WO2008114304A2 - Compositions, method and use of compounds made up of microorganisms for controlling phytopatogenic and/or mycotoxigenic fungi and limiting mycotoxin levels - Google Patents

Abstract

The invention consists of a method for controlling fungal phytopathogens by applying compositions to grape vines and cereals in the field. These compositions are based on antagonistic microorganisms which are not only able to protect plants from phytopathogenic microorganisms, including mycotoxigenic agents, but they are also able to reduce the quantity of mycotoxins present in the edible portions since antagonist microorganisms also control mycotoxigenic fungi, even those already present at the time of application. The preparation of these compositions and their practical agricultural use are also described.

Description

Compositions, method and use of compounds made up of microorganisms for controlling phytopatogenic and/or mycotoxigenic fungi and limiting mycotoxin levels

The present invention consists of a method for using compositions based on microorganisms to control the onset and/or development of infections due to phytopathogenic microorganisms including mycotoxigens; this method also controls the accumulation of mycotoxins in the edible parts of the plant and reduces not only the presence of mycotoxigenic microorganisms and mycotoxins, even those already present at the time of application.

From prior art it is known that strains of yeasts and yeast-like fungi have been successfully applied to agricultural products after harvesting. Using antagonistic microorganisms which possess similar features to both these yeasts and yeast-like fungi to protect stored vegetal products has been successfully used in industrial applications as shown by the commercialization and the various national registrations issued for compositions based on these antagonists. On the other hand, the commercial sales of these yeasts and yeast-like fungi to control phytopathogens responsible for phylloplane diseases are not known, with some exceptions such as a fungal strain of Ampelomyces quisqualis (marketed under the trade name AQlO), which is known to antagonise only powdery mildew. Before AQlO is applied, however, the disease must be present to serve as a base of establishment for it to survive on in order to effectively control the disease; moreover, it does not always achieve satisfactory results.

These yeasts and yeast-like fungi have had limited persistence (i.e. survival) in the environment. Thus, their application has been proposed for the protection of stored vegetal products, where it is possible to apply them under controlled conditions (i.e. temperature, humidity, CO₂) and where UV radiation is almost absent; they have not been recommended or sold, however, for field application on the phyllosphere. In addition, a general selectivity towards single fungal targets for each crop has been noted in the applications described in prior art; therefore, their use as wide-spectrum solutions is highly unlikely as it is necessary to follow complicated application procedures. The applicant's strains, however, possess a wide-spectrum of action against various phytopathogenic fungi affecting fruits and vegetables and are surprisingly polyvalent in the control of leaf pathogens responsible for diseases that affect economically important crops. Among the phytopathogens that have been effectively controlled and which are present in major stored fruit and vegetables such as apples, pears, strawberries, kiwifruit, citrus fruits and wine as well table grapes, there are, for example, Aspergillus spp., Botrytis cinerea, Penicillium expansum, Penicillium digitatum, Penicillium italicum and Rhizopus stolonifer (Lima et al., 1999 - J. Industr. Microbiol. Biotecnol.); among these Aspergillus niger, Aspergillus carbonarius and Penicillium expansum are also mycotoxigenic fungi. Some of the applicant's strains have displayed the ability to colonise fruit surfaces successfully, surviving and resisting for months, thus guaranteeing a remarkable activity against phytopathogenic microorganisms over time. These strains are able to colonise quickly and effectively the fruit wounds thus preventing the attack by phytopathogenic microorganisms which penetrate these wounds, because they are able to resist oxidative stress caused by active oxygen species produced by both the wounded fruit tissues and phytopathogenic fungi such as Botrytis cinerea (Castoria et al., 2003 - Phytopathology). Therefore, their action is carried out by means of pre-colonisation of fruit surfaces as well as of their wounds and by the biosynthesis of enzymes which are able to actively degrade cell walls of phytopathogenic microorganisms and not by means of the biosynthesis of antibiotic substances (Castoria et al., 1997 and 2001-Postharvest Biology and Technology). Interesting activity against Botrytis cinerea e Penicillium expansum by these strains was observed in the cold storage of apples under semi-commercial conditions, even applying the antagonist in the field, before fruit harvesting (Lima et al., 2003 - European Journal of Plant Pathology; Lima et al., 2006 - Postharvest Biol. Technol.).

Some of the applicant's strains have been successful in preventing attacks of phytopathogenic fungi in the field, particularly from powdery mildew (Sphaerotecha fused) on cucurbits (Lima et al., 2002 - IOBC Bull.). These microbial strains were able to survive and maintain populations at high levels after being applied to the leaves of these crops, even under climatic conditions characterised by high temperatures with relatively low levels of humidity. Under these conditions, the effectiveness of the applicant's microbial strains against the powdery mildew was not only comparable to the results achieved by synthetic agrochemicals, especially the triazolic fungicides, but were also superior to results achieved by the biofungicide AQlO (Lima et al., 2002 - Journal of Plant Pathology).

The inventors' strains have also been successful in preventing contamination with the mycotoxin patulin on pome fruit attacked by Penicillium expansum and these strains have been able to transform/biodegrade the mycotoxin into a much less toxic compound (Castoria et al., 2005 - Phytopathology and Castoria et al., 2007).

These microbial strains have been successful in preventing attacks from phytopathogenic fungi, particularly from both Botrytis cinerea and Penicillium expansum on apples, and have shown a synergic increase in their efficacy when combined with naturally derived adjuvants (Lima et al., 2005 - Journal of Food Protection).

The applicant has now confirmed that several of their own strains also allow significant protection from a series of diseases common to grains and cereals (powdery mildew, septoriosis and helminthosporiosis), not only under greenhouse conditions, but also in the field, thus enabling growers to control said phytopathogens either by preventing infections (prophylactic or protective effect) or by fighting the infections already present (therapeutic or curative effect). Similarly, the applicant has successfully carried out tests in the field as well as in greenhouses for controlling diseases that affect numerous other economically important crops such as grapevines, horticultural crops (i.e. melon and zucchini), coffee, soybean and sugar beet. As already reported for fruit, remarkable success has been achieved with these crops as well in controlling mycotoxigens which produce mycotoxins other than patulin; thus providing an interesting solution to an important problem that afflicts agriculture, and for which until now, modern technology was unable to supply wholly satisfactory solutions that also have a low impact on the environment.

Ochratoxin A (OTA), in particular, is potentially carcinogenic to humans and the International Agency for Research on Cancer (IARC) has classified as carcinogen in Group 2B. Ochratoxin A occurs in plants such as cereals, coffee seeds, beans and other pulses (Kuiper-Goodman and Scott, 1989; Pohland et al., 1992; Jørgensen, 1998) as well as in beverages such as wine, grape juice and beer (Majerus, 1996; Pietri, 2001; Zimmerli and Dick, 1996). Wine is considered, after cereals (FAO/WHO, JECFA, 2001), the second major source of OTA in the European Union. Dried grapes may also be a food source of OTA, which frequently occurs at levels as high as 53.6 ppb (FAO/WHO, JECFA, 2001; Stefanaki et al., 2003). The maximum levels of contamination established by the European commission are 2 ppb for both wine and grape juice and 10 ppb for dried grapes (REGULATION EC N. 123/2005, 26 January 2005). OTA contamination is caused by Aspergillus carbonarius in the vineyard as well as by its development in the ensuing stages. Until now, only data from preliminary in vitro tests on the effects of fungicides on the production of OTA have been available (Battilani et al, 2003). Nevertheless, reducing food contamination by fungicides and mycotoxins residues are scientific and social demands expressed by the European Union.

Despite the fact that the applicant has shown through laboratory assessments that it is possible to reduce patulin contamination of food, these observations do not automatically mean that this technology can be extended either to the reducing of other mycotoxins or to applications under practical agricultural conditions in order to reduce mycotoxin levels in crops. Indeed, these tests were carried out under model conditions, unlike normal agricultural practices. Moreover, patulin has a specific chemical structure different from other mycotoxins; each mycotoxin has its own structural features and its own chemical properties, so it is not possible to hypothesize a common catabolic means of detoxifying patulin and other mycotoxins. Surprisingly, the inventors have found that strains of yeasts and yeast-like fungi in their possession are not only able to contribute, to the reduction of infections caused by crop phytopathogens under practical farming conditions, but also to significantly reduce both mycotoxigens and the levels of the relative mycotoxins. Specifically, significant reductions in mycotoxin levels other than patulin, namely ochratoxin A, have been observed.

The applicant's strains are yeasts and yeast-like not pathogenic or harmful to either humans or mammals in general; therefore, they are a sustainable agricultural solution for protecting crops.

Thus, the purpose of the present invention is to solve the problem of crop losses due to phytopathogenic fungi attacks as well as to limit contaminations of vegetal products with mycotoxins. These are not only recognised risk factors for both human and animal health, but also lead to a reduction in the commercial value of the contaminated crops, and, as a consequence, lower profits for farmers.

Another major aspect of this invention concerns a method using compositions based on microorganisms to fight the onset and/or the development of infections caused by phytopathogenic and/or mycotoxigenic microorganisms, whilst also reducing mycotoxin levels which are present in the edible parts of the plants so as to reduce the presence of both mycotoxigenic microorganisms and mycotoxins, even those already present at the time of application. In particular, the present invention concerns a method that uses compositions based on microorganisms to fight the onset and or the development of infections caused on plants by phytopathogenic and mycotoxigenic microorganisms able to produce mycotoxins, in particular other than patulin, that are present on the edible part of the plants, so as to also reduce both the presence of mycotoxigenic microorganisms and micotoxins produced by them, even those already present at the time of application.

Above all, this patent describes a method that uses compositions of compounds based on microorganisms and specific adjuvants to fight the onset and/or the development of infections caused by phytopathogenic and mycotoxigens microorganisms able to produce mycotoxins. In addition, the present invention is able to reduce ochratoxin levels present in the edible parts of the plants so as to also reduce both the presence of mycotoxigenic microorganisms and ochratoxin A, even when already present at the time of application.

The present invention relates to a method using compounds based on yeasts and yeast-like fungi to fight the onset and/or the development of infections on fruit and vegetables such as apples, pears, coffee beans, soybeans, strawberries, kiwifruit, table as well as wine grapes and citrus fruits caused by phytopathogenic and mycotoxigenic fungi such as Aspergillus spp., Botrytis cinerea, Rhizopus stolonifer, Penicillium expansum, Penicillium italicum and Penicillium digitatum, whilst also containing ochratoxin levels present in the edible portions of the plants even if already present at the time of application.

Moreover, one aspect of the present invention concerns a method that uses compounds based on yeast-like fungi to fight the onset and/or the development of infections on table as well as wine grapes caused by the mycotoxigenic fungus Aspergillus carbonarius, which produces ochratoxin A, whilst also reducing ochratoxin A levels present in the edible parts of the plants, even when already present at the time of application. Another unique aspect of this invention involves a method that uses compounds based on yeasts and/or yeast-like fungi to fight the onset and/or the development of diseases affecting cereal crops. This method is not only effective against diseases such as powdery mildew, septoriosis, helminthosporiosis and leaf scald due to Rhynchosporium secalis, but also against mycotoxigenic fungi, such as those belonging to the genera Aspergillus, Fusarium and Penicillium, whilst also reducing mycotoxin levels, especially ochratoxin A levels, present in the edible portions of the plants, even when already present at the time of application. Examples of the cereal crops tested using the method stated in this patent are: wheat, spelt, barley, rye and rice.

Examples of the mycotoxigenic fungi present on these cereal crops are: Aspergillus flavus, Aspergillus parasiticus, Aspergillus ochraceus, Fusarium culmorum, Fusarium crookwellense, Fusarium graminearum, Fusarium proliferatum, Fusarium sporotrichioides, Fusarium Verticilloides and Penicillium verrucosum.

Another unique aspect of this invention describes a method that uses compounds based on yeasts and/or yeast-like fungi to fight the onset and/or the development of diseases affecting corn; diseases such as green mould caused by Penicillium chrysogenum, whilst also reducing mycotoxin levels, other than patulin, especially ochratoxin A levels, present in the edible portions of the plants even when already present at the time of application. The applicant has also found that when the yeasts and yeast-like fungi are applied during the growth season of the crops in the field, they are able to survive at high level of population until harvest, significantly protecting the crop yield (particularly grains and fruit), even during postharvest storage, thus producing a twofold effect of reducing both the mycotoxigens and the mycotoxins which are produced or are already present. In particular, this effect has been observed on cereal crops, where Penicillium chrysogenum and Penicillium verrucosum, both producers of Ochratoxin A, were controlled. . Therefore, a further aspect of the present invention involves a method that uses compounds based on yeasts and yeast-like fungi to fight the onset and/or the development of mycotoxigenic fungi such as those belonging to the genera Aspergillus, Fusarium and Penicillium that infect cereal and fruit yields, pre- and postharvest. These yeasts and yeast- like fungi, by means of a pre-harvest application, are also able to reduce mycotoxin levels, especially ochratoxin A levels present in the edible portions of the plants, even when already present at the time of application. Moreover, the beneficial effects also extend to the harvest itself.

The method of the present invention consists of one or more application of compositions of the above mentioned yeasts and/or yeast-like fungi in the form of a powder or in an aqueous solution, which in turn is diluted with appropriate quantities of water so as to obtain solutions or dispersions, partially or totally insoluble in water, which are then applied once or more times to the aerial parts of the crops. These formulates may contain one or more yeasts and/or yeast-like fungi and are then applied to the plant, either as a suspension of spores or mycelium, or as a mixture of spores and mycelium. These compositions can be readily obtained by drying or lyophilization of the biomass derived from the fermentation of the strains described in this invention, which, in turn, were harvested by the filtration, sedimentation or centrifugation of the fermentation broth. Preferred examples of yeasts and/or yeast-like fungi used in this method include, but are not limited to, strains of Aureobasidium, Rhodotorula and Cryptococcus.

1. Aureobasidium pullulans, specifically, the following strains collected by the University of Molise are the preferred ones: AU14-3-1, AU18-3B, AU34-2 and LS30. The Aureobasidium pullulans strain LS30 has been fingerprinted by molecular methods (De Curtis et ah, 2004) as well as deposited under the Budapest Treaty at the Centraalbureau Voor Schimmelcultures (CBS, P.O. Box 85167-3508 AD UTRECHT, The Netherlands) with the reference number CBS 110902.

2. Rhodotorula glutinis, specifically, the strain LSI l collected by the University of Molise is preferred.

3. Cryptococcus laurentii, specifically, the strain LS28 collected by the University of Molise is preferred.

Therefore, another aspect of the present invention relates to the preparation of compositions based on one or more strains of yeasts and/or yeast-like fungi, included in the genera listed above and obtained by fermentation, separation and desiccation. Preferred mixtures are:

• one or more strains of Aureobasidium pullulans

• one or more strains of Rhodotorula glutinis

• one or more strains of Cryptococcus laurentii

• one or more strains of Aureobasidium pullulans and one or more strains of Rhodotorula glutinis

• one or more strains of Aureobasidium pullulans and one or more strains of Cryptococcus laurentii

• one or more strains of Rhodotorula glutinis and one or more strains of Cryptococcus laurentii

The applicant's strains of yeasts and/or yeast-like fungi proved to be compatible with numerous fungicide compounds commonly used on farm crops, generating unexpected synergies as well as fungicide effectiveness, which made it possible to apply considerably lower dosages of these fungicides, thus resulting not only in a lower impact on the environment, but also in reducing the risk of resistance rise in the phytopathogens to traditional compounds. Mycotoxigenic fungi as well as levels of mycotoxins produced by them also proved to be remarkably lower than expected, thus making it possible to reduce the biomass of yeast-like fungi and/or the dosage of traditional fungicide compounds.

The applicant has found that the composition based on yeasts and/or yeast-like fungi, subject of this invention, can also be used either in mixtures or in alternation with other biocide products, thus generating an unexpected synergic activity which not only helps to control phytopathogenic and mycotoxigenic microorganisms present on the crops, but also contributes to the reduction of mycotoxin levels in the edible portions of the plants, even if present before the application.

These mixtures may be applied either as ready-to-use mixtures or prepared as needed, using the appropriate dosage for each ingredient (yeasts and/or yeast-like fungi and biocide compounds). The use of these compositions produces surprising synergies that may be assessed, for example, by applying either Abbott's or Colby's formula.

The applicant also found that even compounds possessing little or no biocide activity, which the applicant had already reported as synergic in controlling moulds on postharvest food in storage, were able not only to raise the control of phytopathogenic fungi and mycotoxigenic fungi, but also to reduce mycotoxin levels.

Yet another aspect of the present invention relates to the use of compositions based on yeasts and/or yeast-like fungi in combination with active ingredients, either naturally-derived or synthetically produced, whether possessing biocide activity or not, which however, display compatibility with yeasts and/or yeast-like fungi, which form the subject of this method, and generate a synergism measurable according to both Abbott's and Colby's formulas regarding the control of phytopathogens and the reduction of mycotoxigenic fungi and mycotoxins on the edible portions of plants. The applicant had already substantiated and reported the fact that either inorganic salts or carboxylic acids were able to synergise the effects of yeasts and/or yeast-like fungi when applied to control Botrytis rot in pre- and postharvest crops.

The applicant found that alkyl-polyglycosides, such as for example, some monofunctional disaccharides with C8-C18 alkyl groups, sold under the name of Glucopon or Agnique (produced by Cognis) used as surfactants to be incorporated into agrochemical formulations, are unexpectedly able to increase the effectiveness of the compositions based on yeasts and/or yeast-like fungi, subject of the current patent, making it possible to apply them in significantly lower dosages, either when applied alone or in mixtures with other active fungicide compounds.

The applicant has proven that applications of Aureobasidium strains mixed with Glucopon 650, in key phenological stages of plants (pre-bunch closure, veraison and 20 days before harvesting) have evidenced a good survival rate of these microbial strains and, surprisingly, have made it possible to reach levels of effectiveness against not only Botrytis cinerea, but also against secondary rotting grape bunch agents, such as Aspergillus spp. and Penicillium spp, including several mycotoxigenic species, comparable to those achieved with full dosages of synthetic agrochemicals well known for their high activity against Botrytis rot. Similarly, on durum wheat, treatments based on yeasts and/or yeast-like fungi, alone or in combination with reduced dosages of agrochemicals and/or naturally-derived adjuvants, and always in the presence of Glucopon 650, applied in the phenological stage of pre-flowering, have increased survival rates of these microbial strains, and surprisingly, have made it possible to reach significant levels of effectiveness against the main diseases that affect the aerial portions of the plant.

Therefore, another aspect of the present invention relates to the use of compositions of yeasts and/or yeast-like fungi in mixtures with monofunctional disaccharides with C8-C18 alkyl groups, either straight or branched, which may be mixed with a fungicide compound, obtaining a mixture with synergistic effects on phytopathogenic and mycotoxigenic microorganisms. One or more of the following active ingredients is intended when referring to fungicide compounds or compounds able to synergize with these yeasts and/or yeast-like fungi:

(1) Alginic acid.

(2) Corn starch, food additive.

(3) Calcium acetate, food additive.

(4) Calcium ascorbate, food additive.

(5) Calcium chloride, food additive.

(6) Calcium citrate, food additive.

(7) Calcium propionate, food additive.

(8) Calcium silicate, food additive.

(9) Glucopon (also known as Agnique), product sold by Cognis.

(10) Guar gum, food additive.

(11) Locust-bean gum, food additive.

(12) Xanthan gum, food additive.

(13) Soybean oil, food additive, also used as insecticide adjuvant.

(14) Light mineral oils, known as insecticide adjuvants.

(15) Dibasic potassium phosphate, food additive, known also as weak fungicide against powdery mildew.

(16) Fenhexamid, commercial antibotrytic fungicide.

(17) Procymidon, commercial antibotrytic fungicide.

(18) Tetraconazole, commercial triazolic fungicide.

(19) Thiabendazol, commercial wide spectrum fungicide. (20) Trifloxystrobin, commercial methoxyacrylate fungicide.

(21) Ziram Granuflo Fungicide, commercial fungicide.

(22) Sulphur, inorganic antioidium (against powdery mildew) fungicide.

(23) IR5885, a dipeptide compound, corresponding to mixtures of diastereoisomers methyl [S-(R₅S)] [3-(Niso propoxycarbonylvalinyl)-amino]-3-(4-chloro-phenyl) propanoate in any proportion, or in one of the two forms of diastereoisomers S-R or S-S, used individually

(24) IR6141, corresponding to N-( phenylacetyl)-N-2,6-xylyl-R-_.mcthyl alaninate.

(25) Tetraconazole (in its form as a racemic mixture or as an optically active R-isomer).

(26) Salicylic acid (SA) or its derivatives such as acetylsalicylic acid (ASA), copper salts of salicylic acid (SA2CU) or of (SACu) or copper salts of acetylsalicylic acid (ASA2CU).

(27) Copper salt (I) or copper salt (II), such as copper oxychloride, copper hydroxide, the bordeaux mixture, copper sulphate, or a mixture of hydroxide and copper oxychloride (Airone).

(28) Benalaxyl corresponding to N-( phenylacetyl)-N-2,6- xylyl -RS- methyl alaninate.

(29) Metalaxyl corresponding to methyl N-(2- methoxyacetyl)-N-2,6- xylyl -RS alaninate.

(30) Metalaxyl-M corresponding to methyl N-(2- methoxyacetyl)-N-2,6- xylyl -R alaninate.

(31) Oxadixyl corresponding to 2- methoxy -N-(2-oxo-l,3- oxazolidin -3- yl) acet-2',6'- xylidide.

(32) Mandipropamid corresponding to 2-(4- chlorophenyl)-N-[2-(3- methoxy - 4-prop-2- ynyloxy phenyl)ethyl]-2-prop-2- ynyloxy - acetamide.

(33) Iprovalicarb corresponding to O-(l-methyl-ethyl)-N-[2-methyl-l-[[[l-(4- methyl- phenyl) etiljamino] carbonyl]propyl]carbamate. (34) Benthiavalicarb-isopropyl corresponding to O-isopropyl [(S)- 1 - { [( 1 R)- 1 -(6-fluoro- 1,3- benzothiazol-2-il)ethyl]-carbamoyl-2-methylpropyl]carbamate.

(35) Cymoxanil corresponding to l-(2-cyano-2- methoxyimino - acetyl)-3- ethylurea.

(36) Azoxystrobin corresponding to (E)-2-[2-[6-(2- cyanophenoxy)- pyrimidin -4- yloxy]phenyl-3-methyl methoxyacrylate.

(37) Metominofen corresponding to N-methyl-(E)- methoxyimino - (2- phenoxyphenyl) acetamide.

(38) Pyraclostrobin corresponding to methyl N-(2-[l-(4- chlorophenyl) pyrazol-3- yloxymethyl] -phenyl)-N- methoxycarbamate.

(39) Acibenzolar-S-methyl corresponding to methyl benzo( 1,2,3) thiadiazole -7- thiocarboxylate.

(40) Famoxadone corresponding to 5-methyl-5-(4- phenoxyphenyl)-3-( phenylamino) oxazolidinedione -2,4-dione.

(41) Fenamidone corresponding to 4-methyl-4-phenyl- 1 -( phenylamino)-2- methylthio imidazolin-5-one.

(42) Cyazofamide, corresponding to 2-cyano-4-chloro-5-(4-methylphenyl)-l- (N,N- dimethylamino-sulfamoil) imidazol.

(43) Fluazinam corresponding to 3-chloro-N-(3-chloro-5- trifluoromethyl -2-pyridyl)- α,α,α-trifluoro-2,6-dinitro-p- toluidine.

(44) Dimethomorph corresponding to (E,Z)-4- [3 -(4-chlorophenyl)-3 -(3,4- dimethoxyphenyl)- acryloyl] moφholine; or else Flumorph (SYP-L 190) corresponding to (E,Z)-4-[3-(4 fluorophenyl)-3-(3,4- dimethoxyphenyl)-acryloyl]morpholine.

(45) Flumetover corresponding to N₅N- dietylamide of 4- trifluoromethyl -6-3,4- dimethoxyphenyl)-benzoic acid.

(46) Chlorothalonil corresponding to 1,3- dicyano -2,4,5,6- tetra-chloro-benzene. (47) Mancozeb corresponding manganese and zinc salts of ethyleneό/,y(dithiocarbamate) (polymer).

(48) Tolylfluanid corresponding to N- dichlorofluoromethylthio-N',N'-dimethyl-N-p- tolylsulfamide.

(49) Folpet corresponding to N-( trichloromethylthio)phthalimide.

(50) Etridiazole corresponding to ethyl-3- trichloromethyl -1,2,4- thiadiazol ether.

(51) Hymexanol corresponding to 5-methylisoxazol-3-yl.

(52) Propamocarb corresponding to propyl-(3- dimethylaminopropyl) carbamate.

(53) R-3-aminobutanoic acid or RS-3-aminobutanoic acid.

(54) Zoxamide, corresponding to 3,5- dichloro -N-(3- chloro -1 -ethyl- 1 -methyl- 2- oxopropyl)-p- toluamide.

(55) Ethaboxam, corresponding to (RS)-(i-cyano-2-thenyl)-4-ethyl- 2(ethylamino)-5- thiazolecarboxamide .

(56) Fluopicolide, corresponding to 2.6-dicloro-N-[3-cloro-5- (trifluorometil)-2- piridilmetiljbenzammide.

(57) Phosetyl, corresponding to ethyl hydrogen phosphonate.

(58) Phosetyl- Al, corresponding to aluminium salt of ethyl hydrogen phosphonate, commonly known under the commercial name Aliette.

(59) Metominostrobil commercial methoxyacrylate.

(60) Iprodione, procymidone, cyprodinil and pyrimethanil- commercial antibotrytic fungicides.

(61) epoxyconazole, propiconazole, tebuconazole and triazolic fungicides- commercial sterol biosynthesis inhibitors. (62) kresoxim-methyl, picoxystrobin, pyraclostrobin, fluoxastrobin, metominostrobin, orysastrobin, dimoxystrobin and enestroburin, commercial fungicides inhibiting mitochondrial respiration in the fungal cell.

Compounds (23) are described in Italian patent application MI98A002583.

Compound (24) is described in patent application WO 98/26654 A2.

Compound (25) is described in "The Pesticide Manual", 1997, 1 lth edition, British Crop

Protection Council Ed., p. 1174.

Compounds (26) are commercial products and their copper salts are described in Italian patent application MI 2001 A002430.

Compounds (27) are readily available on the market.

Compound (28) is described in "The Pesticide Manual", 1983, seventh edition, British Crop

Protection Council Ed., p. 32.

Compound (29) is described in English patent application GB 1,500,581.

Compound (30) is described in patent application WO 96/01559 Al.

Compound (31) is described in English patent application GB 2,058,059.

Compound (32) is described in patent application WO 01/87822.

Compound (33) is described in European patent application EP 550,788 ed EP 775,696.

Compound (34) is described in European patent application EP 775,696.

Compound (35) is described in "The Pesticide Manual", 1983, seventh edition, British Crop

Protection Council Ed., p. 148.

Compound (36) is described in European patent application EP 382,375.

Compound (37), corresponding to the acronym SSF-126, is described in US patent application

US 5,185,242.

Compound (38) is described in patent application WO 96/01258.

Compound (39) is described in US patent application US 4,931,581. Compound (40) is described in Proceedings "Brighton Crop Protection Conference - Pests and Diseases" 1996.

Compound (41) is described in European patent application EP 629,616.

Compound (42), also called IKF916, is described in European patent application EP 705,823.

Compound (43) is described in European patent application EP 31,257.

Compounds (44) are described respectively in European patent application EP 219,756 and in

Proceedings "Brighton Crop Protection Conference - Pests and Diseases" 2000.

Compound (45) is described in European patent application EP 360,701 ed EP 611,232.

Compound (46) is described in "The Pesticide Manual", 1983, seventh edition, British Crop

Protection Council Ed., p. 120.

Compound (47) is described in "The Pesticide Manual", 1983, seventh edition, British Crop

Protection Council Ed., p. 339.

Compound (48) is described in "The Pesticide Manual", 1983, seventh edition, British Crop

Protection Council Ed., p. 537.

Compound (49) is described in "The Pesticide Manual", 1983, seventh edition, British Crop

Protection Council Ed., p. 599.

Compound (50) is described in "The Pesticide Manual", 1983, seventh edition, British Crop

Protection Council Ed., p. 252.

Compound (51) is described in "The Pesticide Manual", 1983, seventh edition, British Crop

Protection Council Ed., p. 314.

Compound (52) is described in "The Pesticide Manual", 1983, seventh edition, British Crop

Protection Council Ed., p. 471.

Compounds (53) are described in European patent application EP 753,258.

Compound (54) is described in Proceedings "Brighton Crop Protection Conference - Pests and

Diseases 1998". Compound (55) is described in "The e- Pesticide Manual", 2003, thirteenth edition, British

Crop Protection Council Ed.

Compound (56) is described in patent application WO 200111966.

Compounds (57) e (58) are described in "The Pesticide Manual", 1994, tenth edition, British

Crop Protection Council Ed., p. 530.

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564-572. Castoria R, De Curtis F, Lima G, Caputo L, Pacifico S and De Cicco V, 2001. Aureobasidium pullulans (LS30) an antagonist of postharvest pathogens of fruits: study on its modes of action. Postharvest Biol. Technol. 22: 7-17. Castoria R, Morena V, Caputo L, Panfili G, De Curtis F and De Cicco V, 2005. Effect of the biocontrol yeast Rhodotorula glutinis strain LSI l on patulin accumulation in stored apples. Phytopathology 95: 1271-1278. Castoria R., De Curtis F., Lima G., De Cicco V., 1997. β-l,3-glucanase activity of two saprophytic yeasts and possible mode of action as biocontrol agents against postharvest diseases. Postharvest Biol. Technol. 12: 293-300. Castoria R., L. Mannina, L. Maiuro, A. Sobolev, A. Ritieni, R. Ferracane, A. Spina, 2007.

Nuovi dati sul decremento di patulina causato da un lievito di biocontrollo in mele infettate da Penicillium expansum. Rapporti ISTISAN (Istituto Superiore di Sanita)

07/37 ISSN 1123-3117, www.iss.it/publ/rapp/index.php?anno=2007&lang=l&tipo=5. De Curtis F, Caputo L, Castoria R, Lima G, Stea G and De Cicco V, 2004. Use of Fluorescent

Amplified Fragment Length Polymorphism (fAFLP) for Molecular Characterization of the Biocontrol Agent Aureobasidium pullulans strain LS30. Posth. Biol. Technol. 34:

179-186. FAO/WHO, JECFA, 2001. - Safety evaluation of certain mycotoxins in food, WHO Food

Additives Series 47, FAO Food and Nutrition Paper 74; 56^th meeting of the Joint

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2001, 281-415. Ippolito A and Nigro F, 2000. Impact of preharvest application of biological control agents on postharvest diseases of fresh fruits and vegetables. Crop. Prot. 19:715-723. Janisiewicz WJ and Korsten L, 2002. Biological control of postharvest diseases of fruits.

Annu. Rev. Phytopathol. 40, 411-441. Jørgensen K, 1998. Survey of pork, poultry, coffee, beer and pulses for ochratoxin A. Food

Additives and Contaminants 15: 550-554. Kuiper-Goodman T and Scott PM, 1989. Risk assessment of the mycotoxin ochratoxin A.

Biomedical and Environmental Science 2: 179-248. Lima G, Arru S, De Curtis F and Arras G, 1999. Influence of Antagonist, Host Fruit and

Pathogen on the Biological Control of Postharvest Fungal Diseases by Yeasts. Journal of Industrial Microbiology and Biotechnology, 23: 223-229. Lima G, De Curtis F, Castoria R and De Cicco V, 1998. Activity of the yeasts Cryptococcus laurentii and Rhodotorula glutinis against postharvest rots on different fruits. Biocontr.

Sci. Technol. 8: 257-267. Lima G, De Curtis F, Castoria R, De Cicco V, 2003. Integrated control of apple postharvest pathogens and survival of biocontrol yeasts in semi-commercial conditions. Europ. J.

Plant Pathol. 109(4): 341-349. Lima G, De Curtis F, Piedimonte D Spina AM and De Cicco V, 2006. Integration of biocontrol yeast and thiabendazole protects stored apples from fungicide sensitive and resistant isolates of Botrytis cinerea. Postharvest Biol. Technol. 40: 301-307. Lima G, De Curtis F, Piedimonte D, Spina AM and De Cicco V, 2002. Activity of antagonists and natural compounds against powdery mildew of cucurbits: laboratory and field trials.

J. Plant Pathol. 84: 185. Lima G, De Curtis F, Spina AM and De Cicco V, 2002. Survival and activity of biocontrol yeasts against powdery mildew of cucurbits in the field. In: "Biological Control of

Fungal and Bacterial Plant Pathogens", IOBC wprs Bulletin Vol. 25 (10) 2002 ED.: Y

Elad, et al. pp: 187-190. Lima G, Spina AM, Castoria R, De Curtis F, and De Cicco V, 2005. Integration of Biocontrol

Agents and Food-grade Additives for Enhancing Protection of Apples from Penicillium expansum During Storage. J. Food Protection 68: 2100-2106. Majerus P and Otteneder H 1996. Detection and occurence of ochratoxin A in wine and grape juice. Deutsche Lebensmittel-Rundschau 92: 388-390. Pietri A, Bertuzzi T, Pallaroni L and Piva G, 2001. Occurrence of ochratoxin A in Italian wines. Food Addit. Contain. 18:647-654. Pohland AE, Nesheim S and Friedman L, 1992. Ochratoxin A: a rewiew. Pure and Applied

Chemistry 64: 1029-1046. Stefanaki I, Foufa E, Tsatsou-Dritsa A and Photis Dais, 2003. Ochratoxin A concentrations in

Greek domestic wines and dried vine fruits, Food Addit. Contam. 20: 74-83. Zimmerli B and Dick R, 1996. Ochratoxin A in table wine and grape-juice: occurrence and risk assessment', Food Addit. Contam. 13: 655-668.

The following examples are intended to further illustrate the efficacy of the invention at a laboratory and field scale and are not to limit the scope of the invention as defined by the claims.

EXAMPLE 1. Substantiation of the efficacy of Aureobasidium pullulans strains in the control of Aspergillus carbonarius and ochratoxin levels (OTA) on grapes The application of strains of the yeast-like fungus Aureobasidium pullulans as a biocontrol agent on artificially wounded single grape berries so as to create conditions of maximum vulnerability to attacks from phytopathogenic and mycotoxigenic fungi prior to inoculating these grape berries with the phytopathogenic and mycotoxigenic fungus Aspergillus carbonarius, has not only provided a high level of protection from infection, but ochratoxin A concentration is also much lower, even in those few grape berries that were infected, compared to grape berries that were infected but not protected by the strains of the yeast-like fungus. The considerable decrease recorded may be attributed a) to Ochratoxin A degradation and detoxification, i.e. conversion to Ochratoxin α carried out by the ability of yeast-like strains to degrade ochratoxin A as proven in vitro; b) to the interference of these strains in the biosynthesis of Ochratoxin A carried out by the mycotoxigenic fungus; c) to the reduction of biomass growth of infecting ochratoxigenic A. carbonarius. Results

Figure 1 shows that the biocontrol agents Aureobasidium pullulans AU 14-3-1, AUl 8 -3 B and LS30 provide elevated protection even under assay conditions which are extremely favourable to the mycotoxigenic pathogen, corroborated by the fact that the single grape berries had not only been detached from the bunch, but also wounded, kept at humidity and temperature values that are strongly favourable to A. carbonarius

Figure 1 shows the biocontrol activity of strains of Aureobasidium pullulans AUl 4-3-1, AU18-3B and LS30 on wine grape berries (cv Montepulciano) inoculated with Aspergillus carbonarius strain Al 102. Biocontrol activity is expressed as percentages of infected wounds recorded at 3 (A), 4 (B), and 6 (C) days of incubation. The control consists in grape berries which were not pretreated with Aureobasidium pullulans strains before being inoculated with the mycotoxigenic fungal pathogen. The data have been collected from three different experiments. The percentages have been transformed into Bliss angular values before the statistical analysis. Values with the same letter are not significantly different (P=COl) according to Duncan's test.

Figure 2 shows that ochratoxin A contamination was significantly reduced by the treatment using biocontrol agents Aureobasidium pullulans AU14-3-1, AU18-3B and LS30, despite the fact that test conditions were extremely favourable for the pathogens.

Figure 2. Concentrations of ochratoxin A (OTA, white columns) and of ochratoxin α (OTa, black columns) in berries inoculated with mycotoxigenic fungal pathogen Aspergillus carbonarius strain Al 102 and pretreated with strains of Aureobasidium pullulans AUl 4-3-1, AU18-3B and LS30. Each column represents the mean from three different experiments. The values having common letters are not significantly different for P=O.01 according to Duncan's test. The letters within and above the columns refer to OTA and OTa, respectively. Table 1 shows that the ratio between concentrations of Ochratoxin α and those of Ochratoxin A in treatments using biocontrol agents is higher, specifically twice as much for AU18-3B and more than twice as much for AU14-3-1. This stems from the fact that less toxic ochratoxin α is constant in the various treatments, even when ochratoxin A is much less than the control (see Fig.2). This result indicates that these biocontrol agents interfere with the biosynthesis of ochratoxin A by the mycotoxigenic fungus and/or that they degrade (detoxify) ochratoxin A into ochratoxin α.

Table 1. Ratio between concentrations of Ochratoxin α (OTa) and Ochratoxin A (OTA) recorded after 6 days of incubation of wine grape berries infected with Aspergillus carbonarius, pre-treated with the biocontrol agents Aureobasidium pullulans AU14-3-1, AU18-3B and LS30.

*The ratio refers to concentrations expressed as nmol/g of infected grape berries.

Figure 3 shows that the biocontrol agents Aureobasidium pullulans AU14-3-1, AU18-3B and LS30 can grow in the presence of ochratoxin A; Figure 2 shows this is possible because they resist ochratoxin A, despite the elevated concentration to which they are exposed. Figure 3. In vitro growth of Aureobasidium pullulans AU14-3-1 (A), AU18-3B (B) and LS30 (C) strains for 6 days at 23⁰C in Lilly-Barnett culture soil in the presence (black squares) and in absence of (white squares) ochratoxin A 2.5 μM. The data was collected from three different experiments. The values are expressed as CFU/ml and are mean values ± standard deviation from the mean value (n=6).

The exemplary chromatograms of HPLC analyses (Fig.4) of AU 14-3-1 show that biocontrol agents Aureobasidium pullulans AU14-3-1, AU18-3B and LS30 determine in vitro a. noticeable decrease in ochratoxin A and the progressive formation of the less toxic ochratoxin α.

Figure 4 shows the exemplary chromatograms of HPLC of the culture filtrates of Aureobasidium pullulans AU14-3-1 incubated for 6 days at 23⁰C in Lilly-Barnett culture medium in the presence of ochratoxin A 2.5 μM (OTA). Peaks of OTA and ochratoxin α (OTa) are shown at 0 (A), 4 (B) and 6 (C) days from the beginning of the experiments. The experiments were carried out three times.

Figure 5 shows that biocontrol agents Aureobasidium pullulans AU14-3-1, AUl 8 -3 B and LS30 completely degrade ' ochratoxin A, converting it into, ochratoxin α, THUS DETOXIFYING IT, as ochratoxin α is much less toxic. In addition, the degradation activity is complete, though the initial elevated concentrations of ochratoxin A were equal to 2.5 μM, corresponding to 1 μg/ml.

Figure 5. Time-course of in vitro concentrations (μmol) of ochratoxin A (OTA, white squares) and ochratoxin α (OTa, black squares) analysed during 6 days of incubation in Lilly- Barnett culture medium in the absence (A) and in the presence of Aureobasidium pullulans AU14-3-1 (B), AU18-3B (C), and LS30 (D). The initial OTA concentration was 2.5 μM. Data were collected from two different experiments. Values are the means ± standard deviation from the mean (n=6). Conclusions

Biocontrol agents Aureobasidium pullulans AU14-3-1, AU18-3B and LS30 provide a high level of protection even under test conditions that are extremely favourable to the mycotoxigenic pathogen Aspergillus carbonarius. This is substantiated by the fact that not only had the grape berries been detached from the bunch, but they were also wounded and kept at humidity and temperature values that are strongly favourable to A. carbonarius. Ochratoxin A contamination was significantly reduced by the treatment with the biocontrol agents, despite the fact that test conditions were extremely favourable to the pathogen. The ratio ochratoxin α / ochratoxin A in the treatments with biocontrol agents is much higher because the less toxic ochratoxin α is constant in the various treatments, even when ochratoxin A is much less than the control, as in the case of treatments with the biocontrol agents. This result indicates that these biocontrol agents interfere with the biosynthesis of ochratoxin A by the mycotoxigenic fungus and/or that they degrade (detoxify) ochratoxin A into ochratoxin α.

Ochratoxin A is not toxic for the biocontrol agents Aureobasidium pullulans AU14-3-1, AU18-3B and LS30, despite the high initial mycotoxin concentration to which these agents were subjected. They are able to degrade ochratoxin A to less toxic ochratoxin α. Indeed, ochratoxin A was successfully degraded and DETOXIFIED and converted to ochratoxin α by the biocontrol agents Aureobasidium pullulans AU14-3-1, AU18-3B and LS30, despite the initial high concentration to which these agents were subjected. Experimental procedure

Biocontrol of ochratoxigenic Aspergillus carbonarius by A. pullulans strains on wine grapes

Prior to experiments, mature wine grape berries were detached from wine grape bunches, sterilized for 2 minutes in a 1% sodium hypochlorite solution (pH 11.5), washed twice with sterile water and dried on absorbent paper. Berries were then wounded in three equidistant points on the equatorial plan and treated by dipping, alternatively, in suspensions of the four A. pullulans strains LS30, AU14-3-1, AU18-3B (10⁸ cell/ml) for 5 minutes at 80 rpm. Berries were then sprayed with a suspension of A. carbonarius Al 102 conidia (5 x 10⁴ CFU/ml) and incubated on previously ethanol-disinfected grates placed in previously ethanol-disinfected sealed boxes (containing 300 ml sterile water) at 24±1°C and 100% R.H.. Percentages of infected wounds were recorded at 3, 4 and 6 days of incubation. Each treatment consisted of 5 replicates of 10 berries each. Experiments were performed three times. Percentage values were converted into Bliss angular values before statistical analysis. Determination of ochratoxin A and ochratoxin α in wine grape berries The same grape berries used in biocontrol experiments were sampled for comparing the accumulation of OTA and OTa when infection by A. carbonarius occurred in the presence and in the absence of the biocontrol agents. At the end of the biocontrol experiments, infected berries from the different treatments were homogenized with an Ultra-Turrax (IKA-Werke GmbH & Co. KG, Staufen, Germany) at 14,000 rpm. Five gram samples were added to 10 ml of a water solution with 1% (wt/vol) polyethylene glycol and 5% (wt/vol) sodium hydrogen carbonate, mixed vigorously for 3 minutes and then centrifuged at 4000 rpm for 10 minutes. The supernatants were filtered through Whatman GF/A glass microfibre filters. Three milliliters of these extracts were loaded on C₁₈ Sep-Pak RC 500 mg cartridges (Waters, Milford, MA) previously conditioned with methanol (4 ml), water (4 ml) and a 5% sodium hydrogen carbonate solution (2 ml). C₁₈ cartridges were washed with 2 ml of 0.1 M phosphoric acid and 2 ml of water. OTa was eluted with 4 ml of ethyl acetate:methanol:acetic acid (95:5:0.5 vol/vol/vol) whereas OTA was eluted with 2 ml of acetonitrile: acetic acid (98:2 vol/vol) in a separate vial. The purified extract containing OTa was dried under a nitrogen stream, re-dissolved with the purified extract containing OTA and further diluted with 1 ml of distilled water. Aliquots of 50 μl of the purified extracts were analyzed by HPLC with fluorometric detection. The HPLC apparatus was an Agilent 1100 series equipped with a G 1312A binary pump, G 1313A autosampler, G1316A column thermostat set at 30°C, G 132 IA spectrofluorometric detector set at 333 ran (λ_ex) and 460 nm (λ_em) and Agilent Chemstation G 2170AA Windows 2000 operating system (Agilent, Waldbronn, Germany). The separations were performed with a Xterra C₁₈ column (150x4.6 mm - 5 μm) preceded by a guard column with the same packing material (Waters, Milford MA). The mobile phase was an isocratic mixture of acetonitrile:water:acetic acid (99:99:2 vol/vol/vol) eluted at a flow rate of 1.0 ml/min. Mixed standard solutions of commercial OTa (produced by hydrolysis of OTA with carboxipeptidase A -EC 3.4.17.1, Sigma- Aldrich) and commercial OTA were injected in the mobile phase, and peak areas were determined to generate calibration curves for quantitative analyses.

Degradation and detoxification of ochratoxin A to ochratoxin α by strains of of the yeast-like fungus Λureobasidium pidlulans.

Each of the three strains AU14-3-1, AU18-3B, LS30 of Aureobasidium pullulans was inoculated (1 x 10⁵ CFU/ml) in 50 ml of Nutrient Yeast Dextrose Broth medium (NYDB, Nutrient Broth 8 g/1, Yeast extract 5 g/1 and Dextrose 10 g/1,) (Oxoid Ltd, Basingstoke, Hampshire, UK) and grown overnight at 23°C. Cells of each strain were then transferred to three flasks each containing 50 ml of Lilly Barnett medium amended with 40 μl of OTA 1 mg/ml in acetonitrile:water:acetic acid (99:99:2 vol/vol/vol) (final OTA concentration 1.0 μg/ml, corresponding to 2.5 μM), and grown for 6 days at 23°C on a rotary shaker at 160 rpm. Controls consisted of non-inoculated Lilly-Barnett medium, amended with OTA, and of the A. pullulans strains grown in the same medium, amended with 40 μl acetonitrile:water:acetic acid (99:99:2 vol/vol/vol) only. On day 1, 2, 3, 4, 5 and 6 of incubation, the quantities of OTA and OTa were also analyzed by HPLC (see above). The experiment was performed three times and each experiment consisted of three replicates.

Example 2: Use of compositions based on strains of Aureobasidium pidlulans with either adjuvants or fungicides at low dosages to prevent/reduce Botrytis rot infections in wine as well as table grapes

Compatibility tests among strains of the yeast-like fungus Aureobasidium pullulans with various types of adjuvants and/or reduced dosages of synthetic agrochemicals have shown that these substances do not affect the in vitro growth of the microorganisms (e.g. Fig. 6). Experiments carried out on artificial wounds of table grape berries as well as on artificial wounds of other stored fruit pre-treated with preparations of strains of Aureobasidium pullulans and subsequently inoculated with Botrytis cinerea, have shown that the effectiveness of the biocontrol agents (BCAs) is often unexpectedly increased by the addition of adjuvants and/or reduced dosages of various agrochemicals (examples- Figures 7-8). Over a two year period, field trials on wine grapes have demonstrated that the combined use of the biocontrol strains and adjuvants and/or reduced dosages of agrochemicals surprisingly enhances the effectiveness of the BCAs. In particular, treatments with the BCAs as well as adjuvants and/or reduced dosages of agrochemicals, at crucial phenological stages (pre-bunch closure, veraison and 20 days before harvesting) have further confirmed the high rate of survival of the Aureobasidium pullulans strains, and also unexpected levels of effectiveness against Botrytis rot and, as a consequence, against secondary bunch rot agents (Aspergillus spp., Penicillium spp., etc.), which were comparable with those obtained with synthetic antibotrytic fungicides at full dosage (Examples - Figures 9-10).

Hence, preparations based on strains of Aureobasidium pullulans appear to be particularly suitable for use in preventing/reducing the incidence of preharvest and postharvest Botrytis rot. In field trials, treatments based on strains of Aureobasidium pullulans are easily applied and incorporated into the most common control strategies for both grapevine plants and bunches. In addition, the use of preparations based on strains of Aureobasidium pullulans, which is a saprophytic microorganism normally present in vineyards, guarantees high effectiveness and workers' and environmental safety. Further, as demonstrated in laboratory tests, preparations based on strains of Aureobasidium pullulans appear to result in control of both fungicide-sensitive and fungicide-resistant isolates of Botrytis cinerea that are usually widespread in vineyards.

Figure 6 shows the in vitro compatibility, on Basal Yeast Agar (BYA), of Aureobasidium pullulans strain LS30 and the adjuvant Glucopon (pH 7) at various concentrations. Figure 7 shows the progression of infections by Botrytis cinerea on table grape (cv. Italia) berries treated with Aureobasidium pullulans strain LS30 (A), Aureobasidium pullulans strain Au34-2 (B) singly or combined with a fungicide (Procilex: a.i. procymidone) at a low dosages (LD= 15 g/hl) and at full dosage (FD= 150 g/hl). (LS3O_C1 and Au34-2_C1 = 10⁶ Colony- forming units (CFU)/ml; LS30_C2 and Au34-2_C2= 10⁸ CFU /ml).

Figure 8 shows the progression of infections by Botrytis cinerea on artificial wounds of table grape (cv. Italia) berries, treated with. Aureobasidium pullulans strain LS30 alone or combined with either calcium propionate (Ca-Pr) (A) or calcium propionate+soybean oil (SO, 0.25%) (B). LS3O_C1= 10⁶ Colony-forming units (CFU)AnI; LS30_C2= 10⁸ CFU/ml). Figure 9 shows the antibotrytic activity (A) in field trials (year 2005) (McKinney's Index) and the survival (B) of Aureobasidium pullulans (strains LS30 and Au34-2) on wine grape berries, either when the A. pullulans strains were applied alone or combined with an adjuvant (Ad= calcium propionate 0.5%+soybean oil 0.5%), in comparison with the activity of Metscknikowia pulcherrima strain LS 16 and with a commercial fungicide (SWITCH j. Figure 10 shows the antibotrytic activity on wine grape berries in field trials (year 2006) (A and B) of Aureobasidium pullulans strain LS30, and the survival of the antagonist (C) on the berries.

The following diagram illustrates how the antagonist was applied in the frame of pest control strategies in the vineyard:

Treatment Control strategy Pre-bunch Veraison 20 days before closure harvesting

A Biological 1 BCA BCA BCA

B Biological 2 Copper + Sulphur BCA BCA

Integrated-alternate Fenhexamid FD BCA BCA (Fenhexamid used at full dosage: FD)

D Integrated-combined Fenhexamid LD Fenexamide LD + Fenexamide LD +

(Fenhexamid used at low + BCA BCA BCA dosage: LD)

E Test_l : synthetic Fenhexamid FD Fenhexamid FD untreated Anti-Botrytis fungicide

F Test_2: synthetic Pyrimetanil Cyprodinil+ untreated Anti-Botrytis fungicides Fliiidioxonil

G Test_3: Farm schedule No Anti-Botrytis No Anti-Botrytis No Anti-Botrytis with: fungicide fungicide fungicide

*BCA: Concentrations of about 10⁷ viable cell/ml combined with Glucopon 0.1% (pH 7) were applied.

Example 3: use of preparations based on strains of BCAs alone or combined with adjuvants or low dosages of fungicides for the prevention/reduction of powdery mildew and septoriosis on cereal crops in field 21.04.2008 31

Preliminary in vitro compatibility tests between yeast-like Aureobasidium pullulans fungus strain LS30, various types of adjuvants and/or low dosages of synthetic agrochemicals have shown that these substances do not negatively affect growth of the BCA. Levels of effectiveness of BCA preparations against powdery mildew and septoriosis following application at the flag leaf and pre-flowering stages were assessed in field trials carried out on durum wheat over a two-year period (2004 and 2005). The results from both growing seasons show that the effectiveness of the BCAs was significantly enhanced by the presence of adjuvants in their preparations and/or low doses of agrochemicals. Moreover, treatments based on Aureobasidium pullulans and low dosages of fungicides were the most successful in reducing these diseases, displaying levels of activity comparable with those achieved by applications of full doses of fungicides. Specific ecological studies have shown the ability of the strains of Aureobasidium pullulans and of the other BCAs to survive at high rates of population on the exposed surfaces of wheat plants, making it possible to reach high levels of effectiveness against powdery mildew and septoriosis. These results were comparable with ones achieved by using full doses of agrochemicals.

Results obtained in year 2005 reported in Figures HA and HB and in Figure 12 further illustrate the invention. Particularly, Figure 11 shows the activity (McKinney's index of infection) against powdery mildew (A) and against septoriosis (B) on durum wheat plants treated in the field with Rhodotorula glutinis (LSI l), Cryptococcus laurentii (LS28) or Aureobasidium pullulans (LS30) combined with either low dosages of fungicides (LD) or with different additives: calcium chloride (CaCl₂), calcium propionate (Ca-Prop.), humic acids (Hum.Ac), soybean oil (SO). Treatments with water alone and with full doses of fungicides (FD) were used as the controls. Figure 12 shows the population dynamics of strain LS30, expressed in cfu/cm², of leaf surface of durum wheat subjected to the different treatments reported in Figures IA and IB. Time is reported on x axys, whereas the two arrows indicate the time when treatments were applied.

Claims

(1) Compositions based on microorganisms able to control the onset and/or the development of infections caused by phytopathogenic microorganisms and/or mycotoxigens and to reduce mycotoxin levels present on the edible portions so as to achieve the concurrent reduction of the presence of both mycotoxigenic microorganisms and mycotoxins, even those already present at the time of application characterised by the fact that the microorganisms are compositions based on yeasts and/or yeast-like fungi and on specific adjuvants.

(2) Compositions based on microorganisms according to Claim 1 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Aureobasidium.

(3) Compositions based on microorganisms according to Claim 2 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Aureobasidium pullulans.

(4) Compositions based on microorganisms according to Claim 3 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Aureobasidium pullulans codified AU14-3-1.

(5) Compositions based on microorganisms according to Claim 3 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Aureobasidium pullulans codified AU18-3B.

(6) Compositions based on microorganisms according to Claim 3 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Aureobasidium pullulans codified AU34-2.

(7) Compositions based on microorganisms according to Claim 3 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Aureobasidium pullulans codified LS30 (CBS 110902).

(8) Compositions based on microorganisms according to Claim 1 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Rhodotorula.

(9) Compositions based on microorganisms according to Claim 8 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Rhodotorula glutinis.

(10) Compositions based on microorganisms according to Claim 1 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Cryptococcus.

(11) Compositions based on microorganisms according to Claim 10 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Cryptococcus laurentii.

(12) Compositions based on microorganisms according to Claim 1 characterised by the fact that at least two of the yeasts and yeast-like fungi used are strains of Aureobasidium pullulans.

(13) Compositions based on microorganisms according to Claim 12 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Aureobasidium pullulans and another is a strain of Rhodotorula glutinis.

(14) Compositions based on microorganisms according to one of the Claims 3, 4, 5, 6, 7 or 11 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Aureobasidium pullulans and another is a strain of Cryptococcus laurentii.

(15) Compositions based on microorganisms according to either Claims 8 or Claim 9 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Cryptococcus and another is a strain of Rhodotorula.

(16) Compositions based on microorganisms according to either Claim 9 or Claim 11 characterised by the fact that at least one of the yeasts and yeast-like fungi used is a strain of Cryptococcus laurentii and another is a strain of Rhodotorula glutinis.

(17) Compositions based on microorganisms according to any of the preceding Claims characterised by the fact that the adjuvants are synthetic or naturally derived active ingredients, either endowed with or free from biocide activity, and display, however, compatibility with the above-mentioned yeasts and/or yeast-like fungi, used in compositions or alternately, causing not only an unexpected synergetic activity in controlling phytopathogens as well as in reducing the mycotoxins present on a crop, but also in controlling/reducing mycotoxin levels present on the edible portions of the plant, even when they preceded these applications.

(18) Compositions based on microorganisms according to Claim 17 characterised by the fact that said synergic activity is measurable by using Abbott's and Colby's formulas, regarding the control phytopathogens and/or the reduction of mycotoxigens and/or mycotoxins present on the edible portions.

(19) Compositions based on microorganisms according to Claim 18 characterised by the fact that said active ingredients are one or more products selected from the following list: alginic acid, corn starch, calcium acetate, calcium ascorbate, calcium chloride, calcium citrate, calcium propionate, calcium silicate, guar gum, locust-bean gum, xanthan gum, soybean oil, light mineral oils, dibasic potassium phosphate, fenhexamid, procymidon, tetraconazole, thiabendazol, trifloxystrobin, ziram granuflo fungicide, sulphur, IR5885, IR6141, tetraconazole, salicylic acid (SA), acetylsalicylic acid (ASA), copper salts of salicylic acid (SA2Cu) oppure (SACu) or of acetylsalicylic acid (ASA2Cu), a copper salt (i) or else copper salt (ii), such as copper oxychloride, copper hydroxide, the bordeaux mixture, copper sulphate, or else a mixture of hydroxide and copper oxychloride (airone), benalaxyl, kyralaxyl, metalaxyl, metalaxyl-M, mandipropamid, iprovalicarb, benthiavalicarb-isopropyl, cymoxanil, metominofen, pyraclostrobin, acibenzolar-s- methyl, famoxadone, fenamidone, cyazofamide, fluazinam, dimethomorph, fiumorph, o flumetover, chlorothalonil, mancozeb, tolylfluanid, folpet, etridiazole, hymexanol, propamocarb, zoxamide, ethaboxam, fluopicolide, fosetyl, fosetyl-al, metominostrobin, iprodione, procymidone, cyprodinil, pyrimethanil, epoxyconazole, propiconazole, tebuconazole, kresoxim-methyl, picoxystrobin, pyraclostrobin, fluoxastrobin, metominostrobin, orysastrobin, dimoxystrobin, enestroburin.

(20) Method to control the onset and/or the development of infections due to phytopathogens and/or mycotoxigenic fungi, and the mycotoxin levels present in the edible portions of the plant in order to reduce concurrently the presence of mycotoxigenic microorganisms and these selfsame mycotoxins, even when already present at the time of application which uses compositions according to any one of the Claims previously characterised by the fact that said compositions are applied preharvest and the beneficial effects extend then to the crop itself.

(21) Method according to Claim 20 characterised by the fact that it consists in one or more applications of compositions of said yeasts and/or yeast-like fungi on the exposed portions of crops, by means of said compositions appropriately formulated and used according to Claim 11, as powders or aqueous solutions, appropriately diluted, in turn, in quantities of water so as to obtain solutions/dispersants which are partially or totally insoluble in water.

(22) Method according to Claim 21 characterised by the fact that the formulate contain one or more yeasts and/or yeast-like fungi, which are afterwards applied to the plant, either as a mixture of spores or as mycelium, or else as a mixture of spores and mycelium.

(23) Method according to Claim 22 characterised by the fact that said compositions appropriately formulated are obtained either by drying or the lyophilization of the biomass, which comes from the fermentation of Aureobasidium strains.

(24) Method according to Claim 23 characterised by the fact that said biomass is obtained either by filtration, sedimentation or centrifiigation of the fermentation broth of the aforementioned Aureobasidium strains.

(25) Method according to Claim 20 characterised by the fact that said compositions are applied in the presence of monofunctional disaccharides with C8-C18 aliphatic groups, either straight or branched, which may be mixed with a fungicide compound, thus obtaining synergistic mixtures which enhance effectiveness against phytopathogens and/or microtoxigens.

(26) Method according to Claim 20 characterised by the fact that said compositions are applied in the presence of a monofunctional disaccharide with C8-C18 aliphatic groups, called Glucopon 650, and may be mixed with a fungicide compound, thus obtaining synergistic mixtures which enhance effectiveness against phytopathogens and/or microtoxigens.

(27) Method according to Claim 20 that uses compositions based on compounds which are not only based on yeasts and/or yeast-like fungi, but also on specific adjuvants, which are able to control the onset and/or the development of infections caused by phytopathogenic microorganisms and/or mycotoxigens, characterised by the reduction of mycotoxin levels, particularly mycotoxins other than patulin, which are present in the edible portions, by means of the concurrent reduction in mycotoxigenic microorganisms and in said mycotoxins, even when already present at the time of application.

(28) Method according to Claim 20 characterised by the fact that it is carried out either in field or under greenhouse conditions, consisting in one or more applications on plants or on portions of plants, not only in order to reduce phytopathogens and mycotoxigens, but also to be able to reduce mycotoxin levels, even if present before applications.

(29) Method according to Claim 27 characterised by the fact that it is carried out either in field or under greenhouse conditions, consisting in one or more applications on plants or on portions of plants, not only in order to reduce phytopathogens and mycotoxigens, but also to be able to reduce ochratoxin levels, even if present before applications and that uses compositions based on compounds which are not only based on yeasts and/or yeast- like fungi, but also on specific adjuvants, which are able to control the onset and/or the development of infections caused by phytopathogenic microorganisms and/or mycotoxigens, characterised, in particular, by the reduction of ochratoxin levels which are present on the edible portions, by means of the concurrent reduction in mycotoxigenic microorganisms and the afore-mentioned ochratoxins, even when already present at the time of application.

(30) Use of compositions according to one or more of Claims 1-19 characterised by the reduction of mycotoxin levels, particularly mycotoxins other than patulin, which are present in the edible portions, by means of the concurrent reduction in mycotoxigenic microorganisms and in the afore-mentioned mycotoxins, even when already present at the time of application.

(31) Use of compositions according to one or more of Claims 1-19 characterised, in particular, by the reduction of ochratoxin levels present in the edible portions, by means of the concurrent reduction in mycotoxigenic microorganisms and of said ochratoxins, even when already present at the time of application.

(32) Use of compositions according to Claim 30 characterised by the use of compounds based on yeasts and/or yeast-like fungi to control the onset and/or the development of infections in fruit and vegetable crops such as apples, pears, coffee, soybeans, strawberries, kiwifruit, table as well as wine grapes, and citrus fruit, caused by phytopathogenic fungi and/or mycotoxigens, such as Aspergillus spp., Botrytis cinerea, Rhizopus stolonifer, Penicillium expansum, Penicillium italicum and Penicillium digitatum, and also to reduce levels of the mycotoxin ochratoxin A which is present in the edible portions, even when already present at the time of application.

(33) Use of compositions according to Claim 30 characterised by the use of compounds^' based on yeasts and/or yeast-like fungi to control the onset and/or the development of infections in table as well as in wine grapes caused by the mycotoxigenic fungus Aspergillus carbonarius, producer of ochratoxin A, which also reduces levels of the mycotoxin ochratoxin A which is present in the edible portions, even when already present at the time of application.

(34) Use of compositions according to Claim 30 characterised by the use of compounds based on yeasts and/or yeast-like fungi not only to control the onset and/or the development of diseases that affect cereals and grains, in particular, powdery mildew, septoriosis, helminthosporiosis and rincosporiosis, but also to control mycotoxigenic fungi that afflict said cereals, such as microorganisms belonging to the genera Aspergillus, Fusarium and Penicillium, thus also successfully reducing levels of mycotoxins other than patulin, and in particular, reducing levels of the mycotoxin ochratoxin A, which may be present in the edible portions, and even when they already present at the time of application.

(35) Use of compositions according to Claim 33 characterised by the fact that said cereals are: wheat, spelt, barley, rye and rice.

(36) Use of compositions according to Claim 33 characterised by the fact that the mycotoxigens present in said cereals are: Aspergillus flavus, Aspergillus parasiticus, Aspergillus ochraceus, Fusarium culmorum, Fusarium crookwellense, Fusarium graminearum, Fusarium proliferatum, Fusarium sporotrichioides, Fusarium verticilloides, Penicillium verrucosum.

(37) Use of compositions according to Claim 35, characterised by the use of compounds based on yeasts and/or yeast-like fungi to control the onset and/or the development of diseases that affect corn, such as for example, green mould caused by Penicillium chrysogenum, also reducing levels of mycotoxins other than patulin, specifically levels of ochratoxin A, which may be present in the edible portions, even when already present at the time of application.

(38) Use of compositions according to Claim 36, characterised by the use of compounds based on yeasts and/or yeast-like fungi to control the onset and/or the development of mycotoxigens which affect crops producing grains or fruit in the periods pre- or postharvest, such as mycotoxingens belonging to the genera Aspergillus, Fusarium or Penicillium.