西班牙专利ES2671137A1 USE OF 3,4-DIHYDROXYPHENILGLICOL (DHFG) AS A PHY-REGULATOR (Machine-translation by Google Translate,

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专利摘要:
Use of 3,4-dihydroxyphenylglycol (DHFG) as a phytoregulator. The present invention relates to the use of the compound 3,4-dihydroxyphenylglycol (DHFG), compositions comprising it and methods of using same as effective phytoregulators for the treatment of crops and weeds. (Machine-translation by Google Translate, not legally binding)
公开号:ES2671137A1
申请号:ES201631404
申请日:2016-11-03
公开日:2018-06-05
发明作者:Guillermo Rodríguez Gutiérrez；Juan Fernández-Bolaños Guzmán；Aránzazu GARCÍA BORREGO；Juan Antonio ESPEJO CALVO；Antonia M. ROJANO DELGADO；María África FERNÁNDEZ PRIOR
申请人:Tecnofood Id Soluciones SL；Tecnofood Id Soluciones S L；Consejo Superior de Investigaciones Cientificas CSIC；
IPC主号:

专利说明:

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USE OF 3.4-DIHYDROXYPHENYLGLYCOL (DHFG) AS A FITOR
DESCRIPTION
The present invention can be encompassed in the field of biochemistry or plant physiology and agriculture. It refers to the use of the compound 3,4-dihydroxyphenyl glycol (DHFG), compositions that comprise it and methods of use thereof, as effective phytoregulators to enhance the growth of crops, as well as to control the growth of weeds.
STATE OF THE TECHNIQUE
Plants synthesize endogenous growth regulators that participate essentially in their physiology, so much of the current agronomic practices include the use of compounds, mostly synthetic, with properties analogous to these regulators, to modify metabolism and thus increase crop yield, and / or fruit quality, as well as eliminating weeds. For this reason, agrochemical manufacturers are constantly searching for ways to obtain new or better compounds and methods to regulate plant metabolism and consequently increase crop yields.
Many of the growth regulators include compounds such as carboxylic acids, sugar analogs and amines that have the capacity to induce various effects on plants (Alexieva, 1994, Compt. Rend. Acad. Bulg. Sci. 47, 779-82) . In the cultivation of corn it has been shown that treatments with polyhydroxycarboxylic acids increase root growth (Gur et al, 1987, Physiology Plantarum. 69, 633-638) and the formation of root hairs, which favors a better absorption of nutrients.
In relation to the phytoregulatory potential of amines, it is known that they affect essential processes of plants such as flowering, germination, growth and senescence of the leaves, among others. (Shih et al, 1982, I. Plant. Physiol. 70, 15921596). Interestingly, studies have established that certain amines with a regulatory effect on plant growth can also affect the development and physiology of phytopathogenic fungi (Havis et al, 1997, J. Agrie. Food. Chem. 45, 2341-2344). From
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In the same way, it has been reported that sugar analogs can interfere with fungal growth, reducing the damage they may cause (El Gaouth et al, 1995, Plant. Dis. 79, 254-278).
Another type of molecules with phytoregulatory capacity are nitrated derivatives of phenolic acids, particularly o-nitrophenolate, p-nitrophenolate and 5- nitroguayacholate (Górnik and Grzesik, 2005, Folia Horticulturae. 17, 119-127). Although these compounds also have the capacity to affect the development of phytopathogenic fungi, their use has been restricted only to that of phytoregulators to limit their dose due to potential risks. (Official Journal of the European Union, 19.2.2009, 48 / 5-48 / 12).
However, the proliferation and indiscriminate use of synthetic phytoregulators have caused agricultural regulations to be forced to restrict the use of chemical compounds that cause hormonal alterations and dysfunctions in crops or that are toxic. Likewise, the intensive use of chemical pesticides for the control of pests and diseases has caused the causal organisms to develop resistance, forcing the use of increasing doses or the development of more toxic products. These aspects increase the level of risk on the health of ecosystems, the health of farmers and the final consumer.
Thus, in the search for new compounds with phytoregulatory activity, research has been conducted to obtain biomolecules / extracts of interest from by-products of the agro-industry, in order to revalue these by-products and minimize the environmental impact, and allow Development of a new nearby biotechnology industry and / or dependent on the agribusiness supplier of the source of the by-products. On the other hand, biotechnological techniques have been applied, through in vitro laboratory and in vivo bioactivities screening through field trials, of said extracted biomolecules, for the subsequent design of natural phytoregulators (antimicrobial, antifungal, insecticides, acaricides and anti-germinatives for the control of weeds), intended primarily for use in the organic farming and integrated production sectors, which currently have more demand, but also in conventional agriculture in order to reduce the application of phytosanitary products from synthesis chemistry.
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Therefore, it is important to develop biomolecules and / or extracts that understand them as new phytoregulators, non-toxic or of low toxicity, aimed at increasing crop production.
DESCRIPTION OF THE INVENTION
To solve the aforementioned limitations, it is a general object of the invention to provide the use of a compound, preferably obtained as a by-product of the industry, more preferably of the olive oil industry and / or of the table olive industry, a composition that includes it, and a method for its use as an effective phytoregulator.
The inventors have demonstrated on the one hand that 3,4 dihydroxyphenyl glycol (DHFG) is an effective phytoregulator, both for the control of the germination and growth of agricultural crops, as well as a potent herbicide against weeds. In addition, on the other hand, the inventors have shown that this phenol is an important component in the phenolic fraction of the by-products of the olive, both in the olive oil industry and in the table olive industry, thus being able to favor the use and recycling of said by-products. In this sense, the present invention demonstrates that DHFG is a compound called allelochemical given that it provides benefits, especially competitive, in plants, mainly aimed at an increase on germination, growth or development, and on the other hand, it is also capable of induce negative effects on other plants, preferably weeds, inhibiting their germination and preventing their growth. These substances that have such duality are called, as we have mentioned above, allelochemicals. This definition encompasses both harmful and beneficial effects. It is necessary to point out that many substances with allelopathic activity have beneficial effects at very low concentrations and, exceeding a certain threshold, act negatively.
Therefore, the use of DHFG, or an extract rich in said compound, that is to say that it comprises a minimum concentration of at least 1 ppm of DHFG, or alternatively between 0.001-50 g / L of DHFG, obtained from by-products derived from the extraction of olive oil (alpechines, settling waters, vertical centrifuge waters, washing waters, alperujo, pomace, alperujo water, etc.) and / or the olive dressing industry, also known as the industry of the
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Table olives (cooking water, washing water, brines, preservation liquids, etc.) as a phytoregulator has the following advantages:
> Being a natural product that can be obtained physically, it is not toxic, does not contaminate, and does not generate resistance in crops, and less at active concentrations.
> It is highly effective as a herbicide when used in a concentration range between 100 ppm to 50,000 ppm of DHFG (mg DHFG in 1 kg of product).
> It has a high effectiveness as a biostimulant of the germination and growth of crops when used in a range of concentrations between 1 to 1500 ppm of DHFG.
> Use of by-products from the oil mill and / or table olives industry to achieve greater added value to them.
Thus, a first aspect, the present invention relates to the use of 3,4 dihydroxyphenyl glycol (DHFG) as a phytoregulator.
For the purposes of the present invention the term "phytoregulator" refers to any compound capable of inducing the growth of plants, preferably agricultural crops. For the purposes of the present invention, said term also refers to those compounds that are capable of inhibiting weed growth, thus having herbicidal activity.
3.4 DHFG is a compound belonging to the group of phenols and is part of the phenylpropanoid glycoside family. The CAS number of the DHFG is: 2882273-3; and its formula:
image 1
In a particular embodiment, the DHFG compound described in the present invention as a phytoregulator, can be obtained from by-products derived from the
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extraction of olive oil and / or the olive dressing industry. In another more particular embodiment, the DHFG-rich extract obtained from the by-products comprises at least one composition / concentration of DHFG of at least 1 ppm (mg / kg).
For the purposes of the present invention the term "extract rich in DHFG" refers to by-products obtained from the olive oil extraction industry and / or from the olive dressing industry, mixture of said by-products or fractions obtained from of the by-products or mixtures thereof, comprising at least 1 ppm of DHFG, preferably from 1 to 50,000 ppm, or alternatively from
0.001-50 g / L of DHFG.
In another more preferred embodiment, the phytoregulatory activity of the use of DHFG is selected from herbicidal activity against weeds or biostimulant / growth activity of agricultural crops.
For the purposes of the present invention the term "herbicide" refers to any chemical compound capable of controlling or eliminating unwanted plant species.
In another more preferred embodiment, the DHFG compound shows herbicidal activity when used at a concentration of between 100 to 50,000 ppm, more preferably between 500 to 9000 ppm.
For the purposes of the present invention the term crop growth enhancer or biostimulant refers to any compound capable of inducing plant growth and increasing crop yield in general.
In another more preferred embodiment, the DHFG compound shows growth activity of agricultural crops when used at a concentration of between 1 to 1500 ppm, more preferably between 1 to 500 ppm.
In another preferred embodiment, DHFG is used in combination with at least one adjuvant.
For the purposes of the present invention the term "adjuvant" refers to chemicals that help improve the characteristics and properties of a compound or
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chemical substance, so that the final composition is neither toxic nor polluting. These may be any adjuvant that is authorized for use in phytoregulatory formulations, but are preferably selected from oil-based adjuvants and mixtures, organosilicone-based adjuvants and mixtures, non-ionic-based adjuvants and mixtures, polymeric-based adjuvants and mixtures , and adjuvants based on fatty acids and mixtures, and combinations thereof. The adjuvant will be any of those known to the person skilled in the art and used in the technical field of the invention.
In a more preferred embodiment the adjuvants are selected from any of the following: araoil, biopower and / or any combination thereof. In yet another more preferred embodiment, the adjuvants are used at the concentrations determined by the manufacturers thereof.
Another object described in the present invention relates to an extract obtained from by-products derived from the extraction of olive oil and / or from the olive dressing industry, characterized in that said extract comprises a DHFG concentration of at least 1 ppm, preferably between 1 to 50000ppm or alternatively between 0.001 to 50 g / L.
Another object described in the present invention relates to a phytoregulatory composition comprising DHFG, preferably at the concentrations indicated above, and / or an extract as described above.
In another preferred embodiment, the phytoregulatory composition further comprises at least one adjuvant. In another more preferred embodiment, the adjuvant, as indicated above, is selected from any of the list consisting of: oil-based adjuvants and mixtures, organosilicone-based adjuvants and mixtures, non-ionic-based adjuvants and mixtures, polymer-based adjuvants and mixtures, and adjuvants based on fatty acids and mixtures, and combinations thereof. In another more preferred embodiment, the adjuvant is selected from any of the following: araoil and / or biopower.
In another preferred embodiment, the phytoregulatory composition of the invention exhibits herbicidal activity when the concentration of DHFG is between 100 to 50,000 ppm, preferably between 500 to 9000 ppm. In another preferred embodiment, the
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Phytoregulatory composition of the invention exhibits biostimulant growth activity when the concentration of DHFG is from 1 to 1500 ppm, preferably from 1 to 500 ppm.
Another object described in the present invention relates to a method for the control of weeds which comprises administering to an crop an effective dose of DHFG, or of the extract, or of the phytoregulatory composition, as previously described.
For the purposes of the present invention, the term "effective dose" refers to a non-toxic amount of the DHFG compound, as described herein, which is sufficient to provide the desired effect on the cultures, for example, inhibit weed growth or enhance crop growth. The precise and effective amount for each type of activity will depend on the type of crop, the extent to be treated, as well as the type of administration used. Therefore, it is not useful to specify an exact amount effective in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the criteria of the average expert in the field.
For the purposes of the present invention, an effective dose, when it is desired to obtain a herbicidal activity will be about 100 to 50,000 ppm, preferably between 500 to 9000 ppm of DHFG, and an effective dose when a growth-enhancing activity is desired, it will be about 1 to 1500 ppm, preferably between 1 to 500 ppm of DHFG.
In a preferred embodiment, weeds are selected from monocot and dicot. In yet another more preferred embodiment, monocotyledonous weeds are preferably; Lolium rigidum; Lolium multiflorum, and dicotyledonous weeds are preferably: Sinapis arvensis, Chenopodium album and Stellaria media.
Another object described in the present invention relates to a method for inducing the growth of crops comprising administering to an crop an effective dose of DHFG, or of the extract, or of the phytoregulatory composition, as previously described.
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In a preferred embodiment, the cultures are selected from monocot and dicot. In yet another more preferred embodiment, the monocots are preferably Gramineae, more preferably Triticum aestivum (Soft Wheat), Liliaceae, more preferably Allium cepa (Onion var. Galaxia). In another more preferred embodiment, the dicotyledons are preferably Cruciferaes, more preferably Lepidium sativum L. (Water cress); composites, more preferably Lactuca sativa L. (Lettuce var. Cervantes); solanaceaes, more preferably Lycopersicum esculentum L. (Tomato var. Three edges), Solanum tuberosum L. (Potato var. Red Repontiac), Solanum melongena L. (Eggplant var. Redonda Lisa) and Capsicum L. (Pepper var. Italian); Cucurbitaceaes, more preferably Cucumis sativus L. (Cucumber var. Bellpuig), Cucurbita pepo L. (Zucchini var. Diamond) and Citrullus lanatus L. (Watermelon var. Meridian); and Asparagaceaes, more preferably, Asparagus officinalis L. (Asparagus var. Atlas).
In a preferred embodiment of any of the methods described in the present invention, administration of the DHFG compound, of the extract and / or of the phytosanitary composition is administered via spraying, atomization, dispersion, coating, or pouring, depending on the expected results. In yet another more preferred embodiment, once the compound is administered, it can carry out a rapid or slow, preferably slow release.
Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention.
EXAMPLES
The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
Example 1. Obtaining the DHFG compound and / or an extract rich therein.
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From a by-product collected in the olive oil industry such as alperujo, after storage, what are called alperujo waters is generated, which were used in the preparation of extracts and / or concentrates for their chemical and phytoregulatory characterization . These samples of alperujo water were obtained from cooperatives located in Palenciana (Córdoba) and Marchena (Sevilla). Its phenolic content was analyzed and it was determined that it had a good proportion of HT and DHFG, so it can be considered a source to be taken into account for the extraction of polyphenols.
The industrial polyphenol extraction process is carried out following the procedure described in EP1369407B1. Said extraction has been carried out exclusively by physical methods, without the use of organic solvents, so that both the extract and the polyphenol described in the present invention, DHFG, are suitable for use in organic crops. A brief description of the technology used to obtain the phenolic extracts of the present invention is described below.
It was based on a volume of 100 L of fresh vegetable water collected in the company 2013/2014, whose content in the polyphenols of interest is relatively high, with a concentration of HT of 2.45 g / L and DHFG of 0.27 g / L among others , quantified by high performance liquid chromatography (HPLC-UV). The extraction equipment consists of three chromatographic columns and a water treatment equipment with which it is possible to extract and purify three different types of extracts: an extract rich in DHFG, another rich in HT and a third extract that is a mixture of two previous (HT + DGFG). A work cycle was established that includes several steps that are detailed as follows:
1. The first column (C.C1) is loaded with a fixed volume of the source of phenols by dropping it by gravity onto the resin.
2. After loading, the column is washed with water before elution.
3. The download of the column can be done in two steps, and in each one a different extract is obtained.
3.1. In a first phase a mixture of HT and DHFG is obtained with a 10: 1 ratio of both respectively.
3.2. In a second phase an extract rich in HT is collected.
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4. For the purification and separation of the extract rich in DHFG, certain fractions obtained above were passed through a second chromatographic column (C.C2). And, after a series of elutions with water, fractions were obtained that could be concentrated even to dryness using concentration or evaporation equipment with or without vacuum. This results in final extracts with a high biological activity.
The fractions obtained from the C.C2 chromatography column were purified with the help of a third chromatographic column (C.C3). From this step different fractions were obtained from which the richest in DHFG was chosen based on its chromatographic profile, choosing those fractions where the DHFG signal was the majority (concentrations between 0.001 and 50 g / L), to achieve the final extracts of this compound used in the examples described below. These extracts will be prepared at different pHs and different concentrations to analyze their effectiveness as phytoregulators.
Once the three extracts mentioned above have been obtained:
(1) HT rich extract (10-50000 ppm) with various degrees of purity, 50 and 99.8% referred to dry weight;
(2) DHFG-rich extract (1-50000 ppm) with various degrees of purity, 50 and 99.6% referred to dry weight; Y
(3) mixture extract comprising HT + DHFG (in the previous concentrations),
they proceeded to analyze their activity. To do this, tests were carried out in a controlled growth chamber and in the field using monkey and dicot species to control the effect of these compounds on different stages of development, specifically in the pre-emergence or germination stages; early post-emergence or development of the seedling stage; and late post-emergence or plant development until the production stage; of the plants analyzed.
Example 2. Analysis of the phytoregulatory activity of the DHFG compound in pre-emergency tests, both in weeds and in agricultural crops.
9 cm diameter Petri dishes containing filter paper discs received a volume of 4 to 8 ml (depending on the species of plant tested) of a solution
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containing different doses of the phenolic compound of interest isolated with a high degree of purity, greater than 99%, HT, DHFG and HT + DHFG, which ranged between 0 and 400 ppm (0, 50, 100, 150, 200, 250, 300 , 350 and 400 ppm), and on which 15 seeds of each of the crops and plants tested were finally incorporated. Three repetitions of each test were made and for each concentration containing the 15 seeds each to avoid plaque agglutination and thus also be able to study the radicle. As a negative control, Petri dishes were used in which only distilled water was added; and as a positive control a reference herbicide was used, the Flazasulfuron (FZS) that acts both against cultures of mono and dicotyledonous plants, at the same doses used for each of the phenolic compounds (0, 50, 100, 150, 200, 250, 300, 350 and 400 ppm).
The seeded plates were incubated in a controlled growth chamber (Fitotron; Weiss Technik; United Kingdom) with the following conditions: day / night temperature of 27/18 ° C; 14h photoperiod; and 580 pmol m-2 s-1 of light; over a period of 5 to 10 days, depending on species. After that time, the seeds that have germinated successfully are counted and the roots are measured, in order to calculate the germination rate, using the following formula:
IG = [(% GMxLM) / (% GCxLC)] x 100;
where: IG = germination rate; % GM = percentage of germinated seeds in the sample (extract); % GC = percentage of germinated seeds of the negative control (distilled water); LM = average length of the seed roots of the sample and LC = average length of the seed roots of the negative control.
Additionally, the variables were also analyzed: number of germinated seeds, length of plumule (where necessary), roots (length and number), dry weight and wet weight.
The weed species used in these tests are indicated in Table 1, and the agricultural crop species tested are indicated in Table 2.
Table 1. Weed species tested.
Monocotyledonous Dicot
Lolium rigididum (resistance to inh-EPSPS -R to glyphosate-) Sinapis arvensis
Lolium rigididum (multiple resistance to inh- ACCasa and ALS-R to ACCasa and ALS-) Chenopodium album
Lolium multiflorum Middle stellaria
Table 2. Species of agricultural crops tested.
Monocotyledonous Dicot
Graminaea: ■ Triticum aestivum (Soft Wheat) Cruciferae: ■ Lepidium sativum L. (Water cress)
Liliaceae: ■ Allium cepa (Onion var. Galaxy) Compositae: ■ Lactuca sativa L. (Lettuce var. Cervantes)
Solanaceae: ■ Lycopersicum esculentum L. (Tomato var. Three edges), ■ Solanum tuberosum L. (Potato var. Red Repontiac), ■ Solanum melongena L. (Eggplant var. Round Round Lisa) ■ Capsicum L. (Pepper var. Italian)
Cucurbitaceae: ■ Cucumis sativus L. (Cucumber var. Bellpuig) ■ Cucurbita pepo L. (Zucchini var. Diamond) ■ Citrullus lanatus L. (Watermelon var. Meridian)
Asparagaceae: ■ Asparagus officinalis L. (Asparagus var. Atlas)
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With the values obtained for each of the analyzed variables, germination percentage curves have been constructed based on the concentrations used of the compounds HT, DHFG and the mixture of both. The results obtained give an idea of the difference in concentrations that support each of the species tested and the dose that inhibits germination in soil.
The data shown below collect the values necessary to reach the maximum dose 90 (LD90) that refers to the degree of toxicity of a substance indicating that 90% of the population treated with that substance dies, as well as the doses of each extract that induced the growth of plants.
Table 3. LD90 and dose that acts as a growth activator of weeds grown in the presence of the different extracts expressed in ppm of active ingredient. Values> implies that the LD90 has not been reached. When two doses appear it means that results have been obtained for both doses.
LD90 Growth Activating Dose
Weed species HT DHFG HT + DHFG HT DHFG HT + DHFG
L. rigidum (inh- EPSPS) 350 100,> 1000 250 50
L. rigidum (inh- ACCasa and ALS) 1720 50-100 3420 710
S. arvensis 350 100 300
C. album 1800 125> 4000 710
In the particular case of the weed of the species Lolium rigidum (resistant to glyphosate), a greater effectiveness is observed as an antigerminative or herbicide of DHFG against HT and the mixture of both. The 100 ppm dose of DHFG produces a germination reduction of up to 90% compared to the necessary doses of the other compounds tested, 350 ppm for the case of HT and 250 ppm for the case of the mixture of HT + DHFG. The fact that there is a peak of maximum effectiveness at the dose of 100 ppm DHFG, and that at higher doses that effectiveness disappears,
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appearing again at very high doses (> 1000 ppm), it suggests a mechanism of degradation in active radicals that are the cause of these peaks. On the other hand, the increase in germination and foliar mass of said culture at slightly higher doses of 100 ppm of DHFG suggests that said compound has a hormonal nature as is the case with auxinic herbicides. The herbicidal action of HT only appears at relatively high doses of the compound. The HT + DHFG mixture has an increase in foliar mass at 50 ppm, which implies that there is a hormonal growth.
In the tests carried out for the species Lolium rigididum (R to ACCase and ALS) it was demonstrated that a dose of DHFG of 3420 ppm was able to reduce the germination of this species by 90%, while the LD90 for HT and for the mixture of HT + DGHFG was 1720 ppm and 710 ppm, respectively. All the doses of the compounds to which the LD90 was obtained are very high and are not useful for the control of the growth of said weed. However, it was observed that at a dose of 1320 ppm of DHFG there was a weight reduction of 90%. Another curious aspect is that the growth inhibitory effect is repeated again at low doses of 50-100 ppm DHFG. These results show that although DHFG controls the growth of the Lolium rigididum plant (resistant to ACCase and ALS), such control is not as effective as that carried out in the case of the Lolium rigididum plant (glyphosate resistant).
In the case of the Sinapis arvensis species, it had germination problems, so the tetrazolium topographic test was used (Moore R.P. Int Seed Testing Ass Proc. 1969; 34: 233). In said method, the living cells are stained red by reducing a tetrazolium salt, which is colorless, to form a red formazan. Emphasis is placed on the need to know the viability of different parts of the embryo to predict the development of embryos and their conversion into germs that can be counted (Moore R.P. Viability of headquarters. 1973: 94-113). In the case of S. arvensis, as seen in Table 3, it seems that it seems to resist higher doses of the compounds. However, DHFG has the same pattern of action as in the monocot group previously studied (L rigidum). When this plant was cultivated in the presence of each of the compounds studied, it was observed that fungi proliferated to a greater extent in the presence of HT and to a lesser extent with the HT + DHFG mixture, showing that HT induces fungal growth .
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In the case of the species C. album, as observed in Table 3, the mixture of HT + DHFG is more effective as a herbicide against this species than any other compound, since the mixture is able to reduce by one 90% germination of C. album at a lower dose (710 ppm), than that used in the case of DHFG (> 4000 ppm) or HT (1800 ppm). Although it is true that at the dose of 125 ppm of DHFG there is a considerable reduction in germination followed by a decrease in the variables to be studied. However, at that dose of 125 ppm of DHFG the lethal dose LD90 is not reached, which ensures a safe control of the species.
When said weed cultures were treated with the different doses of the herbicide flazasulfuron (the same as the phenolic compounds studied, as indicated above) it was shown that said compound is very effective in the treatment of weeds, both for mono as dicotyledonous, obtaining an LD90 of 90 ppm with said herbicide.
Therefore, these results show that the use of DHFG shows herbicidal activity being able to control the growth of weeds L. rigididum (inh- EPSPS), L. rigididum (inh-ACCasa and ALS), S. arvensis and C. album, when used at doses similar to those used with the herbicide flazasulfuron.
Subsequently, the same test was carried out but in crops of agricultural interest. Table 4 shows the concentration values expressed in ppm of each of the compounds analyzed to reach LD90, as well as the doses of each extract that induced the growth of crops of agricultural interest.
Table 4. LD90 and dose that acts as a growth activator of the different species of agronomic interest tested with the different compounds expressed in ppm of active ingredient. Values> implies that the LD90 has not been reached.
LD90 Growth Activating Dose (ppm)
Species HT DHFG HT + DHFG HT DHFG HT + DHFG
L. sativa L. 7500> 4000 3000 100
L. esculentum L. 460 3560 460
S. tuberosum L. 1790> 4000> 12000 200
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LD90 Growth Activating Dose (ppm)
Species HT DHFG HT + DHFG HT DHFG HT + DHFG
S. melongena L. 1290 3890 780
Capsicum L. 1420 978 970
C. sativus L. 3700> 4000 1760
C. pepo L. 6790> 4000 1720
C. lanatus L. 480 1970 490
A. officinalis L. 7860> 4000 375
A. strain 1375> 4000 495
T. aestivum 750> 4000 3000 100 500 100
The results shown for the species Tríticum aestivum against the different doses of the tested compounds showed that HT affects the germination of this species from the 500 ppm dose, but behaves as a "dirty" compound as it increases the development of fungi in the seeds and therefore induces contamination of the crop.The dose of HT that reduces both germination, as the weight, the number of roots, the length of roots and the length of the formula by 90% (LD90) is equivalent at 750 ppm On the contrary, when the cultures of T. aestivum were treated with the HT + DHFG mixture, the LD90 was 3000 ppm This dose affected the seeds already germinated in every way This mixture acts as an enhancer of germination, weight, root generation, length of these and of the plumule at a dose of 100 ppm In the case of T. aestivum cultures treated with the different doses of DHFG it was observed that the antigenic action Rminative in this species barely reaches 50%, which is an important fact to be used in the control of weeds Lolium spp. on wheat crops. Surprisingly, an increase in germination is observed at doses of 500 ppm, as well as in the fresh weight, said compounds functioning as a growth and germination enhancer in these crops. Root production occurs at lower doses (125 ppm) and the increase in its length at even lower doses (100 ppm). For the development of the formula, 75 ppm is enough to considerably increase its length.
The results summarized in Table 4 for the species Lactuca sativa L. (lettuce var. Cervantes) treated with the different tested concentrations of HT, DHFG or HT + DHFG, have shown that from 4000 ppm of DHFG begins to
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affect the weight, length of the root and cotyledons of these crops. However, the germination LD90 cannot be achieved. At a dose of 100 ppm DHFG, an increase in fresh weight is observed with respect to the control. The treatment with HT in this species induced a fungal contamination due to the extract and the dose that affected both germination and weight, development of roots and cotyledons, was approximately 7500 ppm, being a very high dose. On the other hand, treatment with the HT + DHFG mixture showed that the LD90 was obtained at the dose of 2980 ppm and the dose at which a marked decrease in the fresh weight, roots and length of the cotyledons was observed was 300 ppm
For the Solanum tuberosum L. species (potato var. Red Repontiac) the different doses of DHFG did not affect germination (LD90> 4000 ppm). However, an increase in the increase in root weight formed at a dose of 200 ppm is observed. Treatment with HT at different concentrations showed that the LD90 dose was obtained at 1790 ppm. Said dose also affects the fresh weight of the root formed in the germination stage and it is also observed that rotting occurs at high doses of HT. Treatment of these cultures with the HT + DHFG mixture at different doses showed that the LD90> 12000 ppm, but from a dose of 3000 ppm the effect on germination is pronounced. However, it is observed that there is an increase in root weight in which they have germinated.
As can be seen in Table 4, the behavior of the A. cepa species (onion var. Galaxy) against different doses of DHFG showed a sharp decrease in the germination percentage at very low doses, between 25 and 50 ppm. However, the LD90 for this culture was obtained at doses much higher than 4000 ppm. When said cultures were treated with different doses of HT or HT + DHFG, an LD90 was obtained at a dose of 1375 ppm and 495 ppm, respectively.
The species Lepidium sativum L. (water cress) is a species that is used as an indicator of products that may present phytotoxicity since it is extremely sensitive. The compounds analyzed in the present invention proved to be phytotoxic even at small doses. Specifically, treatment with DHFG induced a sharp decrease in the germination of said plant at doses between 25 and 50 ppm. However, the LD90 for this crop is at doses greater than 4000
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ppm.
Asparagus officinalis L. (Asparagus var. Atlas) did not germinate well in the experiments, so the tetrazolium topographic method was used to determine the live seeds. The LD90 obtained in this test are 7860,> 4000, 375 ppm for HT, DHFG and HT + DHFG, respectively.
In general, the results shown in Table 4 show that treatment with low-dose DHFG is effective against the germination of weeds, while to generate toxic effects in the different species of agricultural interest tested is the compound that You need more concentration. It is quite true that treatment with DHFG shows a germination-promoting effect when used at low concentrations for the cultures of L. sativa L. (lettuce), S.tuberosum L (potato) and T. aestivum (wheat). Thus, DHFG is useful as a phytoregulator, being able to present herbicidal activity against weeds and growth enhancing activity in crops of agricultural interest. In this sense and as previously mentioned, for the particular case of T. aestivum cultures, the antigerminative action of this compound in this species barely reaches 50%, which is an important fact to be used in the control of Lolium spp. on wheat crops. Interestingly, in these cultures of T. aestivum it is observed that the treatment with DHFG at the dose of 500 ppm induces an increase in the germination of said cultures, as well as an increase in the fresh weight of said cultures, thus operating the DHFG as a growth and germination enhancer in these crops. Regarding the rest of the variables analyzed, it is interesting to mention that root production occurs with low doses of DHFG (125 ppm) and the increase in the length thereof at even smaller doses, 100 ppm. For the development of the formula it was enough with 75 ppm of DHFG to considerably increase its length.
As can be seen in Table 4, the treatment of the seeds with HT at the different doses tested was able to control the growth of weeds, but it has the disadvantage of producing fungi and end up contaminating the seeds. In the case of the HT + DHFG mixture, it is observed in the data shown in Table 4 that there are doses that induce the germination of crops but in turn also enhance the growth of weeds, therefore it is not a valid treatment No cash.
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The results obtained for the positive controls (cultures treated with the herbicide FZS showed that the LD90 for different species were in very low doses of the same, approximately 90 ppm, thus recording the effectiveness of said herbicide for both monkey and dicot.
Therefore, HT is able to control the growth of weeds, but has the disadvantage of producing fungi and end up contaminating the seeds. The mixture of HT + DHFG also presents problems since the doses used to control the growth of weeds are at the limit with the doses that prevent the growth of crops, so it is not a useful candidate as a phytoregulator. Instead, the results have shown that DHFG is the main candidate to act as a phytoregulator, being effective as a herbicide, since in addition to presenting large differences between weeds and crops, it has advantages at low doses as a germination activator and of growth
Example 3. Analysis of the phytoregulatory activity of the DHFG compound in early post-emergence tests, both in weeds and in agricultural crops.
Plants in the first stage of growth development were planted in 25 cm3 alveoli with a substrate consisting of a silt: peat mixture (1: 1 ratio) and treated with HT, DHFG or HT + DHFG at different doses. The weed species and agricultural crops tested are those described in Tables Table 5 and Table 6, respectively. The doses tested for each of the treatments have been:
HT: 400, 800, 1600, 3200 and 6400 g active ingredient / ha DHFG: 40, 80, 400, 800 and 1600 g active ingredient / ha
HT + DHFG: 400, 800, 1600, 3200 and 4800 g active ingredient / ha (referred to component HT-ratio HT: DHFG is 1:10).
As a negative control, all the study cultures were used to which no treatment was applied. In the case of analyzing the herbicidal activity in weeds (all species will be used) the herbicide flazasulfuron will be used as a positive control at the same doses as the compounds tested.
After the application of each of the treatments to be tested, the alveoli were introduced into a controlled growth chamber (Fitotron; Weiss Technik; United Kingdom) with the following conditions: day / night temperature of 27/18 ° C; 14 h photoperiod; and light 580 pmol m-2 s-1, during a period of between 20 days, at which time the vegetation was cut and immediately after it was weighed and fresh weight reduction curves were constructed based on the concentrations of each of the extracts tested and the species tested. 10 plants were used as repetitions per treatment and species).
10 Each of the treatments mentioned above were applied via foliar spraying in the case of weed crops, since it is the usual mode of application for this type of vegetation, while in the case of species of agricultural interest, each One of the treatments tested was also applied by washing directly on the stem to confirm if there were differences between both types
15 of application on the final result. Any of the applications known to the person skilled in the art can be used for the compounds of the present invention. In the case of foliar spray, a volume of broth of 400L / ha was prepared, while for washing it was 10,000 L / ha.
Table 5. Weed species tested.
Monocotyledonous Dicot
Lolium spp. (foliar spray) Conyza spp. (foliar spray)
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Table 6. Species of agricultural crops tested.
Monocotyledonous Dicot
Graminaea: ■ Tríticum aestivum (Soft wheat) [Foliar spray] Compositae: ■ Lactuca sativa L. (Lettuce var. Cervantes) [Foliar spray]
Liliaceae: ■ Allium cepa (Onion var. Galaxy) [Foliar spray and direct wash] Solanaceae: ■ Lycopersicum esculentum L. (Tomato var. Three edges) [Foliar spray and direct wash] ■ Solanum tuberosum L. (Potato var. Red Repontiac) [Foliar spray] ■ Solanum melongena L. (Eggplant var.
Round Lisa) [Foliar spray and direct wash] ■ Capsicum L. (Italian pepper pepper) [Foliar spray and direct wash]
Cucurbitaceae: ■ Cucumis sativus L. (Cucumber var. Bellpuig) [Foliar spray and direct wash] ■ Cucurbita pepo L. (Zucchini var. Diamond) [Foliar spray and direct wash] ■ Citrullus lanatus L. (Watermelon var. Meridian) [ Foliar spray and direct wash]
Asparagaceae: ■ Asparagus officinalis L. (Asparagus var. Atlas) [Foliar spray]
The results obtained for the control of weeds using the different concentrations mentioned above of HT, DHFG or HT + DHFG on the Lolium spp species by foliar application showed that exclusively the treatment with DHFG was able to control the growth of said weed. The effective ED90 dose for this species when treated with said extract was 575 g active ingredient / ha, and the 1600 ppm dose achieved absolute control of its growth, while the ED90 dose for HT and HT + DHFG was> 3200 g active ingredient / ha and> 4800 g active ingredient / ha, 10 respectively, so that these treatments were not able to control the growth of this weed (Table 7). In the case of cultures of the Conyza spp species treated with the different concentrations mentioned above of HT, DHFG or HT + DHFG, the same results were obtained as for the Lolium spp species. Exclusively, treatment with DHFG was able to control the growth of this weed. The ED90 for this species when treated with DHFG is at 580 g active ingredient / hectare (Table 7). Absolute control of growth occurred at 800 g active ingredient / hectare.
Table 7. LD90 and dose that acts as a growth activator of the different species with the different compounds (expressed in grams of active ingredient / ha).
LD90 Growth Activating Dose
Species HT DHFG HT + DHFG HT DHFG HT + DHFG
Lolium spp. > 3200 575> 4800
Conyza spp. > 3200 580> 4800
L. sativa L. > 6800> 1600> 4800
L. esculentum L. > 6800> 1600> 4800
S.tuberosum L. 1920> 1600> 4800 400
S. melongena L. > 6800> 1600 4800
Capsicum L. > 6800> 1600> 4800
C. sativus L. > 6800 1600 4800
C. pepo L. > 6800> 1600 2800
C. lanatus L. > 6800> 1600 4800
A. officinalis L. > 6800> 1600> 4800
A. strain > 6800> 1600> 4800
T. aestivum 1520> 1600> 4800 400
5 As can be seen in Table 7, in the case of crops of agricultural interest, it is interesting to note that none of the doses of the compounds used caused phytotoxicity in the crops tested. On the other hand, it is interesting to note that treatment with DHFG induced an increase in weight and vigor in the cultures of T. aestivum and S. tuberosum at doses greater than 400 g active ingredient / ha.
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Thus, the results shown in Table 7 show that the cultures showed vigor and good development against the different doses of the different compounds. Negative effects were observed in some of the crops. The compound that showed the most negative effect was the HT + DHFG mixture, although seeing the dose not 15 is as important as that caused by the DHFG in the cucumber. Keep in mind that HT in foliar spray produces spots on the leaves of plants that appear to be fungi. The most important aspect of the results shown in Table 7 is that DHFG has growth-enhancing activity, which can be observed very well in S. melongena L. crops.
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(eggplant). The doses supported in plants with two to four true leaves are very high because the plant has already developed mechanisms that allow it to resist higher doses.
Example 4. Analysis of the phytoregulatory activity of the DHFG compound in late post-emergence tests, both in weeds and in agricultural crops.
Although in the previous example it has been shown that the effect of treatment with the DHFG compound is less on the induction of growth and germination in already developed plants, it is known that said plants under stress conditions are more susceptible to certain treatments. Therefore, the activity of the DHFG compound has been analyzed when it is administered in the cultures of: Lycopersicum esculentum L. (tomato); Allium cepa (onion); Capsicum L. (pepper); Cucúrbita pepo L. (zucchini); Lactuca sativa L. (lettuce); Solanum melongena L. (eggplant) and Asparagus officinalis L. (asparagus), when these crops were in a late post-emergence stage and also under conditions of abiotic stress (absence of irrigation caused by 2 days without irrigation-1 irrigation-3 days without irrigation-1 irrigation). The application of HT, DHFG and HT + DHFG has been done by spraying in all cases.
Table 8. LD90 of the different species with the different compounds (expressed in grams of active ingredient / ha).
LD90
Species HT DHFG HT + DHFG
L. sativa L. > 6800> 1600> 4800
L. esculentum L. > 6800> 1600> 4800
S. melongena L. > 6800> 1600 4800
Capsicum L. > 6800> 1600> 4800
C. pepo L. > 6800> 1600 2800
A. officinalis L. > 6800> 1600> 4800
A. strain > 6800> 1600> 4800
The cultures have a positive response to the different doses of the different compounds regardless of whether there are affected leaf areas or not. This indicates that the
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damage is minimal since a rapid recovery of them is observed followed by an improvement in its foliar development.
The results shown in Table 8 show that the species analyzed at very high stages of development are not susceptible to the compounds tested, even at very high doses.
Example 5. Analysis of the herbicidal activity of the DHFG compound in pre-emergency stages in the field.
The analysis of the herbicidal activity of the DHFG compound in pre-emergence (germination) stages in the field was carried out in horticultural plots in the town of Huétor-Tajar (Granada, Spain). The DHFG and the HT + DHFG mixture which showed herbicidal activity in the previous pre-emergence examples were tested. The application of these compounds was carried out by foliar spraying using a volume of broth of 400 L / ha, and the doses used have been:
- DHFG: 40, 400 and 800 g of active ingredient / ha
- HT + DHFG: 400, 800 and 1600 g of active ingredient / ha.
As a positive control, the herbicide Flazasulfuron has been used at doses of 100, 200 and 1000 g / ha.
In addition to the exclusive treatment with each of the compounds tested, the herbicidal activity was analyzed by adding an adjuvant to each of them. The adjuvants chosen were Araoil (paraffin oil, Fitalbi, Spain) which is an emulsifiable concentrate with contact insecticidal activity for pest control) and Biopower (alkylene ether sulfate, Bayer, Germany) which is a surfactant adjuvant (non-ionic wetting agent) It is recommended to increase the effectiveness of herbicides that are applied by contact to improve wetting power.
There have been 4 repetitions of each treatment in plots of 2x10 m2. The results show that the commercial herbicide Flazasulfuron is the most effective. In contrast, DHFG and the HT + DHFG mixture without the presence of additives enhance growth. When each of the tested additives is added to said extracts independently, the herbicidal efficacy of said compositions increases considerably, especially when the composition
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it comprises the Biopower adjuvant, which is also expected to mix the two with the adjuvant also to improve.
Additional tests, shown in examples 7 and 8, were carried out to determine the action of adjuvants on the foliar penetrability of the compounds and on the soil stabilization thereof.
Example 6. Analysis of foliar absorption and stability of the DHFG compound and the mixture (DHFG + HT) in pepper plants.
In order to analyze the amount of DHFG and HT + DHFG that the leaves of a plant are able to absorb, as well as the stability of said extracts after absorption, these variables were tested on pepper plant leaves. Briefly, 18 seedlings of Capsicum L. (pepper var. Italian) are transplanted into peat pots, irrigated and allowed to grow for 5 days. Two leaves were chosen from each pot and marked in duplicate.
Starting with DHFG (3.5 g / L), three solutions of 1000, 500 and 100 ppm of DHFG were prepared. In the same way, we proceeded with the DHFG + HT mixture whose concentrations were 1.2 and 13.6 g / L, respectively. The solutions of the mixture were also 1000, 500 and 100 ppm DHFG with respect to HT. These concentrations were chosen based on the stability that the plants presented against these concentrations in the previous examples. In addition, Araoil (paraffin oil that is applied on the 0.5% extract) or Biopower (alkylene ether sulfate applied on the 1% extract) was also applied to each of the compounds tested.
After the 5 days have elapsed and the desired size of each plant is reached, the test is started by applying 0.1 mL of each compound with a syringe for each concentration and each adjuvant, on the leaves of each plant in duplicate. Once the different compounds have been applied with the respective adjuvants, they are allowed to dry for two hours and the leaves of each pot are washed with 10 mL of 50% methanol / water in order to drag the amount of compound that the plant has not absorbed. The petri dish wash is collected and its concentration in HPLC is determined.
Table 9 shows the results as a percentage of active ingredient (DHFG) absorbed by treated sheet and obtained by HPLC and fluorescence.
Table 9. Percentage of 3.4 DHFG absorbed per treated sheet.
Treatment DHFG added (mg) DHFG extracted (mg)% active ingredient absorbed
DHFG 1000 ppm (1) 0.1 0.0984 1.60
DHFG 1000 ppm (2) 0.1 0.0785 21.44
DHFG 1000 ppm + Bio (1) 0.1 0.0513 48.68
DHFG 1000 ppm + Bio (2) 0.1 0.0917 8.24
DHFG 1000 ppm + Ara (1) 0.1 0.0510 48.97
DHFG 1000 ppm + Ara (2) 0.1 0.0381 61.90
HT + DHFG 1000 ppm (1) 0.1 0.0110 8.89
HT + DHFG 1000 ppm (1) 0.1 0.0115 8.85
HT + DHFG 1000 ppm + Bio (1) 0.1 0.0058 9.42
HT + DHFG 1000 ppm + Bio (2) 0.1 0.0059 9.41
HT + DHFG 1000 ppm + Ara (1) 0.1 0.0082 9.17
HT + DHFG 1000 ppm + Ara (2) 0.1 0.0081 9.18
DHFG 500 ppm (1) 0.05 0.0065 86.88
DHFG 500 ppm (2) 0.05 0.0108 78.33
DHFG 500 ppm + Bio (1) 0.05 0.0019 96.02
DHFG 500 ppm + Bio (2) 0.05 0.0034 93.14
DHFG 500 ppm + Ara (1) 0.05 0.0113 77.28
DHFG 500 ppm + Ara (2) 0.05 0.0148 70.32
HT + DHFG 500 ppm (1) 0.05 0.0030 9.39
HT + DHFG 500 ppm (1) 0.05 0.0026 9.47
HT + DHFG 500 ppm + Bio (1) 0.05 0.0019 9.60
HT + DHFG 500 ppm + Bio (2) 0.05 0.0019 9.62
HT + DHFG 500 ppm + Ara (1) 0.05 0.0032 9.34
HT + DHFG 500 ppm + Ara (2) 0.05 0.0037 9.26
DHFG 100 ppm (1) 0.01 0.0015 84.46
DHFG 100 ppm (2) 0.01 0.0004 95.57
DHFG 100 ppm + Bio (1) 0.01 0.0003 96.29
DHFG 100 ppm + Bio (2) 0.01 0.0004 95.83
DHFG 100 ppm + Ara (1) 0.01 0.0015 84.18
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Treatment DHFG added (mg) DHFG extracted (mg)% active ingredient absorbed
DHFG 100 ppm + Ara (2) 0.01 0.0014 85.15
HT + DHFG 100 ppm (1) 0.01 0.00003 9.96
HT + DHFG 100 ppm (1) 0.01 0.00003 9.97
HT + DHFG 100 ppm + Bio (1) 0.01 0.00003 9.96
HT + DHFG 100 ppm + Bio (2) 0.01 0.00003 9.96
HT + DHFG 100 ppm + Ara (1) 0.01 0.00041 9.58
HT + DHFG 100 ppm + Ara (2) 0.01 0.00046 9.53
Table 9 clearly shows how at the concentration of 1000 ppm of DHFG the absorption is much lower in all cases compared to the concentrations of 500 and 100 ppm. In the case of the HT + DHFG mixture, the absorption percentage is maintained in all cases above 10%, regardless of the initial concentration of each active ingredient, showing that more quantity is absorbed by the leaves when It uses the HT + DHFG mix, which when using DHFG only. Also striking is the variability between the samples, and the high absorption of DHFG when used at concentrations of 500 ppm and 100 ppm, both directly, and in combination with the Biopower or Araoil adjuvants. Despite this, it is seen that the use of adjuvants does not dramatically improve the absorption of DHFG, which is absorbed above 80% in concentrations of 100 and 500 ppm.
Example 7. Analysis of the stability of the DHFG compound in wheat crops during germination.
The objective of this test is to determine the stability of a 5 g / L DHFG extract diluted at different concentrations (50, 100, 200, 500, 1000 and 2000 ppm) during the seed germination process of Tríticum durum (Hard Wheat ). These seeds were provided by the Department of Agricultural Chemistry and Edaphology of the University of Córdoba. They are immersed in a 1% sodium hypochlorite solution for 10 seconds to break the shell and facilitate germination, then rinse with distilled water and let dry. Once dried, seven sterile Petri dishes are prepared with separate filter paper discs and 15 seeds are placed
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of wheat in each of them. Then, 5 mL of the different concentrations of DHFG indicated above and a blank with distilled water are added. Petri dishes are sealed with parafilm and left 48 hours at 4 ° C in the dark. After this time they are taken out of the cold chamber and allowed to germinate 3 more days, avoiding the incidence of direct sunlight. After 5 days from the start of the test, samples of the supernatant are taken and the concentration of DHFG in each plate is determined using HPLC-UV at 280 nm. The chromatographic profiles (HPLC) obtained show that no other compounds are formed by degradation or oxidation of DHFG and that it is maintained, verifying the stability of the DHFG compound in the seed without any adjuvant. The concentration of the DHFG compound extracted from each of the germination plates tested is shown in Table 10 below.
Table 10. DHFG concentration extracted from each germination plate of T. durum cultures.
Initial [DHFG] (mg / mL) [DHFG] final (mg / mL)% extracted
0 0 0
fifty 94.78 189.50
100 143.22 143.22
200 198.02 99.01
500 399.06 79.81
1000 977.82 97.80
2000 1913.33 95.70
In Table 10 it can be seen that after 5 days most of the DHFG remains as such in contact with the seed. It should be noted that in two of the concentrations a data has been obtained above the initial value, such as that of 50 and 100 ppm, which may be due to the fact that the solution that impregnates the plate has been able to concentrate or that part of that compound is even endogenous of the plant.
Example 8. Effect of solar radiation on soil samples impregnated with DHFG.
In order to determine the influence of solar radiation on DHFG once impregnated in the farmland, samples of untreated land were taken from
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plots where field studies were carried out in Huetor-Tájar (Granada) and spread in trays of 0.53 x 0.38 m equivalent to 0.2 m2 of surface. Starting from the extract with a concentration of 3.5 g / L of DHFG, solutions of 1000, 500 and 100 ppm were prepared, and 25 mL of each solution was sprayed onto the trays. Said volume was determined knowing that at least 400 L of broth per hectare has been used in the field. In this way, three trays were prepared, one for each extract concentration, divided into two distinct parts, one exposed to solar radiation and the other protected from said radiation when covered with aluminum foil, completely opaque to radiation. A sample was taken at zero time and two more every 24 hours. For each sample, 10g of soil was weighed and extracted cold with 10 mL of a methanol / water mixture (80:20 (v / v)) and with stirring for one hour. Then, the extracted earth was centrifuged and filtered. The concentration of DHFG extracted was determined by HPLC-Fluorescence. Table 11 shows the determined amounts of 3.4 DHFG per gram of land at time 0, 24 and 48 h after application.
Table 11. Amount of DHFG added and extracted in soil samples exposed to solar radiation or in darkness.
[DHFG] mg DHFG g / m2 added mg DHFG g / m2 land 0h mg DHFG g / m2 land 24h mg DHFG g / m2 land 48h
100 ppm light 4.00 0.83 0.78 0.37
100 ppm darkness 4.00 0.81 0.73 0.29
500 ppm light 20.00 7.65 7.65 3.91
500 ppm darkness 20.00 6.66 4.04 2.87
1000 ppm light 40.00 31.20 9.82 1.56
1000 ppm darkness 40.00 22.46 8.73 0.87
It is observed in Table 11 that when DHFG is added to the farmland, a large part of the DHFG compound does not recover in the first extracts and that its concentration in the soil decreases over time, drastically reducing at 48h. It should be noted that at the lowest concentration there are no differences between the trial with and without light, however at the two most concentrations
High differences can be seen, the highest values being those found with solar incidence. The values that remain higher are those corresponding to the concentration of 500 ppm, with a greater decrease in concentration being observed for 1000 ppm. With this test it is verified that DHFG can remain stable, 5 independent of sunlight, on land for a period of 48 hours without the use of adjuvants.
Example 9. Oxidation tests of the DHFG compound at acidic pH and basic pH of the sample.
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To demonstrate that DHFG does not degrade in aqueous solution at both acidic and basic pH, a forced oxidation test at two pHs was performed.
First, an oxidation kinetics of the extract rich in DHFG 15 was carried out under conditions of low concentration at room temperature and with a continuous flow of air, and at the original pH of the sample, acidic pH (6.3). Briefly, it was based on a DHFG extract whose concentration is 0.4 g / L at 96% purity (it is the same extract that has been used in the previous examples for antigerminative tests) and two test tubes with 5 mL were taken of said extract in 20 each tube. A direct oxygen stream was passed through both tubes measuring the concentration of DHFG according to the kinetic profile shown in Table 12.
Table 12. Oxidation kinetics of the DHFG compound at acidic pH 6.3.
Time (h) [DHFG] g / L V final DHFG (mL) g total DHFG
0 0.40 5.00 2.00
0.5 (duplicate 1) 0.40 5.00 2.00
0.5 (duplicate 2) 0.40 5.00 2.00
1 (duplicate 1) 0.46 4.35 2.00
1 (duplicate 2) 0.47 4.25 2.00
2 (duplicate 1) 0.56 3.50 1.96
2 (duplicate 2) 0.57 3.50 1.96
3 (duplicate 1) 0.70 2.85 1.99
3 (duplicate 2) 0.60 3.30 1.98
4 (duplicate 1) 0.81 2.35 1.90
4 (duplicate 2) 0.92 2.15 1.98
Time (h) [DHFG] g / L V final DHFG (mL) g total DHFG
5 (duplicate 1) 1.15 1.70 1.95
5 (duplicate 2) 1.51 1.30 1.96
8 (duplicate 1) Solid 0 2.01
8 (duplicate 2) Solid 0 1.98
As can be seen, the concentration of the DHFG compound increases due to the evaporation effect of the sample, but the amount of DHFG remains constant over time, which can be concluded that with an oxygen current and after five hours of testing , there is no loss of the main component of the extract. The extract was continued to dryness (8 hours), at which time the solid was redissolved to the initial volume and the concentration was determined by observing that nothing of the initial DHFG compound had been lost.
The same test as described above was carried out but at pH 8. For this, 20 mL of the same extract mentioned above was taken and taken to pH 8 with a 10% NaOH solution. One mL of sample was taken in duplicate, through which a stream of oxygen was passed to dryness (Table 13). It was then redissolved with 1 mL of distilled water and its concentration was measured, making the whole test in duplicate.
Table 13. Oxidation kinetics of the DHFG compound at basic pH 8.
Time (h) [DHFG] g / L V final DHFG (mL) g total DHFG
0 0.40 5.00 2.00
0.5 (duplicate 1) 0.40 5.00 2.00
0.5 (duplicate 2) 0.40 5.00 2.00
1 (duplicate 1) 0.48 4.20 2.00
1 (duplicate 2) 0.46 4.25 1.97
2 (duplicate 1) 0.64 3.05 1.95
2 (duplicate 2) 0.64 3.10 1.98
3 (duplicate 1) 0.74 2.60 1.92
3 (duplicate 2) 0.76 2.45 1.87
4 (duplicate 1) 0.90 2.00 1.80
4 (duplicate 2) 0.86 2.10 1.81
Time (h) [DHFG] g / L V final DHFG (mL) g total DHFG
5 (duplicate 1) 2.09 0.85 1.78
5 (duplicate 2) 1.98 0.95 1.88
8 (duplicate 1) Solid 0 1.75
8 (duplicate 2) Solid 0 1.90
As indicated above when the pH is acidic, neither at the basic pH does significant loss of the DHFG compound occur by oxidation when the extract rich in said compound is subjected to basic pH. Therefore, it is concluded that under the conditions tested, acidic pH (pH 6.3) and basic pH (pH 8), no oxidation of the DHFG compound occurs.
Therefore, the present invention demonstrates the phyto-regulatory nature of DHFG as a natural compound, and that the use of adjuvants substantially improves its field activity. The use of said adjuvants, in combination with the DHFG compound, as demonstrated in the present invention exerts a different effect than they should exert and for which they are designed, since DHFG penetrates the plant, remains stable in the seed. and in the soil regardless of the pH or the solar incidence without the help of said adjuvants.

权利要求:
Claims (30)
[1]
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1. Use of 3,4 dihydroxyphenyl glycol (DHFG) as a phytoregulator.
[2]
2. Use according to claim 1 characterized in that the DHFG is obtained from by-products derived from the extraction of olive oil and / or from the olive dressing industry, characterized in that it comprises a concentration of DHFG of between 1 and 50,000 ppm
[3]
3. Use according to any one of claims 1 to 2 wherein the phytoregulatory activity is selected from herbicidal activity against weeds or growth activity of agricultural crops.
[4]
4. Use according to claim 3 wherein the herbicidal activity is carried out when a concentration of between 100 to 50,000 ppm of DHFG is applied.
[5]
5. Use according to claim 4 wherein the herbicidal activity is carried out when a concentration of between 500 to 9000 ppm of DHFG is applied.
[6]
6. Use according to claim 3 wherein the growth enhancing activity is carried out when a concentration of between 1 to 1500 ppm of DHFG is applied.
[7]
7. Use according to claim 6 wherein the growth enhancing activity is carried out when a concentration of between 1 to 500 ppm of DHFG is applied.
[8]
8. Use according to any one of claims 1 to 7 in combination with at least one adjuvant.
[9]
9. Use according to claim 8 wherein the adjuvant is selected from any of the list consisting of: oil-based adjuvants and mixtures, organosilicone-based adjuvants and mixtures, non-ionic-based adjuvants and mixtures, polymer-based adjuvants and mixtures, and adjuvants based on fatty acids and mixtures, and combinations thereof.
[10]
10. Use according to claim 9 wherein the adjuvant is selected from araoil and / or biopower.
[11]
11. Extract obtained from by-products derived from the extraction of olive oil and / or from the olive dressing industry, characterized in that it comprises a concentration of DHFG between 1 and 50,000 ppm.
[12]
12. Phytoregulatory composition comprising DHFG according to any of claims 1 to 7 and / or an extract according to claim 11.
[13]
13. Composition according to claim 12 further comprising at least one adjuvant.
[14]
14. Composition according to any of claims 12 to 13 characterized in that the adjuvant is selected from any of the list consisting of:
5
10
fifteen
twenty
25
30
35
oil-based adjuvants and mixtures, organosilicone-based adjuvants and mixtures, non-ionic-based adjuvants and mixtures, polymeric-based adjuvants and mixtures, and fatty acid-based adjuvants and mixtures, and combinations thereof.
[15]
15. Composition according to any of claims 13 to 14 wherein the adjuvant is selected from araoil and / or biopower.
[16]
16. Composition according to any of claims 12 to 15 characterized in that the concentration of DHFG is between 1 to 50,000 ppm.
[17]
17. Composition according to any of claims 12 to 16 characterized in that it has herbicidal action when the concentration of DHFG is between 100 to 50,000 ppm and growth-enhancing activity when the concentration of DHFG is 1 to 1500 ppm.
[18]
18. Composition according to any of claims 13 to 17 characterized in that it has herbicidal action when the concentration of DHFG is between 500 to 9000 ppm and growth-enhancing activity when the concentration of DHFG is 1 to 500 ppm.
[19]
19. Method for the control of weeds which comprises administering to an culture an effective dose of DHFG according to any of claims 1 to 7, of the extract according to claim 11, or of the composition according to any of claims 12 to 18.
[20]
20. Method according to claim 19 characterized in that the effective dose is administered in the range of 100 to 50,000 ppm.
[21]
21. Method according to any of claims 19 to 20 characterized in that the effective dose is administered in the range of 500 to 9000 ppm.
[22]
22. Method according to any of claims 19 to 21 wherein the weeds are preferably monocotyledonous and / or dicotyledonous.
[23]
23. Method according to claim 22 wherein monocot plants are selected from any of the following species: Lolium rigidum; Lolium multiflorum, and dicotyledons are selected from any of the following species: Sinapis arvensis, Chenopodium album and Stellaria media.
[24]
24. Method according to any of claims 19 to 23 characterized in that the effective dose of DHFG, of the extract or of the composition, is administered via spraying, atomization, dispersion, coating, or pouring.
[25]
25. Method for inducing crop growth comprising administering to an crop an effective dose of DHFG according to any one of claims 1 to 7,
10
fifteen
of extract according to claim 11, or of the composition according to any of claims 12 to 18.
[26]
26. Method according to claim 25 characterized in that the effective dose is administered in the range of 1 to 1500 ppm.
[27]
27. Method according to any of claims 25 to 26 characterized in that the effective dose is administered in the range of 1 to 500 ppm.
[28]
28. A method according to any one of claims 25 to 27, wherein the cultures are preferably monocot cultures and / or dicot cultures.
[29]
29. A method according to claim 28 wherein monocot cultures are selected from any of the following species: Triticum aestivum Allium cepa, and dicot cultures are selected from any of the following species: Lepidium sativum L., Lactuca sativa L ., Lycopersicum esculentum L., Solanum tuberosum L., Solanum melongena L., Capsicum L .; Cucumis sativus L., Cucurbita pepo L., Citrullus lanatus L., Asparagus officinalis L.
[30]
30. Method according to any of claims 25 to 29 characterized in that the effective dose of DHFG, of the extract or of the composition, is administered via spraying, atomization, dispersion, coating, or pouring.

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同族专利:

公开号 | 公开日

ES2671137B1|2019-03-12|

EP3539383A1|2019-09-18|

EP3539383A4|2020-05-27|

WO2018083361A1|2018-05-11|

MA46765A|2019-09-18|

引用文献:

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

ES2315351T3|2001-02-15|2009-04-01|Consejo Superior De Investigaciones Cientificas|METHOD FOR OBTAINING PURIFIED HYDROXYTIROSOL FROM PRODUCTS AND SUBPRODUCTS DERIVED FROM OLIVE OIL.|

ES2341526B1|2008-12-19|2011-06-08|Consejo Superior De Investigaciones Cientificas |PURIFICATION PROCEDURE OF 3,4-DIHYDROXIFENYLGLYCOL FROM VEGETABLE PRODUCTS.|

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ES201631404A|ES2671137B1|2016-11-03|2016-11-03|USE OF 3,4-DIHYDROXYPHENILGLICOLAS A PHYOR REGULATOR|ES201631404A| ES2671137B1|2016-11-03|2016-11-03|USE OF 3,4-DIHYDROXYPHENILGLICOLAS A PHYOR REGULATOR|

PCT/ES2017/070724| WO2018083361A1|2016-11-03|2017-10-31|Use of 3,4-dihydroxyphenylglycolas a plant growth regulator|

MA046765A| MA46765A|2016-11-03|2017-10-31|USE OF 3,4-DIHYDROXY-PHENYL-GLYCOLAS A PHYTO-REGULATOR|

EP17867232.5A| EP3539383A4|2016-11-03|2017-10-31|Use of 3,4-dihydroxyphenylglycolas a plant growth regulator|

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