiologia, Universidade Federal de Viçosa, Brazil 2 Departam ento de Fitopatologia, Universidade Federal de Viçosa, Brazil 1 *Co rre s p o n d in g au th o r: Pereira OL, Departam ento de Fitopatologia, Universidade Federal de Viçosa, Av. Peter Henry Rolfs, s/ n - Cam pus Universitário, Viçosa – MG, CEP. 36570 -90 0 , Brazil Re ce ive d : J une 25, 20 15; Acce p te d : August 28, 20 15; Pu blis h e d : Septem ber 0 2, 20 15 Abstract There are several causes of post-harvest losses in fruits and vegetables, and microbial infections are responsible for the greatest losses that occur during the transport, storage, and sale of these products. Chemical control is the most used method to control post-harvest diseases in fruits and vegetables by directly applying synthetic fungicides to the product to be consumed. However, the indiscriminate use of fungicides may be associated with serious toxicity problems in humans and environmental imbalance. Mycofumigation, which is the use of volatile antimicrobial organic compounds produced by fungi to inhibit microbial growth, has become a promising alternative for controlling phytopathogenic fungi associated with post-harvest diseases in fruits and vegetables. The technique has some advantages relative to traditional disease control methods, for example, it does not require direct contact between the antagonist and the plant product, the antimicrobial volatiles diffuse easily in closed environments, they do not leave residues on the plant product to be consumed, and most of the antimicrobial volatile mixtures exhibit bioactivity against a wide range of microorganisms, including many phytopathogens associated with post-harvest diseases. This review highlights mycofumigation as a method for controlling post-harvest diseases in fruits and vegetables, emphasizing the effects of volatile compounds on phytopathogenic fungi and their potential to be applied during the transport and storage of fresh fruits and vegetables. Keywords: Biofumigation; Muscodor; Antimicrobial volatiles Abbre viatio n VOCs: Volatile Organic Compounds In tro d u ctio n As fruits and vegetables are usually tender and juicy, they can become rich and adequate substrates for microbial growth and, consequently, post-harvest infections. hese infections are usually responsible for the greatest post-harvest losses observed in horticultural products. For example, in citrus fruit, the Penicillium digitatum (Pers.) Sacc. fungus is responsible for more than 90% of post-harvest production losses [1]. Physical and physiological damage favors microbial infections, and fruits’ and vegetables’ natural resistance to disease decreases with maturation, favoring phytopathogen invasion. hese phytopathogens require an entry site to start an infection and may become a serious problem in products stored for long periods of time [2]. Post-harvest decay during the supply chain has been identiied as the greatest cause of post-harvest losses in fruits and vegetables, which results in signiicant economic losses [3]. It is estimated that approximately 20-25% of the fruits and vegetables harvested in developed countries are lost due to action/attack by phytopathogenic microorganisms during post-harvest handling. In developing countries, post-harvest losses are usually higher, especially due to inadequate storage methods and transport diiculties [4]. Fungi are oten involved in the decay of fruits and vegetables. Austin J Biotechnol Bioeng - Volume 2 Issue 4 - 2015 Submit your Manuscript | www.austinpublishinggroup.com Pereira et al. © All rights are reserved his microbial group stands out as important post-harvest diseasecausing agents with the highest frequency and activity, and they are responsible for 80 to 90% of the total losses caused by microbial agents (Figure 1). Many fungal species within the most varied genera have been reported to be associated with post-harvest diseases in fruits and vegetables worldwide: Penicillium Link, Aspergillus P. Micheli, Geotrichum Link, Botrytis P. Micheli, Fusarium Link, Alternaria Nees, Colletotrichum, Dothiorella Sacc, Lasiodiplodia Ellis & Everh, Phomopsis Sacc. & Roum, Cladosporium Link, Phytophthora De Bary, Pythium Nees, Rhizopus Ehrenb, Mucor P. Micheli ex L., Sclerotium Tode, Rhizoctonia D.C. [5-12]. In addition to their potential to cause rot, some fungi that are associated with fruits and vegetables have high potential for mycotoxin production. hese secondary metabolites exhibit bioactivity associated with toxic efects in humans, animals, and plants [13]. Several toxins produced by Aspergillus, Penicillium, and Fusarium species and their toxic efects on humans have been reported [14,15]. Practices have been adopted to reduce the incidence of fungi and consequent damage and losses caused by post-harvest diseases in fruits and vegetables, including manipulation of the storage environment and resistance induction. However, the main method used to control post-harvest diseases in fruits and vegetables is by applying fungicides via spraying or even by immersing the horticultural products in fungicide solution [12,16]. Studies have indicated the eiciency of several fungicides with diferent active ingredients in controlling post-harvest decay in fruits Citation: Gomes AAM, Queiroz MV and Pereira OL. Mycofumigation for the Biological Control of Post-Harvest Diseases in Fruits and Vegetables: A Review. Austin J Biotechnol Bioeng. 2015; 2(4): 1051. Pereira OL Au s tin Pu blis h in g Gro u p led to increased 2,6-dichloro-4-nitroaniline residue levels in plum and nectarine and increased sodium o-phenylphenate residue levels in citrus fruit [24]. Imazil residue was also detected in citrus fruit ater being applied post-harvest, and the residue level was associated with treatment method, where dip-treated fruit exhibited higher quantities of residue than fruit treated with the same fungicide and at the same concentration but by spraying [25]. Intensive pesticide use for disease control has admittedly caused several environmentally related problems, such as contamination of food, soil, water, and animals; toxicity to farmers; resistance of pathogens to certain active ingredients in the pesticides; development of iatrogenic diseases (occurring due to pesticide use); biological imbalance, altering nutrient and organic matter cycling; elimination of beneicial organisms; and reduction of biodiversity, among others [24]. he identiication of these problems has increased the demands for residue-free products, making it necessary to search for disease control/management techniques in fruits and vegetables that do not endanger consumers and to reduce the risk of toxicity to farmers and the environmental imbalance generated by using synthetic fungicides. Myco fu m igatio n H arve s t D is e as e s fo r Co n tro llin g Po s t- Studies involving alternative control of plant diseases have increased signiicantly over the last 20 years, particularly emphasizing biological control as a promising alternative for reducing synthetic fungicide use. he potential of several microorganisms for controlling diferent disease-causing pathogens in fruits and vegetables has been reported [26-29]. Figure 1: Examples of post-harvest diseases of fruits and vegetables. Bitter Rot (A) and blue mold (B), postharvest decay of apple caused by the fungus Colletotrichum spp. and Penicillium expansum respectively; (C) - Decay of nectarine fruitcaused by P. expansum; (D) - Brown Rot of peach caused by Monilinia fructicola; (E) - Black Mold caused by Aspergillus niger on garlic; (F) - Green mold caused by P. digitatum on citrus fruits; (G) - Anthracnose of pepper fruit caused by Colletotrichum sp.; (H) - Decay of table grapes caused by Rhizopus stolonifer and Aspergillus niger. and vegetables. Solutions of borax, sodium bicarbonate, and more recently synthetic fungicides such as sodium ortho-phenyl phenate, imazalil, and thiabendazoleare oten used for controlling post-harvest decay in fruits and vegetables by immersing the fruit in fungicide solution [17,18]. One classic example is the use of 2,6-dichloro4-nitroaniline to control post-harvest decay in peaches, plums, and nectarines [19]. Another very widespread technique involves using benzimidazoles to control post-harvest decay in cherries by application before and ater fruit harvest [20]. Although the use of pesticides such as fungicides has positive aspects, the vast majority of products applied are extremely toxic, endangering human health and environmental balance. Several studies have demonstrated the presence and persistence of fungicide residues in fruits and vegetables [21-23]. he application of fungicides together with high temperatures for controlling post-harvest diseases Submit your Manuscript | www.austinpublishinggroup.com However, the development of commercial products intended for the biocontrol of post-harvest diseases has been limited, most likely due to the long time period necessary to identify, develop, and market the products, in addition to the process’s high inancial cost. Several features characterize a microorganism as an antagonist with potential for the development of commercial products, such as: genetic stability; efective at low concentrations; simple nutritional requirement; capacity to survive under adverse environmental conditions; efective against a wide range of phytopathogens in diferent products; resistant to the chemical products used in the post-harvest environment; compatible with commercial processing procedures; and lack of risk to human health [27]. he vast majority of the studies related to post-harvest biological control involve the use of fungi or bacteria as microbiological control agents. However, the positive efect on disease control/management is oten only observed when the biological agent is directly applied to the fruits or vegetables. his efect may occur mainly due to the main antimicrobial action mechanisms triggered by antagonistic microorganisms, namely competition for space and nutrients, and antibiosis [4,29]. However, some questions have been raised regarding the introduction of antagonists to the human diet and concerns for human health and food security [29]. In addition, the fact that most registered biocontrol products, such as Biosave (Pseudomonas syringae Van Hall), Shemer (Metschnikowia fructicola Kurtzman Austin J Biotechnol Bioeng 2(4): id1051 (2015) - Page - 02 Pereira OL Au s tin Pu blis h in g Gro u p Table 1: Species, host, lifestyle and taxonomic position of ilamentous and yeast fungi reported as VOCs producers. Species Host Lifestyle Site isolation Taxonomic position References Filamentous fungi Muscodor albus Cinnamomum zeylanicum Endophytic Honduras Ascomycota, Sordariomycetes, Xylariales [30] M. kashayum Aegle marmelos Endophytic India Ascomycota, Sordariomycetes, Xylariales [37] M. crispans Ananas ananassoides Endophytic Bolivian Ascomycota, Sordariomycetes, Xylariales [36] M. roseus Grevillea pteridifolia Endophytic Honduras Ascomycota, Sordariomycetes, Xylariales [66] M. oryzae Oryza ruipogon Endophytic Thailand Ascomycota, Sordariomycetes, Xylariales [47] M. musae Musa acuminata Endophytic Thailand Ascomycota, Sordariomycetes, Xylariales [47] M. cinnanomi C. bejolghota Endophytic Thailand Ascomycota, Sordariomycetes, Xylariales [39] M. strobelii C. zeylanicum Endophytic India Ascomycota, Sordariomycetes, Xylariales [38] M. darjeelingensis C. camphora Endophytic India Ascomycota, Sordariomycetes, Xylariales [67] M. tigerii C. camphora Endophytic India Ascomycota, Sordariomycetes, Xylariales [68] M. suthepensis C. bejolghota Endophytic Thailand Ascomycota, Sordariomycetes, Xylariales [47] M. yucatanensis Bursera simaruba Endophytic Mexico Ascomycota, Sordariomycetes, Xylariales [69] M. vitigenus Paullinia paullinioides Endophytic Peru Ascomycota, Sordariomycetes, Xylariales [49] M. equiseti Equisetum debile Endophytic Thailand Ascomycota, Sordariomycetes, Xylariales [47] M. sutura Prestonia triidi Endophytic Colombia Ascomycota, Sordariomycetes, Xylariales [52] M. fengyangensis Actinidia chinensis Endophytic China Ascomycota, Sordariomycetes, Xylariales [48] Hypoxylon sp. Persea indica Endophytic Canary Islands Ascomycota, Sordariomycetes, Xylariales [55] Nodulisporium sp. Myroxylon balsamum Endophytic Ecuador Ascomycota, Sordariomycetes, Xylariales [56] Nodulisporium sp. Lagerstroemia loudoni Endophytic Thailand Ascomycota, Sordariomycetes, Xylariales [57] Myrothecium inunduatum Acalypha indica Endophytic India Ascomycota, Sordariomycetes, Hypocreales [53] Gliocladium sp. Eucryphia cordifolia Endophytic USA Ascomycota, Sordariomycetes, Hypocreales [60] Ascomycota, Sordariomycetes, Hypocreales [70] Bionectria ochroleuca Nothapodytes foetida Endophytic India Ascomycota, Sordariomycetes, Hypocreales [58] Phomopsis sp. Odontoglossum sp. Endophytic Ecuador Ascomycota, Sordariomycetes, Diaporthales [54] Phoma sp. Larrea tridentate Endophytic USA Ascomycota, Dothideomycetes, Pleosporales [71] Gloeosporium sp. Tsuga heterophylla Endophytic USA Ascomycota, Leotiomycetes, Helotiales [59] Oxyporus latemarginatus Capsicum annum Endophytic Basidiomycota, Agaricomycetes [65] Basidiomycota, Agaricomycetes [72] Trichoderma atroviride Schizophyllum commune Saproit Chile Yeast fungi Aureobasidium pullulans Saprophytic Ascomycota, Dothideomycetes, Dothideales [61,62] Saccharomyces cerevisiae Ascomycota, Saccharomycetes, Saccharomycetales [40,41] Candida intermedia Ascomycota, Saccharomycetes, Saccharomycetales [42] Wickerhamomyces anomalus Ascomycota, Saccharomycetes, Saccharomycetales [40] Metschnikowia pulcherrima Ascomycota, Saccharomycetes, Saccharomycetales [40] & Droby), BioNext, AspireTM, Leasafre International (Candida oleophila Kaisha & Iizuka), and Yield Plus [Cryptococcus albidus (Saito) C.E.Skinner], have similar application methods that involve directly applying a cell suspension to horticultural products can generate fear in the population regarding their consumption. Mycofumigation is a diferent biological control strategy for post-harvest diseases in fruits and vegetables that can be an efective alternative to directly applying microorganisms to horticultural products. his strategy consists of the use of antimicrobial Volatile Organic Compounds (VOCs) produced by fungi. Submit your Manuscript | www.austinpublishinggroup.com he concept of mycofumigation started developing with the description of Muscodor albus Worapong, Strobel & W.M.Hes, an endophytic fungus obtained from Cinnamomum zeylanicum Breyne, and its potential for emitting volatile compounds that inhibit the growth and/or promote the death of many plant pathogenic agents [30,31]. A peculiarity of antimicrobial VOCs is that they can difuse in the air, reaching diicult-to-access habitats in closed environments [32]. his property makes antimicrobial VOCs emitted by fungi an additional valuable strategy for post-harvest disease biocontrol. For example, without any direct contact between isolates, the M. albus Austin J Biotechnol Bioeng 2(4): id1051 (2015) - Page - 03 Pereira OL volatiles inhibited growth of a wide range of fungal species, including Aspergillus fumigatus Fresen, A. carbonarius (Bainier) hom, A. lavus Link, A. niger Tiegh, A. ochraceus Wilh, Penicillium verrucosum Dierckx, P. digitatum (Pers.) Sacc. Fusarium culmorum (Wm.G.Sm.) Sacc. F. graminearum Schwabe, Botrytis cinera Pers, Colletotrichum acutatum J.H.Simmonds, Geotrichum candidum Link, Monilinia fructicola (G.Winter) Honey, and Rhizopus sp., important fungal species associated with post-harvest decay and mycotoxin production [31,33,35]. D ive rs ity o f An tim icro bial Vo latile Organ ic Co m p o u n d -Pro d u cin g Fu n gi Ater the discovery of M. albus, many antimicrobial VOCproducing fungal species were identiied (Table 1). he vast majority of these species were isolated from healthy plant tissue, especially from tropical plants commonly used in alternative medicine, such as Ananas ananassoides (Baker) L. B. Sm., Aegle marmelos (L.) Corr., Cinnamomum spp. And Myroxylon balsamum (L.) Harms [30,36-39]. Hitherto, most ilamentous fungi related to antimicrobial volatile emission have belonged to Ascomycota, order Xylariales, and other related ascomycetes are found in the classes Sordariomycetes, Dothideomycetes, and Leotiomycete, all of which are endophytic (Table 1). In a more phylogenetically distant group, the basidiomycetes Oxyporus latemarginatus (Durieu & Mont.) Donkand and Schizophyllum commune Fr. are also related to antimicrobial volatile production, and S. commune is noteworthy because, unlike the others, it was isolated from decomposing material, exhibiting a saprophyticlife style in nature. In addition to ilamentous fungi, some yeasts have the potential for emitting the VOCs described. Aureobasidium pullulans (de Bary & Löwenthal) G. Arnaud, Saccharomyces cerevisiae Meyen ex E.C.Hansen, Candida intermedia (Cif. & Ashford) Langeron & Guerra, Wickerhamomyces anomalus (E.C.Hansen) Kurtzman, Robnett & Bas.-Powers, and Metschnikowia pulcherrima Pitt & M.W.Mill. were reported emitting volatile compound mixtures that inhibit the growth of fungi associated with post-harvest decay in fruits and vegetables [40-42]. he identiication of fungi associated with antimicrobial VOC production has been conducted through morphology studies and mainly by molecular analyses of the internal transcribed spacer (ITS) region sequences of their DNA. For species of the Muscodor genus, identiication and even the proposal of new species have been performed via phylogeny based on ITS region sequencing, accompanied by the volatile compound production proile, as specialized structures in sexual and asexual reproduction have never yet been observed for this genus. his feature is useful for identifying and diferentiating fungal species. An tim icro bial Vo latile Organ ic Co m p o u n d s ( VOCs ) VOCs are solid/liquid carbon-based compounds that easily enter the gas phase via vaporization at 0.01 KPa and temperature close to 20oC, i.e., exhibit high vapor pressure and low water solubility, which allows them to evaporate and difuse easily through the air [16,43]. More than 250 VOCs have been identiied from fungi, occurring Submit your Manuscript | www.austinpublishinggroup.com Au s tin Pu blis h in g Gro u p in the form of mixtures of simple hydrocarbons, heterocyclic hydrocarbons, aldehydes, ketones, alcohols, phenols, thioalcohols, thioesters and their derivatives, including benzene and cyclohexanes [32]. VOCs may be derived from primary and secondary metabolic pathways of microorganisms. he microorganism releases VOCs as products of primary metabolism when it decomposes substrates to extract nutrients necessary for its maintenance. In contrast, in secondary metabolism, VOC production is usually related to competition for resources in nutrient-poor environments [44]. he proiles of volatiles produced by a certain species or isolates may vary, depending on the substrate used for growth, incubation duration, nutrient type present, temperature, and other environmental parameters [32,45]. he same M. albus 620 isolate shows variation in volatile proile composition depending on the nutrient concentration in the growth medium, where the number of volatile compounds detected was higher in culture media that exhibited a greater quantity of the carbon source [46]. he VOCs produced by Muscodor species consist mainly of lowmolecular-weight esters, alcohols, and acids, with diferences between the compound mixtures produced by diferent species of the genus. However, the VOC mixture produced by most Muscodor species has antimicrobial bioactivity [47,48]. Muscodor species vary regarding the VOC mixture emitted. Muscodor crispans Mitch, Strobel, Hess, Vargas & Ezra, for example, do not produce naphthalene or azulene derivatives, compounds observed in other species of the genus Muscodor [36]. In contrast, naphthalene predominates in the VOC mixture emitted by M. vitigenus Daisy, Strobel, Ezra & Hess, and the VOC mixture emitted by this fungus does not exhibit antifungal bioactivity, though it has previously demonstrated lethality in insects [49]. Gas chromatography/mass spectrometry analyses of the VOC mixture produced by M. albus reveal the presence of at least 28 diferent VOCs, representing at least ive classes of organic substances, where the esters contributed the highest percentage in the mixture, followed by alcohols, acids, lipids, and ketones [31]. he antimicrobial action spectra of the compounds emitted by certain species or isolates seem to be afected by the compound mixture emitted by each isolate. Several studies have demonstrated that the volatile mixture among Muscodor species varies, and the action spectrum also varies, with some being more eicient in inhibiting the growth of certain fungi than others [31,37-39,47-49]. An tim icro bial Effe cts o f th e VOCs Pro d u ce d by Fu n gi in Po s t-H arve s t Path o ge n s in Fru its an d Ve ge table s Most studies on the antimicrobial efects of volatiles produced by fungi involve Muscodor species (Figure 2), although the biological functions of the toxic compounds produced are still not well elucidated. Most Muscodor spp. isolates and other antimicrobial volatile-producing species are endophytic.VOC emission by these fungi may act as a defense mechanism for the host plant against pathogen attack, helping the antimicrobial VOC-producing Austin J Biotechnol Bioeng 2(4): id1051 (2015) - Page - 04 Pereira OL Au s tin Pu blis h in g Gro u p compounds emitted by M. albus shows DNA damage in Escherichia coli cells when exposed to VOCs emitted by the fungus, which most likely resulted in the interruption of the replication and/or transcription processes; the compounds also caused morphological changes in the cells, generating increased luidity of the cell membrane [50]. he antimicrobial potential of the compounds emitted by M. albus against diverse microbial groups among fungi, bacteria, and oomycetes has been described in the literature. Growth (in vitro) of B. cinerea, A. fumigatus, Tapesia yallundae Wall work & Spooner, Rhizoctonia solani Kühn, Sclerotinia sclerotiorum (Lib.) de Bary, Candida albicans (C.P.Robin) Berkhout, Pythium ultimumTrow, Verticillium dahliae Kleb, Phytophthora cinnamomi Rands, E. coli, Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus, representative of diverse groups of fungi, oomycetes and bacteria, was inhibited, and their cells died ater exposure to VOCs emitted by M. albus isolates [30,31]. he efects of the VOCs emitted by M. albus 620 were reported (in vitro) against three important fungi frequently associated with post-harvest decay, S. sclerotiorum, B. cinerea and Penicillium expansum Link. he volatiles emitted by the M. albus 620 isolate exhibited signiicant efects in the germination of B. cinerea and P. expansum spores, preventing the conidia of these fungi to germinate and reducing S. sclerotiorum colony diameter growth. For both treatments, the source of M. albus 620 used was rye grain colonized by the fungus, and higher grain weight (0.25 g to 1.25 g/L) in each treatment corresponded to a stronger observed efect, where 1.25 g/L completely inhibited B. cinerea and P. expansum spore generation and S. sclerotiorum growth [51]. he volatiles emitted by M. albus were also tested against important toxin-producing fungi. Conidia of Aspergillus carbonarius (Bainier) hom, A. lavus, A. niger, A. ochraceus, P. verrucosum, F. culmorum, and F. graminearum died or their germination was inhibited (in vitro) when exposed to volatiles produced by M. albus colonizing rye grain at 20oC. When conidia of the same fungi were separately exposed to the compounds most abundant in the compound mixture emitted by M. albus, isobutyric acid and 2-methyl-1-butanol, the same magnitude of efect was not observed [34]. Figure 2: In vitro effect of VOCs produced by Muscodor sp. (2 – upper side of the plate) inhibiting the mycelial growth of A. ochraceus (A2 – bottom of the plate); A. niger (B2 – bottom of the plate); F. semitectum (C2 – bottom of the plate); A. lavus (D2 – bottom of the plate). Control (A1–D1). endophyte survive by preventing colonization of the host plant by microorganisms that compete for the same ecological niche [31]. Toxicity from exposure to M. albus appears to be associated with combined action of the compounds present in the mixture. Each of the ive classes of volatile compounds produced by the fungus (alcohols, esters, ketones, acids, and lipids) had some inhibitory efect against fungi and bacteria when tested alone but did not cause their death. However, they acted synergistically when collectively tested in the mixture, killing a wide range of fungi and bacteria pathogenic to plants and humans [31]. A recent attempt to elucidate the action mechanism of the volatile Submit your Manuscript | www.austinpublishinggroup.com In addition to M. albus, other Muscodor species have also been reported to inhibit the growth of fungi associated with post-harvest decay. VOCs emitted by M. crispans were efective against a wide range of phytopathogens, among which B. cinerea, Colletotrichum lagenarium Caruso & Kuc, Fusarium avenaceum (Fr.) Sacc., F. culmorum, Phytophthora palmivora Butler (Butler), P. ultimum, S. sclerotiorum, G. candidum, A. fumigatus, and Curvularia lunata (Wakker) Boedijn exhibited inhibited colony growth. Additionally, except for the last three, 24-hour exposure to the compound mixture emitted by M. crispans led to cell death [36]. he volatiles emitted by M. strobelii exhibited a broad spectrum of activity against yeasts, bacteria, and ilamentous fungi and, among the fungi tested, the VOCs completely inhibited the growth of Penicillium citreonigrum Dierckx, B. cinerea, and Aspergillus japonicus Saitoater three days of exposure. he mixture of compounds emitted by M. strobelii is diferent from the mixtures of other species of the genus Austin J Biotechnol Bioeng 2(4): id1051 (2015) - Page - 05 Pereira OL Au s tin Pu blis h in g Gro u p Muscodor, exhibiting 4-octadecylmorpholine as the most abundant compound, along with tetraoxapropellan and aspidofractinine-3methanol; the last two compounds are not encountered among the volatiles of the other Muscodor species [38]. assay, the antagonist was inoculated in culture medium deposited at the bottoms of glass boxes containing apples artiicially inoculated with the phytopathogens, thus preventing direct contact between the antagonist and the fruit [62]. Variation in compounds present in the VOC mixture among Muscodor species also occurred in M. sultura, where there is variation in the compound mixture proile compared with other Muscodor species, producing higher abundances of propanoic acid, 2-methyl, and thujopsene. he VOCs emitted by M. sultura exhibited antimicrobial bioactivity against a wide range of fungi, inhibiting the growth of A. fumigatus, B. cinerea, C. lagenarium, Ceratocystis ulmi (Buisman) C. Moreau, Cercospora beticola Sacc., G. candidum, Mycosphaerella ijiensis M. Morelet, P. cinnamomi, P. palmivora, Pythium ultimum, R. solani, S. sclerotiorum, and V. dahliae ater two days of exposure, promoting death of their cells. Many of these species are important phytopathogenic fungi associated with postharvest decay in fruits and vegetables [52]. Other yeasts, such as Candida intermedia, Wickerhamomyces anomalus, and Metschnikowia pulcherrima, have been tested for post-harvest disease control in fruit. Isolates of these yeasts were used to control B. cinera colonization in strawberry and table grape. he VOCs emitted inhibited B. cinera growth in vitro, and the yeasts reduced disease severity when applied in vivo. However, the efect on the inhibition of disease development was more intense ater directly applying yeast suspension to the strawberries inoculated with B. cinera [40,42]. Other Muscodor species, such as M. musae, M. oryzae, M. suthepensis and M. equiseti N. Suwannarach & S. Lumyong, were described together with the antimicrobial potential of VOCs emitted. hese VOCs showed antimicrobial activity against several microorganisms, including important post-harvest phytopathogens, such as A. lavus, B. cinerea, Colletotrichum capsici (Syd. & P. Syd.) Butler & Bisby, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., Colletotrichum musae (Berk.& Curtis) Arx, Penicillium digitatum, and P. expansum, and in most cases, the exposure to the compounds emitted by these Muscodor species inhibited 100% of phytopathogen growth and caused death of their cells [47]. Muscodor species are not the only fungi that have been reported to emitanti microbial volatiles with the potential to inhibit growth and even kill post-harvest phytopathogenic fungi in fruits and vegetables. For Myrothecium inundatum Tode, Phomopsis sp., Hypoxylon sp., Nodulisporium sp., Bionectria ochroleuca (Schwein.) Schroers & Samuels, Schizophyllum commune RF., Gloeosporium sp., and Gliocladium sp., even though these fungi do not exhibit the same efects observed in Muscodor spp. compounds in vitro, the VOCs produced by isolates of these fungi reduced the growth of important fungi associated with post-harvest diseases, such as Aspergillus ochraceus, A. lavus, A. fumigatus, B. cinerea, C. capsici, C. gloeosporioide, C. lagenarium, C. musae, G. candidum, Penicillium digitatum, Penicillium expansum, Phytophthora palmivora, Pythium ultimum, and Sclerotinia sclerotiorum [53-60]. In addition to in vitro assays, some studies have been performed to elucidate the potential of VOCs produced by fungi to control postharvest diseases in fruits and vegetables by mycofumigation of the horticultural product. he VOCs emitted by Aureobasidium pullulans yeast isolates inhibited (in vitro) conidial germination of post-harvest disease-causing phytopathogens in apple. Furthermore, when tested in vivo, the VOCs reduced the incidence of blue mold and bitter rot in apple caused by Penicillium expansum and Colletotrichum acutatum, respectively; however, the greatest efect was observed ater directly applying the antagonists to the fruit [61]. In later tests (in vivo), VOCs of the same isolates signiicantly reduced B. cinerea and P. expansum infection in apple, as observed by the smaller size of damage in the fruit compared with the control treatment; in this Submit your Manuscript | www.austinpublishinggroup.com he potential of volatiles produced by M. albus to control postharvest diseases in fresh fruit by mycofumigation was also studied. Mycofumigation of apple with M. albus culture controlled blue mold (Penicillium expansum) and gray mold (Botrytis cinerea) in apples inoculated with the phytopathogens, without requiring direct contact between the fruit and the M. albus culture. he same was observed in peaches inoculated with Monilinia fructicola, where fumigation with M. albus culture promoted complete control of brown rot in an assay performed using closed plastic boxes. In organic table grape (‘hompson Seedless’ and ‘Red Seedless’ varieties), mycofumigation with M. albus culture in plastic boxes reduced the incidence of postharvest decay [35,63,64]. Mycofumigation with Oxyporus latemarginatus isolate culture also reduced development of gray mold caused by B. cinera in apples [65]. In citrus, mycofumigation with Nodulisporium sp. isolate culture controlled green mold decay in Citrus limon caused by Penicillium digitatum and blue mold decay in Citrus aurantifolia and C. reticulata caused by P. expansum [57]. Co n clu s io n Mycofumigation is a promising alternative for reducing postharvest losses in fruits and vegetables caused by fungi. he method has potential to be applied during the transport and storage of fresh fruits and vegetables, where the presence of antimicrobial VOCs, such as compound mixtures produced by M. albus cultures, may increase the shelf lives of these horticultural products by reducing the incidence of post-harvest diseases. he potential of some fungi to emit VOCs able to inhibit or cause death of important phytopathogenic fungi associated with post-harvest decay, without requiring direct contact with the product to be consumed, together with the wide range of microorganisms sensitive to VOCs from fungal species, makes mycofumigation an interesting method for controlling post-harvest diseases, which, unlike traditional methods, reduces risks to human health and environmental contamination. Ackn o w le d ge m e n t he authors thank the Conselho Nacional de Desenvolvimento Cientíico e Tecnológico – CNPq, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES and Fundação de Amparo a Pesquisa do Estado de Minas Gerais – FAPEMIG for inancial support. Austin J Biotechnol Bioeng 2(4): id1051 (2015) - Page - 06 Pereira OL References 1. Macarisin D, Cohen L, Eick A, Rafael G, Belausov E, Wisniewski M, et al. Penicillium digitatum Suppresses Production of Hydrogen Peroxide in Host Tissue During Infection of Citrus Fruit. Phytopathology. 2007; 97: 1491-1500. 2. Droby S, Chalutz E, Wilson CL, Wisniewski ME. Alternative to the use of synthetic fungicides. Phytoparasitica. 1992; 20: 149-153. Au s tin Pu blis h in g Gro u p control of blue mould using new fungicides and biocontrol yeasts lowers levels of fungicide residues and patulin contamination in apples. Postharvest Biol Tec. 2011; 60: 164-172. 23. 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