ES2470490A1 - Composition for the biological control of powdery mildew in plant cultures based on an ampelomyces strain (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

The present invention relates to a strain of ampelomyces isolated from pumpkin leaves, to its isolation process, to a composition that comprises it as well as to its uses in the biological control in vegetable crops of pests causing the disease known as powdery mildew, preferably in cucurbits (zucchini, squash, etc.) but also in other families such as solanaceae, rosaceae, asteraceae, umbelliferae, fabaceae, fruit or ornamental crops. (Machine-translation by Google Translate, not legally binding)

Description

FIELD OF THE INVENTION

The present invention falls within the field of biotechnology and more specifically within agrobiotechnology. Specifically, it refers to an Ampelomyces strain isolated from pumpkin leaves, to its isolation procedure, to a composition that comprises it as well as to its uses in biological control in plant crops of pests that cause the disease known as oídio, preferably in cucurbitaceae (zucchini, squash, etc.) but also in other families such as Solanaceae, Rosaceae, Asteraceae, Umbelliferae, Fabosaceae, Fruit Trees or Ornamental Crops.

BACKGROUND OF THE INVENTION

Odyde is a disease caused by the fungus of the same name, which acts on leaves and stems in weak plants, normally located in the shade and in areas or times of high humidity. Odes are strict parasites, they have never been cultivated on nutritional media.

The symptomatology is characterized by whitish and powdery spots on the bundle of the leaves of certain plant species that are initially small and more or less circular in shape. As the disease progresses, these spots grow and cover the entire vegetative system, leaves and stems. In cases of exceptionally strong attacks, even the fruits may be affected.

The mildew is common in cucurbit leaves (zucchini, cucumbers, melons and watermelons), although we often find that species of whitish flour that leaves the leaves drying in the rose bushes, in the tender shoots of peaches or nectarines and in the vine, among other plant species.

At present there are certain active agents that are used to treat this disease, such as imazalil, pyrazophos and triforin that act very well as systemic antiodes, but which in the current community review of admitted phytosanitary compounds have not been included. in Annex I of Council Directive 91/414 / EEC of July 15, 1991, on the marketing of plant protection products.

In addition, regarding the treatment of various pests, chemical substances are often not allowed, since when you want to make integrated production in a given crop, biological control or biocontrol is always a priority as a palliative measure of a disease caused For a plague

Therefore, once a type of disease has been identified, only a very limited group of chemicals will be allowed to control it. Thus, in the case of the newspaper, if there is presence of symptoms and / or conditions favorable to the disease, there will be restrictions regarding the limitation of the number of treatments with products of the same chemical family - to avoid the occurrence of resistance, according to FRAC Fungicide Resistance Action Committee recommendations - or not interfere with auxiliary entomofauna, for example. In addition, if organic agriculture is chosen, it is essential to use biological control.

Biological control or biocontrol is a method of control of pests, diseases and weeds that consists of using living organisms in order to control the populations of another organism. It is an agricultural method of pest control (insects, mites, weeds, plant diseases, etc.) that uses predators, parasites, herbivores, microorganisms or other natural means that is an important component of integrated pest control as it is of great economic importance for agriculture.

With regard to plant colonization caused by bacteria, yeasts and fungi, it can be found on the surface of the leaves generating an epiphytic flora that can be eliminated with greater potential thanks to biological control, since this type of agents can adapt more quickly to nutritional changes and above all they are the most adapted to specific environmental conditions.

From an ecological point of view, fungi are the most suitable microorganisms to be able to attack the soil (Blanger RR, Avis TJ 2002. Ecological processes and interactions occuring in leaf surface fungi. In: Phyllosphere Microbiology. SE Lindow, EI Hecht-Poinar and VJ Elliot Editors, American Phytopathology Society St. Paul, Minn. Pp. 193207; Caffi, T., Legler, SE, BugianI, R., Rossi, V. 2013. Combining sanitation and disease modeling for control of grapevine powdery mildew European Journal of Plant Pathology,

135: 817-829; Kiss L. 2003. A review of fungal antagonists of powdery mildews and their potential as biocontrol agents. Pest Management Science, 59: 475-483). Kiss (2003) conducted a review of the ecology, modes of action and efficacy of biological control of

approximately 40 species that were presented as natural antagonists of the soil or tested as potential biocontrol agents.

Of all of them, the one belonging to the genus Ampelomyces is the best known and studied as the natural antagonist of powder. Unlike other fungi, this mycoparito acts directly by invasion and destruction of the cytoplasm of the host. It colonizes and destroys the conidium (asexual structure) and immature ascocarp (sexual structure) of the mycelium of the odicium (Kiss L., Russell JC, Szentiv�nyi O., Xu X., Jeffries P. 2004. Biology and biocontrol potential of Ampelomyces mycoparasites, natural antagonists of powdery mildews fungi. Biocontrol Science and Technology, 14: 635-651).

The mechanism of action of this biological control agent is by parasitization of the pathogen, therefore a certain incidence of the disease is necessary for the antagonist to act. Synthesis of toxins by the genus Ampelomyces has never been detected (Kiss, 2008. Intracellurar mycoparasites in action: interactions between powdery mildew fungi and Ampelomyces. British Mycological Society Symposia Series.

T.B.M. Society: 37-52).

When talking about biological control for myopathies, one must always think that a certain incidence of the disease should be tolerated since these fungi can only act on established infections (Fokkema NJ 1993. Opportunities and problems of control of foliar pathogens with microorganisms, Pesticide Science, 37: 411-416; Gilardi, G., Baudino, M., Garibaldi, A., Gullino, ML 2012. Efficacy of biocontrol agents and natural compounds against powdery mildew of zucchini. Phytoparasitica, 40: 147- 155).

The time that Ampelomyces needs to act against the odeio is between 5-7 days. When a very intense spread or sporulation of the pathogen occurs, Ampelomyces cannot completely control it. It is in these cases when Ampelomyces reduces the severity of the disease and limits the negative effects that the odyium produces on the plants, but does not eradicate the disease.

This mycoparito is more effective when the relative environmental humidity exceeds 80%

(Gilardi G., Manker D.C., Garibaldi A., Gullino M.L. 2008. Efficacy of the biocontrol agents Bacillus subtilis and Ampelomyces quisqualis applied in combination with fungicides against powdery mildew of zucchini. Journal of Plant Diseases and Protection 115: 208-213). The fact of being one of the most studied biocontrol agents has caused a commercial interest for the development of compositions based on this fungus.

In Europe, a product formulated from a strain of Ampelomyces quisqualis M-10 isolated in Israel in 1989 and distributed in Europe under the trade name of AQ10� is included in Annex I.

However, innumerable studies have been carried out with this product to determine its effectiveness against oidium in cucurbitaceous and vine plants, obtaining disparate, inconclusive and not very positive results (DIK AJ, VERHAAR MA, BELANGER RR, 1998. Comparison of three biological control agents against cucumber powdery mildew (Sphaerotheca fuliginea) in semi-commercial-scale glasshouse trials, European Journal of Plant Pathology, 104: 413-423; ELAD Y., KIRSHNER N., YEHUDA N., SZTEJNBERG A. 1998 Management of powdery mildew and gray mold of cucumber by Trichoderma harzianum T39 and Ampelomyces quisqualis AQ10. BioControl, 43: 241-251 SHISHKOFF N., McGRATH MT 2002. AQ10 biofungicide combined with chemical fungicies and AddQ spray adjuvant for control of cucurbit powdery mildew in detached leaf culture. Plant Disease,

86: 915-918; Kiss et al., 2004; ANGELI D., di MARINO E., MAINES L., MESCALCHIN E., 2006. Grapevine powdery mildew and the mycoparasite Ampelomyces quisqualis in Trentino vineyards (nothern Italy). Bulletin OILB / SROP, 29: 89-92; BRAND M., MESSIKA Y., ELAD Y., RAV-DAVID D., SZTEJNBERG A. 2009. Spray treatments combined with climate modification for the management of Leveillula taurica in sweet pepper. European Journal of Plant Pathology, 124: 309-329).

This fact is due to different causes, either that the microorganism does not adapt to the climatology of the area to be treated or that a product with little viability and effectiveness has been formulated over time.

The innovative aspect proposed by the applicant of the present invention in relation to the aforementioned state of the art is the production and formulation of a product for the biological control of powdery mildew, from a new Ampelomyces strain encoded by the inventors with the research code Ampelomyces spp. (22/2005) isolated from zucchini leaves in the province of Lleida, being a product produced and formulated with an indigenous strain isolated in Spain and therefore adapted to the Mediterranean climate, for a future commercial application.

This strain is absolutely stable and efficient in the climatic conditions of the Mediterranean area, being able to be incorporated into a composition with a high degree of efficiency in the biological control of the soil, thus solving the problems of the products available in the prior art as well as the methods of current treatment of this type of disease, being a technically beneficial alternative for the control of hearing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a strain of Ampelomyces with a deposit number in the Spanish Type Culture Collection (CECT) 20749, which is useful for biological control of odyium, more specifically in cucurbitaceae (zucchini, squash, cucumber, melon, watermelon, etc.) and even more specifically in zucchini, but also in other species including other families, such as strawberry, vineyard, apple tree, pear and peach tree. It also refers to a composition comprising this strain with a deposit number in the Spanish Type 20749 Crop Collection and the use of this composition as a biological control agent for plants, preferably for cucurbitaceae and more preferably for zucchini.

The present invention has numerous technical advantages, for example, for cucurbitaceae and other vegetable crops such as strawberry, vineyard, apple tree, pear tree, peach tree etc., it would mean significantly reducing sulfur applications, a product currently widely used in clean farming systems. , but that interferes in a very unfavorable way in the auxiliary fauna (especially �caros) that could be introduced.

The applications of the composition containing strain CECT 20749 in a unique way or by interleaving permitted synthesis oidicides, would imply the technical advantage of eliminating or drastically reducing their residues well below the authorized MRL (Maximum Residue Limit). With the use of the composition object of the present invention, biological control procedures could be available that actually make this type of clean agriculture possible since they currently do not exist.

The progress and improvement that is obtained with the composition object of the present invention in the quality of fruits and vegetables due to the absence of waste and the elimination of the environmental impact that these entail, fully justifies its use and the need that exists today in the state of the art as a method of control of soil in vegetable crops.

In the present invention it is demonstrated how the treatment with the strain Ampelomyces spp. (22/2005) (Strain with access number to the Spanish Type Culture Collection 20749) causes greater biological control of the disease by preventing sporulation of the soil in plant species with respect to plant species without treatment with said strain .

Therefore, a first aspect of the invention relates to an isolated strain of the genus Ampelomyces with a deposit number in the Spanish Type Culture Collection (CECT) 20749, hereinafter "Ampelomyces strain CECT 20749" or "strain CECT 20749" . This strain was deposited with the CECT on 31.3.2009. This fungus is capable of growing in vitro in, for example, but not limited to, the culture medium MEA and PDA (the acronym for "malt extract agar" and "potato dextrose agar" respectively) at a temperature range of 18-25� C in aerobic conditions. The scientific classification of the fungus with deposit number CECT 20749 of the present invention is: Kingdom: Fungi / Phylum: Ascomycetes.

Among the plant species to be treated, one of those with the greatest agronomic interest is the Cucurbitaceae family, which includes species such as zucchini.

The term "cucurbit" in the present invention refers to any plant included within the Cucurbit family, within the order Cucurbital and comprising one or more morphologically similar species phylogenetically related.

The strain of Ampelomyces CECT 20749 was isolated directly from pumpkin leaves infected by o�dio (Podosphaera fusca) following the following procedure:

a) Observation by stereoscopic microscope of the leaves infected by o'dio in search of an area where there are similar structures to pycnidia formed by the possible biocontrol agent,

b) Once the area where these structures are located is delimited, the surface of the sheet is scratched with a scalpel on a slide, these structures are again observed by stereoscopic microscopy and separated on another slide,

c) A drop of ethanol 96� (v / v) is dispersed for 15 seconds on the isolated pycnidia and then the drop is rinsed with sterile water to remove ethanol,

d) The isolated pycnidia are deposited on Petri dishes with MEA medium (the acronym for "malt extract agar") or on PDA medium (the acronym for "potato dextrose agar") and incubated under controlled light conditions. temperature.

With this technique, the strain of Ampelomyces CECT 20749 was isolated, which due to its nutritional requirements requires regular reseeding in fresh medium at a temperature in a range between 18 and 25 � C.

For long-term preservation (approximately 12 months) a suspension of the fungus with glycerol (20%) is carried out and stored at -70�C, its reactivation being necessary before this period of time elapses.

To obtain large quantities of Ampelomyces CECT 20749 it is necessary to find a suitable solid production or liquid production system. Therefore, different studies have been carried out to optimize the production methodologies in both solid and liquid phases. In relation to another aspect, the present invention relates to a composition of Ampelomyces CECT 20749 in solid form with some adjuvants and protectors to ensure the viability and effectiveness of the final product for approximately 12 months. During this period of time and keeping the formulated product at 25C the viability only minimally decreases. There is therefore no need for vacuum packaging.

In relation to another aspect, the present invention relates to the use of the Ampelomyces CECT 20749 strain for biological control of fungal pests that cause damage. The supra-specific nature of the selected strain of Ampelomyces CECT 20749 manages to act as an antagonist colonizing and considering as its vital substrate the different fungi more or less specific for each of the plants, which are causes of the diseases known as o�dio in a generic way, and with it it makes possible its effective use in a wide range of vegetables.

Among the cultures to be treated, the present invention provides a strain of Ampelomyces CECT 20749 and the composition containing it, ready to be used in the biological control of fungal pests that cause damage in both annual or multiannual agricultural crops, whether of low size and tree nature, both outdoors and under greenhouse, and in ornamental crops of plants or trees used in gardening and ornamentation of any kind.

Preferably the crops to be treated are:

Annual agricultural crops including carrots, lettuce and strawberries and in which the annual renewal of vegetation (artichokes) is caused. We consider among them the crops of orchard, both outdoors and under greenhouse, and that can be:

 Cucurbitaceae: among them zucchini [Cucurbita pepo L.], melon [Cucumis melo], cucumber [Cucumis sativus L.], watermelon [Citrullus lanatus (Thunb.)], Pumpkin [Cucurbita maxima], etc. Causal fungi in these species are Podosphaera fusca (Fr.) Braun & Shishkhoff and Golovinomyces cichoracearum (DC).

 Solan�ceas as tomato [Solanum licopersicum] for fresh and processed consumption, eggplant [Solanum melongena L.], pepper [Capsicum annuum], etc. The causal fungi of the soil in these species are Leveillula taurica, Erysiphe cichoracearum.

 Asteraceae (Compounds) especially artichoke [Cynara cardunculus var. Scolymus], Lettuce [Latuca sativa L.], etc. The causal fungi of the soil in these species are Leveillula taurica, Erysiphe cichoracearum.

 Rosaceae such as strawberry [Fragaria xananassa], strawberry [Fragaria vesca L.], raspberry [Rubus idaeus L.], etc. The causal fungi of the soil in these species are Podosphaera aphanis, Sphaeroteca humuli.

 Umbelliferous notably Carrot [Daucus carota L.], etc. The causative fungi of odiium in these species are Erysiphe heraclei.

 Other botanical families such as peas [Pisum sativum] and legumes fabaceas. Causal fungi in these species are caused by Erysiphe polygoni.

Multiannual agricultural crops, as a whole outdoors and deciduous. Include:

  Grape [Vitis vinifera] for winemaking and for table. The causal fungi of the powder in these species are Uncinula nec�tor.

 Bone fruits including peach [Prunus persica L.] and nectarine, apricot tree [Prunus armenica], plum [Prunus domestica], etc. Causal fungi in these species are Sphaerotheca pannosa, Podosphaera tridactila

 Pepita fruit trees include apple tree [Malus domestica], quince [Cydonia oblonga], etc. Causal fungi in these species are Podosphaera leucotricha, Oidium farinosum.

 Nuts fruit trees include hazelnut [Corylus avellana], pistachio [Pistacia vera], etc. The causative fungi of odiium in these species are Phyllictinia sp.

Ornamental crops, and the plants or trees used in gardening and in street ornamentation, the composition of Ampelomyces CECT 20749 will be applied under the same criteria as for the previous crops, being able to specify the uses:

In ornamentals of small size such as rose bushes [Rosa canina L.] where the causal fungi of the odium in these species are odeio caused by Sphaerotheca pannosa. Hydrangeas [Hydrangea macrophylla] where the causal fungi of the odeium in these species are Miicrosphaera polonica, etc., you can follow the indications for horticultural in the greenhouse or outdoors, depending on the conditions of their cultivation. To avoid phytotoxicities, only rapeseed oil or similar is indicated as a supplementary adjuvant.

For outdoor gardening and trees in street ornamentation, the type of treatments at the end and beginning of the season is especially proposed to reduce the inoculation of pathogens. Specifically it can be indicated for deciduous trees.

A special case is the affectation of the banana [Platanus x hispanica or Platanus hybrida, Platanus orientalis x occidentalis, or Platanus acerifolia], and its affectation by powder is caused by Microsphaera alni. Due to their size, treatments to reduce the inoculum of the pathogenic fungus are of maximum interest.

If there is an affectation during the development of the outbreaks, early applications with the formulation of the selected strain of Ampelomyces CECT 20749 allow an acceptable control of the disease since the successive generations of Ampelomyces CECT 20749 continue to colonize the new affected leaf surface that is It is forming.

The nature of the selected strain of Ampelomyces CECT 20749, the particularities of the multiplication method and its exclusive composition, lead to the total safety of applications, both in crops where the harvest is expected to be destined for consumption, as well as in landscaped areas and / or roads where living beings can travel, be people

or animals

The application and use of the selected strain of Ampelomyces CECT 20749 is also an object of the invention. For annual agricultural crops, the application of the composition comprising the selected strain of Ampelomyces CECT 20749 is considered, both in the initial stages of the presence of powder in plants (severity level of 1-5%) looking for a practically preventive effect, as in the most advanced phases in which the oily stains can exceed 50% of the surface and in which a control effect can be achieved and even eradicated. This will recover the functionality of the sheet and its photosynthetic effectiveness.

The number of applications can vary between a single application � 2 successive, depending on the culture, the degree of infestation of the pathogen, and especially according to the conditions of relative humidity, at the time of application and subsequently to it. Under normal conditions, maximum efficiency is achieved by repeating a second treatment between 5 –10 days of the first, especially if the weather has been windy which leads to environmental dryness and / or if the crop has grown strongly, which means a lot of new surface without being treated. A second application is also recommended if the level of affectation by oídio is or very low, or there is a lot of surface with spots of oídio.

Exceptionally, if the crop is in the greenhouse, if it is kept closed at a low temperature and the application is carried out avoiding the central hours of the day, a relative humidity can be achieved that equals or exceeds 60% so that the conditions are the most suitable for the performance of the composition of the selected strain of Ampelomyces CECT 20749 in a single application that ensures the effectiveness of the treatment. Outdoors and away from optimal conditions, applications should be repeated.

For the purpose of the present invention, "pycnide" is understood as a globose or pear-shaped structure, whose plectenchymal wall is internally covered by congenital cells and is open by an oyster. For the purpose of the present invention, "pycnidiospores" means spores caused by pycnidia.

The effective concentration of application is between 1 * 107 and 5 * 106 pycnidiospores / mL of the composition of the selected strain of Ampelomyces CECT 20749 which is achieved with a dose of 6-3 grams of composition per liter of broth (600 - 300 g / Hl), with an expense of 250 to 800 L / Ha of broth, which means between 3000 grams and 750 grams per hectare treated.

In order to promote the best conditions for the performance of the composition object of the present invention of the selected strain of Ampelomyces CECT 20749, the possibility of adding a vegetable oil formula (rapeseed oil or similar, to 2.5 ml / L) in order to ensure wetting, or a derivative of coniferous extract (at 0.5 - 1 ml / L) for the same purpose as above and an additional photoprotective effect. In both cases the products are compatible with the formulation of the selected strain of Ampelomyces CECT 20749.

The application will be carried out by foliar spraying of the broth, either directly with a piston or membrane pump, in manual devices or in tanks coupled to tractors, with pneumatic support (atomizer) or not. In all cases, the total coating of the sheets will be guaranteed by the treatment. In the greenhouse, a nebulizer device could also be used if the dispersion does not exceed 20 g / L, and the temperature of the broth is below 30 ° C.

In the whole of multi-annual agricultural crops, it is proposed to apply the composition of the selected strain of Ampelomyces CECT 20749 for two types of treatments, achieving a direct effect on the affectation by odiium:

a) in spring, in the initial stages of the presence of powder on the leaves (severity level of 1-5%) looking for a practically preventive effect, in the stages close to harvest, in which the spots of o� It can exceed 20% of the leaf surface and in which a control effect can be achieved and even eradicated. This will recover the functionality of the sheet and its photosynthetic effectiveness. This moment of application is especially effective for the pathogenic apple fungus.

b) Causing the decrease of the inoculation of the pathogen, with a post-harvest treatment, with the leaves still functional, prior to its fall, and repeating the application in spring with the budding leaves on the shoots, or the branches with the Freshly opened sheets where appropriate. This strategy allows, in plots large enough to avoid external contamination, to continue treatment campaigns only with sulfur-based formulations preventing the occasional conditions of maximum risk for the onset of new infections.

The effective concentration and doses for applications for multiannual cultures are equivalent to those described for annual crops. Being the volumes of vegetation, in the cases of trees and vineyards the volume of broth will increase proportionally, being able to set between 350 and 1000 L. Also the doses per hectare of formulated can vary between 1 and 3.5 Kg.

On the other hand and not least, in order to integrate the potential biological control agent with other treatments for the control of mildew, mildew and insects for the purpose of the present invention, the compatibility of the selected strain of Ampelomyces CECT 20749 with other active substances used to control these diseases and pests.

Highlight the compatibility of the selected strain of Ampelomyces CECT 20749 with sulfur which is the most commonly used oidicide product today.

In relation to another aspect, the present invention relates to the method of multiplication of the selected strain of Ampelomyces CECT 20749 for its production, which entails the presentation of vital forms that guarantee the rapid and powerful action for the control of fungal pat� target genos, which allows its use in a wide range that more than covers the scope of possible environmental conditions for the development of the set of pathogenic fungi, and that we define in the parameters characterized as follows:

temperature: between 5� and 35� C. relative humidity: equal to or greater than 55%, in the application and in the 3 subsequent hours. insolation: indifferent whether in sunny or shaded areas.

The exclusive composition object of the present invention comprising the strain of Ampelomyces CECT 20749 ensures a comfortable handling and allows its use in the usual conditions for the application of foliar fungicides in terms of methodology and tools, since it does not need special transport conditions since It is done at room temperature. It also does not require prior preparation to incorporate into the application broth due to its perfect dispersion when emptying the dose directly into the water for spraying. Additionally, it has been proven during 12 months of storage that the product formulated and vacuum packed has the same viability as the product formulated and packaged under normal conditions.

It is therefore an object of the invention an isolated fungus of the genus Ampelomyces with the deposit number CECT 20749.

According to a second aspect, the present invention relates to a method of an isolated fungus of the genus Ampelomyces comprising the following steps:

a) .- take pumpkin leaves infected with o�dio (Podosphaera fusca), According to another aspect, the strain of Ampelomyces CECT 20749 isolated is subsequently seeded in fresh medium at a temperature in a range between 18 and 25 � C.

b).

observe by stereoscopic microscope the leaves infected by o�dio

in search of some area where there are similar structures to pycnidia

formed by the possible biocontrol agent,

C).-

delimit the area where these structures are located, called pycnidia,

Y

 scratch in that area  the surface of the blade with a scalpel on a

slides,

d).

observe the pycnidia by stereoscopic microscope and separate in another

slides,

and).

disperse a drop  of ethanol 96� (v / v) for 15 seconds over

pycnidia isolated on the slide and then rinse the drop with

sterile water to remove ethanol,

F).

deposit isolated pycnidia on Petri dishes with MEA medium or medium

PDA and,

g).

incubate the pycnidia of stage f) under controlled light conditions and

temperature obtaining the Ampelomyces CECT 20749 strain.

According to another aspect, the strain of Ampelomyces CECT 20749 isolated is preserved for 12 months in the form of a suspension with 20% glycerol and at a temperature of at least -70�C, its reactivation being necessary before said period of time elapses .

Another important aspect of the present invention is a composition comprising the isolated fungus of the genus Ampelomyces with the deposit number CECT 20749. Preferably, the composition is in solid form and is preserved in the absence of preservatives at temperatures between 20-25 �C. The composition of the present invention is preferably at a concentration between 1 * 107 and 5 * 106 pycnidiospores / mL of Ampelomyces CECT 20749.

Another object of the present invention is the use of the isolated fungus of the genus Ampelomyces with the deposit number CECT 20749 or of the composition comprising it as an agent for biological control against plant infections. Preferably the plant is from the family of cucurbitaceae, Solanaceae, Rosaceae, Asteraceae, Umbelliferae, Fables, fruit trees or ornamental crops.

According to another important aspect, the present invention relates to a method of biological control of the soil of a plant with respect to a control comprising the steps of:

a) .- contacting the affected part of the plant with a composition according to any of claims 5 to 7, and b) .- allowing the composition of any of claims 5 to 7 to act.

Preferably, in step a) the composition is contacted with the plant by spraying an aqueous dispersion containing it. Preferably, the aqueous dispersion contains a concentration between 1 * 107 and 5 * 106 pycnidiospores / mL of the aforementioned composition.

Another object of the present invention is the use of the composition comprising the isolated fungus of the genus Ampelomyces with the deposit number CECT 20749 for biological control against plant infections in an integrated pest control program.

DESCRIPTION OF THE FIGURES

In order to complement the description that has been carried out, as well as to help a better understanding of the features of the invention, according to some examples made, the following are shown here, for illustrative and non-limiting purposes, figures:

Figure 1. Represents the Disease Incidence Index (IIE), which represents the% of surface area occupied per year based on the total surface area of the zucchini plant leaves of the Black Beauty variety grown in the greenhouse. Where CK is the control treatment performed with water and the rest are treatments with Ampelomyces CECT 20749 at different concentrations: 106 pycnidiospores / mL, 5 * 106 pycnidiospores / mL and 107 pycnidiospores / mL. Different letters indicate significant differences between treatments according to a separation of t-Student averages (p <0.05).

Figure 2. Represents the Disease Incidence Index (IIE), which represents the% of surface area occupied by soil based on the total surface area of melon plants grown in the greenhouse. Where CK is the control treatment performed with water and the other is the treatment with Ampelomyces CECT 20749 at a concentration of 107 pycnidiospores / mL. Different letters indicate significant differences between treatments according to a separation of t-Student averages (p <0.05).

Figure 3. Represents the Disease Incidence Index (IIE), which represents the% of surface area occupied by soil based on the total surface area of Brookfield Gala apple leaves in the field. Where CK is the control treatment performed with water and the other is the treatment with Ampelomyces CECT 20749 at a concentration of 107 pycnidiospores / mL. Different letters indicate significant differences between treatments according to a separation of t-Student averages (p <0.05).

Figure 4. Represents the Disease Incidence Index (IIE) that represents the% of surface area occupied by odiium based on the total surface area of Black Beauty zucchini leaves treated with water (CK), with the potential biocontrol agent Ampelomyces CECT 20749 and with the commercial product AQ10� (commercial product containing Ampelomyces quisqualis strain AQ10) prepared at concentrations of 2.5 * 105, 106 and 107 pycnidiospores / mL.

EXAMPLES

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

Example 1: Calculation of application dose of the formulated product

In order to know what dose of the definitive formulation based on the research strain Ampelomyces spp. (22/2005) deposited in the CECT with the number 20749 provides optimum efficacy against the odicium, a trial was carried out on zucchini of the Black Beauty variety grown in the greenhouse. The species of powder that infects this type of crop is Podosphaera fusca.

A total of 80 artificially infected zucchini plants with a strain of Podosphaera fusca 04/05 mildew isolated by the Lleida IRTA center were used. The plants were divided into groups of 20 plants and in each group the following treatments were performed:

Control (CK): Zucchini plants of the Black Beauty variety infected with powder (P. fusca) and treated with water.

 Dosage of Ampelomyces CECT 20749 at 106 pycnidiospores / mL of treatment broth: zucchini plants of the Black Beauty variety infected with powder (P. fusca) and pulverized with Ampelomyces CECT 20749 produced according to the composition of the present invention. Four treatments were carried out with a separation between them of 7 days.

5 Dose of Ampelomyces CECT 20749 at 5 * 106 pycnidiospores / mL of treatment broth: Black Beauty zucchini plants infected with powder (P. fusca) and sprayed with Ampelomyces CECT 20749 produced according to the composition of the present invention . 5 treatments were performed with a separation between them of 7 days.

10 Dose of Ampelomyces CECT 20749 at 107 pycnidiospores / mL of treatment broth: zucchini plants of the Black Beauty variety infected with powder (P. fusca) and sprayed with Ampelomyces CECT 20749 produced according to the composition of the present invention. 5 treatments were performed with a separation between them of 7 days.

15 After the 4 treatments, all the leaves of the zucchini plants were cut and the Disease Incidence Index (IIE) that shows the degree of disease development is evaluated and calculated from the following mathematical formula:

IIE (%) = [[(a * 0) + (b * 1) + (c * 2) + (d * 3)] / 3n] * 100

Where a, b, c, and d correspond to the number of sheets assigned to the scale 20 category of values 0, 1, 2 and 3, and n is the total number of the observed leaves.

The different categories are listed as:

0.- There is no appreciable growth.

1.- Very little mycelium is observed, only a small part of the leaf. Approximately 10-20% of the surface of the sheet.

25 2.- Mycelium is observed. Approximately between 20-60% of the sheet.

3.- Abundant presence of mycelium. Approximately 60 to 100% of the surface of the sheet.

The results obtained are shown in Figure 1. Where treatment with Ampelomyces CECT 20749 of 106 pycnidiospores / mL reduced 78.4% of the disease, the

30 of 5 * 106 pycnidiospores / mL 79.9% and 107 pycnidiospores / mL 87.2%.

In zucchini plants there is a good control of the infection by o�dio (P. fusca) when treating with Ampelomyces CECT 20749. The higher the concentration used the more control there will be of the disease. It is advised to treat between 5 * 106-107 pycnidiospores / mL of treatment broth.

Example 2: Trials in melon and apple tree with Ampelomyces strain CECT 20749

Being Ampelomyces CECT 20749 an isolated strain of pumpkin leaves assumes a better adaptation of the biocontrol agent to this type of plants. However, we wanted to evaluate its effectiveness in other types of crops.

The effectiveness of the possible biological product in melon and apple tree was evaluated. The species of mildew that causes the disease in melon is also P. fusca, but in apple trees the causative agent of the infection is another species specifically Podosphaera leucotricha.

In melons, the greenhouse test was carried out on 40 plants artificially infected with the Podosphaera fusca 04/05 strain isolated by the Lleida IRTA center. The plants were divided into groups of 20 plants and in each group the following treatments were performed:

Control (CK): melon plants infected with powder (P. fusca) and treated with water. Dose of Ampelomyces CECT 20749 at 107 pycnidiospores / mL of treatment broth: melon plants infected with powder (P. fusca) and sprayed with Ampelomyces spp. 22/2005 produced according to the claimed composition. Four treatments were carried out with a separation between them of 7 days.

After the 4 treatments, all the leaves of the melon plants were cut and the Disease Incidence Index (IIE) was evaluated according to the mathematical formula explained in Example 1 which shows the degree of disease development.

The results are shown in Figure 2 and indicate that Ampelomyces CECT 20749 at a concentration of 107 pycnidiospores / mL is able to significantly reduce infection by melon mildew by 57.6%.

In apple trees, the attack by hate is produced by Podosphaera leucotricha. This test was carried out in a field of apple trees, located in the municipality of Lleida, of the naturally infected Brookfield Gala variety. A total of 30 trees with presence of the disease were selected.

The chosen trees were divided into two groups of 15 and in each group the following treatments were performed:

Control (CK): Brookfield Gala apple trees infected by o�dio (P. leucotricha) and treated with water.

Dose of Ampelomyces CECT 20749 at 107 pycnidiospores / mL of treatment broth: Brookfield Gala apple trees infected by o�dio (P. leucotricha) and foliarly sprayed with Ampelomyces CECT 20749 produced according to the claimed composition. Four treatments were carried out with a separation between them of 7 days. Prior to the treatments, 20 branches (10 of each face) were chosen from each selected tree to monitor the disease.

After the 4 treatments, all the leaves of the selected branches were cut and the Disease Incidence Index (IIE) was evaluated according to the mathematical formula explained in Example 2C showing the degree of infection development. The results are shown in Figure 3 and indicate that Ampelomyces CECT 20749 at a concentration of 107 pycnidiospores / mL is able to significantly reduce ear infection in apple trees by 60%.

Example 3: Compatibility tests of the Ampelomyces CECT 20749 strain with other agents

The compatibility of the Ampelomyces CECT 20749 strain with various synthetic active substances of oidicide, mildewide and insecticide effect was evaluated in order to incorporate the biological control agent into an integrated pest control program.

The methodology consists in making a dilution bank from a suspension of the microorganism with water. Subsequently, the mycoparasite is put in contact with a solution of the active substance tested prepared at the commercial concentration for a time not less than 30 minutes and the dilution bank is re-performed. If the concentration of the microorganism decreases in a logarithm, the active substance is considered incompatible with Ampelomyces CECT 20749.

Kind of product

Product trade name Active matter Dose (ppm) Compatibility

Oidicide

Stroby WG Kresoxim methyl 300 Incompatible

Laitri

Miclobutanyl 800 Incompatible

Laicon

Polyoxin B 2500 Compatible

Fungistop

Sulfur suspension 6000 Compatible

Flint

Trifloxystrobin 150 Incompatible

Mildicide

festival Cimoxanil + Cuprocalcium Sulfate 4000 Incompatible

Chelai flow

Folpet 4000 Incompatible

Cuneb Forte

Potassium Phosphite 2500 Compatible

24 AI

Copper hydroxide 3500 Compatible

Copper Lainco

Copper oxychloride 4000 Compatible

Lainco Broth

Cuprocalcium Sulfate 10000 Incompatible

Insecticide

Laotta Abamectin 1000 Compatible

Eina

Azadirachtin 1000 Compatible

Clarnet

C / orpirijos 2000 Incompatible

Cascade

Fluphenoxuron 1000 Compatible

Plainsman

Imidacloprid 1000 Compatible

AI 162

L cihalotrina 800 Compatible

Bulldog

Pyridaben 1000 Compatible

Spintor 480SC

Spinosad 250 Compatible

Table 3 shows the compatibility of Ampelomyces CECT 20749 with different active substances with oidicidal, mildicidal and insecticidal action.

Table 3 also shows the compatibility results, where one of the

15 the most notable is the compatibility of Ampelomyces CECT 20749 with sulfur, the use of which is very widespread in Spain. This fact offers us a possibility of integrating the two treatments for a possible control of the disease.

Comparative Example 4: Comparison between the effectiveness of Ampelomyces CECT 20749 and the commercial product AQ10�

20 In Zucchini leaves of the Black Beauty variety artificially infected with the 04/05 strain isolated in the IRTA of Lleida, the effectiveness of the Ampelomyces CECT 20749 strain was compared with the commercial product AQ10� (commercial product containing Ampelomyces quisqualis strain AQ10) at different treatment concentrations: 2.5 * 105 pycnidiospores / mL, 106 pycnidiospores / mL and 107 pycnidiospores / mL.

The effectiveness was evaluated after 7 days of having performed the treatments and the Disease Incidence Index (IIE) was calculated according to the mathematical formula explained in Example 1 that shows the degree of infection development.

The results obtained indicate the low effectiveness of AQ10� in reducing the

5 infection compared to the effectiveness of Ampelomyces CECT 20749 (Figure 4). The treatments with AQ10� resulted in a 5.5% reduction in disease by treating zucchini leaves at a concentration of 2.5 * 105 pycnidiospores / mL, 11.5% at a concentration of 106 pycnidiospores / mL and 12% in the treatment of 107 pycnidiospores / mL.

With treatments with Ampelomyces CECT 20749 disease reductions

10 were 61.5% in the treatment at 2.5 * 105 pycnidiospores / mL, 78% at 106 pycnidiospores / mL and 91.5% at the concentration of 107 pycnidiospores / mL.

Claims (14)

1.- Isolated fungus of the genus Ampelomyces with the deposit number CECT 20749. 2. Method of isolation of the fungus of claim 1 characterized in that it comprises the following steps:

to).

take pumpkin leaves infected with powder (Podosphaera fusca),

b).

observe by stereoscopic microscope the leaves infected by o�dio

in search of some area where there are similar structures to pycnidia

formed by the possible biocontrol agent,

C).-

delimit the area where these structures are located, called pycnidia,

Y

 scratch in that area  the surface of the blade with a scalpel on a

slides,

d).

observe the pycnidia by stereoscopic microscope and separate in another

slides,

and).

disperse a drop  of ethanol 96� (v / v) for 15 seconds over

pycnidia isolated on the slide and then rinse the drop with

sterile water to remove ethanol,

F).

deposit isolated pycnidia on Petri dishes with MEA medium or medium

PDA and,

g).

incubate the pycnidia of stage f) under controlled light conditions and

temperature obtaining the Ampelomyces CECT 20749 strain.

3. Isolation method according to claim 2, characterized in that the Ampelomyces CECT 20749 strain isolated is subsequently re-seeded in fresh medium at a temperature in a range between 18 and 25 � C. 4. Isolation method according to any of claims 2 or 3 characterized in that the Ampelomyces CECT 20749 strain isolated is preserved for 12 months in the form of a suspension with 20% glycerol and at a temperature of at least -70�C, being necessary its reactivation before this period of time elapses. 5. Composition comprising the fungus according to claim 1. 6. Composition according to claim 5 characterized in that it is in solid form. 7. Composition according to any of claims 5 or 6, characterized in that it is preserved in the absence of preservatives at temperatures between 20-25�C. 8. Use of the fungus of claim 1 or of the composition according to any of claims 5 to 7 as an agent for biological control against odyde infections in 5 plants. 9. Use according to claim 8 wherein the plant is of the family of cucurbitaceae, solanaceous, rosy, asteric, umbelliferous, fabulous, fruit or ornamental crops. 10.- Method of biological control of the soil of a plant with respect to a control comprising: 10 a) .- contacting the affected part of the plant with a composition according to any of claims 5 to 7, and b) .- let the composition of any of claims 5 to 7 act. 11. The biological control method according to claim 10, characterized in that in step a) the composition is contacted with the plant by spraying an aqueous dispersion containing it. 12. The biological control method according to claim 11, characterized in that the aqueous dispersion contains a concentration between 1 * 107 and 5 * 106 pycnidiospores / mL of the composition of claims 5 to 7. 13. Use of the composition according to any of claims 5 to 7 for biological control against plant infections in plants in an integrated pest control program. : n "uuQ nuQrEQ" a "* u "Qn to Qn! 100 90 to 80 70 60 fifty 40 30 b b or 10 0 CK 106 5 * 106 107 TreatEuQnto FIG. one : n "QrEQ" a "* uuQ n "Qnuua wn! 90 80 70 60 50 40 30 20 10 0 to TreatEuQnto FIG. 2 CK CECT 20749 AQ10 CECT 20749 AQ10 CECT20749 AQ10 (255 * 105pic / mL) (255 * 105pic / mL) (106 pic / mL) (106 pic / mL) (107 pic / mL) (107 pic / mL) FIG. 4 SPANISH OFFICE OF THE PATENTS AND BRAND Application No.: 201430566 SPAIN Date of submission of the application: 04/16/2014 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

TO

WO 2014044723 A1 (FOND EDMUND MACH) 03.23.2014, claims. 1-13

TO

US 5190754 A (SZTEJNBERG ABRAHAM) 02.03.1993, claims. 1-13

TO

ANGELI DARIO et al. Existence of different physiological forms within genetically diverse strains of Ampelomyces quisqualis. Phytoparasitic 2012. VOL: 40 No: 1 Pgs: 37-51 ISSN 0334-2123 (print) ISSN 1876-7184 (electronic) Doi: doi: 10.1007 / s12600-011-0197-x, summary. 1-13

TO

GOPALAKRISHNAN C et al. Management of okra powdery mildew using Ampelomyces quisqualis. Journal of Biological Control. 2009. VOL: 23 No: 3 Pgs: 325-327 ISSN 0971-930X (print) ISSN 0970-5732 (electronic), summary. 1-13

TO

ANGELI DARIO et al. Is the mycoparasitic activity of Ampelomyces quisqualis biocontrol strains related to phylogeny and hydrolytic enzyme production. Biological Control 2012. VOL: 63 No: 3 Pages: 348-358 ISSN 1049-9644 (print) ISSN 1090-2112 (electronic) Doi: doi: 10.1016 / j.biocontrol.2012.08.010, summary. 1-13

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of completion of the report 13.06.2014

Examiner I. Rueda Mol�ns Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201430566 CLASSIFICATION OBJECT OF THE APPLICATION A01N63 / 04 (2006.01) C12N1 / 14 (2006.01) C12R1 / 645 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A01N, C12N, C12R Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, BIOSIS State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201430566 Date of Completion of Written Opinion: 06.13.2014 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 1-13 Claims IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims 1-13 Claims IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201430566 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

WO 2014044723 A1 (FOND EDMUND MACH) 03/27/2014

D02

US 5190754 A (SZTEJNBERG ABRAHAM) 02.03.1993

D03

ANGELI DARIO et al. Existence of different physiological forms within genetically diverse strains of Ampelomyces quisqualis. Phytoparasitic VOL: 40 No: 1 Pgs: 37-51 ISSN 0334-2123 (print) ISSN1876-7184 (electronic) Doi: doi: 10.1007 / s12600-011-0197-x 2012

D04

GOPALAKRISHNAN C et al. Management of okra powdery mildew using Ampelomyces quisqualis. Journal of Biological Control. VOL: 23 No: 3 Pgs: 325-327 ISSN 0971-930X (print) ISSN 0970-5732 (electronic), summary. 2009

D05

ANGELI DARIO et al. Is the mycoparasitic activity of Ampelomyces quisqualis biocontrol strains related to phylogeny and hydrolytic enzyme production. Biological Control VOL: 63 No: 3 Pages: 348-358 ISSN 1049-9644 (print) ISSN 1090-2112 (electronic) Doi: doi: 10.1016 / j.biocontrol.2012.08.010, summary. 2012

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement  NOVELTY AND INVENTIVE ACTIVITY (articles 6 and 8 of Law 11/1986) In claims 1, 5-7 of the patent application a fungus of the genus Ampelomyces is claimed with the deposit number CECT 20749 and a composition comprising said fungus. In claims 2-4 of the patent application the method of isolation of said fungus is claimed. In claims 8, 9 and 13 of the patent application, the use of said fungus or of said composition is claimed for biological control against odyde infections. In claims 10-12 of the patent application, a method of biological control of powder is claimed, which uses the fungus object of the invention. Any of the documents cited, D01-D05, reflects the use of different Ampelomyces strains for biological control of the soil. The Ampelomyces fungus with the deposit number CECT 20749 object of the invention is an indigenous strain isolated in Spain, as indicated on page 5 of the description of the patent application. The strain object of the invention has a high degree of efficacy as reflected in the incidence rates of the disease shown in the figures of the patent application. None of the documents cited reflects an Ampelomyces strain that has identical or similar characteristics to the strain object of the invention. Therefore, claims 1-13 of the patent application present novelty and inventive activity, as established in articles 6 and 8 of Law 11/1986. State of the Art Report Page 4/4