Control of ochratoxin A-producing fungi in grape berry by microbial antagonists: A review

Accepted Manuscript Control of Ochratoxin A-Producing Fungi in Grape Berry by Microbial Antagonists: A Review Hongyin Zhang, Maurice Tibiru Apaliya, Gustav K. Mahunu, Liangliang Chen, Wanhai Li PII:

S0924-2244(15)30115-1

DOI:

10.1016/j.tifs.2016.03.012

Reference:

TIFS 1788

To appear in:

Trends in Food Science & Technology

Received Date: 24 September 2015 Revised Date:

11 March 2016

Accepted Date: 26 March 2016

Please cite this article as: Zhang, H., Apaliya, M.T., Mahunu, G.K., Chen, L., Li, W., Control of Ochratoxin A-Producing Fungi in Grape Berry by Microbial Antagonists: A Review, Trends in Food Science & Technology (2016), doi: 10.1016/j.tifs.2016.03.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Control of Ochratoxin A-Producing Fungi in Grape Berry by Microbial Antagonists: A

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Review

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Hongyin Zhang1*, Maurice Tibiru Apaliya1, Gustav K. Mahunu 1, Liangliang Chen 1, Wanhai Li 1

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212013, Jiangsu, P. R. of China

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School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang

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*Corresponding author.

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Email address: [email protected]

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Tel.: +86-511-88780174; Fax: +86-511-88780201.

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ABSTRACT

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Background: Ochratoxin A (OTA) remains a challenge in the face of continuous efforts to

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produce quality and wholesome wine and table grape berries to meet food safety standards.

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However, the use of chemicals to control postharvest diseases is a public concern with increasing

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consumer awareness of the dangers associated with the consumption of fungicide-treated

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commodities. Synthetic fungicides are well-known for their hazardous effects on human health

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and potential contamination to the environment. Moreover, pathogens are noted to have

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developed resistance to these chemicals because of their continuous use and abuse.

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Scope and Approach: This review focused on the efficacies, potentials and developmental trends

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of microbial antagonists in the control and biodegradation of OTA-producing fungi in grapes and

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wine. It outlined the steps and challenges in the development of bioproducts. It also recounted the

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successes and developments of biocontrol products to date.

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Key findings and conclusions: OTA production in grapes is caused by the genera Aspergillus

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and Penicillium, with the species Aspergillus carbanarius as the dominant cause across the globe.

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The prevalence of OTA-producing fungi in grape vineyards are influenced by temperature, water

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activity (aw), pH, relative humidity and type of cultivar. Biological control agents (BCAs) have

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proved successful to control and/or degrade OTA-producing fungi, among which antagonistic

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yeasts play the leading role. In addition, biocontrol products such as BioSave, Yieldplus,

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Serenade and Aspire have been developed. Preharvest application is acknowledged to be the best

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for grapes even a day before harvest because postharvest treatment affects the bloom of the

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grapes.

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KEYWORDS: Grapes; Fungi; Biological control; Postharvest decay; Ochratoxin A;

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detoxification

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1. Introduction OTA is among the most important mycotoxin contaminants of foodstuffs and beverages

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due to its potent immunotoxic, teratogenic, nephrotoxic and genotoxic properties to humans

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(Pfohl-Leszkowicz, 2007). OTA has been categorized as a possible human carcinogen (group 2B)

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according to the International Agency of Research on Cancer (IARC, 1993). Notably, Aspergillus

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ochraceus and Penicillium verrucosum were considered to be the main cause of OTA in tropical

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and temperate regions respectively (Mantle, 2002). However, apart from P. nordicum and P.

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verrucosum, the genus Aspergillus is considered the most important species accountable for the

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presence of OTA in food. This genus is reported to have more than 100 species, with a complex

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taxonomy under continuous revision. It was last revised and published based on broad

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phylogenetic analysis (Peterson, 2008). OTA is a common contaminant of a wide range of food

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and food products which include cereals and its derived products (Jørgensen & Jacobsen, 2002),

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grapes, cocoa beans, spices, nuts, olives, grapes, beans coffee beans and figs (Battilani, Pietri,

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Bertuzzi, Languasco, Giorni, & Kozakiewicz, 2003). Grapes and its derived products are the

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second most contaminated with OTA after cereals. Table 1 list of OTA-fungi found in grapes.

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Regarding meat, OTA has been reported in non-ruminants such as pigs, poultry, rabbits and rats

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(Fink-Gremmels, 2008). Ruminants on the other hand, tolerate OTA because of the protozoan

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normal flora of their rumens which degrades OTA into OTAα. Nonetheless traces of OTA have

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been found in meat from ruminants and dairy milk.

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Based on the health risks associated with OTA, the European Union in 2006, passed a

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legislation recommending the maximum permissible limits of OTA in a number of produce (De

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Curtis, de Felice, Ianiri, De Cicco, & Castoria, 2012). Numerous researches have been carried out

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including chemical (Bellí, Marín, Sanchis, & Ramos, 2006), physical (Var, Kabak, & Erginkaya,

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2008) and microbiological (Péteri, Téren, Vágvölgyi, & Varga, 2007) to look for ways to

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eliminate or minimize OTA contaminants from foods. However, the drawback of chemical and

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physical methods pose a challenge to be used effectively for the control of OTA-producing fungi.

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For instance, physical methods such as filtration or adsorption could reduce sensorial attributes

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such as color, taste, aroma and other desirable properties (Grazioli, Fumi, & Silva, 2006).

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The use of synthetic chemicals can effectively control the growth and proliferation of

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OTA- producing fungi and contamination in food. However, the use of synthetic fungicides could

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lead to environmental pollution and development of resistance by toxigenic fungi and other major

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plant pathogens. Again, the use of synthetic chemicals have been noted to have cumulative and

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residual effects that are harmful to human life (Zhang, Wang, Zheng, & Dong, 2007). Thus, under

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the current circumstances, BCAs remain the only promising and efficient method to control OTA

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and other plant pathogens without harmful effect to the environment, human and development of

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resistance by pathogens.

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Accordingly, there has been increasing interest, by researchers during the last decade to

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employ biological methods to control OTA in foods, by researching into yeast, bacteria and non-

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toxic fungi for their ability to inhibit the growth of OTA producing fungi, as well as detoxify

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OTA through binding or degradation to limits acceptable by legislation (2-10 µg/L) (Bleve,

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Grieco, Cozzi, Logrieco, & Visconti, 2006). Moreover, as highlighted by Wilson and Wisniewski

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(1994), biological control with microbial antagonists proved to be a viable alternative either

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singly or in combination with other eco-friendly methods to minimize the use of synthetic

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fungicides. In choosing a microbial antagonist therefore, it is recommended to take a closer look

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at the points stated herein: (a) the level of disease control by the antagonist have to be high ( 95-

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98%), (b) the possibility of commercializing the antagonist in the market, (c) the food safety

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requirements regarding the usage of the microbial antagonist and (d) the market value of the fruit

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to justify the application or usage of the microbial antagonist (Chalutz & Droby, 1998). In line

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with these, several microbial antagonists have been investigated to determine their efficacy

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against numerous postharvest fungal pathogens such as

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Botrytis spp., Monilia spp., and Rhizopus spp (Droby et al., 2002; Zahavi et al., 2000). According

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to Chalutz and Droby (1998); Spadaro and Droby (2016), yeasts appear to have most of the

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qualities stated above, henceforth in the recent past, studies have been centered on the

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identification and selection of antagonist yeast strains. Indeed,

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Control Agents involve the use of bioeffector methods to control pests, and plant diseases by

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employing microorganisms) method has proved to be very successful in the control of fruit rots

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and mycotoxin producing fungi (Mahunu, Zhang, Yang, Li, & Zheng, 2015).

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Aspergillus spp., penicillium spp.,

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no single BCA (Biological

In view of the above, there is the need for a concerted effort to harmonize and integrate a

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mixture of non-competitive and complementary BCAs, which are likely to have broad spectrum

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of postharvest disease control. The objectives of this review were to: (1) assess the removal of

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OTA from wine using microbiological methods. (2) recount the successes attained after the

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emergence of microbial antagonists to control pre- and postharvest OTA-producing fungi in grape

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berry and the way forward, (3) assess the efficacies and commercial potentials of BCAs in the

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control of plant diseases and biodegradation of OTA

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2. OTA detoxification pathway

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OTA is a 7-carboxy-5-chloro-8-hydroxy-3, 4-dihydro-3 R -methylisocoumarin, linked

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through its 7-carboxy group to L -β -phenylalanine by an amide bond (Van der Merwe, Steyn, &

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Fourie, 1965). OTA is very stable at high temperature thus, industrial processing of raw materials

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of feed and food containing OTA does not eliminate it and the toxin remains an integral part in

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the end-product. The bio-synthesis pathway of OTA has still not been totally explicated.

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Nevertheless, it is clear that the pathway encompasses some critical steps, such as the bio-

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synthesis of isocoumarin group by the catalyzing action of polyketide synthase (PKS), the

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catalyzing reaction by the enzyme peptide synthetase linked with the amino acid phenylalanine by

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the aid of the carboxyl group, and the chlorination step.

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However, the order of these reactions are not yet well-defined. It has been reported that

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carboxypeptidase A activity enzymes have the ability to degrade OTA (Amézqueta, González-

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Peñas, Murillo-Arbizu, & de Cerain, 2009) (Fig. 1) though there are some toxicity challenges

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regarding the use of enzymes to degrade OTA in produce because of their undesirable effects on

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non-targeted microbes on the quality of wine. Some bacteria (Streptococcus, Bacillus and

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Bifidbacterium) and fungi (Alternaria, Penicillium and Botrytis) have been revealed to be able to

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degrade OTA (Petruzzi, Sinigaglia, Corbo, Campaniello, Speranza, & Bevilacqua, 2014). OTA

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production to a very large extent depends on intrinsic factors such as the nutritional composition,

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the type of food matrix, water activity, plant type and moisture content.

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3. Preharvest application of BCAs

Generally, microbial control agents can be applied either at preharvest or postharvest

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depending on which method is effective in controlling or suppressing the pathogen (OTA

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producing fungi) (Sharma, Singh, & Singh, 2009). Nonetheless, preharvest application of BCAs

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allows for a better colonization of the fruit wound and surface thus, protecting the fruits during

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storage (Ippolito & Nigro, 2000). BCAs encounter variable and harsh environmental conditions

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on the field and these threaten their survival than those conditions encountered during storage

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(Ippolito & Nigro, 2000). This reduce the number of microbial control agents that are suitable for

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prehavest application. For prehavest application to be successful, the microbial antagonist should possess the

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ability to anchor or attach itself to the fruit surface, as persistent attachment by the antagonist

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offers a better colonization and prevents wind or rain from detaching or dislodging it (Dickinson,

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1986). For instance, as a way of attaching itself, the yeast-like fungi (A. pullulans) produce slime,

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predominantly made of extracellular polysaccharides on the phylloplane which aids its adhesion

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(Meng & Tian, 2009). It is well established that, quiescent infections at the field during growth

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mark the beginning of most postharvest diseases. These latent infections can occur at any stage of

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the produce and will remain so until maturity and ripening stages where they manifest upon the

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least opportunity. Battilani, Pietri, Bertuzzi, Languasco, Giorni, & Kozakiewicz (2003) emphasized

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that, grape berries can be contaminated at early veraison (the onset of ripening) period. However,

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the harvesting period is considered the most crucial period for fungi infection and development.

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This observation was found in Spanish berries (Bellí, Marín, Coronas, Sanchis, & Ramos, 2007).

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Grape berries wounded by insects such as Lobesia botrana and grape fruit fly, and fungal

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pathogens such as Erysiphe nector as well as mechanical damage may facilitate the entry of

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Aspergillus and penicillium spp in berry tissues since these opportunistic pathogens take the least

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advantage to colonize wounds. This is especially the case when the berries are fully ripe or near

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maturity. Preharvest application is therefore necessary for yeasts to colonize and curtail disease

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incidence on the field before postharvest application. Preharvest spraying with BCAs and or

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synergic compounds to reduce storage rot of grapes appear to be considered a good method as

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postharvest liquid treatment is not desirable for this fruit, because its bloom could be damaged

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(Zahavi et al., 2000).

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Moreover, spraying with BCAs prior to harvesting is considered to be a prospective

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technology aimed at significantly reducing the occurrence of decay in fruits and vegetables during

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transportation and storage (Amézqueta, González-Peñas, Murillo-Arbizu, & de Cerain, 2009). In a

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similar vein, pre- and postharvest applications with chitosin greatly improved grape berry

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resistance by inducing the defense related enzymes like 3-glucanase, chitinase, Phenylalanine

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ammonia-lyas (PAL), Peroxidases (POD) , superoxide dismutase (SOD) and Polyphenol oxidase

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(PPO) in table grapes (Meng, Li, Liu, & Tian, 2008). In vitro and in sito findings of the antifungal

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activities of chitosan have also been noted. Prehavest treatment with chitosan has reduced the

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propagules of filamentous fungi like Aspergillus and penicillium spp.

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Thus, the effectiveness of preharvest applications with BCAs and chitosan is due to the

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antifungal features of chitosan as well as its adhesive features and capabilities to trigger the

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defense mechanism in the produce. Preharvest spraying with A. pullulans strain L47 four times

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before harvest led to a significant decrease in both the incidence and prevalence of mold of table

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grapes and the population dynamics of the yeasts were also noticed to have increased (Schena,

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Ippolito, Zahavi, Cohen, Nigro, & Droby, 1999). Furthermore, Preharvest spraying of table grapes

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10 days before harvest using C. laurentii significantly minimized decay at storage possibly

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through initial colonization and suppression of saprophytes and other fungi pathogens (Meng,

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Qin, & Tian, 2010). The use of BCAs in the future to control ochratoxins producing fungi will

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very much depend on their effectiveness on the field and the cost of production (Ponsone, Chiotta,

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Palazzini, Combina, & Chulze, 2012).

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Prevention of OTA in the vineyard is the core aim of the farmer and the food industry. A.

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carbonarius is a saprophyte that is found on the top horizon of soil beneath vineyards and grows

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in injured berries caused by abiotic and biotic factors. This fungus, is believed to be blown onto

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grape berries upon which it develops and cause damage to bunches (Leong, Hocking, Pitt, Kazi,

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Emmett, & Scott, 2006). Thus, these saprophytes are constantly present on soils waiting for the

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least opportunity to invade the host. At large, injured grapes are proned to fungal diseases such as

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gray rot, black rot and downy mildew (Covarelli, Beccari, Marini, & Tosi, 2012). It has been

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reported by Varga and Kozakiewicz (2006) that the presence of OTA in wine may perhaps be

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reduced by about 80% when recommended vineyard establishment and management practices

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such as training, trellising, canopy management, harvesting, handling, diseases and pests control

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are strictly adhered to. These practices are very essential in ensuring the quality of both table and

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wine grapes. Up till now, GAPs are the best methods to control OTA contamination (Ponsone,

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Chiotta, Palazzini, Combina, & Chulze, 2012).

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It has been reported by Battilani, Giorni, Bertuzzi, Formenti, and Pietri (2006) that

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regional data on weather conditions have now been integrated into a model to facilitate the use of

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Geographical Information Systems (GIS) together with environmental information to aid the

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prediction of the year and periodicity in which ecological conditions show low or high risk of

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OTA contamination of grapes. Therefore, vineyard farmers need to be alert, in order to take

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advantage of this technology to boost their production and to produce OTA-free grapes to meet

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the demands of the ever enlightened consumer. Generally, preharvest management practices

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should ensure that mechanical damage and injuries caused by insects are kept at the barest

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minimum. Time of harvesting is an important factor that determines the level of infection.

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Usually, grapes harvested early minimize the risk of infection than late harvest. Care must be

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taken during harvesting to avoid bruises or injuries as these will facilitate the entry of pathogens.

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4. Postharvest application of BCAs

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Several literature reviews suggested that, the use of microbial antagonists during

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postharvest (storage phase of fruits) is more promising than preharvest due to stable

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environmental conditions (Droby, Wisniewski, Macarisin, & Wilson, 2009; Janisiewicz &

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Korsten, 2002). Postharvest application offers a stable environment for harvested fruits and

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vegetables and is conducive for BCAs to thrive better than field application. It is therefore

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advantageous in terms of time and ease of application, effectiveness, and cost benefit analysis. In

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view of the above, postharvest application of BCAs have a greater potential of achieving success

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compared to preharvest.

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When contamination of the grapes persists after the preharvest phase, the toxin has to be

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managed in postharvest. At this phase, handling, storage and processing are the critical points that

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need attention. Postharvest pathogens can be suppressed by the application of BCAs through

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postharvest dips or sprays. For instance, postharvest dip treatments containing the yeast

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Saccharomyces cerevisiae significantly reduced OTA produced by A. niger and A. ochraceus

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without any effect on the quality of coffee (Velmourougane, Bhat, Gopinandhan, &

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Panneerselvam, 2011). Similarly, the yeast Trichosporon mycotoxinivorans detoxified OTA

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through the cleavage of the phenylalanine moiety to produce the derivative OTAα. This is

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because T. mycotoxinivorans can to be freeze-dried, fermented and stabilized without losing its

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efficacy. Its postharvest delivery for OTA detoxification appears to be feasible.

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Postharvest detoxification of OTA involves two phases, inhibition of the pathogens

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shortly after harvest, and bioremediation of the toxins with the application of microbial

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antagonists. Suppression of mycotoxins during postharvest storage was discovered in some

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actinimycetes strains to produce compounds that blocked the biosynthesis pathway of mycotoxins

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(Medeiros, Martins, Zucchi, Melo, Batista, & Machado, 2012).

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However, Postharvest control treatment is not recommended for grape berries because of

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its thin waxy pericarp and succulent mesocarp, which are vulnerable to damage. Therefore,

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preharvest treatment is highly advisable even one day before harvest can greatly control

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postharvest rot of grapes and improve their shelflife (Meng & Tian, 2009). Even though

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postharvest is not recommended for grapes, Meng and Tian (2009) reported in their findings that

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C. laurentii combined with chitosan drastically reduced the incidence of natural diseases of table

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grape berries stored at 0 o C followed by 20 oC. It was subsequently observed that a PAL activity

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was much higher in the prehavest treatment than that of the control.

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5. Microbiological method of OTA detoxification in wine

The occurrence of OTA in wine is ubiquitous. Generally, red wine has higher amount of

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OTA than other wines as a result of the time of contact between grape juice and berry skin during

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maceration (Petruzzi, Bevilacqua, Baiano, Beneduce, Corbo, & Sinigaglia, 2014; Quintela,

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Villarán, de Armentia, & Elejalde, 2013). Globally, wine is an important alcoholic beverage so

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the presence of OTA poses risk to the safety of consumers, and threatens the progress of the wine

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industry. As stated earlier, OTA cannot be detoxified through industrial processing because of its

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thermo stability.

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exclusion of moldy grapes and/or bunches during winemaking can reduce or eliminate OTA toxin

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but economically this might not be feasible.

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In view of this, during maceration, it resists destruction. Practically, the

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The main methods through which microbiological antagonists detoxify OTA are by

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adsorption and degradation. Microbial antagonists can degrade OTA in two ways, the hydrolysis

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of the OTA amide bond to a non-harmful OTAα and L-β-phenylalanine and secondly, the

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hydrolysis of the lactone ring resulting in an opened lactone OTA form. Various types of lactic

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acid producing bacteria and yeasts have been employed to detoxify OTA from wines with some 11

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degree of success. Pediococcus parvulus isolated from Douro wine successfully detoxified OTA

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during malolactic fermentation (Abrunhosa et al., 2014) because some P. parvulus strains have

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probiotic features. The degradation of the OTA by P. parvulus UTAD 473 was observed in grape

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must. The OTA was noted to have biodegraded via hydrolysis of the OTA amide bond and

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subsequent discharge of L-β-phenylalanine moieties and OTAα. A number of lactic acid

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producing bacteria have been reported to have detoxified OTA through adsorption by their cell

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walls in juice and must. Some of them include Oenococcus oeni RM11 and L. plantarum CECT

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748T (Del Prete, Rodriguez, Carrascosa, de las Rivas, Garcia-Moruno, & Muñoz, 2007). The

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polysaccharides and peptidoglycan were believed to be involved in the toxin binding.

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The use of yeasts as winemaking recipe for the detoxification of OTA was initially

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conducted and comprehensively reported by (Bejaoui, Mathieu, Taillandier, & Lebrihi, 2004).

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They indicated that both heat- and acid-treated cells bound significant amounts of OTA than

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viable cells in synthetic grape juice. Viable yeasts adsorbed 35% of the OTA, while heat- and

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acid-treated yeasts cells detoxified the OTA up to 90.80 % and 73 % respectively. They explained

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that, heating could alter the surface features of the cell, by denaturing the proteins and also

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forming Millard reaction products. Concerning the acid-treated yeast, they observed that, the acid

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might have affected the polysaccharides through the release of monomers, which fragmented into

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aldehydes following the breaking of the glycosidic bonds. The discharged products triggered

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higher adsorption sites than viable yeast cells (Piotrowska, Nowak, & Czyzowska, 2013).

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Some macromolecules such as β-D-glucans and mannoproteins present in the cell walls of

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the commercial yeast S. cerevisiae are good biomass that serve as adsorbent materials (Ringot,

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Lerzy, Chaplain, Bonhoure, Auclair, & Larondelle, 2007). This yeast is made up of an inner cell

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wall composed of chitin and β-1,3-glucan, which is 50-60 % of the cell wall dry weight and outer

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layer of β-1,6-glucan heavily glycosylated with

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partially polar components that are discharged in the course of alcohol fermentation usually at the

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end. At wine pH, mannoproteins become negatively charged and establish polar and non-polar

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interactions with OTA (Ringot, Lerzy, Chaplain, Bonhoure, Auclair, & Larondelle, 2007).

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Thenceforth, Hocking, Su-lin, Kazi, Emmett, and Scott (2007) observed that OTA cannot be

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biologically transformed by yeasts during fermentation, rather the toxin is adsorbed onto the

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biomass mainly composed of mannoproteins and glucans.

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mannoproteins. These mannoproteins are

Conversely, Angioni et al. (2007) Observed that some yeast reduced OTA in wine through

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other means rather than adsorption but could not suggest the possible pathway for the degradation

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because L-β- phenylalanine and OTAα were not found. They however, could not find the

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products of the interaction. Temperature, ethanol concentration and pH are among the major

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factors that affect autolysis in a perfect system. Petruzzi, Corbo, Sinigaglia, and Bevilacqua

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(2014) used two commercial and three wild S. cerevisiae strains and a commercial cell wall to

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detoxify OTA. A temperature of 25 and 30 °C, 5, 10, and 15% ethanol and pH of 3.0 and 3.5 were

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the parameters used in the research. The authors revealed that, the treatments degraded more of

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the OTA at 30 °C, pH 3.0 and 5 % ethanol, indicating that, the above physico-chemical

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parameters influence OTA detoxification. Some other equally important factors that influence

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OTA detoxification using microbial antagonists are the kind of substrates, the type of strains,

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flocculence, toxin concentration, cell dimension and cell sedimentation kinetics (Petruzzi,

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Bevilacqua, Baiano, Beneduce, Corbo, & Sinigaglia, 2014)

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Detoxification is genetically controlled by an inherent polygenic trait of wine yeasts.

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Some strains have the ability to release the toxin after adsorption (Bevilacqua, Petruzzi, Corbo,

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Baiano, Garofalo, & Sinigaglia, 2014; Piotrowska, Nowak, & Czyzowska, 2013), possibly due to

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the type of hydrogen and hydrophobic interaction. Recently,

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Beneduce, Corbo, & Sinigaglia (2014) proposed that yeast strains with dual purpose of serving as

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a functional starter culture and health related need to be focused on. Thus, yeasts and some lactic

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acid producing bacteria have great future for the detoxification of OTA without any effect on the

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organoleptic and functional characteristics of wine, unlike chemical and some physical methods

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which have effects on the nutritional and functional properties. This explains why microbial

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antagonists are gaining prominence in their use. Selection of microbial antagonist strains to

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detoxify mycotoxins should therefore be based on biological mechanisms and starter culture

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functions. Further research is therefore necessary for the isolation and identification of new yeast

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strains with the potentials of achieving this dual task.

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Petruzzi, Bevilacqua, Baiano,

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6. Factors affecting the bioactivity of microbial antagonists

Environmental factors such as temperature, moisture, and pH are critical in affecting

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fungal growth and development during pre- and postharvest handling (Liu et al., 2012; Ponsone,

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Chiotta, Palazzini, Combina, & Chulze, 2012) . Fungi can be classified based on their tolerance to

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temperature as mesophilic (able to thrive in moderate temperatures), thermophilic (thrives at

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relatively high temperatures), and psychrophilic (capable of living at relatively low temperatures).

309

Therefore, these environmental factors and their interactions between diverse fungi play important

310

roles in mycotoxin production. Climatic conditions favor ochratoxigenic Aspergillus spp than

311

penicillium spp which have been observed to be present and responsible for OTA production

312

(Abarca, Accensi, Bragulat, Castella, & Cabanes, 2003) (Fig. 2).

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In addition, JØrgensen (2005) indicated that, Aspergillus spp have the capability of

314

growing in variable climatic conditions and in food produce and this shows their global

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prevalence as OTA producers. Among the Aspergillus section Nigri, A. carbonarius was noted to

316

be highest OTA producer than black Aspergilli (Battilani, Giorni, & Pietri, 2003). Another report

317

by Bejaoui, Mathieu, Taillandier, and Lebrihi (2006) showed that, the number of A. carbonaruis

318

correlates with OTA production and A. carbonarius is known to be the common OTA producer

319

of grapes in France due to their abundance in grapevines. Furthermore, earlier studies highlighted

320

their invasive character and ability to colonize and permeate grape berries even without damage to

321

skin (Battilani & Pietri, 2002). A. carbonarius was also stated to be the main OTA producer in

322

Spanish grapes (Abarca, Accensi, Bragulat, & Cabanes, 2001) and Italian grapes (Battilani,

323

Giorni, & Pietri, 2003). A. carbonarius is also observed to be aggressive due to its potential

324

intrinsic toxigenic character.

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Location of a vineyard is another essential factor that influence the prevalence of OTA-

326

producing fungi. As stated above, OTA production is dependent on the latitude of the area,

327

therefore, the lower the latitude, the greater the occurrence and concentration (Battilani, Magan,

328

& Logrieco, 2006). Thus, the geography of a region, influences the climatic conditions and

329

subsequently mold growth and contamination in a correlated manner. In the Mediterranean

330

countries, A. carbonarius were noticed to be the principal contributor of OTA contamination of

331

grapes as well as in Australia (Leong, Hocking, Pitt, Kazi, Emmett, & Scott, 2006).

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According to several investigations conducted, grape products from the Mediterranean

333

Basin of Southern Europe (Battilani, Giorni, & Pietri, 2003) and North Africa (Filali et al., 2001)

334

were more contaminated by OTA.

335

researchers (Stefanaki, Foufa, Tsatsou-Dritsa, & Dais, 2003) to correlate with the production

336

region in the Mediterranean countries such as Greece and Italy. They observed that, in Southern

337

Europe, grapes growing in vineyards were more prone to fungi growth and development resulting

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Additionally, OTA levels have been found by some

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338

in higher potential of infestation by OTA-producing fungi and contamination. As mentioned by

339

Covarelli, Beccari, Marini, & Tosi (2012), grape wines produced in Mediterranean Basin seem to

340

be highly contaminated than grape wines from other European countries. From the foregoing experimental findings, geographical location and environmental

342

conditions have serious influence on OTA-producing fungi and the subsequent occurrence of

343

OTA in grapes. For instance, the level of OTA production recorded its peak at a temperature

344

range of 15-20 oC with A. carbonarius and 20-25 oC with A. niger (Esteban, Abarca, Bragulat, &

345

Cabañes, 2004) . On the other hand Battilani, Giorni, and Pietri (2003) also observed that, A.

346

niger reached their highest growth level and tolerated temperatures greater than 37 oC. In sharp

347

contrast, Bellí, Ramos, Coronas, Sanchis, and Marín (2005) reported that A. carbonarius grew

348

faster at a maximum temperature of 30 oC than other temperatures (15 oC, 20 oC, 35 oC and 37

349

o

350

0Æ95 and 0Æ99 aw.

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C) levels and the growth rate increased with increasing aw, with maximum growth rate being

In a lab-scale experiment conducted by De Curtis, de Felice, Ianiri, De Cicco, and

352

Castoria (2012), they revealed that ochratoxigenic A. carbonarius strain A1102 was completely

353

controlled or inhibited by two strains of the yeast-like fungus A. pullulans AU34-2 and LS30 and

354

Metschnikowia pulcherrima LS16 at 60% relative humidity and temperature 20 °C. For these

355

reasons, environmental conditions play important role, due to the influence they have on BCAs

356

survival and the interaction among the trio: pathogen, host plant and antagonists (Lahlali,

357

Hamadi, & Jijakli, 2011). In an in vitro assay, two antagonistic yeasts Cryptococcus laurentii LS

358

28 and Rhodotorula glutinis LS-11 demonstrated high antagonistic abilities against Botrytis

359

cinerea and Penicillium expansum in the presence of hydrogen peroxide (H2O2) and superoxide

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360

anion (ܱଶ.ି), though Cryptococcus laurentii LS 28 showed more resistance to the reactive oxygen

361

species (ROS) stress (Castoria, Caputo, De Curtis, & De Cicco, 2003). Bellí, Marín, Sanchis, and Ramos (2004) observed that the optimum aw for black

363

Aspergilli to grow appears to be 0.98, compared to the aw of grapes on the field. As mentioned by

364

Covarelli, Beccari, Marini, and Tosi (2012), aw has an effect on the growth of toxigenic producing

365

fungi in grapes, with the prime value between 0.930-0.987. According to Kapetanakou, Panagou,

366

Gialitaki, Drosinos, and Skandamis (2009), the minimum aw required to produce toxin in A.

367

carbonarius is 0.85, A. ochraceus is 0.87 and 0.90 with the case of A. niger. Other factors noted

368

to significantly influence fungi growth and development are the application of fungicides, time

369

and rate of application as well as storage environment of harvested grapes. In terms of hydrogen

370

ion concentration (pH), Penicillium and Aspergillus spp are more tolerant to acid and alkaline pH

371

respectively (Wheeler, Hurdman, & Pitt, 1991). Black Aspergillus spp colonize injured berries

372

because grape berry moth (Lobesia botrana) is constantly present in vineyards and is a vector that

373

carries spores to grapes (Cubaiu, 2009).

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Regardless of the environmental factors which affect the bioactivity of BCAs, some

375

researches at both pre- and postharvest, demonstrated to be successful in the control of OTA.

376

Largely, all the microbial antagonists used were phyllosphere or carposphere yeast antagonists

377

(Table 2). For instance, A. pullulans strains LS30 and AU34-2 degraded OTA to less toxic

378

ochratoxin α in both synthetic medium and fresh grape must (De Curtis, de Felice, Ianiri, De Cicco,

379

& Castoria, 2012). Furthermore, other experiments conducted by Dimakopoulou et al. (2008) using

380

Phyllosphere yeast (A. pullulans Y-1) were successful in the control of sour rot disease 99%

381

(2005) to 90% (2006) caused by A. carbonarius in wine producing vineyards. Additionally,

382

studies conducted by Ponsone, Chiotta, Combina, Dalcero, and Chulze (2011) demonstrated that

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native yeasts strains Kluyveromyces thermotolerans provided effective control of A. niger and A.

384

carbonarius in grape vineyards in Argentina by reducing both growth and OTA accumulation to

385

50%. Some strains however, were effective in controlling the growth factors but could not remove

386

the toxin produced. Similarly, Dimakopoulou et al. (2008) reported that, one strain of

387

pullulans application effectively reduced sour rot disease incidence caused by A. carbonarius in

388

grape berries. In order to maintain the quality and shelf life of grape berries, both preharvest and

389

postharvest treatments are necessary under the present circumstances. Henceforth, a successful

390

microbial antagonist should be prolific and capable of surviving under unfavorable climatic

391

conditions which otherwise would be detrimental to the pathogen (Droby, Chalutz, Wilson, &

392

Wisniewski, 1992).

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7. Commercial possibilities of biocontrol products

While strenuous efforts are being by posthavest pathologists, microbiologists, postharvest

397

biologists, biotechnologists, entomologists and several related specialists to establish and develop

398

biocontrol products with equal or better effectiveness than synthetic pesticides, concerns have

399

been raised about their acceptability and compliance. For instance, Uri (1998) noted that,

400

biopesticides development and usage in the future will excel alongside the backdrop of ecological

401

effects accompanying the use of traditional pesticides and government policies promulgated to

402

mitigate their effects.

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403

This notwithstanding, significant gains have been achieved with regard to the

404

development and commercialization of biocontrol products. For instance, the first biological

405

control product to be manufactured and patented was Bacillus subtilis strain B-3, for the control

406

of stone fruits in the United States (Pusey & Wilson, 1984). Similarly, as reported by Sharma, 18

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Singh, & Singh (2009), a pilot test conducted using B. subtillis in commercial trials for the control

408

of peach rot containing bioagents was effectively combined with wax usually used in packing

409

lines. Many biological products have been manufactured and commercialized. For instance,

410

BioSave produced from a saprophyte strain of Pseudomonas syringae by Ecoscience’

411

Corporation, Orlando, US, tested effective and is recommended for the control of gray and blue

412

mold diseases of pear and apple. The P. syringae applied on a commercial parking line and

413

assessed for decay development, proved successful after three months (Janisiewicz & Korsten,

414

2002).

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Aspire is another biocontrol product from Ecogen-Israel Partnership Limited produced

416

from the yeast Pichia guilliermondii (Janisiewicz & Korsten, 2002). A pilot test carried out on

417

this product in large packing lines revealed that it was combined with 10-fold-diluted

418

thiobendazole and it yielded 100% inhibition of citrus diseases after havest. Other products such

419

as AQ-10 developed from Ampelomyces quisqualis in the US has also been successfully used to

420

control powdery mildew in grapes. Also, a biocontrol product named Serenade made from B.

421

subtilis in the Ecogen, Inc., USA has proved effective in controlling brown rot in grapes (Sharma,

422

Singh, & Singh, 2009). BCA products such as Shemer has also been introduced and is very

423

effective against some postharvest disease of fruits such as grapes, strawberry, citrus, potatoes,

424

peach, and other pome fruits. ‘Avogreen’ a biocontrol product developed from B. subtilis in

425

South Africa was demonstrated to control anthracnose and Cercospora species of avocado in the

426

field (Janisiewicz & Korsten, 2002).

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In spite of these remarkable achievements, however, no single biocontrol product has

428

showed complete control of postharvest diseases or has been effective compared to some of the

429

well-known synthetic pesticides. These, raise concerns in the food industry as both farmers and

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consumers demand food free of chemical residues. In view of this, there has been several findings

431

advocating for the combination of two or more BCAs (Droby, Wisniewski, Macarisin, & Wilson,

432

2009) in the control of postharvest diseases and mycotoxins including OTA. Such combinations

433

of BCAs can provide a broader spectrum of control than a single BCA.

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Even though numerous microbial antagonists have been identified and used in the

435

development of biocontrol products in these extensive investigations, only a few of them are

436

available in the market. This causes scientists to examine prudently for the impediments and

437

restrictions in the development of BCAs as commercial products. The areas highlighted to cause

438

transformational advances include enhancement of efficacy, delivery and persistence (Glare et al.,

439

2012).

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For these years, some biocontrol products have been developed for control of postharvest

441

diseases of fruits and vegetables (table 3), and there is great potential for the development and

442

commercialization of more biocontrol products in the near future as the field is relatively young

443

and research is moving at a fast pace. Moreover, the demand for safe food keep increasing and

444

this would encourage the use of more bioproducts. Presently, in the USA, a number of biocontrol

445

products are being traded for the control of postharvest diseases, while others are in the course of

446

being registered. Some of these companies are relatively small and privately owned firms with

447

limited product-line such as AgBiochem manufacturer of Galltrol (McSpadden Gardener &

448

Fravel, 2002). Others however, are publicly owned with diverse product lines and have

449

considerable capitalization values running into millions of dollars (Eden Bioscience).

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The challenges however, are related to the processes involved in the development and

451

commercialization of biocontrol products. Screening of antagonistic microorganisms for

452

commercialization and control of plant pathogens is a complex procedure. The isolation and

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development of BCAs involves several steps (Fig. 3). The importance of each step and the

454

impact on the process of determining, developing and commercializing a bioproducts has

455

certainly changed over time. Köhl, Postma, Nicot, Ruocco, and Blum (2011) reported that,

456

numerous factors that have been considered in the screening process apart from the antagonist

457

efficacy, include possibility of growth in fermenters for mass production, environmental

458

characteristics necessary for good field performance, toxicological issues, legal property rights

459

and marketing. Therefore an antagonist considered effective but produces only few cells in agar

460

plates might not be ideal for mass production since it is likely to be expensive or even impossible.

461

In addition, understanding the mechanisms involved and the potential strains that are responsible

462

for biocontrol activity is also important. Available experience so far has not found adverse health

463

effects associated with bioproducts, though, rules that can be formulated to properly address the

464

risk associated with them are lacking. In this regard, further research is needed to establish

465

possible health hazards associated with the use of bioproducts if any. Once an antagonist strain

466

has been selected for development after identification, bioassays and pilot tests, patenting has to

467

be considered. Even though, obtaining and maintaining patents is costly and only a few has been

468

registered as active substance for biocontrol.

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With regard to mass production and formulation of microorganisms, significant progress

470

has been made over the past few years. For instance, the discovery of new formulations of

471

bioproducts has improved storability, compatibility with other equipment and efficacy (Hynes &

472

Boyetchko, 2006). Moreover, the development and enhancement of solid state fermentation

473

technologies for filamentous fungi has brought about the discovery of many new bioproducts

474

(Kiewnick, 2010). The progress in formulation and fermentation, has also contributed to increases

475

in the quality of biocontrol products.

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Ecological and toxicological impacts are among the key steps in the registration process

477

for BCAs (Strasser & Butt, 2005). Considering the fact that good knowledge and understanding

478

of the behavior of microbial antagonists is critical to the determination of their potential side

479

effects. The registration procedure is of equal importance to all the steps in the process of

480

developing and commercializing biocontrol products. The registration procedure remain a

481

challenge to the commercialization of bioproducts due to regulatory processes that are responsible

482

for the delays, the scope of which is beyond this review. However, the average time that it takes

483

to register a BCA is about 26 months (range12-60) in the US whiles in the EU is more than 80

484

months (63-104) (Kiewnick, 2010). Despite the above mentioned challenges, there is worldwide

485

interest in the commercialization of biocontrol products and many national and multinational agro

486

companies have boosted their investment reach (Fravel, 2005)

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8. Future trends

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In this review the authors discussed the successes of microbial antagonists used to control

490

OTA-producing fungi and/or detoxify OTA in grape berries and wine. Indeed, biological control

491

strategies have emerged as the preferred methods to control OTA-producing fungi and other

492

mycotoxins caused by pathogens in fruits and vegetables. BCAs have been found to be effective

493

against OTA-producing fungi among which various strains of yeast antagonists remain dominant.

494

This is because of their ability to thrive under variable climatic conditions, to adhere to fruit

495

surfaces, to detoxify OTA through cell wall adsorption, and to combine with small doses of low

496

risk pesticides among others. Prehavest applications of microbial antaginists to control OTA-

497

producing fungi in grape berries are promising.

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Researchers continually work on many microbial antagonists in vivo and in vitro to assess

499

their antagonistic activities in order to select which to use based on their consistent efficacy after

500

field trials. Despite this progress, one of the challenges accounting for the failure of microbial

501

antagonists to be commercialized is the small marketing to justify the development and

502

registration cost. Many microbial antagonists, after identification and characterization, are often

503

not patented or developed for these reasons. The small market size for biocontrol products could

504

be due to their small spectra of action on pathogens. Another reason is the prolonged time and

505

complex production procedure required to develop and register a bioproduct as illustrated in the

506

text. Moreover, the inconsistency and ineffectiveness of bioproducts are the main problems facing

507

their acceptance.

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Notwithstanding the above mentioned challenges, there are prospects for bioproducts. The

509

availability and commercialization of some biocontrol products in the market are indications that

510

they are cost effective and will thrive against all odds. As found in literature, more bioproducts

511

are at various stages of development and registration. Funding for fundamental and applied

512

researches is continuously being provided by donors like US Department of Agriculture and other

513

multi-national agencies throughout the world to unearth innovations in BCAs. Presently, the

514

focus on biocontrol products should be centered on the acceptability and adoptability around the

515

world rather than the replaceability of fungicides.

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The use of adjuvants such as food additives, salts, plant extracts, essential oils, etc, to

517

enhance the performance of microbial antagonists are among attempts to improve the viability

518

and consistency of BCAs. However, further research is urgently needed to determine the

519

mechanisms of action on microbial antagonists in order to develop more viable bioproducts. The

520

performance of most BCAs depend on environmental parameters because they are living

23

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organisms. The ecology of both the microbial antagonist and the targeted pathogen must be

522

understood in the development of BCAs. The manipulation of the epiphytic community could be

523

a more practicable approach to obtain an effective control of pathogens. This is because the

524

virulence of the pathogens to a large extent are influenced by temperature, relative humidity, aw

525

and biotic factors in the preharvest environment. Further research on the environmental factors

526

that limit the performance of microbial antagonists is needed in order to obtain good formulations.

527

The prospects of biological control industry would depend on research, innovative

528

marketing strategies, more education and product marketing (Group, 1998) . In view of these,

529

scientists, producers and consumers must accept the fact that BCA development and

530

commercialization will progress slowly. Generally, government policies are in favor of the use of

531

BCAs because they are safe according to records. With the ever increasing consumer awareness

532

about the risks of mycotoxins in fruits and vegetables, legislations have been instituted to protect

533

and guarantee the safety of consumers. Finally, in this review the authors recommend further

534

studies into any possible molecular mechanism(s) involved in exogenous compound-antagonistic

535

yeast-pathogen interactions on OTA contents of grape berries.

536

Acknowledgments

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This work was supported by the National Natural Science Foundation of China (31271967),

539

the Research Fund for the Doctoral Program of Higher Education of China (20123227110015),

540

the Technology Support Plan of Jiangsu Province (BE2014372), and the Technology Support

541

Plan of Zhenjiang (NY2013004).

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799 800 801 802

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Table 1. OTA-producing fungi found in grapes.

References (Bellí et al., 2007), (Gil-Serna et al., 2009), (Esteban et al., 2004), (Selma et al., 2008), (Battilani & Pietri, 2002),(Varga & Kozakiewicz, 2006)

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Fungi Aspergillus carbanarius Aspergillus ochraceus Aspergillus niger Penicillium nordicum Penicillium verrucosum

RI PT

808

M AN U

809 810 811 812 813

817 818 819 820 821 822 823 824

EP

816

AC C

815

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814

825 826 827 38

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Table 2. Microbial Antagonists that have been successfully used to control and degrade OTA-

830

producing fungi and contaminants in grape berry. Reference (s) (Schena et al., 1999)

Aspergillus niger A. carbonarius (sour rot) A. carbonarius (sour rot) Aspergillus niger Aspergillus carbonarius A. carbonarius (sour rot) Aspergillus niger A. carbonarius (sour rot)

(Castoria et al., 2001) (Schena et al., 2003) (Schena et al., 2003) (Bleve et al., 2006)

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Pathogen/disease Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus. niger

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(Zahavi et al., 2000)

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Microbial Antagonists A. pullulans strain L-47 A. pullulans strain LS236 A. pullulans strain LS250 Candida guilliermondii strain A42 A. pullulans LS-30 A. pullulans 533 A. pullulans 547 Issatchenkia terricola Metschnikowia pulcherrima Candida incommunis A. pullulans AU34-2

(Bleve et al., 2006)

831

AC C

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(Bleve et al., 2006), (De Curtis, et al., 2012) Streptomyces aureofaciens Aspergillus niger (Perrone et al., 2007) Rhodotorula glutinis (Haggag & Abdall, 2012) A. pullulans Y-1 Aspergillus carbonarius (Dimakopoulou et al., 2008) (sour rot) Metschnikowia pulcherrima Aspergillus carbonarius (De Felice et al., 2008) LS16 Kluyveromyces thermotolerans A. carbonarius (sour rot) (Ponsone et al., 2011) RCKT4 and RCKT5 Aspergillus niger Lachancea thermotolerans 751 Aspergillus carbonarius (Fiori et al., 2014) Candida intermedia 235 Aspergillus carbonarius Candida zemplinina M3 Aspergillus carbonarius, (Zhu et al., 2015) Aspergillus ochraceus Saccharomyces cerevisiae Aspergillus carbonarius, M114 and C297 Aspergillus ochraceus (Zhu et al., 2015) Pichia kluyveri M117 Aspergillus carbonarius, Aspergillus ochraceus (Zhu et al., 2015) Metschnikowia aff. fructicola Aspergillus carbonarius, (Zhu et al., 2015) M179 Aspergillus ochraceus 832 833 834

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Table 3. Biocontrol products developed for control of postharvest diseases of fruits and vegetables. Antagonist

Target Disease/causative agent Botrytis bunch rot (B. cinerea). Brown rot (M. fructicola), Powdery mildew (P. leucotricha), Peach rot (M. fructicola) Late blight (P. infestans) Powdery mildews (U. necator). (P. xanthii), (P. aphanis)

Serenade

Bacillus subtilis QST 713

USA

AQ-10

Ampelomyces quisqualis strain M-10

Netherland

Shenmer (water dispersible granules)

Metschnikowia fructicola

USA

Biosave

Pseudomonas syringae ESC-10 ESC-11

USA

Aspire

Pichia guilliermondii

Blue mold (P. expansum) and (B cinerea)

South Africa

Avogreen

Bacillus subtilis

Anthracnose, Cercospora spot, (blotch) leaf spot (C. purpurea)

South Africa

Yield plus

Italy

EP

Sonata

Amylo-X (water dispersible granules)

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Gray mold (B. cinerea), Sour rot (A. niger), Blue mold (P. expansun and P. italicum), Rhizopus decay (R. stolonifer) Brown rots (M. fructigena) Blue mold (P. expansum) Gray mold (B. cinerea) Mucor rot (M. piriformis) Green mold (P. digitarum) Sour rot (G. candidum)

Mode of application Pre & Postharvest

Pear Citrus Apple Avocado

Preharvest application (Foliar spray)

spray or drench in pre and postharvest

Pre & Postharvest

Pre & postharvest Pre & Postharvest (drip/wax) ESC-11 Preharvest

Cryptococcus albidus Bacillus pumilus QST 2028

Gray mold (B. cinerea) Powdery mildew, downy mildew, blight, and soyabean rust

Apple pear Grapes Strawberry Pome fruits

Preharvest

Bacillus amyloliquefaciens

gray mold (Botrytis cinerea), Fire blight (Erwinia amylovora),gray mold(Botrytis cinerea)

Grapes Apple Pear strawberry

Preharvest

AC C

USA

SC

USA

Fruit/vegeta bles Grapes Apple Tomato Onion Citrus Peach Pear Grapes Apples Aubergine Tomatoes Strawberry Cucumber Grapes Strawberry Citrus Potatoes Peach Pepper Tomatoes Raspberry Citrus Apple potato Cherry Avocado

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Bioproduct

TE D

835 836

837 838

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839 840 841

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842 843

Fig. 1. OTA converted to ochratoxin α by carboxypeptidase A. (Stander, Steyn, van der Westhuizen, & Payne, 2001)

SC

844 845 846 847

Fig. 2 A. Fresh red grapes

B. Grapes infected with Aspergillus ochraceus

849 850

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Fig. 3. Flow diagram for the development and commercialization of a microbial biocontrol agent. Source: (Kiewnick, 2010)

851

855 856 857 858 859 860

EP

854

AC C

853

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852

861 862 863

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866 867

Fig. 1.

868

872 873 874 875 876 877

EP

871

AC C

870

TE D

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878 879 880 42

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881 882

M AN U

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883

887 888 889 890 891 892 893

EP

886

Fig. 2.

AC C

885

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894 895

43

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Isolation

897 898 Identification characterisation

Bioassays

900

Pilot tests

RI PT

899

Selected strain

901 902

Mass production

SC

Toxicology & environmental impact

903

M AN U

904

Patent

Specific analytical methosd

Storage & formulation

905 906 907

Registration for use

911 912 913 914 915

EP

910

Fig. 3.

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Biological control agents have proved successful to control OTA-producing fungi.




Mechanisms of action of BCAs against OTA-producing fungi are reviewed.




Environmental conditions and location affect the growth of OTA-producing fungi.




Preharvest applications of BCAs are promising means to reduce OTA- producing fungi.

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