WO2009087179A2

WO2009087179A2 - Amendment controlling soil born pest and pathogen, use and using method of such amendment

Info

Publication number: WO2009087179A2
Application number: PCT/EP2009/050143
Authority: WO
Inventors: Luca Lazzeri; Giovanna Curto; Onofrio Leoni; Gina Rosalinda De Nicola; Lorena Malaguti; Giampiero Patalano
Original assignee: Cerearltoscana S.P.A.
Priority date: 2008-01-07
Filing date: 2009-01-07
Publication date: 2009-07-16
Also published as: WO2009087179A3; ITBO20080010A1

Abstract

An amendment controlling soil born pest and pathogen includes at least one oil emulsified in water, or in an aqueous liquid, and at least an ingredient assembly containing at least a glucosinolate and at least an enzyme selected in the group consisting of: glucosidasic enzymes, and thioglusidasic enzymes; and to include an emulsified or dissolved liquid proteinic hydrolyzed agent.

Description

AMENDMENT CONTROLLING SOIL BORN PEST AND PATHOGEN, USE AND USING METHOD OF SUCH AMENDMENT.

TECHNICAL FIELD

The present invention refers to agriculture and garden products, in particular refers to an amendment controlling soil born pest and pathogen specially fit for fertilizing and protecting plants from attack of pathogenic and soil born pests.

BACKGROUND ART

The recent application of Montreal protocol includes the no use of the methyl bromide, a synthesis fumigant product at high effectiveness in the control of some soil pathogenic as nematodes, as compound inserted in the list of those responsible of the reduction of the ozone layer. At the same time the registration procedure of others analogous synthesis compounds, in community area, had a significant hardening causing the suspension of the use authorization for some compounds.

Therefore, in the last few years, the operators and the market were interested in defence techniques of agricultural cultivations for reducing or eliminating the use of synthesis products.

Recently, in this field pellets have been produced with vegetal amendment, however having the disadvantage of being applied to the culture only as soil treatments during the pre-plant, to avoid possible phytotoxicity phenomena; it follows that the pathogenic and soil born pests limitation could be insufficient during the cultivation, such as due to a high initial infection.

Is therefore evident the strategic importance of having formulates, in addition to the pellet products, of vegetal origin that can be applied also during the culture development and fit for making a fertilizing action and soil amendment.

DISCLOSURE OF THE INVENTION An aim of this invention is to propose a fertilizer fit to supply also an eco-efficient amendment action as option of the synthesis active principles through an oil formulation, natural nitrogenous components and Brassica flours with cytotoxic activity for some soil pathogens and parasites. The compound can also be distributed onto the culture in the field, for example with sub foliage or drip irrigation.

Another aim is to propose a formulation of an entirely renewable-based product having nematocidal, insecticide and fungicide activity and composed by an emulsion of a vegetal oil and a natural liquid proteinic hydrolyzed compound containing in suspension of Brassica seed flours containing glucosinolate. The product can be commercialized in phials of one or more litres without no special precaution because characterised by absent or limited human toxicity.

BEST MODE OF CARRYING OUT THE INVENTION

Vegetable oils, obtained by pressing or solvent extracting from seeds, have already today some agriculture applications, predominantly as animal or some insect repellent, considering their asphyxiating effect. The oil used in the formulation object of the present patent application will be rich of fat acid with long or medium chain preferably extracted from Brassica seeds.

Natural proteinic hydrolyzed compounds are naturally produced liquids characterised by different concentrations of total nitrogen on average included between 1 and 30% generally obtained by hydrolysis of vegetal or animal proteins. In the last few years, such products have found application in the growing techniques of reduced environmental impact as fertilizing liquids even if they are characterised by a nitrogen content very often lower in respect to the similar synthesis solid fertilizers.

Flours used in the formulation derive from of Brassica seeds and are on average characterised by glucosinolate content between 50 and 250 μmole for gram of seed. The glucosinolates are thioglucoside present in nature with over 120 kinds, not activate by themselves. The glucosinolates are easily hydrolizable with water from the endogenous enzyme myrosin action, with formation of multiple products (isothiocyanates, thiocyanates, nitriles, epithionitriles, thiones etc.) varying in function of the conditions in which the enzymatic hydrolysis and the glucosinolate precursor occurs, and which have differentiated biologic or cytotoxic activity. Between these, the isothiocyanates are the much more active compounds. These molecules, in fact, in function of their chemical structure, of the dose and of the reference system, have shown in the last few years antitumour activity, antimicrobial activity in the upper organisms, as well as property insecticide, insect-repellent, nematocidal, repellents, fungal toxic when used against insects or pathogenic fungi of plants in the soil.

The formulation object of patent, consists in a hydrolyzed proteinic/oil mixture in which, before or after the addition of water in a proportion variable between 98% and 20%, a dispersion of Brassica flour is added characterised by an high content of glucosinolate. In formulating the hydrolyzed proteinic/oil mixture an appropriate emulsifying agent can be added to facilitate and stabilize the oil dispersion in the aqueous solution.

The doses of the components of the formulated compound are hereafter described: a) vegetable oil: minimum of 0.1% of the aqueous solution; b) hydrolyzed proteinic liquid: minimum of 0,1 % of the aqueous solution c) emulsifying agent: minimum of 0,5% of the oily fraction; d) Brassica flour: minimum with 50 μmole of glucosinolate for gram of seed and applied with minimum dosing of 0.02 g L^"1;

Before use, the formulated compound must be emulsified with an aqueous solution that can contain other compounds (salts, organic solvents, etc.) present in variable percentages.

The invention is illustrated in the following examples in which reference is made to the six enclosed tables.

Example 1

In laboratory, the valuation of the timing release of isothiocyanates from Brassica flours in suspension in a proteinic-water hydrolyzed-oil emulsion. It has been operated in screw Pyrex flask iso: 5,7 ml of natural proteinic hydrolyzed and 3,6 ml of vegetable oil containing an emulsifying agent have been mixed with 100 ml of water. Two modalities of preparation of the formulated compound have been compared: 1,5 g of flour of Brassica carinata of fine grinding have been dispersed in the proteinic- oil hydrolyzed mixture before adding water (formulated compound 1) or after adding water (formulated compound 2) maintaining everything in a continuous agitation and with room temperature. Every 10 minutes has been taken 1 ml of emulsion-flour mixture and moved into a vial of 10 ml closed with screw plug and pierceable septum. The vial has been kept in agitation for 5 minutes before carrying out the gas- chromatographic analysis with head space technique. The analysis has been carried out with a chromatograph VARIAN SATURN 3800 having a flame ionization detector (FID) and column J&W DB23(30 m, 0,25 mm id and 0,25 μm df). The analysis conditions have been the following: gas of nitrogen transport to the flow of 5 ml min-1, injector temperature 200°C, detector temperature 280⁰C, analysis in isothermic conditions at 50⁰C, injection of 200-500 μl of sample in splitless modality. The amount of isothiocyanate has been valued on the base of a previously defined calibration line using a formulated compound prepared with known quantity of pure isothiocyanate rather than from flours. The release in time of the isothiocyanate has been quoted in diagram as % yield considered on the basis of the theoretic value esteemed on the basis of the glucosinolate contents of flours (figure 1).

The results show a greater yield in isothiocyanate in the formulation with dispersion of the flour after the addition of water (formulated compound 2). In fact, with this formulation the release of isothiocyanate produced by the enzymatic degradation of the glucosinolate present in the flour reaches in 70 minutes the maximum value of about the 80% with respect to the theoretic value and remains constant in the course of time.

When the flour is dispersed in the proteinic-oil hydrolyzed mixture before adding water (formulated compound 1) the release of isothiocyanate has a similar course, but the added maximum value is only 70% of the theoretic and the time needed for reaching it is longer (80 minutes).

Example 2 Evaluation, in laboratory, of the release during time of isothiocyanates from Brassica flours when contained in a permeable system rather than dispersed in the medium.

It has been operated in screw Pyrex flask iso: 5,7 ml of natural proteinic hydrolyzed and 3,6 ml of vegetable oil containing an emulsifying agent have been mixed with 100 ml of water. In the mixture have been immersed 1,5 g of flour at fine grinding contained in a porous bag maintaining everything under agitation and at room temperature. Two filters of different porosity have been compared (filter 1 and filter 2) and has been used as reference the flour in free form. The release of isothiocyanates, followed in the course of time by means of analysis GC of the head space as in the example 1, is been much more slower with respect to the free flour, reaching the maximum value only after 110 minutes with a yield of 18% with the filter 1 and of 45% with the filter 2 (figure 2).

Example 3

Evaluation, in laboratory, of the release of isothiocyanates from Brassica flours in a proteinic- water oil-hydrolyzed emulsion after removal of the flour by filtration with 120 mesh filter.

It has been operated in iso screw Pyrex flask: 5,7 ml of natural proteinic hydrolyzed and 3,6 ml of vegetable oil containing an emulsifying agent have been mixed with 100 ml of water. In this emulsion it has been dispersed 1,5 g of flour of Brassica carinata at fine grinding and placed in continuous agitation at room temperature.

The flour has been moved away by filtration with a 120 mesh filter and the filtered emulsion has been immediately analyzed for the quantification of present isothiocyanates. The trial has been repeated more times considering different times of emulsion-flour homogenization (25 min, 45 min, 70 min and 90 min) and the middle value of isothiocyanate quantity determined for each time reported in time dependent drawing (figure 3).

Moreover, it has been also checked if the release of isothiocyanates went on in the emulsion after removal of the flour supposing a process with two speeds a) fast solubilisation of glucosinolate and myrosin action in the emulsion, b) transformation, slower, glucosinolate into isothiocyanates. Therefore we have considered the time of emulsion-flour homogenization of 25 minutes from hydrating. The slurry has been therefore filtered (120 mesh filter) and the emulsion free from flours has been again placed into a continuous agitation taking, at prefixed times, sub-samples to evaluate the concentration of isothiocyanates that has been after reported in time dependent diagram. Also in this case, the free form flour as reference has been used.

The results of these tests show that the hydrolysis of the glucosinolate continues in the emulsion also after that at 25^th minutes the flours have been moved away. The percent yield, normally lower of 10 points with respect to the reference one (example 1), is practically the same of the trials made with time of emulsion-flour homogenization much more longer (figure 3).

Example 4

Evaluation at laboratory level, of the release during time of isothiocyanates from Brassica flours in proteinic oil-hydrolyzed emulsion due to addition of other flours of the family of Brassica at high content of myrosin action (disoiled flour of seeds of Sinapis Alba) at variable quantity.

It has been operated in screw Pyrex iso flask: 5,7 ml of natural proteinic hydrolyzed and 3,6 ml of vegetable oil containing an emulsifying agent have been mixed with 100 ml of water. In this mixture have been dispersed 1,5 g of flour with greater granulometry with respect to examples 1, 2 and 3 and various amounts (0, 5, 10 and 15% of the total) of a second flour at high content of myrosin activity. The dispersion has been maintained in a continuous agitation and at room temperature. The release of isothiocyanates from the flour with larger granulometry with respect of that used in the previous examples is happened more slowly (about 25% of theoretic after minutes 100) and is strongly accelerated by adding flour at high content of myrosin action reaching the maximum (beyond the 80% of theoretic) after about 50 minutes (figure 4).

Example 5 Evaluation at laboratory level of the release in the course of time of isothiocyanates from Brassica flours due to addition of different quantities of ascorbic acid, as activator of the myrosin enzyme.

The test has provided the use of ascorbic acid added to the formulation after solubilisation in water. It has been operated in screw Pyrex iso flask: 5,7 ml of natural proteinic hydrolyzed and 3,6 ml of vegetable oil containing an emulsifying agent have been mixed with 100 ml of a solution of ascorbic acid realizing different concentrations of acid with respect to the used flour (0.25, 2.6 and 5 mg g-1). In this mixture have been dispersed 1,5 g of flour at greater granulometry as in the example 4 and the dispersion has been placed under agitation and maintained at room temperature. The release of isothiocyanate has been followed during time by GC as in the previous examples.

The results show that the addition of minimum quantity of ascorbic acid (2.6 mg g-1 of flour) has determined a fast release of isothiocyanate reaching the maximum value in about 40 minutes (figure 5).

Example 6

Evaluation of the activity of the formulated compound described in example 1 on galligen nematodes (Meloidogyne incognita).

The tests have been made in jar using the standard dose of Brassica carinata flour (33O g L-I of oil) dispersed in a proteinic hydrolyzed oil emulsion in water. The treatment has been repeated eight and thirty-six days after the transplanting of tomato seedlings cv. UC82, variety notoriously sensible to nematodes attack. The infesting level of the soil has been valued 6 and 76 days after the transplanting, valuing the number of L2 in the soil and the attack intensity onto the tomato roots (table VI).

The results show a good response of plants to the treatment with the formulated compound object of patent application. The three different formulations, in fact, have shown in the first sample a clear decrease of the number of larvae in the soil, joined, above all, for that with triple dose flour, at a less infesting index. In the sample after 71 days from the first treatment and 41 from a second treatment, the infesting index of the vases treated with the formulated compound is clearly resulted lower with respect to the untreated one, while the number of larvae is resulted superior in the trial with single dose flour. The formulated compound with triple dose of flour has therefore shown a significant reduction of the infesting level of the roots that, moreover, shown a greater growing of side roots, showing some phyto-stimulating action of the same formulated compound and in particular of that with triple dose flour.

An advantage of this invention is to supply a fertilizer fit to fulfil also an amendment environmentally friendly action and alternative to the synthesis active principles through an oil formulation, natural nitrogenous components and Brassica flours at cytotoxic activity referring to some soil pests and pathogens.

Other advantage is to supply a formulated compound that can be distributed also onto the culture in field, for example with drop or sub-leaves watering arrangements.

Other advantage is to supply a formulation of a product with entirely renewable base with nematocidal, insecticide and fungicide activity.

Additional advantage is to supply a marketable formulated compound without the aqueous fraction in phials of one or more litres without any special precaution because characterised by limited or absent human toxicity.

It is important to observe that the effect of combining the Brassica seed flour with the oil emulsion in water unexpectedly provides a synergic effect higher than the sum of the effects supplied by the flour solution or by the oil separately supplied. It is plausible that at least a part of such synergic effect is due to the fact that the oil retards the flour reactions in water allowing the development of the active principles directly in the soil, powering and prolonging the effects.

Also the combination of the proteinic hydrolyzed with the emulsion provides an unexpected synergic effect probably due to the fact that the insecticide and/or nematocidal effect simultaneously to the delivery of nitrogenous substances determines an additional effect bio-stimulating onto the cellular growing of the cultivations for reasons that, at the moment, are object of further studies. TABLE VI

Sample after 28 days from the Sample after 71 days from the first treatment first treatment and 41 days after the second treatment

Treatments L2 lOOcc Infesting index L2 lOOcc Infesting index

Non treated 45.8 2.6 9.0 3.5

Formulated compound with 8.8 2.1 40.7 2.1 single flour dose

Formulated compound with

7.4 1.7 6.7 1.1 triple dose of flour

Formulated compound with triple dose of 10.5 2.1 15.7 1.5 flour in envelope

Evaluation of the effectiveness in the control of Meloidogyne incognita on tomato roots of two treatments with different formulated compound made at 37 and 71 days after the transplanting on soil at high infesting content.

Claims

1) Amendment with soil born pest and pathogen control, characterised in that has at least one emulsified oil in water or in an aqueous liquid and at least an ingredient assembly containing at least one glucosinolate and at least one enzyme chosen in the group consisting of:

- glucosidasic enzymes, and

- thioglusidasic enzymes; and to include an emulsified or dissolved liquid proteinic hydrolyzed agent.

2) Amendment according to claim 1 characterised in that the at least one oil is of vegetal origin.

3) Amendment according to any of the previous claims characterised in that the assembly of ingredients consists of at least one flour of seeds.

4) Amendment according to claim 3 characterised in that the seeds are of Brassica.

5) Amendment according to claim 3 characterised in that the seeds are of Brassica carinata and/or Brassica verna and/or Eruca sativa.

6) Amendment according to any of the previous claims characterised in that has an emulsifying agent.

7) Amendment according to any of the previous claims characterised in that the minimum percentage of vegetable oil in the aqueous solution is of about 0.1%.

8) Amendment according to any of the previous claims characterised in that the minimum percentage of liquid proteinic hydrolyzed agent in the aqueous solution is of about 0.1 %.

9) Amendment according to claim 6 and anyone of the preceding claims characterised in that the minimum percentage of emulsifying liquid agent in the aqueous solution is of about 0.5 %. 10) Amendment according to claim 4 and anyone of the preceding claims characterised in that the minimum dosing of Brassica flour in the aqueous solution is ofabout 0.02 g lΛ

11) Amendment according to claim 4 and anyone of the preceding claims characterised in that the Brassica flour is of the type containing at least 50 μmole of glucosinolate for seed gram.

12) Amendment according to any of the previous claims characterised in that has moreover at least one between salts and organic solvents dissolved into water or aqueous liquid.

13) Use of the amendment of any claims from 1 to 12 characterised in that it is employed as amendment with cytotoxic effect for plant pathogenic.

14) Use of the amendment of any claims from 1 to 12 characterised in that it is employed as amendment with fungitoxic effect for plant fungal pathogenic.

15) Use of the amendment of claim 5 or any remaining claims from 1 to 13 characterised in that it is employed as amendment with insecticide or nematocidal effect.

16) Method of use of the amendment of any claims from 1 to 12 characterised by emulsifying the oil into water or into the aqueous liquid and by_. dissolving the solid components, obtaining the amendment in liquid form, before use.

17) Method of use according to claim 16 characterised bv filtering the amendment in liquid form before its delivering.

18) Method of use according to claim 16 or the 17 characterised by supplying the amendment by means of sub-leaves or drop irrigating system.