[0001] The invention relates to a yeast strain and a method for fighting fungal infections in fungi of agricultural and commercial interest, in particular edible mushrooms of the Pleurotus ostreatus species, using the aforesaid strain.

[0002] The invention can be therefore applied industrially in the agronomic field of the cultivation of edible mushrooms.

[0003] The Pleurotus ostreatus ( Pleurotus ostreatus (Jacq.) P. Kumm.) basidiomycete, commonly known as the“oyster mushroom”, is an edible mushroom, is amongst the most cultivated in the western world and is susceptible to viral, bacterial and fungal infections (Bellettini M. B., Bellettini S., Menino Destefanis Vitola F., Assumpcao Fiorda F., Maccari A. Jr., Soccol C.R. (2017). Residual compost from the production of Bactris gasipaes Kunth and Pleurotus ostreatus as soil conditioners for Lactuca sativa‘Veronica’ . Semina: Ciencias agrarias, Landrina, 2, 581-594). In recent years, the production of P. ostreatus has been seriously compromised by the spread of a serious pathology, known as green mould and caused by ascomycetes fungi belonging to the genus Trichoderma, namely Trichoderma pleuroti (Park M.S., Seo G.S., Lee K.H., Bae K.S., Yu S.H. (2005). Characterization of Trichoderma spp. associated with green mold of oyster mushroom by PCR-RFLP and sequence analysis of ITS regions of rDNA. Plant. Pathol. J., 21, 229-236; Bellettini M., Florida F. (2016) Production pests and diseases in mushroom Pleurotus spp crops. Apprehendere, Guarapuava, page 152) and Trichoderma pleuroticola (Komoh - Zelazowska M., Bissett J., Zafari D., Hatvani L., Manczinger L., Woo S., Lorito M., Kredics L., Kubicek C.P, Druzhinina I.S. (2007). Genetically closely related but phenotypically divergent Trichoderma species cause world-wide green mould disease in oyster mushroom farms. Appl. Microbiol. Biotechnok, 73, 7415-7426).

[0004] The Prochloraz imidazole fungicide is considered to be one of the most effective synthetic active principles that are able to hamper the development of Trichoderma spp. on P. ostreatus. For example, in Italy, the integrated production procedures established by the Emilia-Romagna Region allow only the use of Prochloraz against green mould in mushroom bed.

[0005] A drawback that is connected with the constant use of the aforesaid fungicide is the possible onset of resistance phenomena in the pathogenic agent.

[0006] Another drawback consists of the fact that Prochloraz is potentially harmful for the operators administering the fungicide to the crops, the consumers of the so treated mushrooms and the environment. In particular, Prochloraz has been deemed to be able to act as an endocrine disrupter (Vinggaard A.M., Hass U., Dalgaard M., Andersen H.R., B o n c fc Id- J ø g e n s e n E., Christiansen S., Laier P., Poulsen M.E. (2006) Prochloraz: an imidazole fungicide with multiple mechanisms of action. Int. J. Androl. 29 (1): 186-92).

[0007] The need is thus felt to overcome the drawbacks disclosed above and, in particular, to make available an environmentally sustainable alternative to the use of synthetic pesticide.

[0008] An object of the invention is to improve the known methods for fighting the fungal diseases that afflict mushrooms of agricultural interest, in particular edible mushrooms.

[0009] Another object is to improve the known methods for hindering the infections by ascomycetes of the Trichoderma spp. genus, in particular Trichoderma pleuroti and/or Trichoderma pleuroticola, in edible basidiomycetes, in particular Pleurotus ostreatus.

[0010] A further object is to make available a method that enables the infections by Trichoderma pleuroti and/or Trichoderma pleuroticola in Pleurotus ostreatus to be fought, which method enables the use of pesticides to be avoided, thus reducing the risk of harmful effects for the operators, the consumers and the environment.

[0011] In a first aspect of the invention, a yeast strain (L8 strain of Aureobasidium pullulans) is provided, as defined in claim 1.

[0012] In a second aspect of the invention, a method as defined in claim 7 is provided.

[0013] Owing to these aspects, a yeast strain (L8 strain of Aureobasidium pullulans ) and a method are made available that enable the drawbacks of the prior art to be overcome. In fact, the yeast strain identified and studied by the Inventors enables the fungal infections to be fought that attack edible mushrooms. More specifically, the L8 strain of Aureobasidium pullulans is able to hamper the spread of infections by ascomycetes of the Trichoderma spp. genus, in particular Trichoderma pleuroti and Trichoderma pleuroticola, in basidiomycetes of the Pleurotus ostreatus species. The strain according to the invention can thus be effectively used to protect the crops (mushroom beds) of Pleurotus ostreatus from the infections of the aforesaid ascomycetes.

[0014] The discovery of the strain according to the invention, as well as the experimental verification of the properties of the aforesaid strain, have enabled a method to be devised by means of which it is possible to fight the infections by Trichoderma spp. in Pleurotus ostreatus without using synthetic pesticide, like Prochloraz, and thus reducing the risk of harmful effects for the operators, the consumers and the environment.

[0015] The use of the strain according to the invention is based on a surprisingly unexpected technical effect, namely the ability of the aforesaid microorganism to inhibit the growth of Trichoderma pleuroti and Trichoderma pleuroticola and, at the same time, to stimulate the growth of Pleurotus ostreatus. The mode by which the aforesaid unexpected technical effect was discovered by the Inventors will be illustrated with greater detail below.

[0016] The invention can be understood and implemented better with the attached drawings that illustrate an embodiment thereof by way of non-limiting example, in which:

[0017] Figure 1 shows a sequence of ribosomal DNA of the strain according to the invention, compared with a corresponding sequence of a reference strain;

[0018] Figure 2 shows a sequence of DNA that is contained exclusively in the strain according to the invention;

[0019] Figure 3 is a schematic drawing illustrating an in vitro antagonism test performed with the strain according to the invention;

[0020] Figure 4 is a photograph showing two Petri dishes used for performing the in vitro antagonism test of Figure 3;

[0021] Figure 5 is a graph illustrating the percentage of inhibition of the mycelial growth of ascomycetes that is induced by the strain according to the invention during the in vitro antagonism tests;

[0022] Figure 6 is a photograph showing the effect of volatile metabolites, which are produced by the strain according to the invention, on the Trichoderma pleuroticola ascomycete;

[0023] Figure 7 is a photograph showing the effect of volatile metabolites, which are produced by the strain according to the invention, on the Pleurotus ostreatus basidiomycete;

[0024] Figure 8 is a graph, illustrating the percentage of inhibition of the mycelial growth of ascomycetes that is induced by volatile metabolites produced by the strain according to the invention;

[0025] Figure 9 is a photograph showing the effect of non-volatile metabolites, which are produced by the strain according to the invention, on the Trichoderma pleuroticola ascomycete; [0026] Figure 10 is a graph, illustrating the percentage of inhibition of the mycelial growth of ascomycetes that is induced by non-volatile metabolites produced by the strain according to the invention;

[0027] Figure 11 is a graph, illustrating the trend of the index of colonization of a growth substrate, by the Pleurotus ostreatus basidiomycete, in different treatment conditions of the substrate;

[0028] Figure 12 is a photograph showing the different effects of the presence or absence of the strain according to the invention in a mushroom bed of Pleurotus ostreatus.

[0029] In the context of the present description and the attached claims, the use of the term“mushroom” in the singular does not exclude that the strain and the method according to the invention are able to provide the desired technical effect (hampering the spread of fungal diseases in edible mushrooms) when they are used for treating a plurality of mushrooms.

[0030] In the context of the present description and the attached claims, the terms in each of the following pairs are considered to be synonyms and are used in reciprocally interchangeable manner: “ Aureobasidium pullulans” and “A. pullulans”, “ Pleurotus ostreatus” and“P. ostreatus”;“ Trichoderma pleuroti” and“7. pleuroti”,“ Trichoderma pleuroticola” and“G. pleuroticola” “volatile metabolites” and“VOCs”;“non-volatile metabolites” and“NVOCs”;“conidia” and“spores” (understood as agamic spores).

[0031] Aureobasidium pullulans (De Bary) Arnaud is commonly known as“black yeast” and many names have been attributed to it over the last 90 years because of the variety of environments in which it can develop. The following Table 1 shows the scientific classification of this yeast:

[0032] Table 1

[0033] Aureobasidium sp. is comprised in the epiphytic microbial flora of fruits and leaves of plants that are typical of temperate regions. Over the last thirty years, the use of Aureobasidium sp. as a biological control agent against fungal pathogens of the fruits, in particular in the post-harvest phase, gave rise to a certain interest. In fact, among the numerous microorganisms that are potentially effective in reducing fruit rot, yeasts or yeast-like fungi are particularly interesting for various reasons. First of all, the biological activity of these microorganisms is not based on the production of antibiotic substances, as on the other hand occurs for many bacteria. Secondarily, these microorganisms do not produce negative impacts from an environmental and toxicological point of view. Lastly, these microorganisms are easily cultivatable on a large scale, through the use of inexpensive and easily obtainable substrates.

[0034] Among the antagonistic microorganisms, A. pullulans has shown optimal antagonistic abilities against fungal pathogens linked to pre- and post-harvest (Zhang D., Spadaro D., Garibaldi A., Gullino M.L., 2010. Potential biocontrol activity of a strain of Pichia guilliermondii against grey mold of apples and its possible modes of action. Biol Control, 57, 193-201; Mari et al. 2012, cited previously; Di Francesco A., Roberti R., Martini M., Baraldi E., Mari M. (2015) Activities of Aureobasidium pullulans cell filtrates against Monilinia laxa of peaches. Microbiol. Research., 181, 61-67, Di Francesco A., Ugolini L., D’Aquino S., Pagnotta E., Mari M. (2017). Biocontrol of Monilinia laxa by Aureobasidium pullulans strains: insights on competition for nutrients and space. Internat. J Food Microbiol., 248, 32-38).

[0035] The strain according to the invention - namely the L8 strain of A. pullulans - was isolated from the surface of cv.“Redhaven” peaches cultivated and harvested in an organic peach orchard of the University of Bologna located in Cadriano (Mari M., Martini C., Guidarelli M., Neri F., 2012. Postharvest biocontrol of Monilinia laxa, Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains. Biol Control, 60, 132-140). The strain was selected from several others because of its marked aptitude to hamper the development of post-harvest pathogenic fungi on fruit.

[0036] The L8 strain was deposited on 16 May 2018 with the DBVPG collection (Industrial Yeasts Collection, Borgo XX Giugno, 74, 06121 Perugia, Italia) with number 41P. The L8 strain is grown in the laboratory on an NYDA agarized substrate and the optimum growth temperature is 25°C. The NYDA agarized substrate is produced by dissolving in 1000 ml of distilled water the following ingredients: 8 grams of Nutrient Broth, 5 grams of Yeast Extract, 10 grams of dextrose and 15 grams of agar.

[0037] The L8 strain was identified through observations under the optical microscope and molecular techniques with the sequencing of the non-coding region of ribosomal DNA (ITS = Internal Transcribed Spacer) and by creating specific SCAR (Sequence Characterized Amplified Region) primers (Di Francesco A., Calassanzio M., Mari M., Ratti C., Folchi A., Baraldi E. (2018). Molecular characterization of the two postharvest biological control agents Aureobasidium pullulans LI and L8. Biol. Control, 123, 53-59).

[0038] It has been pointed out that the results of the observations under the microscope of the yeast cells are comparable with the observations reported in the morphological recognition keys (Barnett J.A., Payne R.W. (2000) Yarrow D. Yeasts: characteristics and identification. Cambridge University Press, UK pp. 1139) of the species A. pullulans.

[0039] The sequencing results have confirmed the identification on a morphological basis. In fact, the F8 strain is 100% homologous to a strain of A. pullulans deposited in Gene bank (CBS 584.75T, accession number FJ150906). Figure 1 shows the 100% homology verifiable between the ITS sequence of the F8 strain of A. pullulans and a corresponding ITS sequence of the CBS 584.75T strain of A. pullulans. The ITS sequence of the F8 strain of A. pullulans has also been disclosed in a Sequence Fisting (complying with the Standard ST.25 WIPO), which is attached to the present patent application and in which the aforesaid ITS sequence is named SEQ ID NO: 1.

[0040] Through the use of specific primers (SCAR markers) it was possible to distinguish the F8 strain from a population of A. pullulans of national and international origin. The aforesaid primers in fact amplify a fragment of l37bp (base pairs) contained exclusively in the F8 strain and shown in Figure 2 (in which the fragment corresponds to the polynucleotide sequence portion comprised between the two arrows). This l37bp fragment has been further disclosed in the aforesaid Sequence Fisting, in which the fragment is named SEQ ID NO: 2.

[0041] Moreover, through the use of the Droplet digital PCR it was possible to verify how many yeast cells correspond to a DNA nanogram on the basis of a single gene of this yeast. As the aforesaid primers amplify with at least 5ng of DNA of this yeast, the corresponding detected number of cells is 215.

[0042] The antimicrobial activity exhibited by the F8 strain of A. pullulans against the main pathogenic agents that are responsible for green mould in P. ostreatus resulted to be extremely interesting. Both in vitro and in vivo tests were performed, through which it was possible to demonstrate the efficacy of the L8 strain in hampering the mycelial growth of T. pleuroti and T. pleuroticola without interfering with the growth of the target fungus P. ostreatus. In particular, it was observed that the L8 strain is not only able to inhibit the growth of T. pleuroti and T. pleuroticola , but is also able to stimulate the growth of P. ostreatus.

[0043] Although the action mechanism has not yet been fully explained by which the L8 strain is able to play a role as an antagonist microorganism, as well as to stimulate the growth of P. ostreatus, in the light of the experimental evidence gathered by the Inventors it appears to be scientifically correct to attribute (at least partially) this effect to active (volatile and non-volatile) metabolites produced by the strain according to the invention.

[0044] In particular, it is hypothesized that the L8 strain is able to hamper, and thus contain, the development of the mycelium of Trichoderma spp. by competition for the substrate and creating an environment that is less favourable to the growth of Trichoderma spp. owing to the production of the aforesaid metabolites, which would be able to cause an increase in the substrate pH. Nevertheless, the metabolites produced by the L8 strain and the action mechanism of the latter are still the object of study by the Inventors.

[0045] By way of non-limiting example of the invention, the following experimental tests are disclosed below: in vitro antagonism tests (Example 1); in vivo antagonism tests (Example 2); use of the L8 strain in mushroom bed (Example 3).

[0046] In the following examples, the species T. pleuroti and T. pleuroticola were also called collectively“ Trichoderma spp.”. In the in vitro tests, two different strains of T. pleuroti (named as“strain 1” and“strain 2”) and two different strains of T. pleuroticola (named“strain 1” and“strain 2”) were used.

[0047] Example 1 - In vitro antagonism tests

[0048] The efficacy of the L8 strain against Trichoderma spp. was tested in vitro by evaluating: the interaction between Trichoderma spp. and the L8 strain in dual growth; the activity of volatile metabolites (VOCs) produced by the L8 strain against Trichoderma spp.; the activity of non-volatile metabolites (NVOCs) produced by the L8 strain against Trichoderma spp.

[0049] The activity of the aforesaid volatile and non-volatile metabolites was evaluated, besides against Trichoderma spp., also against P. ostreatus.

[0050] 1.1) Interaction between Trichoderma spp. and L8 strain in dual growth [0051] The dual interaction L8 vs. Trichoderma spp. (strain 1 and strain 2 of T. pleuroti, strain 1 and strain 2 of T. pleuroticold) was evaluated through growth on PDA (Potato Dextrose Agar) solid substrate. Mycelial discs of Trichoderma spp. (0 6 mm) were removed from a colony in active growth (7 days) and were inoculated in a Petri dish (0 90 mm) on PDA at a distance of about 30 mm from the edge of the dish. The L8 strain was inoculated using the smear technique at a distance of 30 mm from the edge of the Petri dish. After incubation of the dishes at 25°C for 7 days, it was proceeded to measure the diameter of the colony of Trichoderma spp. along a line passing from the centre of the mycelial disc to the colony of the L8 strain. The reciprocal position (on the Petri dish) of the inoculation zones of Trichoderma spp. and of the L8 strain is shown schematically in Figure 3. The control consisted of colonies of Trichoderma spp. grown in the absence of the L8 strain on the same substrate. Five repetitions were performed for each thesis.

[0052] In the photograph of Figure 4, two Petri dishes are shown, in which the dual growth test disclosed above was performed by using the L8 strain and Trichoderma pleuroticola. The two Petri dishes highlight the ability of the L8 strain to hamper the mycelial development of Trichoderma pleuroticola : on the left, the Petri dish of the control (devoid of the L8 strain) is shown and on the right the Petri dish is shown on which both Trichoderma pleuroticola and the L8 strain have been inoculated. By comparing the two Petri dishes, it is possible to note that in the dish on the right the growth of the colony (mycelial growth) of Trichoderma pleuroticola was clearly inhibited near the adjacent (linear) growth of the L8 strain.

[0053] Figure 5 is a graph illustrating the percentage of inhibition of the mycelial growth (calculated according to a method that is known to the persons skilled in the art) of T pleuroti (strains 1 and 2) and of T pleuroticola (strains 1 and 2) in dual growth. The graph reports (from left to right) the % values of growth inhibition calculated for: strain 1 of T. pleuroti, strain 2 of T. pleuroti, strain 1 of T. pleuroticola and strain 2 of T. pleuroticola.

[0054] As shown in the graph of Figure 5, the L8 strain reduced the growth of the colonies of T pleuroti and T. pleuroticola by 20% (strain 1 of T pleuroti), by 30 - 40% (strain 2 of T pleuroti ; strain 2 of T. pleuroticola ) and by 45% (strain 1 of T. pleuroticola ) with respect to the control (Petri dish devoid of the L8 strain).

[0055] 1.2) Activity of volatile metabolites produced by the L8 strain against Trichoderma spp. [0056] The activity of the volatile metabolites produced by the L8 strain towards the growth of Trichoderma spp. and P. ostreatus was tested according to the method disclosed by Di Francesco et al (Di Francesco A., Ugolini, L., Lazzeri, L., Mari, M. (2015). Production of volatile organic compounds by Aureobasidium pullulans as a potential mechanism action against postharvest fruit pathogens. Biol. Control, 81, 8-14). Petri dishes containing NYDA (Nutrient Yeast Dextrose Agar) agarized substrate were inoculated with 100 pl of a cell suspension of the L8 strain (10 cells ml ) and incubated for 48h at 25°C. Petri dishes containing PDA substrate were inoculated separately with mycelial discs of Trichoderma spp. (strain 1 and strain 2 of T. pleuroti strain 1 and strain 2 of T. pleuroticola ) and of P. ostreatus (strain POK12; strain POSP). Controls were also prepared, namely Petri dishes inoculated separately with mycelial discs of Trichoderma spp. and of P. ostreatus and not superimposed on corresponding Petri dishes inoculated with the L8 strain.

[0057] The Petri dishes inoculated with the mycelial discs were superimposed on the Petri dishes inoculated with the L8 strain. The reciprocally superimposed dishes were sealed with Parafilm ^® tape and incubated for 5 days at 25 °C together with the controls. In this manner, the growth of Trichoderma spp. and of P. ostreatus was subjected to the action of possible volatile metabolites produced by the L8 strain. The pH of the agarized substrate containing Trichoderma spp. and P. ostreatus was measured with a pH microelectrode (5033 pH electrode, Crison, Spain) before and after the incubation period with the agarized substrates containing the L8 strain.

[0058] In the photograph of Figure 6, as an example, the effect of the volatile metabolites on the mycelial growth of Trichoderma pleuroticola is shown: on the left, a Petri dish of the control (incubation and growth in the absence of the volatile metabolites produced by the L8 strain) is shown and on the right a Petri dish is shown that was incubated in the presence of the volatile metabolites produced by the L8 strain. By comparing the two Petri dishes it is possible to note that, in the dish on the right, the growth of the colony (mycelial growth) of Trichoderma pleuroticola has been significantly hampered by the volatile metabolites produced by the L8 strain.

[0059] Figure 8 is a graph illustrating the percentage of inhibition of the mycelial growth (calculated according to a method that is known to the persons skilled in the art) of T. pleuroti (strains 1 and 2) and of T. pleuroticola (strains 1 and 2) with respect to the control, as a result of the volatile metabolites produced by the L8 strain, in the test disclosed above. The graph reports (from left to right) the % values of growth inhibition calculated for: strain 1 of T. pleuroti, strain 2 of T. pleuroti, strain 1 of T. pleuroticola and strain 2 of T. pleuroticola. The volatile metabolites produced by the L8 strain are able to reduce by about 40% on average the mycelial growth in the tested strains of T. pleuroti and T. pleuroticola.

[0060] In the photograph of Figure 7 the effect of the volatile metabolites on the mycelial growth of P. ostreatus is shown. From left to right there are shown: POK12 strain in the absence of the volatile metabolites produced by the L8 strain; POK12 strain in the presence of the volatile metabolites produced by the L8 strain; POSP strain in the absence of the volatile metabolites produced by the L8 strain; POSP strain in the presence of the volatile metabolites produced by the L8 strain. It is possible to note a greater mycelial growth in the Petri dishes incubated under the effect of the volatile metabolites produced by the L8 strain.

[0061] In fact, an increase in biomass (average biomass increase of 4.2% and 3.8%) was detected in the dishes that were incubated with the two strains of P. ostreatus and subjected to the action of the volatile metabolites produced by the L8 strain. The following Table 2 reports the biomass of P. ostreatus after 5 days of incubation in the presence of the volatile metabolites produced by the L8 strain:

[0062] Table 2

Moreover, in the growth substrate of P. ostreatus an increase in the pH value of about 1.2 times was detected, as a result of the volatile metabolites produced by the L8 strain.

The following Table 3 reports the pH values of the substrate after colonization by the mycelium of P. ostreatus in the presence of the volatile metabolites produced by the L8 strain: Table 3

[0063] 1.3) Activity of non-volatile metabolites produced by the L8 strain against Trichoderma spp. [0064] The activity of the non-volatile metabolites produced by the L8 strain against Trichoderma spp. (strain 1 and strain 2 of T. pleuroti strain 1 and strain 2 of T. pleuroticold) was tested with the techniques described by Lundberg and Unestan (Lundberg A, Unestan T. (1980). Antagonism against Fomesannosus. Comparison between different test methods “in vitro” and“in vivo”. Mycopathol.70: 107-115) and Dennis and Webster (Dennis C., Webster J. (1971). Antagonistic properties of species groups of Trichoderma I. Production of non-volatile antibiotics. Trans. Brit. Mycol. Soc., 57, 25-39). 100 pl of a suspension of cells (1x10 cells ml ) of the L8 strain were inoculated on a sterile cellophane disc that had been previously placed in contact with the surface of a Petri dish containing NYDA substrate. The dishes were incubated at 25°C for 48 h (to enable possible non-volatile metabolites to be released into the substrate by the L8 strain), then the cellophane was removed and a mycelial disc of Trichoderma spp. (0 6 mm) was inoculated in a central zone of the substrate of the aforesaid Petri dish. After 3 days of incubation at 25°C for 48 h, the diameter of the colony of Trichoderma spp was measured. The control consisted of colonies of Trichoderma spp. (strain 1 and strain 2 of T. pleuroti; strain 1 and strain 2 of T. pleuroticola ) grown in the absence of the non volatile metabolites produced by the L8 strain on the same substrate. Five repetitions were performed for thesis.

[0065] In the photograph of Figure 9, as an example, the effect of the non-volatile metabolites on the mycelial growth of Trichoderma pleuroticola is shown: on the left, a Petri dish of the control (incubation and growth in the absence of the non-volatile metabolites produced by the L8 strain) is shown and on the right a Petri dish is shown in the substrate of which non-volatile metabolites produced by the L8 strain have been released. By comparing the two Petri dishes, it is possible to note that, in the dish on the right, the growth of the colony (mycelial growth) of Trichoderma pleuroticola has been significantly hampered by the non-volatile metabolites produced by the L8 strain.

[0066] Figure 10 is a graph illustrating the percentage of inhibition of the mycelial growth (calculated according to a method that is known to the persons skilled in the art) of T. pleuroti (strains 1 and 2) and of T. pleuroticola (strains 1 and 2) with respect to the control, as a result of the non-volatile metabolites produced by the L8 strain, in the test disclosed above. The graph reports (from left to right) the % values of growth inhibition calculated for: strain 1 of T. pleuroti , strain 2 of T. pleuroti , strain 1 of T. pleuroticola and strain 2 of T. pleuroticola. The non-volatile metabolites produced by the L8 strain are able to reduce by about 50% on average the mycelial growth in the tested strains of T. pleuroti and T. pleuroticola.

[0067] Example 2 - In vivo antagonism tests

[0068] The in vivo test was made by using fragments of pasteurized straw as a substrate. The substrate was inoculated with the commercial“Spoppo” strain of P. ostreatus. The inoculated substrate was placed in trays made of plastics that were bored laterally and having dimensions of 16 x 10 x 6.5 cm. Untreated (control) witnesses were also made in which only water was added to the substrate inoculated with the P. ostreatus strain. Two strains of Trichoderma spp. were then inoculated separately, namely a strain of T. pleuroti and a strain of T. pleuroticola , by adding 5 ml of conidial suspension per tray. For the purpose of simulating two different degrees of intensity of fungal infection, two different concentrations were made, namely 10 and 10 conidia/ml, for the T. pleuroti strain and for the T. pleuroticola strain.

[0069] Subsequently, treatments were performed separately with the L8 strain and with the Prochloraz synthetic fungicide. The L8 strain was inoculated at a concentration of 1x10 cells ml . The Prochloraz fungicide was administered at a concentration of 0.25 ml/l and at a concentration of 1.25 ml/l (the latter concentration corresponds to the field dose). Each tray was finally placed inside a closed polyethylene bag to enable the action of possible volatile metabolites produced by the L8 strain.

[0070] The trays were placed in a growth chamber at 25±2°C and a relative humidity of 72%, in the dark for 20 days and subsequently with dark-light alternation (12 h and 12 h). After four weeks from the start of the test, the colonization of the substrate by the P. ostreatus mycelium was verified visually, by using the modified McKinney scale (1923): 0 = 0% colonization; 1 = 1-10% colonization; 2 = 11-25%; 3 = 26-50%; 4 = 51-70%; 5 = over 70% colonization. The colonization index of the substrate for each performed treatment was then calculated (through a method that is known to the persons skilled in the art).

[0071] Figure 11 is a graph illustrating the pattern of the colonization index (0-5) of the substrate by P. ostreatus. The graph reports (from left to right) the results corresponding to the following situations:

- no treatment (administration of water), in the absence of T. pleuroti and T. pleuroticola ;

- treatment with the L8 strain, in the absence of T. pleuroti and T. pleuroticola ; - treatment with Prochloraz (doses: 0 ml/l, 0.25 ml/l and 1.25 ml/l) and L8 strain, in the presence of T. pleuroti (1x10 conidia/ml);

- treatment with Prochloraz (doses: 0 ml/l, 0.25 ml/l and 1.25 ml/l) and L8 strain, in the presence of T. pleuroti (lxlO ⁵ conidia/ml);

- treatment with Prochloraz (doses: 0 ml/l, 0.25 ml/l and 1.25 ml/l) and L8 strain, in the presence of T. pleuroticola (1x10 conidia/ml);

- treatment with Prochloraz (doses: 0 ml/l, 0.25 ml/l and 1.25 ml/l) and L8 strain, in the presence of T. pleuroticola (lxlO ⁵ conidia/ml).

[0072] The results of the in vivo test highlighted that, in the absence of Trichoderma spp., the treatment with the L8 strain allowed a colonization of the substrate by the P. ostreatus mycelium similar to the colonization of the substrate observed in the non-treated witness or control (administration of water). The treatment with the L8 strain caused an increase in the colonization of the substrate by P. ostreatus in comparison with the infected control (namely, the substrate inoculated with Trichoderma spp and treated with Prochloraz at a 0 ml/l dose). This was evident both in the presence of T. pleuroti and in the presence of T. pleuroticola, at both the used concentrations (1x10 conidia/ml; 1x10 conidia/ml).

[0073] Example 3 - Use of the L8 strain in mushroom bed

[0074] Pleurotus ostreatus is a basidiomycete that develops suitably on organic materials of vegetable origin. The most used material for growing P. ostreatus is the pasteurized wheat straw. The L8 strain was added to the cultivation substrate (wheat straw) of P. ostreatus immediately after the pasteurization and by spraying, in particular through a distribution by spraying of 2 litres of water suspension of cells of L8 strain (concentration: 10 cells x ml ). The so treated cultivation substrate was inoculated with P. ostreatus and divided into 80 bales, which were placed in the cultivation greenhouses of a mushroom bed. 40 days after the treatment and the inoculation, the carpophores (fruiting bodies) of P. ostreatus developed regularly, namely the symptoms of the disease (infection by Trichoderma spp.) were not detected. On the contrary, in the bales that were not treated with the L8 strain and inoculated with P. ostreatus, the carpophores did not develop (symptom of the fungal infection). The photograph of Figure 12 shows (on the left) a ball treated with the L8 strain, in which the production of carpophores of P. ostreatus is evident, and a non-treated ball (on the right), in which symptoms of green mould and absence of carpophores are noted. [0075] From what has been disclosed above, it is clear that the strain and the method according to the invention enable the fungal infections to be fought effectively that attack edible mushrooms, in particular the infections by Trichoderma pleuroti and Trichoderma pleuroticola ascomycetes in mushroom beds of the Pleurotus ostreatus basidiomycete.