Review: Utilization of antagonistic yeasts to manage postharvest fungal diseases of fruit

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International Journal of Food Microbiology 167 (2013) 153–160

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International Journal of Food Microbiology

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Review

Review: Utilization of antagonistic yeasts tomanage postharvest fungaldiseases of fruit

Jia Liu a,⁎, Yuan Sui a, Michael Wisniewski b, Samir Droby c, Yongsheng Liu a,d,⁎⁎a School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, Chinab U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS), Kearneysville, WV 25430, USAc Agricultural Research Organization (ARO), The Volcani Center, Bet Dagan 50250, Israeld Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University,Chengdu 610064, China

⁎ Correspondence to: J. Liu, Hefei University of Techno230009, China. Tel.: +86 131 3551 9768; fax: +86 551 6⁎⁎ Correspondence to: Y. Liu, Hefei University of Techno230009, China. Tel.: +86 187 0983 2886; fax: +86 551 6

E-mail addresses: [email protected] (J. Liu), liuyongsh

0168-1605/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.ijfoodmicro.2013.09.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 May 2013Received in revised form 2 September 2013Accepted 10 September 2013Available online 18 September 2013

Keywords:Antagonistic yeastMode of actionPostharvest diseaseStress tolerance

Significant losses in harvested fruit can be directly attributable to decay fungi. Some of these pathogenic fungi arealso the source of mycotoxins that are harmful to humans. Biological control of postharvest decay of fruits,vegetables and grains using antagonistic yeasts has been explored as one of several promising alternatives tochemical fungicides, the use of which is facing increasingly more stringent regulation. Yeast species have beenisolated over the past two decades from a variety of sources, including fruit surfaces, the phyllosphere, soil andsea water, and their potential as postharvest biocontrol agents has been investigated. Several mechanismshave been proposed as responsible for their antagonistic activity, including competition for nutrients andspace, parasitism of the pathogen, secretion of antifungal compounds, induction of host resistance, biofilm forma-tion, and most recently, the involvement of reactive oxygen species (ROS) in defense response. It has beenrecognized that a biocontrol system is composed of a three-way interaction between the host (commodity),the pathogen and the yeast, all of which are affected by environmental factors. Efficacy and consistent perfor-mance in controlling postharvest diseases are the hurdles that must be overcome if the use of yeast biocontrolagents and other alternatives are to be widely used commercially. Therefore, attempts have been made tocombine alternative treatments in order improve their overall performance. The current review provides abrief overview of the topic of the use of yeasts as postharvest biocontrol agents and includes information onthe sources from which yeast antagonists have been isolated, their mode of action, and abiotic stress resistancein yeast as it relates to biocontrol performance. Areas in need of future research are also highlighted.

© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1532. Sources of yeast antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1543. Mode of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1554. Enhancing biocontrol efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575. Commercial application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1586. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

logy, Tunxi Road No. 193, Hefei291 9399.logy, Tunxi Road No. 193, Hefei290 [email protected] (Y. Liu).

ights reserved.

1. Introduction

Postharvest losses of fruits, including pome fruit, stone fruit, berryfruit and citrus, can be quite significant if handling, processing, and stor-age conditions are not optimal. Losses representing up to 25% of totalproduction in industrialized countries andmore than 50% in developing

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Table 1Characteristics of yeasts for the biocontrol of postharvest spoilage fungi on fruits.Adapted and modified from Barkai-Golan (2001) and Wilson and Wisniewski (1989).

Genetically stableEffective at low concentrationsNot fastidious in its nutrient requirementsCapable of surviving under adverse environmental conditions (including low/hightemperature, oxidative stress and controlled atmosphere storage)

Effective against a wide range of pathogens on a variety of fruitsAmenable to production on an inexpensive growth mediumPreparable in a formulation that can be effectively stored and dispensedCompatible with commercial processing proceduresResistant to pesticides, friendly to environment and non-pathogenic to host commodityNot produce metabolites that are deleterious to human healthUnable to grow at 37 °C and not associated with infections in humans

154 J. Liu et al. / International Journal of Food Microbiology 167 (2013) 153–160

countries have been reported (Nunes et al., 2012). The high levels ofdecay caused by fungal pathogens can be directly attributable to thelarge amount of nutrients and water, low pH, and the decrease in in-trinsic decay resistance of fruit after harvest (Droby et al., 1992).Fungal species within the genera Penicillium, Botrytis, Monilinia,Rhizopus, Alternaria, Aspergillus, Fusarium, Geotrichum, Gloeosporiumand Mucor represent postharvest pathogens responsible for manyof the most important postharvest diseases (Barkai-Golan, 2001).

In addition to economic considerations, infected produce representsa potential health risk since several fungal genera, such as Penicillium,Alternaria and Fusarium, produce mycotoxins that pose a human healthhazard. For example, Penicillium expansum, the causal pathogen ofpostharvest blue mold in a variety of fruits, produces a number of toxicsecondary metabolites including patulin, citrinin, and chaetoglobosins,all of which are potential carcinogens (Andersen et al., 2004). Althoughthe use of synthetic chemical fungicides is the principal method ofcontrolling postharvest diseases and the resulting rots they cause,stricter regulatory policies are being imposed on their use. There isalso strong consumer demand to reduce the use of potentially harm-ful chemicals in their food supply. For these reasons, alternativemethods for managing postharvest diseases have been increasinglyexplored during the last 25 years. The reader is referred to the followingreports for a comprehensive review of the subject: Droby et al. (1992,2009), Janisiewicz and Korsten (2002), Sharma et al. (2009) andWisniewski et al. (2007).

Biological control using microbial agents has been reported amongseveral alternatives to be an effective approach, to the use of syntheticchemical fungicides for managing postharvest fruit decay (Droby et al.,2009; Spadaro and Gullino, 2004). In this regard, the use of antagonisticyeasts has been especially emphasized since the production of toxicsecondary metabolites (antibiotics) is generally not involved in theirinhibitory activity (Wisniewski and Wilson, 1992). Considerable infor-mation is also availablewith respect to the commercial scale productionof yeast including fermentation, formulation, storage, and handling(Wisniewski et al., 2007).

The original source (fruit surfaces) for the isolation of yeast antago-nists (Wilson andWisniewski, 1989; Wilson et al., 1993) has expandedto other environments, such as sea water (Hernández-Montiel et al.,2010; Wang et al., 2008) and Antarctic soil samples (Vero et al., 2013).The main intent in exploring these new sources has been both to iden-tify yeasts that can thrive in stressful environments and to discoverisolates with novel modes of action.

Yeasts used to manage postharvest diseases in orchards and pack-inghouses encounter a variety of stressful conditions that can affecttheir viability and efficacy, including extreme temperature, low hu-midity, oxidative stress, lack of nutrients, and adverse pH. Therefore,abiotic stress tolerance is an essential attribute for yeasts used asbiocontrol agents. Several methods, including physiological manipu-lation (Abadias et al., 2001;Mokiou andMagan, 2008), stress adapta-tion (Liu et al., 2011a, 2012), and the use of exogenous anti-stresssubstances (An et al., 2012; Liu et al., 2011b) have been employedto enhance stress tolerance and improve biocontrol efficacy.

A suitable formulation and large-scale testing are fundamentalsteps in the process of developing a commercial biocontrol product.Pilot scale studies of biocontrol yeasts using both liquid and dryformulations of yeasts have been conducted (Long et al., 2007;Patiño-Vera et al., 2005; Melin et al., 2007; Mokhtarnejad et al.,2011; Torres et al., 2003). In the current review, a brief overviewof research that has led to a more comprehensive understanding ofpostharvest biocontrol systems is presented and discussed with anemphasis on the most recent literature. Information on methodsand sources for isolating and screening yeast antagonists, the per-formance and efficacy of yeast under different environmental condi-tions, and the potential for commercializing yeast as postharvestbiocontrol agents are presented. Areas in need of research are alsohighlighted.

2. Sources of yeast antagonists

In an early attempt to develop postharvest biocontrol agents, Puseyand Wilson (1984) reported the ability of a strain (B-3) of Bacillussubtilis to control brown rot on peach caused by Monilinia fructicola.It was subsequently discovered that the antibiotic, iturin, produced bythe bacterium was the main factor responsible for the control ofbrown rot. The use of an antibiotic-producing microorganism on foodraised significant concerns and so Wilson et al. (1993) developed a se-lection strategy for identifying potential postharvest biocontrol agentsbased on the in vivo selection of non-antibiotic producing yeast strainsby applying wash water of fruits into surface wounds and isolatingmicrobes from wounds that were protected against infection fromartificially inoculated pathogens. This selection and screening strate-gy was later used by many different laboratories to identify potentialyeast antagonists.

Once a promising yeast antagonist is isolated in pure culture, itis identified using morphological and physiological characterization(Chanchaichaovivat et al., 2007) and/or by DNA sequencing of con-served regions of ribosomal DNA (Kurtzman and Droby, 2001). A num-ber of antagonists have been identified using this strategy and evaluatedon the criteria presented in Table 1. Based on these selection criteria, theyeast antagonist, Candida oleophila (strain I-182), isolated from thesurface of tomato fruit, became the first-generation of yeast-basedpostharvest biocontrol products commercialized under the trade name‘Aspire’ by Ecogen, Inc. (Wisniewski et al., 2007). The product was avail-able on the market for a few years but is no longer available; however,this potential of this yeast species has been explored by other researchers(Bastiaanse et al., 2010;Massart et al., 2005). A commercial yeast biocon-trol product, based on Metschnikowia fructicola, is marketed in Israelunder the trade name “Shemer” by Bayer, Inc.

Yeasts that are naturally present on and apparently endemic tofruit surfaces represent the major group of yeasts utilized to managepostharvest diseases. However, antagonists have also been isolatedfrom other sources, such as the phyllosphere, roots, soil, and seawater. For example, the phyllosphere yeast, Rhodotorula glutinis(strainY-44), isolated from leaves of tomato, has been reported tosuppress gray mold (Botrytis cinerea) on both leaves and fruits of tomato(Kalogiannis et al., 2006). Kloeckera apiculata (strain 34-9), isolated fromcitrus roots, has been reported to be effective in controlling Penicilliumitalicum and B. cinerea on citrus and grapes, respectively (Long et al.,2005). The psychrotrophic yeast, Leucosporidium scottii (strain At17), iso-lated fromAntarctic soil, was identified as a good biocontrol agent againstboth blue and gray mold of apple caused by P. expansum and B. cinerea,respectively (Vero et al., 2013). Compared to yeasts isolated from fruitsurfaces, marine yeasts typically have higher osmotolerance and there-fore may potentially be more suitable for use under conditions whereyeast are exposed to abiotic stress (Hernández-Montiel et al., 2010;Wang et al., 2010). The marine yeast, Rhodosporidium paludigenum, iso-lated from the East China Sea, effectively inhibits P. expansum on pearfruit, and Alternaria alternata on Chinese winter jujube (Wang et al.,

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155J. Liu et al. / International Journal of Food Microbiology 167 (2013) 153–160

2010). Suzzi et al. (1995) reported on the postharvest biocontrol activ-ity of natural wine yeasts isolated from grape berry. Among the mostpromising antagonists, exhibiting a wide spectrum of activity, weretwo strains of Saccharomyces cerevisiae and one of Zygosaccharomyces.Table 2 provides a partial list of yeasts isolated from different sourcesand identified as good antagonists.

3. Mode of action

As outlined in Fig. 1, postharvest biocontrol systems are complexand are influenced by a variety of parameters. Biocontrol yeasts areapplied to fruit in commercial packinghouses by spraying, dipping anddrenching, common practices also used for the delivery of chemicalfungicides. Prior and subsequent to the application of antagonisticyeasts on harvested commodities, environmental factors such astemperature, oxidative stress, pH and water activity can influencetheir viability and hence efficacy (Teixidó et al., 1998; Liu et al.,2011a, 2012). Once yeast cells come in contact with a fruit surface,they will also occupy surface wounds inflicted during harvesting andsubsequent handling. In the wounds, yeasts grow rapidly during thefirst 24 h and as a result deplete available nutrients and physically occu-py the wound space. This can have a direct effect on the germination offungal spores. Therefore, in the first 24 h, the most important mode ofaction of yeast antagonists is competition for nutrients and niche exclu-sion (Li et al., 2008; Liu et al., 2012). After 24 h, other modes of actioncan play a significant role in concert with nutrient competition andniche exclusion to bring about decay management (Droby et al., 2000,2009; Wisniewski et al., 2007). Wounded fruit tissues respond to vari-ous yeast elicitors (mainly cell wall components and other secretedcompounds) by activating host defense mechanisms (Chan et al.,2007; Droby et al., 2002), regulating yeast population density throughchanges in pH, the production of oxidative compounds (Hershkovitzet al., 2012; Macarisin et al., 2010), and inducing a change in yeastmorphology (Fiori et al., 2012). In turn, yeasts adhere to host tissues(Scherm et al., 2003) or pathogen cell walls (Chan and Tian, 2005;Wisniewski et al., 1991) forming biofilms, secrete fungal cell wall

Table 2Representative antagonistic yeasts from difference sources used for management ofpostharvest diseases.

Source Yeast species Reference

Fruit surfaceTomato Candida oleophila (I-182) Wilson et al. (1993)Grape Metschnikowia fructicola

(NRRL Y-27328)Kurtzman and Droby(2001)

Grape Saccharomyces cerevisiae(strains N.826 & N.831)

Suzzi et al. (1995)

Grape Zygosaccharomyces (N. F30) Suzzi et al. (1995)Citrus Pichia guilliermondii (US-7) Droby et al. (1993)Orange Candida saitoana (240) El-Ghaouth et al. (1998)Apple Candida ciferrii (283) Vero et al. (2002)Apple Candida sake (CPA-1) Viñas et al. (1998)Apple Cryptococcus laurentii Qin et al. (2004)Apple Rhodotorula glutinis Qin et al. (2004)Peach Pichia membranaefaciens Fan and Tian (2000)Lemon Cystofilobasidium infirmominiatum Vero et al. (2011)

PhyllosphereTomato leaves Rhodotorula glutinisY-44 Kalogiannis et al. (2006)

RootCitrus root Kloeckera apiculata 34-9 Long et al. (2005)

SoilAntarctic soil Leucosporidium scottii At17 Vero et al. (2013)Orchard soil Pichia caribbica Zhao et al. (2012)

SeaEast China Sea Rhodosporidium paludigenum Wang et al. (2008)Mar de Cortes Debaryomyces hansenii Hernández-Montiel

et al. (2010)

degrading enzymes (Bar-Shimon et al., 2004; Castoria et al., 1997),and deplete iron needed for pathogen growth (Saravanakumar et al.,2008; Sipiczki, 2006). The role of reactive oxygen species (ROS) in bio-control systems especially has been demonstrated to play an importantrole in biocontrol systems (Castoria et al., 2003; Liu et al., 2012;Macarisin et al., 2010). The importance of any one mode of action canvary between biocontrol systems (yeast–pathogen–host). Modes ofaction of yeast antagonists against specific fungal pathogens, otherthan competition for space and nutrients (the basic mode of action formost of antagonistic yeasts), are listed in Table 3.

The ability of antagonistic yeasts to attach to the hyphae of fungalpathogens and their ability to secrete lytic enzymes play an importantrole in biocontrol activity. The first discovery of these attributes wasthe report of the ability of the yeast antagonist, Pichia guilliermondii,to become firmly attached to the hyphae of B. cinerea and secreteβ-(1–3) glucanase which degraded the cell walls of the pathogen(Wisniewski et al., 1991). Zhang et al. (2012) more recently demon-strated the antifungal activity of an alkaline serine protease by theyeast-like fungus, Aureobasidium pullulans (strain PL5), and docu-mented its role in the mode of action of this biocontrol agent.

Antagonistic yeasts have also been reported to act as a bio-elicitor offruit host resistance (Droby and Chalutz, 1994). With the developmentof DNA microarrays and high-throughput sequencing technologies,global changes in gene expression in both host tissues and yeasts havebeen reported that provide a better understanding of how biocontrolsystems operate. For example, a microarray analysis of the response ofcherry tomato to the antagonistic yeast, Cryptococcus laurentii, indicatedthat genes involved in metabolism, signal transduction, and stress re-sponse were up-regulated while genes related to energy metabolismand photosynthesis were suppressed. The changes in gene expressionin tomato fruit inducedbyC. laurentiiwere associatedwith increased re-sistance to pathogen infection (Jiang et al., 2009). In grapefruit, applica-tion of the yeast, Metschnikowia fructicola, up-regulated pathogenesis-related (PR) genes and genes that are part of a MAPK cascade involvedin defense signaling and down-regulated antioxidant genes such asperoxidase, superoxide dismutase and catalase. The collection of genesup-regulated by M. fructicola in grapefruit was consistent with aninduced resistance response and it was postulated that the inducedresponse played a role in the efficacy ofM. fructicola against postharvestpathogens such as Penicillium digitatum (Hershkovitz et al., 2012).A proteomic study by Chan et al. (2007) indicated that Pichiamembranaefaciens induced antioxidant and PR proteins in peach fruit,and it was again suggested that these proteins played an essential rolein the control of P. expansum in this biocontrol system.

Hershkovitz et al. (2013) conducted a transcriptomic analysis, usingRNA-Seq, to examine changes in gene expression inM. fructicolawhen itwas exposed to citrus tissues and the postharvest pathogen P. digitatum.Results indicated that more than 250 genes exhibited expression re-sponses specifically associated with the yeast–citrus vs. the yeast–pathogen interaction. Genes related to transmembrane, multidrugtransport, and amino acid metabolism were induced in the yeast–pathogen interaction, while expression of genes involved in oxidativestress, iron homeostasis, zinc homeostasis and lipidmetabolismwas in-duced in the yeast–fruit interaction. Collectively, these reports indicatethat different gene/protein profiles are involved in different antagonis-tic yeast–host–pathogen interactions, demonstrating the dynamics ofdifferent biocontrol system and how “omic” technologies can provideinsights into the modes of action of antagonistic yeasts.

Several different yeast species in the genusMetschnikowia, includingMetschnikowia pulcherrima (Janisiewicz et al., 2001; Piano et al., 1997;Spadaro et al., 2002), M. fructicola (Kurtzman and Droby, 2001), andMetschnikowia andauensis (Manso and Nunes, 2011), have been usedas postharvest biocontrol agents. Sipiczki (2006) reported the activityof a siderophore as a mechanism by which Metschnikowia yeasts canstrongly antagonize the growth of various filamentous fungi, yeasts,and bacteria by depleting iron in the growth medium. Saravanakumar

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Environment

Biocontrol Yeast

Fungal Pathogen Fruit Host

Efficacy

Pathogenesis Resistance

Phytotoxin, suppression of oxidativeburst, host pH modification, proteincarbonylation

Antifungal compounds, PR proteins,oxidative burst, host systemicresistance

Fig 1. Diagram of possible interactions between host, pathogen and antagonist and their environment.

156 J. Liu et al. / International Journal of Food Microbiology 167 (2013) 153–160

et al. (2008) determined that a siderophore plays an active role in vivo.They demonstrated that M. pulcherrima (strain MACH1) produced thesiderophore, pulcherrimin, that scavenged iron and reduced the growthof B. cinerea, A. alternata and P. expansum both in vitro and in apple fruit.Therefore, iron content in the fruit may be an important parameterinfluencing biocontrol efficacy in some biocontrol systems.

When applied to fruit surfaces and wounds, antagonistic yeastsencounter ROS-generated oxidative stress that can affect their viabilityand/or performance (Castoria et al., 2003; Macarisin et al., 2010).Castoria et al. (2003) investigated the relationship between oxidativestress resistance and the fitness of antagonistic yeasts as postharvestbiocontrol agents and suggested that the ability of a yeast to tolerate

Table 3Representative modes of action involved in the tritrophic interactions (antagonistic yeast–fung

Mode of action Antagonistic yeast

Induction of host defense Candida oleophila (I-182)Candida saitoanaPichia membranaefaciensPichia caribbicaCryptococcus laurentii

Attachment & lytic enzyme secretion Pichia membranaefaciens

Cryptococcus albidus

Pichia guilliermondii (87)Adjustment of population density Candida oleophila (I-182)Morphology change Saccharomyces cerevisiae (M25)

Pichia fermentansIron depletion Metschnikowia isolates from noble-rotted grap

Metschnikowia pulcherrima

ROS tolerance Cryptococcus laurentii LS-28

Metschnikowia fructicolaCystofilobasidium infirmominiatumCandida oleophila (I-182)

Alleviation of oxidative damage of fruit host Pichia membranaefaciensCryptococcus laurentiiCandida guilliermondiiRhodotorula glutinis

Induction of ROS production in host Candida oleophila (I-182)Metschnikowia fructicola (277)

Note: As the competition for nutrients and space is the basic attrubite, it is not listed in this tab

the high levels of ROS produced in apple fruit in response to woundingmay be an essential characteristic of an effective biocontrol agent. Thisdiscovery raised questions about the role of ROS in biocontrol systems.Liu et al. (2012) later confirmed the importance of oxidative stresstolerance in antagonistic yeasts. In that study, they demonstrated thatexposing the yeast, C. oleophila (strain 182), to a sublethal level of oxida-tive stress increased stress tolerance of the yeast to subsequent lethallevels of oxidative andheat stress andalso resulted in ahigher level of bio-control efficacy compared to non-stress-adapted yeast. Additionally, ex-posure of biocontrol yeasts to glycine betaine induced the activation ofthe antioxidant system in these yeasts (Liu et al., 2011b; Sui et al., 2012)increased oxidative stress tolerance, and biocontrol efficacy in apple fruit.

al pathogen–host fruit) in postharvest biocontrol systems.

Fungal pathogen Host fruit Reference

Penicillium digitatum Grapefruit Droby et al. (2002)Botrytis cinerea Apple El-Ghaouth et al. (2003)Penicillium expansum Peach Chan et al. (2007)Rhizopus stolonifer Peach Xu et al. (2013)N/A Tomato Jiang et al. (2009)Monilinia fructicolaPenicillium expansumRhizopus stolonifer

Apple Chan and Tian (2005)

Monilinia fructicolaPenicillium expansum

Apple Chan and Tian (2005)

Botrytis cinerea N/A Wisniewski et al. (1991)N/A Grapefruit McGuire (2000)Penicillium expansum Apple Scherm et al. (2003)N/A Apple vs. peach Giobbe et al. (2007) & Fiori et al. (2012)

es Botrytis cinerea N/A Sipiczki (2006)Botrytis cinerea Apple Saravanakumar et al. (2008)Alternaria alternata ApplePenicillium expansum AppleBotrytis cinereaPenicillium expansum

Apple Castoria et al. (2003)

Penicillium expansum Apple Liu et al. (2011a)Penicillium expansum Apple Liu et al. (2011b)Penicillium expansumBotrytis cinerea

Apple Liu et al. (2012)

Monilinia fructicola Peach Xu et al. (2008)

N/A Apple Macarisin et al. (2010)

le.

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In addition to the effect of ROS on yeasts, yeast antagonists also influ-ence ROS production and defense signaling in fruit tissues. An oxidativeburst, during which large quantities of ROS are generated, is one of theearliest host responses to pathogen attack (Mellersh et al., 2002;Nanda et al., 2010). Xu et al. (2008) reported that four different antago-nistic yeasts were able to stimulate both antioxidant gene expressionand antioxidant enzyme activity in peach fruit tissues and that the up-regulation of these genes/proteins was associated with lower levels ofprotein carbonylation (an indication of oxidative damage to proteins)in response to ROS produced by M. fructicola. Furthermore, Macarisinet al. (2010) reported that yeast cells themselves underwent a signifi-cant oxidative burst when applied to fruit and induced cells in thefruitmesocarp to produce elevated levels of H2O2,whichmay act direct-ly as a defense compound or to stimulate the host defense responsepathway. Understanding the role of ROS in specific biocontrol systems,as well as how environmental stimuli affect ROS levels, may lead tothe selection ofmore effective antagonists and newmethods of optimiz-ing their activity.

Two attributes that play a prominent role in the efficacy of antago-nistic yeasts as biocontrol agents are the ability to reach a specific celldensity at the site of infection and also the ability of the yeast to attachthemselves to both plant host tissues and pathogen spores and hyphae.When these events take place, a fundamental change in gene expressionand metabolism occurs in the antagonistic yeast that often leads to bio-film formation (Droby et al., 2009). This structural change in the mor-phology of the yeast population represents an attribute that greatlycontributes to their ability to inhibit postharvest rots by reducing theamount of physical space available for pathogen development and in-terfering with the flow of nutrients and/or germination signals fromthe host to the pathogen spores. Scherm et al. (2003) reported thatthe ability of a strain of S. cerevisiae (M25) to form a biofilm in liquid cul-ture was directly associated with its ability to act as a biocontrol agent.Only yeast cells collected from the biofilm phase were effective ininhibiting P. expansum infection in apple fruit, apparently by enhancingthe ability of the yeast to effectively colonize fruit wounds. Not allchanges in morphology, however, are positively associated with biocon-trol activity. Giobbe et al. (2007) reported that Pichia fermentanswas ca-pable of effectively inhibiting infection of apple byMonilinia but formedpseudo-hyphae when administered to peach fruit and became patho-genic causing substantial amounts of fruit decay. A further study on theresponse of P. fermentans to the twodifferent fruit hosts, identified differ-entially expressed genes in the yeast that were associated with changesin yeast morphology (single cell on apple fruit vs. pseudohyphae onpeach fruit) (Fiori et al., 2012). Results indicated that genes involved instress response, glycolysis, amino acid metabolism, and alcoholic fer-mentation were more highly expressed after 24 h of growth on peachfruit. Understanding how the application of antagonistic yeast to fruitinduces changes in cell morphology and the ability to form a biofilm isimportant for improving biocontrol efficacy and crucial to assessing thecommercial potential of a specific antagonistic yeast strain. Attempts todevelop a yeast-based product that is effective on one commodity butpathogenic on another commodity would be futile and emphasizes theneed for broad studies and risk assessment in developing any commer-cial product.

4. Enhancing biocontrol efficacy

During large-scale fermentation and formulation of a commercialproduct, yeasts are exposed to a wide array of stressful environmentalconditions such as high temperature, freeze/spray drying (desiccation),and oxidative stress. Therefore, enhancing stress tolerancemay representa useful strategy for improving both the viability and performance ofantagonistic yeasts in commercial preparations. Teixidó et al. (1998)demonstrated that increasing the intracellular content of total polyolsand sugars in Candida sake by the addition of glycerol, glucose or tre-halose to the growth medium led to an improvement in the water

stress tolerance and biocontrol efficacy of C. sake when used to con-trol P. expansum on apple fruit. Similarly, increased levels of intracel-lular trehalose in C. laurentii, brought about by culturing the yeast ina trehalose-containing medium, improved viability and biocontrolefficacy after the yeast was freeze dried and used at low tempera-tures or under controlled atmosphere conditions (Li and Tian, 2006).

As previously mentioned, transient exposure to a sublethal stresscan often induce tolerance to a more extreme stress andmay providecross-protection against other stresses. Thus, this approach also rep-resents a potential strategy for improving the efficacy of formulatedbiocontrol products. Salt-adapted R. paludigenum exhibited higherviability than non-adapted cells under low water activity and freez-ing stress (Wang et al., 2010). A mild heat shock pretreatment(30 min at 40 °C) improved the tolerance of M. fructicola to subse-quent high temperature and oxidative stress (Liu et al., 2011a). Inboth of the above reports, stress adaptation improved both subse-quent stress tolerance and biocontrol activity of the yeasts. Thus,pre-adaptation of yeast to abiotic stress may have practical implica-tions for improving the efficacy and reliability of yeast biocontrolproducts used to manage postharvest spoilage of fruits under awider array of environmental conditions.

A commercial preparation of a biocontrol agent needs to retainefficacy and possess adequate shelf life. Both dry and liquid formula-tions have been commonly used to prepare yeast biocontrol products(Melin et al., 2006). The advantages of a dry formulation are longershelf-life under non-refrigerated conditions, protection of the productfrom contamination during storage, and ease of product shipping, distri-bution and storage (Li and Tian, 2006;Melin et al., 2007). Advantages ofa liquid formulation are lower costs of manufacturing the product sincedrying and the addition of fillers are not needed, and the need for rehy-dration is eliminated. Both dehydration and rehydration often result inhigh mortality (Abadias et al., 2003b; Eleutherio et al., 1993). An exog-enous protectant is often included in both types of formulations toameliorate the impact of environmental stress on cell viability andbiocontrol efficacy. Exogenous trehalose at the concentration of 5%or 10% markedly increased the cell viability of two antagonistic yeasts,C. laurentii and R. glutinis, after freeze-drying (Li et al., 2008). Duringstorage of a liquid formulation, oxidative stress may be one of themajor factors responsible for decreased cell viability. The antioxidantL-ascorbic acid enhanced the effect of sugar protectants (trehaloseand galactose) on the viability of C. laurentii and P. membranaefaciensin liquid formulations (Liu et al., 2009).

Combination of yeast with other antimicrobial compounds has alsobeen demonstrated to be an effective method for improving biocontrolperformance. Qin et al. (2003) reported that salicylic acid enhanced thebiocontrol efficacy of R. glutinis against P. expansum and A. alternata insweet cherry fruit. Salicylic acid (SA) at low concentrations had little ef-fect on the growth of the yeast or the pathogens but induced the activityof defense-related enzymes including polyphenoloxidase, phenylala-nine ammonia-lyase, and β-1,3-glucanase. The authors concluded thatSA enhanced the biocontrol efficacy of the antagonistic yeast by induc-ing biochemical defense responses in the fruit host rather any fungitoxiceffect on the pathogens. Additional studies confirmed the ability of SA toenhance biocontrol activity in other biocontrol systems (Farahani andEtebarian, 2012; Yu and Zheng, 2006; Yu et al., 2007a; Zhang et al.,2010). Another plant growth regulator and defense activator, methyljasmonate, has also been utilized to enhance the biocontrol efficacy ofantagonistic yeasts on various fruits, such as apple (Ebrahimi et al.,2012), pear (Zhang et al., 2009), peach (Yao and Tian, 2005) and loquat(Cao et al., 2009). The natural compound, chitosan (poly-β-(1 → 4)N-acetyl-D-glucosamine), together with its derivatives, has been reportedto control postharvest diseases through its antifungal properties andability to elicit host defense responses (Bautista-Baños et al., 2006;Terry and Joyce, 2004). Based on these properties, chitosan has beendemonstrated to be a an effective additive for improving the perfor-mance of biocontrol yeasts, such as Candida saitoana (El-Ghaouth

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et al., 2000) and C. laurentii (Meng et al., 2010; Yu et al., 2007b). Anothergroup of chemicals used alone or in combinationwith biocontrol agentsare inorganic salts andminerals, such as calcium chloride (Gholamnejadand Etebarian, 2009; Tian et al., 2002; Wisniewski et al., 1995), ammo-niummolybdate (Wan et al., 2003), sodium bicarbonate (Conway et al.,2004; Karabulut et al., 2003; Janisiewicz et al., 2008; Wan et al., 2003),silicon (Farahani et al., 2012; Qin and Tian, 2005) and borate (Cao et al.,2012). These compounds typically have a direct inhibitory effect on path-ogens but little effect on the viability of antagonistic yeasts at the sameconcentrations.

5. Commercial application

In order to be successful, a biocontrol product needs to provide anacceptable and consistent level of control of target diseases in the targetcommodity under commercial processing and storage conditions. Theseconditions may vary significantly for different commodities and indifferent packinghouses (Droby et al., 2009). Pilot studies, semi-commercial, and large-scale commercial tests requiring large amountsof formulatedproduct are needed to obtain the data necessary to evaluatethe efficacy of a biocontrol agent (Abadias et al., 2003a). It is important forthese tests to be conducted in packinghouses at different locations underconditions of natural infection (Long et al., 2006, 2007). Regulatoryapproval of a formulation by government agencies is also required toproduce a commercial product. The application package for approvalmust contain a third-party evaluation of the safety of the formulatedproduct to human health, as well as efficacy data. The registration ofbiocontrol products for postharvest use in the USA is the responsibilityof the Environmental Protection Agency (EPA) and on average has re-quired about two years. In contrast, the registration process in Europetakes almost seven years (Nunes, 2012).

Although many different yeasts, isolated from a variety of sources,have been reported as good postharvest biocontrol agents, only a fewyeast-based biocontrol products are available in the market: Shemer™(M. fructicola, Bayer, Germany), Candifruit™ (C. sake, Sipcam-Inagra,Spain), and Boni-Protect™ (the yeast-like fungus, A. pullulans, Bio-protect, Germany). These products are registered for use on severaldifferent commodities and for several different pathogens. The abilityto control different rots on different commodities is essential for theeconomic viability of a postharvest biocontrol product. For example,Shemer, based on a heat-, oxidative stress- and osmo-tolerant strainof M. fructicola NRRL Y-27328 (Droby et al., 2009; Kurtzman andDroby, 2001; Liu et al., 2011a), has been shown to be effective againstrots caused by Botrytis, Penicillium, Rhizopus and Aspergillus on straw-berry (Karabulut et al., 2004), grape, sweet potato, carrot and citrus(Blachinsky et al., 2007).

6. Conclusions

Pesticide residues in fresh fruits and vegetables have been and willcontinue to be one of the main concerns of regulatory agencies andconsumers. Therefore, reducing or eliminating the pre- and postharvestuse of synthetic chemical fungicides by developing alternativemanage-ment strategies remains a high research priority. This review article hasprovided a brief overview on the use of yeast antagonists as a viablealternative to synthetic, chemical fungicides. For amore comprehensivereview of the subject, the reader is referred to published reports:Abano and Sam-Amoah (2012), Droby et al. (2009), Janisiewicz andKorsten (2002), Nunes (2012), Sharma et al. (2009), Spadaro andGullino (2004), Wilson and Wisniewski, M. (1989) and Wisniewskiet al. (2007). It is anticipated that the continuing withdrawal of keypostharvest fungicides from the market, due to exclusion by regula-tory agencies or the high-cost of re-registration, will lead to an absenceof effective tools for reducing postharvest losses. Hence, the use of bio-control products is expected to gain momentum in the coming yearsand become more widely accepted as a component of an integrated

approach to managing postharvest diseases. Many challenges need tobe addressed in order to develop a commercially successful postharvestbiocontrol product. These include: a) the improvement and enhance-ment of biocontrol efficacy under commercial conditions; b) the de-velopment of high quality, economical methods of fermentation andformulation; c) themaintenance of cell viability and biocontrol efficacy inthe formulated product; d) the identification of yeast antagonists that ex-hibit a wide spectrum of activity against several different pathogens ondifferent commodities; e) the establishment of an effective marketingoutlet, preferably by a multinational based company; and f) developinga fundamental understanding of how biocontrol systems operate andhow the environment affects the interactions between the host, patho-gen, and biocontrol agent as outlined in Fig. 1.

Acknowledgments

The authorswish to thank themany scientists involved in postharvestbiocontrol research for their research efforts and thoughtful discussionsof this topic over the past twenty-five years and apologize to themany in-vestigators whose specific results could not be cited due to space limita-tions but whose work framed the questions and ideas that are discussedin this review. This work was supported by the Key Project from theGovernment of Anhui Province (No. 2012AKKG0739), the NationalScience and Technology Key Project of China (Nos. 2011CB100401 &2007AA10Z100), the National Natural Science Foundation of China(No. 31171179), and Advanced Program of Doctoral Fund of Ministryof Education of China (No. 20110181130009).

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