WO2017109802A1 - Strains of clonostachys roseae (anam. gliocladium roseum) and their use in the limitation of the growth of pathogenic fungi and as plants growth promoters - Google Patents

Abstract

Methods for containment of phytopathogenic fungi, induction of stress resistance and bio-stimulation of farm plants, comprising the application of strains of Clonostachys Rosea fungi, characterized in that said strains comprise at least a strain selected among: CRrO, deposit number 252/2015 BGr GNLWT; CRM, deposit number 253/2015 BGr GNL1; CRr2, deposit number 254/2015 BGr GNL2.

Description

STRAINS OF CLONOSTACHYS ROSEAE (ANA . GLIOCLADIU ROSEU ) AND

THEIR USE IN THE LIMITATION OF THE GROWTH OF PATHOGENIC FUNGI AND AS PLANTS GROWTH PROMOTERS

Field of the invention

The present invention describes the use of three strains (CRrO, CRM and CRr2) of antagonistic fungi belonging to Clonostachys roseae species for containment of phytopathogenic fungi and parasitic phanerogams in many cultivated species. The strains of antagonistic fungi CRrO, CRM and CRr2 have been deposited at the International Deposit Authority, Budapest, on 10/12/2015 (Corvinus University - National Collection of Agricultural and Industrial Microorganisms) with the following deposit numbers: 252/2015 BGr GNLWT (Clonostachys sp. - WT); 253/2015 BGr GNL1 (Clonostachys sp. - CRM); 254/2015 BGr GNL2 (Clonostachys sp. - CRr2); the strains selected find application both against diseases of root system and against pathogens of the aerial part of plants. In addition, they can promote growth and are able to improve production qualitatively and quantitatively.

In particular, as it is explained in detail in the following examples, CRrO, CRM and CRr2 strains, each one applied alone, are able to promote growth in plants of bean and tobacco and to limit damages caused by R. solani (see examples 1 and 2); in association with iprodione in reduced doses (see example 3) they are able to obtain, while opposing to R. solani, better results than those obtained by the sole fungicide iprodione in reduced doses; CRM and CRr2 strains can also be recovered selectively after treatment on plants of bean, wheat and tobacco (see examples 4 and 5); a mixture of the three strains is able to contrast many phytopthogenic fungi among which: Rhizoctonia, Phytophthora, Sclerotica, Fusarium, Pyrenochaeta, Phoma, Verticillium and parasitic plants as Orobanche spp., on many crops as: broccoli, fennel, tomato, asparagus and wheat (see examples 6 to 11).

State of the art

At the state of the art there are known many applications of BCA (biological control agents) for controlling many phytopthogenic agents belonging to Rhizoctonia, Botrytis, Phytophtora, Pythium, Sclerotinia and Fusarium genera. Anyway, according to the known state of the art, BCA of fungal origin cannot be used together with synthetized fungicides in strategies of integrated plant protection, since fungicides control not only phytopathogenic fungi but also antagonistic fungi.

At the state of the art it is possible to use many formulations available on the market, among which: Biogard (Ampelomyces quisquialis), Contans {Coniothynum minitans), Remedier (Thchoderma asperellum and T. gamsii), Serenade max (Bacillus subtilis), Amylo X (B. amyloliquefaciens), Flocter (B. firmus).

Anyway, notwithstanding the different mechanisms of action used by the various microorganisms present in the known formulations (parasitism, competition for substrate and production of toxic metabolites, production of volatile substances, etc.), biologic pesticides have some limits yet, as for example a lower efficacy than synthetized fungicides in some environmental conditions, a slowness in reaching the desired effects, the possibility of only a preventive use and not when the pathology has developed, a lower shelf-life and application modes which, sometimes, are less easy. On the contrary, synthetized compounds have some undeniable advantages (rapid effect, efficacy also in post-infection stage, ample spectrum of action and an easier preparation and application), even if they pose problems of environmental and toxicity nature for humans and useful species. In economic terms, such factors result in a series of disadvantages for the user of BCA, who is forced to buy more products to fight against many phytopathogenic agents, otherwise controllable by means of a single chemical product, and has to carry out more treatments and to equip oneself for a correct shelf-life and application of the bio-pesticides.

ft solani K(ihn (Teleomorph: Thanetephorus cucumeris (Frank) Donk) is a cosmopolite pathogen of many cultivated species. It is known as agent of rot of plants, of "sore shin" and target spot on leaves. Rot of plants and "sore shin" are widely diffused and cause losses higher than 15%. In addition, isolates belonging to AGs 1 to 5 of ft solani have been reported as agents of diseases of tobacco (Shew and Lucas, 1991); therefore, in many areas it is needed to control ft. solani in seedbed and outdoor (Levin, 2004, Caiazzo et al. 2005). So far the development of this pathogen has been contrasted by using synthetized fungicides.

Recently, symptoms of rhizoctonia have also become frequent in particular high profitability crops, as beans; damages are often remarkable, and thus the application of fungicides has become a diffused practice with its respective environmental problems.

Antagonistic capacities of C. rosea fungus are known, which is yet used as agent of containment in the battle against Botrytis cinerea on strawberry, tomato and raspberry crops (Sutton et al, 1997) and to R. solani on tobacco (Lahoz et al., 2002). Such fungus, which can be found in many geographical (tropical, desert and temperate) areas is linked to many organisms (fungi, nematodes, plants) and can also be linked to roots, leaves, fruits and seeds of many species of plants. For C. rosea many mechanisms of action have been reported (Sutton at al. 1997, Lahoz et al. 2004).

Anyway, to the inventors' knowledge C. rosea has never been used in association with synthetized fungicides, nor strains of C. rosea resistant to Iprodione, Fenexamide and Boscalid have been selected, which are able to keep unaltered the characteristics of the wild strain, as it is clear from the following example 2. In fact, the main limit today concerning the use of fungi as bio-control agents in the field of programs of integrated plant protection which provide the use of synthetized fungicides and BCA together consists in the fact that the application of fungicides reduces or eliminates totally the population of the BCA used.

Aim of the invention

Therefore, a first aim of the present invention is to provide strains of C. rosea which can be used in association with synthetized fungicides, in order to reduce synthetized fungicides quantities to be used with respect to the exclusive use of synthetized fungicides, and to sidestep the development of mechanisms of resistance by pathogens at the same time.

In particular, aim of the present invention is to provide fungal strains of C. rosea resistant to the active substances: Iprodione, Fenexamide and Boscalid, which can be used in programs of integrated plant protection. This allows to contain some pathogens extremely harmful for farm crops, belonging to many fungal genera, among which: Rhizoctonia, Phytophthora, Sclerotinia, Fusarium, Pyrenochaeta, Phoma, Verticillium, Orobanche, thus reducing the use of synthetized fungicides both in terms of number of applications and doses to be administered.

Another aim of the present invention is to provide crop substrates for nursery gardening and plants cultivation, which can be prepared industrially, containing at least one of the selected strains of C. rosea (CRrO, CRM , CRr2), so that said substrates are given control properties of many plant parasites among which those belonging to Verticillium, Fusarium, Pyrenochaeta and Orobanche genera and which are able to improve development and to induce stress resistance in plants.

The present invention provides also curing mixtures to be applied to seeds of many cultivated species among which, wheat (grain in general), and horticultural ones for controlling many parasites of plants among which those belonging to Rhizoctonia, Phytophthora, Sclerotica, Fusarium, Pyrenochaeta, Phoma, Verticillium genera, said mixtures comprising at least one of the selected strains of C. rosea (CRrO, CRM , CRr2) and being able to improve development and to induce stress resistance in plants which will be born from the cured seeds.

Yet the present invention provides mixtures comprising at least one of the selected strains of C. rosea (CRrO, CR , Crr2) and applicable both by drench and drip irrigation both to plants in nursery garden and during cultivation (outdoor/greenhouse) in soil or out of soil of many cultivated species, among which asparagus, cabbage, fennels, tomatoes etc... for controlling many parasites of plants among which those belonging to Rhizoctonia, Phytophthora, Sclerotinia, Fusarium, Pyrenochaeta, Phoma, Verticillium, Orobanche genera and which are able to improve development of plants and to induce stress resistance in plants. Lastly, the present invention provides a formulation comprising at least one active substance of synthesis among Iprodione, Fenexamide and Boscalid, and fungal isolates belonging to at least one of the selected strains of C. rosea (CRrO, CR , CRr2) useful in treatment of pathogenic fungi.

Brief description

The invention described in the following reaches these aims since it provides three strains of C. rosea (CRrO, CRM and CRr2) which can be used, alone or in mixture, as agents of containment of crops pathogens, inducers of improvement of development and stress resistance to plants.

These strains, whose deposit numbers at IDA, Budapest, have been reported yet, are: (i) resistant to active substances as iprodione, fenexamide and boscalid and effective in containing R. solani and B. cinerea (CRM and CRr2); (ii) mutant with unaltered characteristics of the wild strain; (iii) effective against R. solani on bean and tobacco by combined application of reduced doses of synthetized fungicide and antagonist resistant strains; (iv) able to induce systemic resistance to R. solani in tobacco plants; (v) useful in contrasting R. solani, Erysiphie Orontii, Sclerotica spp, Fusarium spp, Rizoctonia spp, Phytophtora spp, P. lycopersici, Phoma spp, Verticillium spp, and the agents responsible for take-all disease and fusarium ear rot of corn, Orobanche.

In addition to the desired resistance to fungicides, the isolates obtained by in vitro screening in presence of target fungicides have kept all the characteristics of the wild strain. The techniques used have not made use of genetic transformations of the strain used, but the variants have been obtained by classic techniques. The two isolated strains are also recognizable by means of traditional and molecular markers.

In the following there are reported some examples of application of the two strains CRM and CRr2, not limiting the aims of the invention. It is to be precised that the tests were carried out in the Centra di Saggio Bioagrites authorized by MiPAF, under the D.L. 17/03/95 n. 195, to carry out tests in compliance with the good practice for outdoor test performance. The data collected was analysed statistically by means of the XLSTAT 2008 program - Comparison between k samples (Kruskal-Wallis, Friedman, ...); for each measurement different letters on columns correspond to statistically significantly different values. The significance from a statistical point of view was determined by means of the Tukey's test for P = 0,05.

Example 1 : capacity of CRrO of promoting growth and containing R. solani attacks on bean and tobacco

Aims: a) evaluation of the capacity of a population of isolated strains of C. rosea of promoting growth of plants of bean and tobacco; b) evaluation of the antagonistic activity of isolated strains of C. rosea in containing R. solani on bean and tobacco. 1.1 - Activity of growth promotion and antagonism

6 tests have been carried out: 3 for bean and 3 for tobacco. For these assays 6 isolates of C. rosea were used, all of wild type. The inoculation of the antagonists consisted in the addition of 200 ml for plant of a 2 x 10⁶ conidial suspension containing the antagonistic strains after seeding of bean (variety Controne Bean) and after transplantation on tobacco (White burley variety). Treatments were repeated three times in a month with the same methodology. The parameters evaluated 20 days after the last application were: dry weight of plants, height of plants and roots for tobacco, while for bean it was evaluated also the production of grain for plant.

For outdoor R. solani control two tests for bean and as much for tobacco were carried out in fields naturally infected by the CRA CAT pilot farm of Scafati. To such aim it was used the same method as above with the only difference that the pathogen was in the soil. The parameters detected were: gravity of symptoms for tobacco (according to Mc Kinney) and incidence rate of dead plants for bean. Each plot was formed by 4 rows of 5 meters each and by 4 replications for both the crops and each experiment.

1.2 - Statistical analysis and results

All the parameters evaluated have shown that the wild type isolate of C. rosea (CRrO) has shown a significantly higher performance of growth promotion than the other 5 isolates naturally occurring and previously isolated from different geographical areas (fig. 1-3), used as comparison in the present study.

In addition to the other characteristics, the isolate CRrO has shown its capacity of containing the disease, which proved to be of great interest. In figure 4 it is shown the percentage of plants affected by R. solani while varying the strain of C. rosea used; in figure 5 it is shown the respective index of gravity. Therefore, from the data it is clear how the selected isolate CRrO comprises in itself all the positive characteristics for use on large scale both for growth promotion and efficacy in containing diseases, even if it is not resistant to synthetized fungi, as instead the two strains CRM and CRr2 are.

Example 2 - Antagonistic capacities of the strain of wild type (wt - wild type) CRrO of C. rosea and selection of strains resistant to active substances iprodione, fenexamide and boscalid with the same characteristics of wt

Aims: a) obtaining isolates of C. rosea resistant to the active substances iprodione, fenexamide and boscalid; b) checking if isolates of C. rosea resistant to the active substances iprodione, fenexamide and boscalid keep the characteristics of the wild type isolate of origin; c) checking if mutants keep the antagonistic capacities of the wild type.

Example 2.1 - Obtaining strains of C. rosea resistant to the active substances: iprodione, Boscalid and fenexamide starting from a population of wild type isolates For obtaining resistant isolates a pool of Wild type isolates of C. rosea was tested for sensibility to the active substances Ipropdione, Boscalid and Fenexamide, by using the method of the poisoned agar. In all the experiments carried out it was used malt extract agar (MEA) (oxoid CM 59). The test was carried out by arranging a 5 mm cylinder of the fungal mycelium, withdrawn from 10 days old colonies, on 3 Petri dishes for each one of the 7 different concentrations of the fungicide (0,001 to 1000 mg/l). After 4 and 10 days of incubation, the radial growth of the colonies of C. rosea was measured at the different fungicide concentrations assayed. The data was expressed as growth percentage with respect to the one detected on control dishes without fungicide.

The relative toxicity was expressed as ICso, defined as the concentration causing a 50% reduction in radial growth. To such aim the transformation in probit of data was carried out (Finney, 1971). The isolates with higher ICso were bred on dishes containing 2000 and 5000 ppm of fungicides. Colonies survived to treatment and grown on the poisoned substrate were transferred again on poisoned substrate to confirm resistance.

2.2 - Evaluation of the biologic characteristics

To evaluate if the resistant strains kept the same characteristics as the wild one, there were evaluated: a) growth after 5 days at 27°C by using 4 Petri dishes for each isolate strain and b) capacity of producing conidia measured on 20 mm² of mycelium collected from colonies grown on MEA for a week and suspended in 5 ml of distilled water. The conidial concentration was determined by using the globule-counting chamber. No significant difference was found between the isolates, object of the study, both concerning growth at 27°C and capacity of producing conidia (table 1).

Table 1

Example 2.2.1 - Quantification of activity of N-Acetyl β-D-glucosaminidase, β-D- N-N'-diacetylchitobiosidase and endochitinase.

In order to evaluate the biochemical characteristics of the resistant isolates the activity of some enzymes involved in the antagonism process was evaluated. The evaluation of activity of N-Acetyl β-D-glucosaminidase, β-D-N-N'- diacetylchitobiosidase was carried out by following the protocol reported by Tronsmo and Harman (1993). The endochitinase activity instead was carried out by following Reissig et al. (1955). All the isolates were put in culture by using as exclusive carbon source 5 g of colloidal chitin or as an alternative the lyophilised, triturated, spun and sterilised mycelium of R. solani.

The isolates resulted in an activity which was not statistically different of evaluated enzymes. It was interesting the different response in N-Acetyl β-D- glucosaminidase and andochitinase activity, obtained on chitin in comparison with the growth on mycelium of ft solani; this latter induced a greater activity thus indicating affinity of C. rosea with this source of chitin. In fig. 9 it is reported the quantification of activity of N-Acetyl β-D-glucosaminidase (A) and β-D-N-N'- diacetylchitobiosidase (B) for the various strains, and in fig. 10 the quantification of activity of endochitinase.

Example 2.3 - Tests in greenhouse

Two tests were carried out in greenhouse to evaluate the antagonistic efficacy towards R. solani of the three isolates of C. rosea (wild type CRrO, CRM and CRr2). The inoculum of R. solani was prepared by growing the fungus for 10 days at 25°C in Erlenmayer on sterilised seeds of millet; in the following a quantity equal to 0,5% by weight was added to the sterile soil. The inoculation of the antagonists consisted in the addition of 15 ml of a 2 x 10⁶ conidial suspension after seeding of bean (variety Controne Bean), to each hole of the containers in polystyrene with 24 holes used for seeding. To evaluate the efficacy the number of healthy plants was counted 15 days after seeding. The number of fully healthy plants was counted to make an estimate which considered only the cases of full success of treatment. Data highlighted that the isolates CRM and CRr2 resistant to fungicides produced a number of fully healthy plants (187 and 192) totally equal to the one obtained with the Wt (CRrO), as it is clear from data of figure 11 , where it is shown the number of healthy plants while varying treatment (RT 27 = R. solani AG4; Iprod = Iprodione).

2.4 - Statistical analysis and results

The activity of the active substances has been expressed as percentage of inhibition and the relation between concentration and inhibition has been linearized and IC50 calculated according to Finney (1971 ). Data was analysed by using ANOVA and the averages separated by Tukey's test (p = 0,05).

The IC50 measurement (40,8 mg/l) has shown that CRrO is sensible to iprodione as well as to fenexamide and Boscalid. Only 2 colonies (CRM and CRr2) of the 576 ones used in the screening with high IC50 have shown resistance to all the three active substances used.

The IC50 measured for the two mutant strains (CRM and CRr2) was greater than 5000 mg/l (fig. 6), which is very higher than concentrations to which the fungicide has to be used normally in the praxis.

Example 2 - Conclusions

It is generally observed that the fungal strains in which resistance to one or more active substances is induced in laboratory, often have characteristics altered with respect to the starting isolate, among which: virulence level, production of extracellular pectic enzymes, capacity of forming spores and production of pigments (Van Tuyl, 1977). Contrary to what happens in the isolated strains known at the state of the art, the isolates resistant to iprodione, fenexamide and Boscalid, object of the present invention (strains CRM and CRr2), have kept unaltered antagonistic capacities and essential biologic and biochemical properties.

The application of -synthetized fungicides associated with the use of isolates resistant to the biologic agent C. rosea (CRM and CRr2) allows to reduce the fungicide intake on crops while keeping high levels of efficacy in treatment of R. solani, as it is clear also from the following example.

Example 3 - control of R. solani on tobacco in float system and bean in greenhouse by using reduced doses of fungicide and c. rosea strains resistant to iprodione

Two tests were carried out on plants of tobacco in float system; the wild type isolate CRrO of C. rosea and two resistant isolates CRM and CRr2 and two doses of the fungicide iprodione (1 ,4 and 0,46 g a.i./m²) were used in association with R. solani or not. The inoculum of R. solani was prepared by growing the fungus for 10 days at 25°C in Erlenmayer on sterilised seeds of millet: in the following a quantity of R. solani equal to 0,5% by weight was added to the sterile soil. The inoculation of the antagonists consisted in the addition of 15 ml of a 2 x 10⁶ conidial suspension. To evaluate the efficacy the number of healthy plants was counted a month after treatment.

Also in case of plants of bean two tests were carried out in greenhouse. The same isolates of C. rosea used in the previous test (wild type CRrO, CRM and CRR2) were used and two doses of iprodione (1 ,4 and 0,46 g a.i./m²) in association with the inoculation of R. solani or not were combined in a factorial experiment with 4 replications. The inoculum of R. solani was prepared, also in this case, by growing the fungus for 10 days at 25°C in Erlenmayer on sterilised seeds of millet; in the following a quantity equal to 0,5% by weight was added to the sterile soil. The inoculation of the antagonists consisted in the addition of 15 ml of a 2 x 10⁶ conidial suspension after seeding of bean (variety Controne Bean), to each hole of the containers in polystyrene with 24 holes used. To evaluate the efficacy the number of healthy plants was counted 15 days after seeding.

Results - Tests in greenhouse

Data obtained confirmed that the contemporaneous application of reduced doses of fungicide and BCA resistant isolates, CRM and CRr2, produced fully healthy plants with values 187 and 192, respectively. Even if these data is, in absolute value, lower than the one highlighted from the use of normal doses of fungicide, it is significantly higher than the one obtained by only using reduced doses of fungicide (see fig. 11 ).

Example 3 - Conclusions

The application of low doses of fungicide associated with the use of isolates (CRM and CRr2) resistant to the biologic agent C. rosea has lead to an equal level of containment with respect to the fungicide applied in full dose, while the results of fungicide in reduced doses were not exciting, thus indicating a synergistic action between the two methods.

Finally, by considering that in many cases the application of reduced doses of pesticides can induce easier resistance to the same by pathogens, the contemporaneous application of systems using different mechanisms of action can reduce such possibility and can contribute to the reduction of quantities of the active substances of synthesis used at the same time.

Example 4 - capacity of producing systemic resistance to Erysiphe orontii in tobacco by CRM strain Aims: a) evaluating if the isolate CRM is able to induce biochemical variations in the aerial part, after application to roots; b) evaluating if the variations obtained in the aerial part influence the development of £ orontii in leaves.

4.1 - Inoculation of the CRM strain on plants of tobacco

Seeds of tobacco of Burley 64 variety were disinfected and arranged in sterile soil. After 30 days the plants were transplanted in alveolar containers with 24 holes and kept in growth chambers at 25°C for a photoperiod of 12 h. Two containers were inoculated with 10 ml of a conidial suspension (2 x 10⁶) of CRM for each plant. 12 plants for each of the 4 inoculation times fixed: 0, 1 , 4, 8 and 12 days, were subjected to analysis of the main enzymatic activities correlated to ISR response.

4.2 - Determined enzymatic activities

On the inoculated plants and on the not inoculated control ones the activities of the following enzymes were measured: 1 ,3-P-glucanase (EC 3.2.1.6), 1 ,4-β- glucosydase (EC 3.2.2.21 ), N -acetyl-p-D-glucosaminidase (EC 3.2.1.30), nonspecific chitinase e β-d-NN'-diacetyl-chitobiosidase (Tronsmo and Harman, 1993). In addition, peroxidases were determined electrophoretically (Gianinazzi et al. 1970).

In figure 12 it is shown the variation of the enzymatic activity in leaves of tobacco treated with a conidial suspension of C. rosea (CRM) on roots in four different times of inoculation or in not treated (control) ones, (a) 1 ,3-p-glucanase, (b) chitinase, (c) 1 ,4- -glucosydase, (d) N-acetyl- -D-glucosaminidase; in figure 13 it is shown the activity of β-d-N-N'-diacetylchitobiosidase in leaves of tobacco in four different times after inoculation with isolate CRM on roots or without inoculation (control). In fig. 14 the electrophoretic patterns are shown which are obtained by PAGE of peroxidase isoenzymes in leaves of Tobacco in four different times after inoculation on roots with isolate CRM or not inoculated (control).

4.3 - Assay of efficacy of ISR on £ orontii

35 days old plants of Tobacco were transplanted individually in pots 30 cm in diameter. Half of the plants (20) were inoculated only on leaves with conidia of £ orontii. 20 plants were inoculated, with £. orontii on leaves as well, but 24 h before, 8 and 14 days after inoculation with £ orontii also on roots with a conidial suspension quantity equal to 2 x 10⁶ of CRM strain. To evaluate the effect of treatment with resistance inducer, £ orontii colonies were counted for leaf and the percentage of leaf surface affected was estimated, compared to the witness inoculated only with the oidium agent.

Table 2

Example 4 - Conclusions

CRM has induced many metabolic variations and this is the first time it is observed on whole plants after application on roots of an isolate of C. rosea (CRM). Therefore, this strain results to be an optimal biocontrol agent BCA thanks to its useful characteristics: safety of use for operators and animals, environmental adaptation capacity, environmental safety, no pathogenic relations for cultivated plants. CRM isolate has induced a clear reduction of symptoms of oidium on plants of Tobacco thus influencing the gravity of symptoms more than their incidence rate, indicating that the mechanism of protection induced is postinfection.

Example 5 - Selective recovery of isolated strains CRM and CRr2 of C. rosea resistant to fungicides

Aim: selectively recovering isolates of C. rosea (CRM and CRr2) from roots of bean and wheat after application to seeds in pre-seeding stage

5.1 Inoculation of seeds and determination of the number of colonies recovered on roots of bean and wheat

100 seeds of bean were inoculated by means of curing treatment with conidia of CRM and CRr2 and others 5 isolates of C. rosea of different origins (CRr01 to CRr05). At the end of preparation, the talc powder used as dispersing agent in preparing the curing mixture contained about 1 x 10⁵ active conidia per gram of powder of the two isolates of test, while for the other isolates it was 0,5 x 10^s to 2 x 10⁵. The seeds were seeded outdoor with a row for each isolate. After 80 days the roots of beans were cut and brought in laboratory. The roots were cut in 4 mm little pieces and 100 pieces randomly chosen for each one of 3 replications were used for isolation. The substrates used were potato dextrose agar and potato dextrose agar additioned with 1000 ppm of each one of the 3 fungicides iprodione, fenexamide and boscalid to which CRM and CRr2 isolates are resistant. With the mixture of the selected strains of C. rosea (CRr01 , CRM , CRr2) a quantity of grain maize was cured, useful to seed 1 hectare of grain in two different areas. Seeding was carried out on 26^th November 2014 and 10^th January 2015. At the end of cultivation, June 2015, 200 plants for plot were withdrawn, from which the root was separated and then the same was done for beans. In this case the test served to show the affinity of the isolate to roots of wheat.

5.2 Results for bean

From data of table 3, it is clear that according to what detected in previous examples, isolates of C. rosea CRM and CRr2 can be found on roots of plants of bean inoculated with a percentage of 65,3 for CRM and 71 ,2 for CRr2, definitely higher than the ones recorded for isolates CRr01 1 to 5, used for comparison. Moreover they are the only ones which, confirming the resistance obtained with previous tests, are able to grow on substrate poisoned with fungicides.

Table 3

5.3 - Results for wheat

From the 200 fragments of roots, colonies of C. rosea were found equal to 13,5% for the seeding of 1^th area carried out on 26^th November 2014, and equal to 13% for the seeding of 2^nd area carried out on 10^th January 2015.

Example 5 - Conclusions

Selectively recovering isolates (CRM and CRr2) of C. rosea from roots of bean after application of curing to pre-seeding seeds highlighted further not only the possibility to recover the same on a selective means, thus confirming the possibility to recognize them surely thanks to their characteristics, but allows also to overcome the main limit of today's technique which consists in the impossibility to use a BCA of fungal origin together with fungicide treatments.

Data of test carried out on wheat shows that, even if the application of isolates occurred at the time of curing of the grain maize, it was possible to find the fungus active yet on roots of wheat of plants at the end of crop cycle in both the cultivation areas.

Example 6 to 1 1

In the following examples, if not otherwise specified, the fields chosen for tests had been previously analysed to check the presence of phytopathogenic fungi useful to reach the aims of the same tests (evaluation of efficacy in the use of antagonists towards fungi of rhizosphere of crucifers). In addition, phytopathogenic fungi were bred in laboratory on substrates specific for the single species. The evaluation of the activity of control towards diseases due to telluric fungi was carried out on each single plant at the end of crop cycle by counting the number of plants infected with respect to the whole.

The presence of phytopathogenic fungi was checked by isolation on axenic substrates (selective PDAs/MEAs). The same substrates, containing iprodione were used to check the presence of the antagonistic fungi used in the various studies. The data collected was analyzed statistically by means of the XLSTAT 2008 program - Comparison between k samples (Kruskal-Wallis, Friedman, ...); for each measurement different letters on columns correspond to statistically significantly different values.

Example 6: Control of Fusarium spp., Sclerotinia spp., Rhizoctonia spp., Phytophthora spp., Pyrenochaeta spp. and Phoma spp., on different species of outdoor horticultural plants by drench applications of a mixture of strains of C. rosea (wild type CRrO, and CRM and CRr2 resistant to fungicides).

6.1. Test on plants of broccoli (Parthenon variety)

Two tests were carried out on plants of broccoli (Brassica oleracea var. botrytis L.) by using the wild type isolate CRrO of C. rosea and two resistant isolates CRM and CRr2 in mixture.

The antagonists were inoculated by immersing alveolar containers containing the plants (at BBCH 13-14 stage), immediately before the outdoor transplantation, in a suspension containing the three fungal strains of C. rosea (CRrO, CRM and CRr2), so that 30000 UFC of each single fungal strain were absorbed for plant. In 2015 there were carried out two summer-fall tests, in agro Dauno and agro Nocerino - Sarnese respectively. The units forming colonies (conidia, mycelium, sclerotia, etc ..) were distributed in the soil in the respective plots by fert-irrigation 8 days before the transplantation. The tests were arranged by using a split plot experimental scheme with 4 replications for each thesis compared, for a total number of 250 plants for replication.

Results of tests

Data obtained confirmed that the contemporaneous application of the three fungal strains (CRrO, CRM and CRr2) of C. rosea in mixture gives the plants of broccoli a very high protection towards Sclerotinia spp. and Rhizoctonia spp. The efficacy of control of rot diseases caused by Sclerotinia spp. and rhizoctonia was statistically better in both tests, for P = 0,05, with respect to the control not treated. In fig. 16 it is shown the percentage of plants with symptoms of Sclerotinia while varying treatment, in fig. 17 the percentage of plants with symptoms of Rhizoctonia.

6.2 - Tests on plants of fennel (var. Valentino)

Four tests on plants of fennel (Foeniculum vulgare Mill.) were carried out by using the wild type isolate CRrO of C. rosea and the two resistant isolates CRM and CRr2 in mixture. The antagonists were inoculated by immersing alveolar containers containing the plants (at BBCH 13-14 stage), immediately before the outdoor transplantation, in a suspension containing the three fungal strains of C. rosea (CRrO, CRM and CRr2), so that 30000 UFC of single fungal strain were absorbed for plant.

Four tests were carried out, one in winter-spring cycle (December 2014 - May 2015, the other one in summer-fall cycle (July - December 2015). The units forming colonies (conidia, mycelium, sclerotia, etc..) were distributed in the soil in the respective plots by fert-irrigation 8 days before the transplantation. The tests were arranged by using a split plot experimental scheme with 4 replications for each thesis compared, for a total number of 250 plants for replication.

Results of tests

Data obtained confirmed that the contemporaneous application of the mixture of the three fungal strains (CRrO, CRM and CRr2) of C. rosea gave the plants of fennel a very high protection towards Sclerotinia spp. Rhizoctonia spp. Phoma spp. and Fusarium spp. The efficacy of control of rot diseases caused by Sclerotinia spp. Rhizoctonia spp. Phoma spp. and Fusarium spp. was statistically better in the four tests, for P = 0,05, with respect to the control not treated. On the contrary, the efficacy of treatment with the mixture of antagonistic strains resulted to be comparable, for P = 0,05, to the one obtained with the Tellus standard. In fig. 18 it is reported the percentage of plants with symptoms of Phoma spp. , in fig. 19 with symptoms of Sclerotinia spp., in fig. 20 of Rhizoctonia spp. and in fig. 21 of Fusarium spp.

6.3 - Tests on plants of tomato (var. Roma)

5 tests on tomato (Solanum lycopersicum, L.) were carried out by using the wild type isolate CRrO of C. rosea and the two resistant isolates CRM and CRr2 in mixture. The antagonists were inoculated by immersing alveolar containers containing the plants (at BBCH 13-14 stage), immediately before the outdoor transplantation, in a suspension containing the three fungal strains of C. rosea (CRrO, CRM and CRr2), so that 30000 UFC of each single fungal strain were absorbed for plant. The tests were carried out in 2015 in agro Nocerino Sarnese, in Fiano of Nocera Inferiore (SA). The units forming colonies (conidia, mycelium, sclerotia, etc..) were distributed in the soil in the respective plots by fert-irrigation 8 days before the transplantation. The tests were arranged by using a split plot experimental scheme with 4 replications for each thesis compared, for a total number of 80 plants for replication. The evaluation of activity of control towards diseases due to telluric fungi was carried out on each single plant (by using a scale 0-10, where 0 = no damages; 10 = 100% of root surface with symptoms of necrosis/rot/corkiness) and it was evaluated at the end of crop cycle. As an alternative it was evaluated the number of infected plants with respect to the whole.

The presence of phytopthogenic mycete in plants of tomato was checked by isolation on axenic substrates (selective PDA). The re-isolations of C. rosea (CRrO, CRM and CRr2) were carried out on PDA containing iprodione.

Results of tests

Data obtained confirmed that the application of the three fungal strains (CRrO, CRM and CRr2) of C. rosea in mixture gave the plants of tomato a very high protection to Sclerotinia spp. Rhizoctonia spp. Fusarium spp., Verticillium spp. and Pyrenochaeta spp. The efficacy of control of rot diseases by Sclerotinia spp. rhizoctonia, corky root by Pyrenochaeta lycopersici, tracheomycosis by Fusarium spp. and Verticillium spp. was statistically better in all the tests realized, for P = 0,05, with respect to the control not treated. On the contrary, the efficacy of treatment with the mixture of antagonistic strains resulted to be comparable, for P = 0,05, to the one obtained with the Enovit metile and Tellus standards. In fig. 22 it is shown the average index of disease for root internal cork, in a scale 1 to 10 while varying treatment; in fig. 23 it is reported the percentage of plants with symptoms of Fusarium, in fig. 24 with symptoms of Verticillium, in fig. 25 of Sclerotinia spp. And in fig. 26 of Rhizoctonia.

Example 6 - Conclusions

The applications in nursery garden or pre-transplantation of the mixture of 3 strains of C. rosea (CRrO, CRM and CR2) to plants of different horticultural species by drench brought to a level of containment of the various diseases of the root system higher or equal to the one obtained by the use of traditional (i.e. Enovit metile) or biologic standard pesticides (i.e. Tellus). A single application in nursery garden of antagonistic fungi and their subsequent development in root zhyzosphere allows to reduce the number of treatments with high costs saving in terms of plant protection, environmental and operator protection.

Application by drench to plants of horticultural species of the mixture of strains of C. rosea and the following colonization of the cube of crop substrate before the transplantation allows also to diffuse in the agricultural environment (soil) antagonistic fungi of various phytopathogenic agents, thus improving plant health characteristics and in particular the soil fungistasis.

Example 7 - control of Verticillium spp., Fusarium spp. and P. licopersici on plants of tomato and asparagus in greenhouse by means of application to crop substrate of a mixture of strains of C. rosea (wild type CRrO and CR , CRr2 resistant to fungicides)

Prior to report the results of specific tests, it is to be precised that the crop substrate according to the present invention can be prepared with various materials of organic nature, among which: not composted simple plant amendment (i.e. coconut fibre, rice husk, wood fibre), green composted amendment, acid peat, neutral peat, humidified peat. These can be used alone, mixed between each other, or with the addition of other organic materials, materials of mineral origin, specific action products, correctives and fertilizers. In the substrate a mixture is mixed comprising one or more of the three isolates of C. rosea (CRrO, CR , CRr2) with concentration variable according to the crop which will receive the same.

7.1 - Test on plants of tomato (var. Roma)

3 tests on tomato were carried out in greenhouse by using the wild type isolate CRrO of C. rosea and two resistant isolates CRM and CRr2 in mixture. The inoculation of the antagonists consisted in the addition of 15 ml of a conidial suspension to the crop substrate previously sterilized, in order to have a quantity of conidia equal to 50000 UFC in each plastic plot 18 cm in diameter which received three plants of tomato in the following. The plants were transplanted 5 days after treatment at the 2° stage, pair of true leaves (BBCH 14 growing stage). Phytopthogenic fungi were bred in laboratory on substrates specific for the single species. The units forming colonies (conidia, mycelium, sclerotia, etc..) were distributed in pots by fert-irrigation 15 days after transplantation of the plants of tomato at 4° stage, pair of leaves (BBCH 18 growing stage). The presence of phytopathogenic mycete in plants of tomato was checked by isolation on axenic substrates (selective PDAs). The re-isolations of C. rosea (CRrO, CRM and CRr2) were carried out on PDA containing iprodione.

To evaluate the efficacy both the number of healthy plants three months after transplantation and the percentage of area affected by the disease on the root system (P. lycopersici) were counted.

The tests were arranged by using a split plot experimental scheme with 3 replications for each thesis compared, with a total number of 30 plants for replication. The evaluation of the activity of control towards diseases due to telluric fungi was carried out on each single plant (by using a scale 0-10, where 0 = no damages; 10 = 100% of root surface with symptoms of necrosis/rot/corkiness), at the end of crop cycle, or counting the number of plants with symptoms with respect to the whole.

Results of tests

Data obtained confirmed that the contemporaneous application of the three fungal strains (CRrO, CRM and CRr2) of C. rosea in mixture gave the plants of tomato a very high protection to Fusarium oxysporum f.s. lycopersici, Verticillium albo-atrum and Pyrenochaeta lycopersici. The efficacy of control of diseases of root internal cork by P. lycopersici and tracheomycosis by F. oxysporum f.s. lycopersici and V. albo-atrum was statistically better, for P = 0,05, with respect to the control not treated. In fig. 27 there are shown the percentages, while varying treatment, of plants with symptoms of Fusarium, in fig. 28 with symptoms of Verticillium and in fig. 29 the index of disease of root internal cork.

7.2 - Tests on asparagus

A test on asparagus was carried out in greenhouse by using the wild type isolate CRrO of C. rosea and two resistant isolates CRM and CRr2 in mixture. The inoculation of the antagonists consisted in the addition of 15 ml of a conidial suspension to the crop substrate previously sterilized, in order to have a quantity of conidia equal to 50000 UFC in each plastic pot 18 cm in diameter which received five plants of asparagus in the following. The plants were transplanted 5 days after treatment at the 3° stage, leaf (BBCH 13 growing stage).

Phytopthogenic fungi were bred in laboratory on substrates specific for the single species. The units forming colonies (conidia, mycelium, sclerotia, etc..) were distributed in pots by fert-irrigation 15 days after transplantation of the plants of asparagus at 4° stage, leaf (BBCH 14 growing stage). The presence of phytopathogenic mycete in the plants of asparagus was checked by isolation on axenic substrates (selective PDAs). The re-isolations of C. rosea (CRrO, CRM and CRr2) were carried out on PDA containing iprodione. To evaluate the efficacy the number of healthy plants three months after transplantation was counted.

The tests were arranged by using a split plot experimental scheme with 3 replications for each thesis compared, with 50 plants for replication. The evaluation of the activity of control towards infections by F. oxysporum f.s. asparagi was carried out by counting the number of plants with symptoms with respect to the whole.

Results of tests

Data obtained confirmed that the contemporaneous application of the three fungal strains (CRrO, CRM and CRr2) of C. rosea in mixture gave the plants of tomato a very high protection to F. oxysproum f.s. asparagi.

The efficacy of control of tracheomycosis by F. oxysporum f.s. asparagi resulted to be statistically better, for P = 0,05, with respect to the control not treated (see fig. 30). In fig. 30 it is shown the percentage, while varying treatment, of plants with symptoms of Fusarium.

Conclusions The application to the crop substrate of the mixture of the three isolates of C. rosea (CRrO, CRM and CRr2) allowed to control effectively, in controlled environment, the diseases due to F. oxysporum f.s. lycopersici, V. albo-atrum and P. lycopersici on tomato and F. oxysporum f.s. asparagi on asparagus. In particular, in addition to the check, the level of containment of the various diseases (tracheo-fusarium and root internal cork) resulted to be higher than the one reached by using the biologic standard (Tellus).

It is interesting to note that the application to substrate of the mixture of the three isolates of C. rosea (CRrO, CRM and CRr2) allows to put on the market a ready crop substrate, containing antagonists useful to control the rhizosphere of the horticultural plants, thus increasing the added value of the same crop substrate. This implies also the elimination of treatments in nursery garden, with the consequent improvement of the nursery garden supply chain both from an economic and technical point of view.

A single application to crop substrate of antagonistic fungi and their following development on rhizosphere allows to protect the plants of tomato or asparagus both in nursery garden and outdoor, thus eliminating or reducing strongly the interventions with conventional pesticides, with consequent high costs saving and environmental and human health protection.

Example 8 - capacity of the mixture of C. rosea isolates (wild type CRrO + fungicide resistant CRM and CRr2), applied by means of curing or encrusting of seeds (bio-curing), of improving quali-quantitative performances in the production of wheat (bio-stimulation) and in containing attacks of phytopathogenic mycete which cause take-all and fusarium ear rot.

Aims:

A) evaluating the antagonistic activity of isolates of C. rosea towards phytopathogenic mycete causing take-all and fusarium ear rot of corn.

B) evaluating the bio-stimulant activity towards wheat of the mixture of strains of C. rosea wild type CRrO + two isolates resistant to fungicides CR1 and CR2.

Two test on hard wheat (Triticum durum desf.) var. Quadrato were carried out by using the isolates of C. rosea wild type CRrO and two resistant insolates CRM and CRr2 applied in mixture to seeds by means of the technique of curing. The two tests were carried out with an interval of one month the one after the other one: one started in the first decade of December 2014, the other one in the first decade of January 2015. The inoculation consisted in curing, prior to seeding, the maize with a suspension containing the three fungal strains of C. rosea (CRrO, CRM and CRr2) so that a quantity equal to 10 x 10⁶ UFC of each strain was absorbed on the surface of maize.

In particular, the antagonistic fungi were applied by using a pilot system of seeds curing. Both adjuvants and substances able to keep the fungal conidia vital for not less than 6 months after treatment were added to the mixture. The tests were arranged on a surface of about one hectare of soil for thesis and inside each thesis 12 areas of assays were individuated of one square meter each.

The evaluation of the activity of control towards diseases due to telluric fungi responsible for take-all and fusarium ear rot of corn was carried out on each single plant or ear both by insulating mycete responsible for the diseases on axenic selective substrate and by observing the damage caused by the same. In particular, the following parameters were detected:

- % of maize infected by F. graminearum (analysis carried out separately on bare maize and whole maize);

- % of plants of wheat infected by the fungal agents responsible for take-all [Ophiobolus (Ophiobolus graminis), Cercosporella (Cercosporella herpotricoides), Rhizoctonia (Rhizoctonia cerealis) and those belonging to the Fusarium genre (F. nivale, F. culmorum, F. graminearum)].

In order to evaluate the bio-stimulating and regulating activity of the biochemical functions induced by the mixture of the three strains of C. rosea, the following parameters were detected: weight of ears for m²; average weight of ear; weight of maize for m²; diameter of stalk; height of plants with or without ear; SPAD. The qualitative parameters detected were: proteins, gluten, yellow index; % relative humidity; hectolitre weight.

Results of tests - aim A

Evaluation of infection by take-all

The measurement intended to evaluate the incidence rate of take-all disease, the % thus expressed was determined by analysing 100 plants for each area of assay, for a total number of 1200 plants analysed for thesis. In figs. 31 and 32, for the two tests carried out in two different farms, there are reported the percentages of plants infected by mycete responsible for take-all disease, while varying treatment. Measurement of the quantity of maize infected by F. graminearum The measurement intended to quantify the maize infected by F. graninearum. The % expressed was determined by isolating mycete on selective substrate in 4 replications, each consisting in 100 maize for thesis.

Aim A) evaluating the bio-stimulating activity towards wheat of the mixture of strains of C. rosea wild type CRrO + two fungicide resistant isolates CRM and CRr2

The results obtained show clearly that the application of the three fungal strains (CRrO, CRM and CRr2) of C. rosea in mixture to the seeds by means of the technique of curing (bio-curing) gave the plants of hard wheat a better vegetative and productive performance. The bio-stimulating activity resulted in greater production of maize, in an improvement of the biometric characteristics of the plants and in a strong improvement of the product characteristics of maize, as reported in the appended tables and graphics. All the parameters show statistically significant differences, for P = 0,05, with respect to the control not treated. In fig. 33 there are shown the percentages of maize infected by F. graminearum, evaluated both on bare maize and whole maize for the two tests carried out in two different farms.

Aim B) evaluating the antagonistic activity of isolates of C. rosea towards phytopathogenic mycete causing take-all and fusarium ear rot of corn

The data obtained shows clearly that the application of the mixture of the three fungal strains (CRrO, CRM and CRr2) of C. rosea to the seeds by means of the technique of curing (bio-curing) gave the plants of hard wheat a better vegetative and productive performance. The antagonistic activity towards phytopathogenic mycete for wheat Ophiobolus (Ophibolus graminis), Cercosporella (Cercosporella herpotericoides), Rhizocotnia (Rhizoctonia cerealis) and those belonging to Fusarium genre (F. nivale, F. culmorum, F. graniearum) resulted to be optimal for the control of take-all disease of wheat and fusarium ear rot. The results are reported in figs. 34 to 42. In fig. 34 there are shown the average heights of plants with no ear while varying treatment, in fig. 35 the heights with ear; in fig. 36 the average weight of wheat ear, in fig. 37 the average of maize weight (g/mq); in fig. 38 the average of chlorophyll index (SPAD), in fig. 39 the average of diameter of wheat stem, in fig. 40 the average of weight of ears of wheat at harvesting (g/mq); in fig. 41 the hectolitre weight expressed in Kg/hi, in fig. 42 the product parameters of grain maize. Conclusions

The application to seeds of wheat (grain in general) of the mixture of the three strains of C. rosea (CRrO, CRM and CRr2) brought to a level of containment of the various diseases of the root system, stalk and ear higher or equal to the one reached by using traditional (chemical) pesticides.

A single application to seeds of antagonistic fungi and their following development in the root rhizosphere allows to reduce the number of applications of treatments with high costs saving in plant protection, greater environmental health and protection of human health in general and of farm operators in particular. The bio- curing of seeds allows also to diffuse antagonistic fungi of many phytopathogenic agents in the agricultural environment (soil), thus improving their agronomic characteristics and in particular the fungistasis of the soil.

The bio-curing treatment with the mixture of three strains of C. rosea (CRrO, CRM and CRr2) allows to substitute the chemical curing treatment, with the consequence that the thus treated seeds can be used also in biologic or integrated agriculture.

The bio-curing treatment with the mixture of three strains of C. rosea (CRrO, CRM and CRr2) gives the plants of wheat an improvement in vegetative performances, as it is highlighted in the results concerning the measured biometric parameters (weight of ears for m², average weight of ear, bio-max at harvesting, weight of maize for m², diameter of stalk, height of plants with or without ear, SPAD). The treatment of bio-curing with the mixture of three strains of C. rosea (CRrO, CRM and CRr2) gives also the plants of wheat an improvement of quali-quantitative characteristics, as it is highlighted in the results concerning the qualitative parameters of maize (proteins, gluten, yellow index, % relative humidity, hectolitre weight).

Example 9 - capacity of the mixture of strains CRrO/CRM/CRr2 of modifying biochemical processes in wheat by modulating the production of proteins responsible for systemic resistance acquired and photosynthetic metabolism, by means of bio-curing of seeds

Test on hard wheat (var. Quadrato)

A test on hard wheat (T. durum desf.) var. Quadrato was carried out by using the mixture of C. rosea isolates wild type CRrO and two resistant isolates CRM and CRr2, applied to seeds by means of the technique of curing. The inoculation of the antagonists consisted in curing maize, prior to seeding, with a suspension containing the three fungal strains of C. rosea (CRrO, CRM and CRr2) so that 10 x 10⁶ UFC of each strain were absorbed on the surface of the same. In particular, wheat maize of the var. Quadrato were biologically cured in thesis 1 (CRrO/CRr1/CRr2 strains) and remained not treated in thesis 2. The seeds of the two theses were then arranged in a container with sterile soil and incubated in a growing chamber at 25°C with a photoperiod of 10 hours.

In particular, the antagonistic fungi were applied to maize by using a pilot system of seeds curing. Both adjuvants and substances able to keep fungal conidia vital for not less than 6 months after treatment were added to fungal mixture. From the originated plants samples of leaves were withdrawn 90 days after seeding, and precisely in the stage of end of heading. The test was carried out in three replications, each one containing 100 seeds.

The leaves were powdered in liquid nitrogen and kept at -70°C. For protein extraction, leaves thus powdered were subjected to washing with ethanol and deionised water. After centrifugation, the pellet obtained was dried in speed- vacuum and re-suspended in 1 % (w/v) SDS, 20% (v/v) glycerine, 40 mM DTT, 50 mM Tris-HCI, pH 7,5. The protein content was determined according to Bradford method. Aliquots of extracts obtained from samples not treated and treated with the mixture of the three strains of C. rosea were precipitated with chloroform/methanol and the protein pellets were dried and subjected to two- dimensional electrophoresis. The passages in IEF (isoelectrophocusing) were realized on IPG Dry-strips 13 cm in a pH non-linear gradient 3-11 (GE-Heathcare), the proteins were separated bi-dimensionally by SDS-PAGE in a polyacrylamide gel 13% (w/v). Gels were stained with Coomassie and analysed by means of software ImageMaster 2D Elite (Amersham Biosciences). The protein spots were excised from gel, digested with trypsin and analysed in MALDI-ToF MS. The identification of proteins was realized by means of the research engine MASCOT, by using the databases NCBInr protein and Swiss-Prot TrEMBL and the peptide mass fingerprinting (PMF).

In figs. 43 and 44 there are shown: a bi-dimensional analysis of the proteins extracted from leaves of wheat coming from the two theses compared: control (not treated) and biologically cured with the mixture of the three strains of C. rosea, and the individuation of proteins whose expression is regulated by treatment of grain maize with the mixture of the three strains of C. rosea. The up-regulated proteins are circled in black, the down-regulated ones are circled in white.

With respect to control (not treated), In the samples biologically cured with C. rosea, 11 spots are individuated corresponding to 8 different proteins with a significant difference in the expression level; 6 proteins result to be up-regulated and 2 proteins result to be down-regulated.

On the basis of their function, such proteins were subdivided in two groups:

1. N° 6 proteins involved in mechanisms of defence/stress responses

2. N° 2 proteins Involved in the photosynthetic metabolism

Identification of proteins

table 4 - Up-regulated proteins from pathogenesis (defence/stress response):

Table 5: Down-regulated proteins involved in the photosynthesis

Conclusions

The results highlight how the biologic curing carried out with c. Rosea (strains CRrO/CRr1/CRr2) activates different molecular mechanisms of defence in seed and plant, typical of SAR (systemic acquired resistance). The plants can be sensitised (activated) to react faster and more effectively after attacks by pathogens and insects. Another interesting aspect highlighted, from a biochemical-functional point of view, is represented by the down-regulation of two among the most important proteins for photosynthesis and photo-respiration: the oxygen-evolving enhancer protein-2 and ribulose bisphosphate carboxylase. This would show how, by reduction of carbon fixation and glucose synthesis, the system activates an energy saving form to be intended to the activation of mechanisms of defence.

Therefore, among the advantages deriving from the biological curing of seed there are enlisted: more resistant plants, reduced environmental impact (soil contamination, water beds...), possibility of use in biologic productions, minor risks linked to their use for operators, lower costs.

The mixture of strains of C. rosea (CRrO/CRr1/CRr2) induced many metabolic variations, in particular the up-regulation of proteins from pathogenesis (defence/stress responses) linked to SAR, and the down-regulation of proteins involved in photosynthesis. It is the first time that such phenomenon is demonstrated on hard wheat after application to seeds.

Therefore, the strains of C. rosea (CRrO/CRr1/CRr2) are optimal candidates for production of biocontrol agents (biologic pesticides) since they are resistance induction agents in plants thanks to the characteristics of stimulators of systemic acquired resistance.

Example 10 - capacity of the mixture of strains CRrO/CRr1/CRr2 of modifying biochemical processes in tomato by modulating the production of proteins responsible for systemic acquired resistance, energy and photosynthetic metabolism, by application in drip irrigation

10.1 - Test on tomato (var. Perfect peel)

A test on tomato (L esculentum) was carried out by using a mixture of the isolates of C. rosea wild type CRrO and two resistant isolates CRM and CRr2 applied to plants by drip irrigation. The inoculation of the antagonists consisted in carrying out 3 applications by means of drip irrigation on plants of tomato at BBCH 12-13 development stage of a suspension containing the three fungal strains of C. rosea (CRrO, CRM and CRr2), 10-12 days the one after to the other one (up to reach a stage of development of plants BBCH 16-17). The test was realized in three replications, each one made up of 9 plants. The plants were treated by means of application of a conidial suspension containing 2 x 10⁶ UFC of each one of the three fungal strains in mixture. For each plant, a volume of application equal to 100 ml was used. Plants were bred in phytocell in controlled thermo-hygrometric conditions, with a photoperiod equal to 16 daily hours, 12000 lux, 24°C and 8 nightly hours at 16°C. Samples of leaves were withdrawn from plants and analysed 15 days after the last application, in number of 2 samples for replication. The leaves were powdered in liquid nitrogen and kept at -70°C. For protein extraction, leaves thus powdered were subjected to washing with ethanol and deionised water. After centrifugation, the pellet obtained was dried in speed-vacuum and re-suspended in 1 % (w/v) SDS, 20% (v/v) glycerine, 40 mM DTT, 50 mM Tris-HCI, pH 7,5. The protein content was determined according to Bradford method. Aliquots of extracts obtained from samples not treated and treated with the mixture of the three strains of C. rosea were precipitated with chloroform/methanol and the protein pellets were dried and subjected to two-dimensional electrophoresis. The passages in IEF (isoelectrophocusing) were realized on IPG Dry-strips 13 cm in a pH nonlinear gradient 3-11 (GE-Heathcare), the proteins were separated bi-dimensionally by SDS-PAGE in a polyacrylamide gel 13% (w/v). Gels were stained with Coomassie and analysed by means of software ImageMaster 2D Elite (Amersham Biosciences). The protein spots were excised from gel, digested with trypsin and analysed in MALDI-ToF MS. The identification of proteins was realized by means of the research engine MASCOT, by using the databases NCBInr protein and Swiss-Prot T rEMBL and the peptide mass fingerprinting (PMF).

In fig. 45 it is shown a bi-dimensional analysis of the proteins extracted from leaves of tomato coming from the two theses compared: control (not treated) at left, treated by leaf drip irrigation with the mixture of the three strains of C. rosea, at right; in fig. 46 it is shown a table for identification of the proteins expressed differentially (up- and down-regulated) in leaves of tomato treated with the mixture of the three strains of C. rosea.

With respect to control (not treated), in samples treated via drip irrigation with C. rosea, 30 spots are individuated corresponding to 30 different proteins with a significant difference in the expression level; 22 proteins result to be up-regulated and 8 proteins result to be down-regulated.

On the basis of their function, such proteins were subdivided in four groups:

1. N° 18 proteins involved in mechanisms of defence/stress responses (up- regulated)

2. N° 3 proteins involved in energy metabolism (up-regulated) 3. N° 7 proteins involved in photosynthetic metabolism (down-regulated)

4. N° 2 proteins, one being up-regulated and the other being down-regulated, not identified yet.

Conclusions

The results highlight how the treatment via drip irrigation with C. Rosea (strains CRrO/CRr1/CRr2) activates different molecular mechanisms of defence in the plant of tomato, typical of SAR (systemic acquired resistance); linked to energy metabolism (beta subunit of ATP synthethase), linked to photosynthetic metabolism. The mixture of strains of C. rosea (CRrO/CRr1/CRr2) induced many metabolic variations, in particular the up-regulation of proteins from pathogenesis (defence/stress responses) linked to SAR and of proteins linked to energy metabolism, and the down-regulation of proteins involved in photosynthesis. It is the first time that such phenomenon is demonstrated on tomato after the application to plants via drip irrigation.

Another interesting aspect highlighted, from a biochemical-functional point of view, is represented by the down-regulation of two among the most important proteins for photosynthesis and photo-respiration: the oxygen-evolving enhancer protein-2 and ribulose bisphosphate carboxylase. This would show how, by reduction of carbon fixation and glucose synthesis, the system activates an energy saving form to be intended to the activation of mechanisms of defence. Therefore, the strains of C. rosea (CRrO/CRr1/CRr2) are optimal candidates for production of biocontrol agents (biologic pesticides) since they are resistance induction agents in plants thanks to the characteristics of stimulators of systemic acquired resistance.

Example 11 : control of Orobanche spp. on horticultural species (fennel and broccoli) outdoor by drench application of a mixture of strains of C. rosea (wild type CRrO and two fungicide resistant isolates CRM and CRe2)

Example 11 - test on plants of broccoli (Marathon F1)

A test on plants of broccoli (Brassica oleracea var. botrytis L.) Marathon F1 was carried out by using the mixture of 3 isolates of C. rosea: the wild type CRrO and two fungicide resistant isolates, CRM and CRr2. The inoculation was carried out by immersing alveolar containers containing the plants (at BBCH 13-14 stage), immediately before the transplantation outdoor, in a suspension containing the three fungal strains of C. rosea (CRrO, CRM and CRr2), so that 30000 UFC of each single fungal strain are absorbed by each plant. It was carried out a fall-winter test in 2015, in agro Dauno, in Foggia, and precisely in Cervara. The field chosen for the test had been previously cultivated with tomato, in the spring-summer cycle, and there had been found very strong infestations of Orobanche spp. The tests were arranged by using a split plot experimental scheme with 4 replications for each thesis compared, for a total number of 250 plants for replication. The crop system was at single row. The evaluation of the activity of control towards parasite Orobanche spp. was carried out on each plot at the end of crop cycle by calculating the average of the number of turions and the number of broccoli for plot infested by Orobanche spp. The presence of antagonistic fungi was checked by isolation on axenic substrate (selective PDAs) containing iprodione.

In fig. 47 it is shown the average of the number of turions for plot while varying treatment; in fig. 48 it is shown the percentage of plants of broccoli infested by orobanche. The data obtained confirmed that the contemporaneous application of the three fungal strains (CRrO, CRM and CRr2) of C. rosea in mixture gives the plants of broccoli a very high protection towards Orobanche spp. with an efficacy statistically higher, for P = 0,05, with respect to the control not treated.

11.2 Test on plants of fennel (Orbit F1)

A test on plants of fennel (Foeniculum vulgare) was carried out by using the mixture of 3 isolates of C. rosea: the wild type CRrO and two fungicide resistant isolates, CRM and CRr2. The inoculation was carried out by immersing alveolar containers containing the plants (at BBCH 13-14 stage), immediately before the transplantation outdoor, in a suspension containing the three fungal strains of C. rosea (CRrO, CRM and CRr2), so that 30000 UFC of each single fungal strain are absorbed by each plant.

It was carried out a fall-winter test in 2015, in agro Dauno, in Foggia, and precisely in Cervara. The field chosen for the test had been previously cultivated with tomato, in the spring-summer cycle, and there had been found very strong infestations of Orobanche spp. The tests were arranged by using a split plot experimental scheme with 4 replications for each thesis compared, for a total number of 250 plants for replication.

The evaluation of the activity of control towards parasite Orobanche was carried out on each plot at the end of crop cycle, by calculating the average of the number of turions and the number of plants of broccoli for plot infested by Orobanche spp. The presence of antagonistic fungi was checked by isolation on axenic substrate (selective PDAs) containing iprodione.

In fig. 49 it is shown the average of the number of turions for plot while varying treatment; in fig. 50 it is shown the percentage of plants of fennel infested by orobanche. The data obtained confirmed that the contemporaneous application of the three fungal strains (CRrO, CRM and CRr2) of C. rosea in mixture gives the plants of broccoli a very high protection towards Orobanche spp. with an efficacy statistically higher, for P = 0,05, with respect to the control not treated.

Conclusions

The experiments carried out in Agro Dauno, where the parasitic phanerogam "Orobanche spp." is endemic by now for many species cultivated among which solanaceae, brassicaceae and umbrellifereae, highlighted clearly that the mixture of strains of C. rosea (wild type CRrO and two fungicide resistant isolates CRM and CRr2) performs a clear action of containment on the species used in tests (fennel and broccoli). The use of the mixture of strains of C. rosea (wild type CRrO and two fungicide resistant isolates CRM and CRr2) by means of treatment to plants in nursery garden, or immediately before the transplantation for immersion of alveolar containers in a conidial suspension, allows to protect plants for the whole crop cycle of the same.

Therefore, the mixture of strains of C. rosea (wild type CRrO and two fungicide resistant isolates CRM and CRr2) can be used as bio-herbicide for controlling the various species of parasitic phanerogam on many cultivated species.

To the inventors' knowledge, it is the first time that a mixture of strains of C. rosea is used as bio-herbicide for controlling the parasitic plants of cultivated species. The use of the mixture of C. rosea (wild type CRrO and two fungicide resistant isolates CRM and CRr2) as bio-herbicide in nursery garden or immediately before the transplantation of horticultural plants allows remarkable costs saving for the reduction of damages caused by the parasite, also and above all for the absence of products both of synthetic and natural origin useful for Orobanche spp. containment.

Claims

1. Method for containment of phytopathogenic fungi in crops comprising the application of strains of Clonostachys Rosea fungi, characterized in that said strains comprise at least a strain selected among:

- CRrO, deposit number 252/2015 BGr GNLWT;

- CRM , deposit number 253/2015 BGr GNL1 ;

- CRr2, deposit number 254/2015 BGr GNL2.

2. Method according to claim 1 , characterized in that said crops comprise one or more of the following ones: tobacco, bean, wheat, broccoli, fennel, tomato, asparagus, grain.

3. Method according to claim 1 or 2, characterized in that said phytopathogenic fungi comprise one or more among the following ones: Rhizoctonia, Phytophthora,

Sclerotica, Fusarium, pyrenochaeta, phoma verticillium, orobanche.

4. Method according to any one of the preceding claims, characterized in that said strains comprise at least a strain selected among:

- CRrO, deposit number 252/2015 BGr GNLWT;

- CR , deposit number 253/2015 BGr GNL1 ;

- CRr2, deposit number 254/2015 BGr GNL2.

used in association with a synthetic fungicide.

5. Method according to claim 4, characterized in that said synthetized fungicide is a fungicide comprising at least one of the following active substances: iprodione, fenexamide and boscalid.

6. Method according to any one of the preceding claims, characterized in that said strains of Clonostachys rosea fungi comprise a mixture comprising:

- CRrO, deposit number 252/2015 BGr GNLWT;

- CRM , deposit number 253/2015 BGr GNL1 ;

- CRr2, deposit number 254/2015 BGr GNL2.

7. Crop substrate comprising a mixture comprising one or more of said isolated strains of C. rosea (CRrO, CRM , CRr2).

8. Curing mixtures to be applied to seeds of cultivated species for controlling parasites of plants belonging to Rhizoctonia, Phytophthora, Sclerotica, Fusarium,

Pyrenochaeta, Phoma, Verticillium genera, said mixtures comprising at least one of said isolated strains of C. rosea (CRrO, CRM , CRr2).