ES2920198B2 - Aspergillus tubingensis strain and its use for the isolation of nematicidal compounds - Google Patents

Description

DESCRIPTION

Aspergillus tubingensis strain and its use for the isolation of nematicidal compounds

field of invention

The present invention belongs to the technical field of compounds for controlling pests in crops, more specifically nematicidal compounds.

The isolation of compounds from a new strain of the species Aspergillus tubingensis , to which the present invention also relates, is described.

Other aspects of the invention are compositions that comprise nematicidal compounds isolated from the new strain of the species Aspergillus tubingensis, and the procedure for controlling nematodes in crops.

Background of the invention

The damage that nematodes cause to the main crops is known, both in greenhouses and agricultural plantations in open fields, as well as in gardens.

In order to control the disease caused by nematodes, chemical control can be used on them. Chemical control of nematodes is carried out through treatment with chemical agents such as carbamates, organophosphates and similar agents. Although chemical nematicides, mainly organophosphates or carbamates, achieve high control of phytopathogenic nematodes, they are highly toxic when they remain in the soil, accumulate in crop plants and harm human and animal health. They also cause environmental pollution due to the negative actions they exert against soil microorganisms, which are beneficial for the growth of crops, and gradually cause the soil to become sterile.

Therefore, the use of chemical pesticides has been avoided due to problems such as environmental pollution, destruction of the ecosystem, pesticide poisoning of humans and animals, and the demand for products that respect the environment has also increased in parallel.

Along these lines, there is a growing demand for the development of nematicides that are safer for the environment and that can replace the chemical pesticides mentioned above.

In recent years, among environmentally friendly methods for controlling nematodes, methods that use microorganisms have become increasingly numerous.

Studies have shown that microorganisms are capable of producing various metabolites that act directly on pathogens, reducing their negative effects on crops.

European patent EP3282007 refers to the strain of Aspergillus niger F22 and its nematicidal activity against plant parasitic nematodes and specifically against Meloidogyne spp. This document does not describe any of the active compounds produced by strain F22.

The present invention describes a new strain of Aspergillus tubingensis, its culture broth and its crude extract, and identifies the compounds with nematicidal activity produced by it.

Description of the invention

The present invention relates to the isolation of nematicidal compounds from a new strain of Aspergillus tubingensis.

The new strain of Aspergillus tubingensis is an endophytic microorganism. The first aspect of the invention is the new strain of the species Aspergillus tubingensis, (strain of the invention or strain of Aspergillus tubingensis of the invention referenced as strain BP3 in the examples), which was deposited on December 15, 2020 in the Collection Spanish Type Crops (CECT) (Burjassot, Valencia, Spain) and to which the accession number CECT 21186 was assigned.

Other aspects of the invention are the fermentation broth and the crude extract extracted from the strain of the invention.

The fermentation broth of the strain of the invention is obtained from a culture of the strain of the invention by a procedure that comprises:

a) cultivate the strain of Aspergillus tubingensis of the invention, until the mycelium develops;

b) ferment the mycelium developed in a) in a liquid medium;

c) separate the mycelium to obtain the fermentation broth.

The crude extract of the strain of the invention is obtained when, after steps a)-c) described above, an extraction is carried out from the fermentation broth.

Therefore, another aspect of the invention is the procedure for obtaining the crude extract that includes the steps of:

a) cultivate the strain of Aspergillus tubingensis of the invention, until the mycelium develops;

b) ferment the mycelium developed in a) in a liquid medium;

c) separate the mycelium to obtain the fermentation broth;

d) perform an extraction from the fermentation broth obtained in c) to obtain the crude extract.

This strain has nematicidal activity, so another aspect of the invention refers to a nematicidal agent that includes the fermentation broth and/or a crude extract of the strain of the invention.

The invention also includes a method for controlling nematodes that comprises the following step, contacting a plant, seed, seedling, or substrate with the strain and/or the fermentation broth and/or a crude extract of the strain of the invention.

From this strain of the invention, a series of compounds were isolated and their nematicidal activity was verified.

Therefore, another aspect of the invention is the use of the strain of the invention to obtain the compound of formula (I), and/or the compound of formula (II), and/or the compound of formula (III). and/or the compound of formula (IV);

and the salts and/or solvates thereof.

Also part of the invention is the procedure for obtaining the compound of formula (I) and/or the compound of formula (II) and/or the compound of formula (III) and/or the compound of formula (IV), a procedure that includes the steps of:

a) cultivate the strain of Aspergillus tubingensis of the invention, until the mycelium develops;

b) ferment the mycelium developed in a) in a liquid medium;

c) separate the mycelium to obtain the fermentation broth;

d) perform an extraction, from the fermentation broth obtained in c) to obtain the crude extract;

e) isolating the compound of formula (I) and/or the compound of formula (II) and/or the compound of formula (III) and/or the compound of formula (IV).

The compounds of formula (I), formula (II), formula (III) and formula (IV) have nematicidal activity, therefore, another aspect of the invention is the use of said compounds as nematicides.

With the compounds of formula (I) and/or formula (II) and/or formula (III) and/or formula (IV) nematicidal compositions can be formulated and therefore another aspect of the invention is a composition for the control of nematodes comprising as active ingredient at least one of the compounds selected from: compound of formula (I), compound of formula (II), compound of formula (III) and compound of formula (IV) or a salt or a solvate of these , acceptable in agriculture.

Finally, an aspect of the invention is a method for controlling nematodes that comprises the step of contacting a plant, seed, seedling, or substrate with the compound of formula (I) and/or the compound of formula (II) and /or the compound of formula (III) and/or the compound of formula (IV).

Detailed description of the invention

As has been said, one aspect of the invention is the strain of the Aspergillus tubingensis species of the invention, deposited in the Spanish Type Culture Collection (CECT) (Burjassot, Valencia, Spain) with the accession number CECT 21186.

As said before, the crude extract of the strain of the invention is obtained from a culture of the strain of the invention by a procedure that comprises:

a) cultivate the strain of Aspergillus tubingensis of the invention, until the mycelium develops;

b) ferment the mycelium developed in a) in a liquid medium;

c) separate the mycelium to obtain the fermentation broth;

d) carry out an extraction of the fermentation broth to obtain a crude extract.

The procedure for obtaining the fermentation broth includes steps a) to c).

In embodiments of the invention the strain can be grown in a liquid medium or in a solid medium. Preferably in step a) the culture is carried out in solid PDA medium in the dark.

Preferably in step b) the liquid medium is a modified Czapek-Dox culture medium as described in example 1. Preferably it is fermented between 4 and 14 days, more preferably between 5 and 8 days.

Preferably, the separation of step c) is carried out by filtration.

In step d) the solvent can be selected from: dichloromethane, ethyl ether and ethyl acetate. In particular, the solvent of step d) is ethyl acetate.

As has been said, the crude extract of the strain and the culture broth of the invention have nematicidal activity. In particular, they have nematicidal activity against nematodes, particularly the genus Meloidogyne and, more particularly, Meloidogyne javanica .

Nodule-forming nematodes of the genus Meloidogyne are one of the main pathogens of plant species and have a worldwide distribution. They are obligate endoparasites, polyphagous in nature, and infect more than 3000 cultivated plant species, causing severe damage with consequent economic losses. The infection is It occurs when the second-age juvenile (J2), mobile and infective, is attracted by the root system of the host plant. Throughout its life cycle the nematode remains inside the root and the infection affects the growth of the plant, causes wilting, increases its susceptibility to other pathogens and in certain conditions can cause its death.

As has been said, the procedure for obtaining the compound of formula (I) and/or the compound of formula (II) and/or the compound of formula (III) and/or the compound of formula (IV) also forms part of the invention. ), procedure comprising steps a) to e) previously described and a last step f) where step f) is to isolate the compound of formula (I) and/or the compound of formula (II) and/or the compound of formula (III) and/or the compound of formula (IV).

Preferably, isolation is carried out by fractionation techniques.

In embodiments of the invention, the fractionation techniques can be selected between: vacuum liquid chromatography, column chromatography, column chromatography using different solid phases (silica gel, sephadex LH-20) and high-performance liquid chromatography (HPLC), eluting with different polarity gradients using different combinations of organic solvents.

As another aspect has been said, the invention refers to a composition for the control of nematodes that comprises as an active ingredient at least one of the compounds selected from: compound of formula (I), compound of formula (II), compound of formula (III) and compound of formula (IV) or a salt or solvate thereof, acceptable in agriculture.

The composition of the invention may additionally comprise various vehicles and agents that facilitate its conservation, handling and application.

The vehicles are selected from: solid vehicles, liquid vehicles, gaseous vehicles.

In embodiments of the invention, the agents can be selected from surfactants and auxiliaries for the formulation of compositions of the invention.

For example, an additive can be added to the composition to formulate forms such as concentrates, wettable powders, flowable liquids (e.g. suspension in water, emulsion in water, among others), powders, aerosols.

Examples of solid carriers that fall within the scope of the invention are fine powders or granules of clays (e.g. kaolin clay, diatomaceous earth, synthetic hydrated silicon oxide, bentonite, Fubasaml clay, acid clay), talcs, ceramics and other inorganic minerals, such as, for example, sericite, quartz, sulfur, active carbon, calcium carbonate, hydrated silica, commercial fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium chloride.

Examples of liquid carriers that fall within the scope of the invention are water, alcohols (e.g., methanol, ethanol), ketones (e.g., acetone, methyl ethyl ketone), aromatic hydrocarbons (e.g., benzene, toluene, xylene, ethylbenzene, methylnaphthalene), aliphatic hydrocarbons (e.g. hexane, cyclohexane, kerosene, diesel oil), esters (e.g. ethyl acetate, butyl acetate), nitriles (e.g. acetonitrile, isobutyronitrile), ethers (e.g. diisopropyl ether , dioxane), acid amides (e.g., N, N-dimethylformamide, N, N-dimethylacetamide), halogenated hydrocarbons (e.g., dichloroethane, trichloroethane, carbon tetrachloride), dimethyl sulfoxide, vegetable oils (e.g., oil soybean, cottonseed oil).

Examples of gaseous carrier that fall within the scope of the invention are spray agent, including butane gas, LPG (liquefied petroleum gas), dimethyl ether, carbon dioxide gas.

Examples of surfactants that fall within the scope of the invention are: alkyl sulfates, alkyl sulfonate salts, alkyl aryl sulfonates, alkyl aryl esters, polyoxyethylene compounds thereof, polyethylene glycolic esters, polyhydroxy alcohol esters and derivatives. of sugar alcohol.

Examples of auxiliary agent for formulation as fixing agent and dispersing agent that are included in the invention are casein, gelatin, polysaccharides (e.g., starch powder, gum arabic, cellulose derivative, alginic acid), lignin derivatives, bentonite, sugars, synthetic water-soluble polymers (e.g., polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acids).

Examples of stabilizers that fall within the scope of the invention are PAP (isopropyl acid phosphate), BHT (2,6-di-tert-butyl-4-methylphenol), BHA (mixture of 2-tert-butyl-4methoxyphenol and 3-tert-butyl-4-methoxyphenol), vegetable oils, mineral oils, surfactants, fatty acids or esters thereof.

The fermentation broth, the crude extract and the compositions of the invention can be used together with at least one other additional active ingredient. Examples of additional active ingredient are insecticides, acaricides, fungicides, herbicides, plant growth regulators, fertilizers, soil conditioners and animal baits.

Examples

Example 1: Crop preparation

It began with carrying out a culture in solid PDA medium (Potato, dextrose and agar) of a sample of the strain of the fungus Aspergillus tubingensis of the invention (called BP3 in the examples) preserved at -80°C. The seeded plate sealed with parafilm was incubated at 25°C in the dark in a culture chamber for 8 days. Subsequently, several replicas were prepared that were grown under the same conditions to have material and start fermentation in a liquid medium.

Preparation of pre-inocula.

250 mL Erlenmeyer flasks were prepared with a volume of 50 mL of modified Czapek-Dox culture medium. [NaNO ³ (2 g/L), KH ² PO ⁴ (5 g/L), MgSÜ ⁴ (0.5 g/L), FeSO ⁴ (0.01 g/L), ZnSÜ ⁴ (0.003 g/L ), yeast extract (1 g/L) and glucose (60 g/L)]. The flasks were autoclaved for 25 minutes at 121°C. Each of them was inoculated under sterile conditions under a laminar flow hood by scraping the mycelium from a plate to which 10mL of sterile water had been added to the surface of the colony. Once the scraping process was completed, the incubation was carried out in a culture chamber at 25°C and continuous shaking in an orbital shaker (120 rpm) for 3 days.

Fermentation process

Fermentation was carried out in 500 ml flasks with a volume of 200 mL of culture medium for 5, 8 and 12 days under the same shaking and temperature conditions described. A volume of 10 mL of the pre-inocula culture per flask was used as inoculum.

Obtaining the crude extract

The crude extracts were obtained from 5, 8 and 12 day cultures of strain BP3, the mycelium was separated from the medium by vacuum filtration on a Büchner funnel. The pH value of the liquid medium was recorded (pH=4.31) and a liquid-liquid extraction was performed with acetate. ethyl as organic solvent. The process was repeated three times drying the organic phases over Na ² SO ⁴ . Removal of the solvent under reduced pressure in a rotary evaporator led to obtaining the crude extracts with yields of 1.3, 2.8 and 2.4 g/L, respectively.

Example 2. Test of the biological activity of the extract obtained in Example 1

Biological activity assay

The nematode species used as the target species in the biological activity test of the extract was Meloidogyne javanica.

Nematicidal activity: In vitro tests with infective juveniles (J2)

Sterile 96-well U-bottom cell culture plates were used. 5 μl of the extract at a concentration of 20 μg/μl and 95 μl of water with approximately 100 J2 were added to each well. The final concentration of the extract and/or treatment to be tested was 1 μg/μl. 4 repetitions were carried out per treatment. The controls included 95 μl of water with nematodes plus 5 μl of aqueous solution with the same solvent used (DMSO+Tween 0.6%) for the treatments. The distribution of the samples was carried out in groups of 4 at random and leaving the outer row and column filled with water to maintain humidity and avoid the edge effect. The plate was incubated in the dark at 24°C for 72 hours.

After the incubation period, dead and live nematodes were counted under a stereoscopic microscope. Nematicidal activity data were presented as J2 mortality percentage corrected according to the Scheider-Orelli formula.

The extracts of Example 1 were active on M. javanica with 100% mortality.

Example 3. Biodirected fractionation of the crude extract

Techniques used for fractionation and chemical characterization

Column chromatography (CC)

For normal phase chromatography, silica gel and a hexane-ethyl acetate-methanol gradient of increasing polarities (5 cm diameter, 15 cm h, Si 35-70 um) were used.

Preparative HPLC Chromatography

An HPLC pump (Varian ProStar 210) and an Interchrom KR5/25M column were used. The flow rate was 20 ml/min collected in 10 ml tubes at half capacity with a Gilson FC203B collector.

Size exclusion chromatography

Sephadex LH-20 (Pharmacia Fine Chemicals) was used as a support for the stationary phase and a mixture of solvents n-hexane:CH ² Cl ² :MeOH in different proportions, depending on the polarity of the sample to be chromatographed, as eluent.

Thin Layer Chromatography (TLC)

The monitoring of the chromatographic columns was carried out by thin layer chromatography, using commercial chromatographs of silica gel 60 F254 with aluminum base (20 x 20 cm, art. 5554) and neutral aluminum oxide 60 F254 with aluminum base (20 x 20 cm, art. 5550), purchased from Merck. The same solvents and polarities were used in the elution system as in the chromatographic columns. The development of the chromatographs was carried out using the oleum/vanillin reagent to detect the compounds, by spraying on the chromatographs and heat.

OLEUM: is composed of a mixture of sulfuric acid (4%), acetic acid (80%) and distilled water (16%). The plates were sprayed with this reagent and visualized by applying heat (110°C-130°C).

VANILLIN: 0.5 g vanillin, 100 ml sulfuric acid/ethanol (40:10).

Mass coupled gas chromatography (GC-MS)

For the qualitative and quantitative determination of the compounds present in the essential oils and volatile fractions, gas chromatography coupled to mass spectrometry (GC-MS) was used as a methodology; a Shimadzu GC-2010 equipment coupled to a gas detector was used. Shimadzu GCMS-QP2010-Ultra masses with an electron impact ionization source at 70 eV and using a Simple Quadrupole analyzer. Helium was used as carrier gas. Chromatography was carried out with a Teknokroma TRB-5 (95%) Dimethyl-(5%) diphenylpolysiloxane capillary column, 30 m x 0.25 mm ID and 0.25 ^m phase thickness. The working conditions used were: injection in Split mode injecting 1 ^l of sample with a split ratio (20:1), injector temperature 300°C, temperature of the transfer line connected to the mass spectrometer 250°C and ionization source temperature 220°C. The initial column temperature was 70°C, heating to 290°C at 6°C/min and leaving at 290°C for 15 min. The mass spectra and retention time were used to identify the compounds by comparison with those found in the Wiley database (Wiley 275 Mass Spectra Database, 2001), while the relative area % of all were used for quantification. the peaks obtained in the chromatograms.

In general, NMR spectra (1H and 13C) were obtained with Bruker Advance-AMX-500 spectrometers (1H 500 MHz/13C 125 MHz). A Micromass LCT Premier mass spectrometer was used for high resolution MS. For the IR spectra, a Perkin Elmer model 1600 FT-IR spectrometer was used and the optical activities [a]o were measured in a Perkin Elmer, model 343 polarimeter using the D line of a sodium lamp.

Biodirected fractionation of the crude extract

300 mg of the crude extract obtained after 5 days of culture were fractionated on a Sephadex-LH-20 column with a mixture of n-hexane:DCM:MeOH (2:1:1). Compound 2 of formula (II) (45 mg) was isolated.

The fractionation of the crude extract obtained after 8 days (400 mg) was carried out on a Sephadex-LH-20 chromatographic column with increasing mixtures in polarity of nhexane:DCM:MeOH (3:1:1)-(2:1: 1) and allowed the identification of compounds ( 3 ) (9 mg) and ( 4 ) (11 mg) from the active fractions.

Fractionation of the crude extract from 12 days of culture was carried out on a silica gel flash chromatographic column (5 cm diameter, 15 cm h, Si 35-70 um) with 4 grams of the extract in 250 grams of silica. A gradient elution was carried out with 4 volumes of 500 ml for each polarity using mixtures of n-Hexane: AcOEt (20:80) as eluent; AcOEt (100): AcOEt: MeOH (95:5) and AcOEt: MeOH (50:50) to obtain 8 fractions. The active fractions BP3 (B) and BP3 (E, F) were purified by high performance liquid chromatography (HPLC) (Varian ProStar 210) with an Interchrom KR5/25M column and a flow rate was 20 ml/min, which allowed the identification of compound 1 in the active fraction BP3 (B)—4 (6.4 mg). Additionally, compound 2 was identified in the active fractions B2 and B3 with a relative abundance of 40.5% and 28.6% respectively (GC-MS analysis).

Chemical characterization of compound 1

Chemical characterization by spectroscopy (1H NMR, 13C NMR) of the active fraction B4 of the 12-day culture allowed the identification of compound 1.

Compound 1 was obtained as a colorless oil. Its molecular formula was determined as C ¹⁶ H ²⁸ O ⁴ by HRESIMS [M+Na]+, m/z 307.1882, which shows three degrees of unsaturation in its molecule. Its IR spectrum showed two absorption bands at Dmax 3459 cm ^-1 and 1640 cm ^-1 due to hydroxyl groups and a carbonyl group, respectively. The ¹³ C NMR spectrum in combination with the two-dimensional ¹ H- ¹ H COZY and HSQC spectra presented characteristic signals of five methyl groups, five methines, two of them attached to oxygen at 72.1 (C-11) and 86.9 (C-7 ) and one olefinic at 132.8 (C-5), as well as the signals of three methylene groups. In addition, quaternary carbons were observed, one attached to oxygen at 87.4 (C-10), an olefinic carbon at 133.8 (C-6), and another from a carbonyl group at 181.3 (C-1). Analysis of these spectroscopic data and the correlations observed in the HMBC and NOESY experiments confirmed a tetrahydrofuran base structure for compound 1 and allowed it to be identified as asperic acid ( 1 ) (Varoglu M. et. al. 2000. J Nat Prod. 63, 41-34).

Compound 1 has been described previously, but its nematicidal activity has not. This compound in the present invention has been referred to as formula (I).

Asperic acid (1) . [α] _D +4.05 (c 0.37, CHCl3); IR (film) Dmax 3459, 2094,1640, 1530, 1423, 1342, 1330,1131 cm-1; ¹ H NMR (CDCl ₃ , 500 MHz): 50.94 (3H, d, J = 6.7 Hz,H-14), 1.12 (3H, d, J = 6.5 Hz, H-12), 1.15 (3H, d, J = 6.7 Hz, H-13), 1.18 (3H, s, H-16), 1.38 (1H, m, H-3a), 1.56 (1H, ddd, J = 18.9, 7.8, 4.1 Hz, H-9a) , 1.62 (3H, d, J = 1.4 Hz, H-15), 1.69 (1H, m, H-3b), 1.88 (2H, m, H-8), 2.20 (1H, td, J = 12.5, 7.7 Hz, H-9b), 2.41 (1H, ddd, J = 9.2, 7.2, 5.7 Hz, H-2), 2.48 (1H, tt, J = 10.0, 3.6 Hz, H-4), 3.78 (1H, m , H-11), 4.33 (1H, dd, J = 10.7, 5.3 Hz, H-7), 5.16 (1H, d, J = 9.7 Hz, H-5.); ¹³ C NMR (CDCl ₃ , 125 MHz): 5 181.3 (C-1), 133.8 (C-6), 132.8 (C-5), 87.4 (C-10), 86.9 (C-7), 72.1 (C -11), 40.7 (C-3), 37.1 (C-1), 30.7 (C-8), 30.2 (C-9), 29.9 (C-4), 24.5 (C-16), 21.3 (C- 14), 17.5 (C-12), 16.7 (C-13), 11.1 (C-15); HR-ESIMS m/z 307.1882 [M Na]+ (calculated for C ^1a H ²⁸ O ⁴ Na, 237.1103).

Chemical characterization of compound 2

The compound showed a molecular formula C ¹¹ H ¹⁸ O ⁴ established through HR-ESIMS ([M+Na]+, m/z 237.1102. The chemical structure was determined by comparison of the 1H and 13C NMR spectroscopic data with those published previously for (+)-hexylitaconic acid (Isogai, A. et al. 1984 , Agr. Biol. Chem., 48, 2607-2609). The positive optical rotation (aD 13.09) indicated that it is the S stereoisomer whose configuration absolute was determined by vibrational circular dichroism (Nakahashi et al. 2009, Bioorg Med Chem Lett.

19, 3027-3030).

(+)-hexylitaconic acid-(2) . [[α] _D +13.09 (c 0.1, CHCl3); ¹ H NMR (CDCl ₃ , 500 MHz): 50.88 (3H, t, J = 6.8 Hz, H-10), 1.29 (4H, m, H-8 and H-9), 1.34 (4H, m, H- 6 and H-7), 1.74 (1H, dq, J = 14.3, 7.9, 6.6 Hz, H-5), 1.94 (1H, dq, J = 14.8, 7.7, 7.1 Hz, H-5), 3.41 (1H , t, J = 7.4 Hz, H-2), 5.83 (1H, s, H-11a), 6.54 (1H, s,H-11b), 10.98 (2H, br s, 2 X COOH); ¹³ C NMR (CDCl3, 125 MHz): 5 179.8 (C-1), 171.9 (C-4), 137.3 (C-3), 129.7 (C-11), 46.9 (C-2), 31.6 (C- 8), 29.8 (C-5), 29.0 (C-7), 27.3 (C-6), 22.6 (C-9), 14.0 (C-10); HR-ESIMS m/z 237.1102 [M Na]+ (calculated for C11H18O4Na, 237.1103).

Chemical characterization of compound 3.

Its molecular formula was determined as C ¹² H ²⁰ O ⁵ by HR-ESIMS ([M+Na]+, m/z.

267.1203). The ¹ H and ¹³ C NMR spectrum indicated the presence of a structure related to that of the compound ( 2 ). The most significant difference is the presence of signals corresponding to a hydroxyl group of a secondary alcohol deduced from the ^1H and ^13C NMR signals at 3.70 5 ^h (1H, q, J=5.8 Hz) and 68.5 5 ^c , respectively. , and an additional signal from a methoxyl group at C-1 (3.66 5 ^h and 52.4 5 ^c ). These data allowed the identification of compound 3 as the 1-methyl ester of 9-hydroxy-hexylitaconic acid (Klembe C. et al 2004 , J. Nat. Prod, 67, 1058-1063).

The duplicity of the signals of some carbons in its ¹³ C NMR spectrum indicated that it is a mixture of stereoisomers at the C-2 of the 1-methyl ester of 9-hydroxyhexylitaconic acid.

9-hydroxy-hexylitaconic acid 1-methyl ester (3) . ¹ H NMR (CDCl ₃ , 500 MHz): 5 1.14 (3H, d, J = 6.2 Hz, H-10), 1.36 (6H, m, H-6, H-7 and H-8), 1.69 (1H , m, H-5a), 1.87 (1H, m, H-5b), 3.48 (1H, t, J = 7.4 Hz, H-2), 3.66 (3H, s, -OCH ³ ), 3.70 (1H, q, J = 5.8 Hz, H-9), 5.73 (1H, s, H-11a), 6.32 (1H, s, H-11b); ¹³ C NMR (CDCl ₃ , 125 MHz): 5175.8 (-COOCH ³ ), 169.5 (C-1), 140.8 (C-3), 127.1 (C-11), 68.5 (C-9), 52.4 (-OCH ³ ), 48.10 (C-2), 48.05 (C-2), 39.9 (C-8), 32.09 (C-5), 32.07 (C-5), 28.62 (C-6), 28.60 (C-6 ), 26.6 (C-7), 23.5 (C-10);HR-ESIMS m/z 267.1203 [M Na]+ (calculated for C12H2OO5Na, 267.1208).

Chemical characterization of compound 4

The molecular ion at m/z 267. 1201 [M+Na]+ according to a molecular formula C12H2OO5 and the ^1H NMR spectrum signals indicated that compound 4 is an isomer of compound 3 . The comparison between the ¹ H and ¹³ C NMR spectra of both compounds showed the disappearance of signals from the methyl group (1.14 5 ^h and 23.5 5 ^c ) and the presence of a new triplet at 3.54 5 ^h (2H, t, J= 6.6 Hz) and 68.5 5 ^c due to a terminal hydroxymethylene group in the aliphatic chain. The correlations observed in the HMBC and COZY spectra confirmed the position of the new hydroxyl group at C-10. These data coincide with those published for asperitaconic acid A and allow the identification of compound 4 as the 1-methyl ester of 10-hydroxy-hexylitaconic acid (Ding L. et al. 2018 , J Antibiot, 71, 902-904) .

10-hydroxy-hexylitaconic acid 1-methyl ester (4) . [a] ^ü +2.3 (c 0.30, CHCl3); ¹ H NMR (CDCl ₃ , 500 MHz): 5 1.33 (6H, m, H-6,H-7 and H-8), 1.52 (2H, q, J = 7.2 Hz, H-9), 1.69 (1H , m, H-5a), 1.87 (1H, m, H-5b), 3.48 (1H, t, J = 7.5 Hz, H-2), 3.54 (2H, t, J = 6.6 Hz, H-10) , 3.65 (5H, s,-OCH ³ ), 5.75 (1H, s, H-11a), 6.33 (1H, s, H-11b); ¹³ C NMR (CDCl ₃ , 125 MHz): 5 175.7 (C-1), 169.3 (C-4), 140.6 (C-3), 127.2 (C-11), 62.9 (C-10), 52.4 (- OCH ³ ), 48.0 (C-2), 33.5 (C-9), 32.1 (C-5), 30.2 (C-7), 28.5 (C-6), 26.7 (C-8);HR-ESIMS m /z 267.1201 [M Na]+ (calculated for C12H2OO5Na, 267.1208).

Table 1. Nematicidal activity of compounds 1 - 4 , expressed as mean and standard error (from four repetitions) of the corrected mortality percentages of J2 of M. javanica.

Example 4. Nematicidal activity in vivo

Nematodes:

The population of Meloidogyne javanica was maintained in a greenhouse on highly susceptible tomato plants (Marmande variety), with periodic inoculations. At 60 days after one of the inoculations, the infected roots of the tomato plants were processed and the egg masses were manually isolated from the galls under the stereoscopic microscope. The inoculum of the infective juveniles (J2) was obtained for three days at from the incubation of egg masses in water at 25°C.

Treatment:

BP3 crude extract (4-5D). A treatment solution was prepared at 0.5 mg/mL (in water and 1% ethanol). Controls consisted of distilled water and 1% ethanol.

Procedure:

The treatment was applied to the substrate (75 ml per 600 g of sandy clay soil) which, after being homogenized, was transferred to 800 mL pots (5) and moistened with water to saturation. The control consisted of 5 pots with untreated soil. Each pot was then inoculated with 1000 eggs and juveniles of M. javanica and left to incubate for 5 days in a plant growth chamber (25 ± 2 °C, 60%RH). After the incubation period, a 21-day-old tomato seedling (var. Marmande) was transplanted into each pot and kept under the same controlled conditions for 45 days with the application of a fertilizer solution every 10 days.

At 45 days after inoculation, the plants were lifted. The root systems were washed and weighed and the whole roots were processed to determine the following parameters:

Nodulation index (NI) : Roots were visually analyzed for nodulation index on a scale of 0 to 5, where 0 = no galls (healthy, root system without infestation), 1 = 25% of the root system with galls 2 = 26-50% of the root system with galls), 3 = 51 -75% of the root system with galls, 4 = 76-90% of the root system with galls, 5 = >91% of the root system with galls (Hussey and Janssen,2002).

Table 2 shows the results of the In planta test. The root systems of the treated plants presented very low nodulation rates, unlike those of the infested control plants.

Table 2 Incidence of M. javanica J2 infection in the roots of treated and control tomato plants.

Likewise, the treatment applied had no phytotoxicity effects and no significant differences were observed regarding the weight or appearance of the treated root systems compared to the control.

Claims (12)

1. Strain of the species Aspergillus tubingensis deposited in the Spanish Type Culture Collection (CECT) with the accession number CECT 21186. 2. Fermentation broth extracted from the strain defined in claim 1. 3. Procedure for obtaining a fermentation broth that includes: a) cultivate the strain of Aspergillus tubingensis defined in claim 1, until the mycelium develops; b) ferment the mycelium obtained in a) in a liquid medium; c) separate the mycelium to obtain the fermentation broth. 4. Crude extract extracted from the strain defined in claim 1. 5. Procedure for obtaining the crude extract that includes the steps of: a) cultivate the strain of Aspergillus tubingensis defined in claim 1, until the mycelium develops; b) ferment the mycelium obtained in a) in a liquid medium; c) separate the mycelium to obtain the fermentation broth; d) perform an extraction from the fermentation broth obtained in c) to obtain the crude extract. 6. Nematicidal agent comprising the fermentation broth and/or a crude extract of the strain defined in claim 1. 7. Method for controlling nematodes that comprises the step of contacting a plant, seed, seedling, or substrate with the strain and/or the fermentation broth and/or a crude extract of the strain defined in claim 1. 8. Use of the strain defined in claim 1 to obtain the compound of formula (I), and/or the compound of formula (II), and/or the compound of formula (III) and/or the compound of formula (IV);

and the salts and/or solvates thereof. 9. Procedure for obtaining the compound of formula (I) and/or the compound of formula (II) and/or the compound of formula (III) and/or the compound of formula (IV), which comprises the steps of: a) cultivate the strain of Aspergillus tubingensis defined in claim 1, until the mycelium develops; b) ferment the mycelium obtained in a) in a liquid medium; c) separate the mycelium to obtain the fermentation broth; d) perform an extraction, from the fermentation broth obtained in c) to obtain the crude extract; e) isolating the compound of formula (I) and/or the compound of formula (II) and/or the compound of formula (III) and/or the compound of formula (IV). 10. Use of the compounds of formula (I) and/or formula (II) and/or formula (III) and/or formula (IV) as nematicide. 11. Nematicidal compositions comprising as active ingredient at least one of the compounds selected from: compound of formula (I), compound of formula (II), compound of formula (III) and compound of formula (IV) or a salt or a solvate of these, acceptable in agriculture. 12. Method for controlling nematodes that comprises the step of contacting a plant, seed, seedling, or substrate with the compound of formula (I) and/or the compound of formula (II) and/or the compound of formula (III). ) and/or the compound of formula (IV).