Lipochitooligosaccharides stimulating arbuscular-mycorhisal symbiosis

FIELD: chemistry.

SUBSTANCE: group of inventions relates to application of lipochitooligosaccharide of formula for stimulation of plant mycorhisation, where n=2 or 3, R1 represents lipid substituent, which represents fatty acid chain, containing from 16 to 18 carbon atoms, which can be saturated or mono- or di-unsaturated, and R2 represents H or SO3H. Also claimed is mixture of lipochitooligosaccharides for stimulation of arbuscular-mycorhisal plant symbiosis, for stimulation of plant seed germination or for stimulation of plant seed germination or for stimulation of plant root system development and containing in effective amount lipochitooligosaccharide of formula (I), where R2 represents H, and lipochitooligosaccharide of formula (I), where R2 represents SO3H.

EFFECT: group of inventions effectively stimulates plant seed germination, of arbuscular-mycorhisal plant symbiosis and plant root system development.

9 cl, 16 dwg, 9 ex

 

The present invention relates to lipoperoxidation involved in arbuscular-mycorrhizal symbiosis, and to methods of their use.

It is believed that the arbuscular mycorrhiza (AM) has established a symbiotic relationship with plant roots more than 400 million years ago, since the appearance of the earliest land plants, suggesting that AM fungi help the plants in the development of the land (Remy et al., 1994). This group of fungi, recently renamed the Glomeromycota, is one of the most widely used, and AM relationships are found throughout the plant Kingdom, including angiosperms and gymnosperms plants, pteridophytes and some bryophytes (Smith and Read., 2008). Among angiosperms at least 80% of the species are able to form AM symbioses; the only notable exceptions are the Brassicaceae and Chenopodiaceae. AM fungi are able to transport rare or poorly soluble mineral nutrients such as phosphorus, zinc and copper, from the soil to the plant, which in turn provides the fungus with carbohydrates. This exchange of nutrients can be vital when soil fertility and water availability is reduced, that is, in conditions that severely limit agricultural production in most regions of the world (Smith and Read, 2008).

Dragonprime known symbiotic relationships between plants and soil microorganisms is ritually symbiosis. Unlike arbuscular-mycorrhizal symbiosis, which is widely distributed among plants, izobilnyi symbiosis is found only in legumes, and mushrooms instead participate in nitrogen-fixing bacteria, collectively called rhizobia, which belong to several genera, including Rhizobium, Bradyrhizobium, Azorhizobium, and Sinorhizobium. Izobilnyi symbiosis leads to the formation of certain structures nodules on the roots of leguminous host plant. The nodules provide a suitable environment for rhizobia, allowing them to fix molecular nitrogen and provide a host plant associated with nitrogen. Initiation bean-ritorialjnogo symbiosis depends on symbiotic signals that produce both partner symbiont. The signals emitted by the plant, usually are flavonoids secreted in the composition of root exudates. These flavonoids interact with izobilnyi transcription factors family NodD, which activate the transcription of genes klubenkoobrazovaniya (nod genes) involved in the development of bacterial signal molecules called Nod factors (Denarie et al., 1996). Nod-factors have a common basic structure, including the chitin skeleton of four or five residues N-acetylglucosamine, linked by beta-1,4-linkages, N-acylated by nereguliruemaia the end OST the rigid fatty acids of different lengths and degrees of unsaturation. This basic structure can be further N-methylated by nereguliruemaia the end, and can also be O-substituted by nereguliruemaia and/or by reducing the end. This variety of substituents provides a wide variety of Nod-factors with different structures (different structure Nod-factors described in Denarie et al., 1996; D Haeze et al., 2002). Specificity bean-ritorialjnogo interaction (i.e. this type rhizobia forms nodules on certain kinds of leguminous plants is the result of this diversity.

Using genetic analysis of pathway signaling Nod-factors in the roots of the model legume plant Medicago truncatula was identified a number of genes involved in this pathway (Stacey et al., 2006). Growing evidence indicates that receptors Nod-factors are receptorgamma kinase with extracellular sugar binding LysM domains, such as the products of the genes NFP and LYK3 from M. truncatula. The interaction of the Nod-factor with its receptor triggers the subsequent signaling cascade, which includes a rapid influx of calcium ions, calcium spike and expression of certain genes nedelino. These subsequent events affect specific genes encoding proteins involved in calcium signaling, such as DMI1, DMI2 and DMI3 from M. truncatula, encoding respectively kayany channel, rizatriptan kinase, is terasul leucine-rich repeats, and CA2+/calmodulin-dependent protein kinase, as well as genes encoding proteins involved in the control of gene expression, such as NSP1 and NSP2, which encode transcription factors.

Although AM fungi from the point of view of agriculture and ecologically extremely important cellular and molecular mechanisms that control the formation of mycorrhizal symbiosis, much less well known than the mechanisms involved in izobilnyi symbiosis.

M. truncatula has been shown that nodule and mycorrhizal programs have at least three common component (Catoira et al. 2000), namely, the products of the genes DMI1, DMI2 and DMI3 involved in calcium signaling.

However, events like the preceding and following calcium signaling, are still poorly characterized in the case arbuscular-mycorrhizal symbiosis, particularly those who are involved in the early signaling and lead to mutual recognition of plant and fungal partners. The study of these events is complicated by the fact that the fungal partner is an obligate symbiont, which cannot be grown in pure culture in the absence of living plants, as well as the lack of genetic tools available for this group of fungi (Harrison, 2005). However, recently it was shown that between the symbionts to the physical interaction is the transfer of the diffusible signals. From plants in the composition of root exudates can be emitted substances apocarotenal family, strigolactone and stimulate branching hyphae germinating spores of AM fungi, giving the signal for switching the physiological state of the fungus on the active precipitiously growth (Akiyama et al., 2005; Besserer et al., 2006). It was also reported about the existence on the part of the fungus diffusible substances produced by AM fungi and is able to activate the responses of plants associated with the program endomycorrhizal (Kosuta et al., 2003; Weidmann et al., 2004; Navazio et al., 2007). More specifically, a series of experiments performed on M. truncatula, recently showed that AM fungi produce diffusible substances that can stimulate the expression of various responses of plants. Three species of Gigaspora and one species of Glomus were able through cellophane membrane to cause induction of the expression of the symbiotic gene MtENOD11 in the roots of seedlings (Kosuta et al., 2003). Three fungal pathogen did not cause a similar response that speaks in support of the hypothesis of the induction response of a particular signaling molecule of the AM fungus. In a similar way AM fungus Glomus intraradices has been shown to be activated through the membrane transcription of plant genes, the expression of which depends on symbiotic gene DMI3 (Weidmann et al. 2004). In addition, it was found that the diffusion signal of AM fungi caused temporary higher is their cytosolic levels of calcium in cell cultures of soybean and implemented to increase regulation of genes related DMI1, DMI2 and DMI3 (Navazio et al., 2007).

Olah et al. (2005) reported that Nod factors from Sinorhizobium meliloli, ritorialjnogo of symbiont M. truncatula, were able to stimulate memorization and formation of lateral roots in M. truncatula. Stimulation of the formation of lateral roots was also observed when using diffusible factors from arbuscular-minoritiy mushrooms (factors ICC), but not Nod-factors of izobilnyi species {Sinorhizobium fredii and Rhizobium leguminosarum), which can cause the formation of nodules in species of the genus Medicago. It was also reported that all the genes path signaling Nod factor identified to date, in particular NFP gene encoding the presumed receptor Nod-factor, and DMI3 genes and NSP1 were necessary to stimulate the formation of lateral roots Nod-factors, but not factors of the ICC, which was required only genes DMI1 and DMI2. Based on these observations, these authors proposed a model explaining the stimulation of minoritatii and formation of lateral roots of legumes factors ICC and Nod-factors. According to this model, the factors of the ICC and Nod-factors that are recognized by different receptors on the cell surface, activate a common signaling path DMI1/DMI2/DMI3; in the case of factors of the ICC, DMI1 and DMI2 were sufficient to stimulate the formation of lateral roots, while DMI3 was required for the stimulation of minoritatii. Olah et al. also discussed the possibility of the reduction of the chemical nature of the factors of the ICC. They put forward the hypothesis that these factors most likely are not the auxin-like substances, since their effect on root development was different from that observed under the influence of these substances. They also suggested that their structure should be different from the structure of the Nod factors as receptor NFP, apparently, can distinguish between them.

It is therefore evident that although the existence of diffusible factors ICC" allocated to AM fungi and is able to activate the responses of plants recognized in the art, the chemical nature of these factors has not been identified so far.

The authors of the present invention was able to clear the factors ICC of exudates as minorityowned roots and germinating spores of the AM fungus Glomus intradices. They also determined their chemical structure and showed that they effectively stimulate root growth and root colonization of AM fungus.

Factors ICC, refined by the authors of the present invention is a mixture of sulfated and desulfation of lipoperoxidation (LHO); and they Nod-factors have the same chitin backbone of residues N-acetylglucosamine, linked by beta-1,4-linkages, N-acylated by nereguliruemaia the end of the balance of fatty acids. However, the factors of the ICC have a simpler structure compared with Nod-factors. Its the only O-substitution, which was observed in the factors of the ICC, is O-sulfation at the reducing end of the molecule. No other On-substitution, such as O-carbarnoyl on nereguliruem late or On-fucosyl on reducing the end, were not detected. The only N-substitution on the end nereguliruemaia GlcNAc residue at the factors of the ICC, purified from Glomus intradices is conventional acylation of fatty acids, mainly oleic (C18:1) and palmitic acids (C16:0). In contrast, the N-substitution of Nod-factors more difficult. Often it's a double substitution of the N-methyl group and N-acyl group (often Aksenova acid), as in strains of rhizobia that form nodules on most tropical legumes and legume subfamily Mimosoideae. N-methylation is determined by widespread izobilnyi genome nods (Denarie et al., 1996). Alternatively, the N-acylation of certain polyunsaturated fatty acid is the rule among rhizobia that form nodules on legumes of temperate latitudes, related to the Galegoid clade (Denarie et al., 1996). In fact, LHO having the structure as simple as characterized by the inventors factors ICC were observed among the Nod factors produced by various strains of rhizobia studied so far (Denarie et al., 1996; D Haeze et al., 2002).

According to this invention, a method of gaining the factors of the ICC from the mushroom group Glomeromycota, while this method includes obtaining exudation from plant roots, minorityowned specified by the fungus, or from germinating spores specified mushroom, the extraction of these exudates butanol and collection butanole extract containing the specified lipophilicity.

According to a preferred implementation variant of the present invention, the method includes the further steps of solid-phase extraction of the specified butanole extract using reversed-phase chromatography on C18 with sequential washes of 20%, 50% and 100% acetonitrile and collecting fractions, elyuirovaniya 50% wage acetonitrile containing these factors ICC.

According to a further preferred implementation variant, the method includes the further steps of purification of the specified faction, elyuirovaniya 50% wage acetonitrile, using reversed-phase high-performance liquid chromatography on reversed-phase C18 column using a linear gradient from 20% to 100% acetonitrile and collecting fractions, elyuirovaniya 3.0-48% acetonitrile that contains sulfated lipophilicity, and/or faction, elyuirovaniya in 64-72% acetonitrile that contains desulfation lipophilicity.

According to a particular implementation variant of the present invention, the specified mushroom from the group Gloeromycota represents Glomus intraradices.

However, fungal factors ICC can also be obtained from other species of Glomeromycota, producing them using the above stages of extraction or options.

"The factor ICC" is defined in this description as lipohyalinosis, which corresponds to the following formula (I):

where n=0, 1,2, 3, 4 or 5, preferably 2 or 3;

R1is a lipid Deputy containing from 12 to 22, preferably 14 to 20, carbon atoms, which may be saturated, or mono-, di-, tri-, Tetra-, Penta - or hexanediamine;

R2represents H or SO3H.

Lipid substituent R1preferably is a chain of fatty acids. R1may also be an aromatic analogue chain fatty acids, as analogues of Nod-factor, described, for example, Grenouillat et al. (2004), or in PCT WO/2005/063784.

Preferably, R1is a chain of fatty acids synthesized by the fungus arbuscular mycorrhizal fungi, in particular chain saturated or mono - or dimensional fatty acids containing 16 or 18 carbon atoms. Preferably, if the specified chain fatty acid is unsaturated, it included at least one double bond in the CIS-conformation (e.g., C18:1 oleic acid). No restriction is that the list of examples of preferred chain fatty acid comprises C16:0, C18:0, C16:1ω5, C16:1ω7, C18:1ω5, C18:1ω7, C18:1ω9, 18:2ω6,9, C20:from 0, C20:1ω9 and C20:4ω6, 9, 12, 15.

Factors ICC can also be characterized and distinguished from lipoperoxidation related structures, such as Nod-factors, their biological properties. These biological properties can be tested using the appropriate biotests. In particular, you can use the biotests, based on the ability factors of the ICC to stimulate the formation of lateral roots in model legumes M. truncaluta. More specifically, while the factors of the ICC, like Nod-factors, can stimulate the formation of lateral roots of wild type plants, but not defective in symbiosis mutants dmi1, dmi2 and dmi3, factors ICC can also, in contrast to the Nod factors stimulate the formation of lateral roots from defective in symbiosis nsp1 mutant.

If necessary, also accessible the biotests, allowing to distinguish desulfation factors ICC from sulfated factors ICC, (for example, when necessary, be divided into mushroom extract fraction containing desulfation factors ICC from those that contain sulfated factors ICC): for example, sulfated factors ICC is able to induce gene expression MtENODll growing in the roots of M. truncatula, while desulfation factors ICC can cause branching of the root hair and the OECS pea seed.

Factors ICC can be cleaned from fungi, as described above. They can also be obtained by chemical synthesis and/or produced in genetically modified bacterial cells. For example, chitooligosaccharides frame, sulfated or not, can be synthesized in recombinant bacteria, as described, for example, Samain et al. (1997, 1999) for the synthesis of precursors of Nod-factor, and subsequently allerban on free amino group nereguliruemaia limit sugar, as described, for example, Ohsten Rasmussen et al. (2004). You can also use a mutant strain of bacteria of the Rhizobiaceae, producing factors ICC instead of Nod-factors, for example, a strain that is genetically modified to Express only those of the structural genes of biosynthetic pathways Nod, which are involved in the synthesis chitooligosaccharides skeleton, and those that are involved in N-acylation nereguliruemaia end of glucosamine corresponding fatty acid C16 or C18, perhaps those involved in O-sulfation of the reducing end glucosamine, as described, for example, Ardourel et al. (1994) or Lugtenberg et al. (1995).

The present invention also covers mixtures of different factors ICC formula (I). and in particular a mixture of sulfated and desulfation factors ICC, including one or more lipoperoxidation formula (I) such that R2before the hat is H, and one or more lipoperoxidation formula (I) such that R2represents the SO3H. Lipophilicity the above-mentioned mixture may also differ among themselves by the number of residues N-acetylglucosamine and/or the nature of the substituent R1(e.g., length and/or degree of unsaturation chain fatty acids).

A mixture of factors ICC the present invention can, for example, be obtained by extraction of the factors of the ICC arbuscular-mycorrhizal fungi, as described above, and collection of mushroom extract. They can also be obtained by producing various factors ICC separately and mixing them.

Purified or synthetic lipophilicity, and more specifically, purified or synthetic factors ICC formula (I) or mixtures thereof, described herein can be used to stimulate microzooplankton and thus have a wide range of applications in agriculture, horticulture and forestry for most cultivated plants, are capable of forming mycorrhiza and as a consequence possessing receptors of the factor of the ICC.

In addition to their use for stimulating arbuscular-mycorrhizal symbiosis, purified or synthetic factors ICC or their mixtures can also be used:

- to stimulate the germination of seeds that may be useful for the treatment of seeds with a wide spec is rum applications in agriculture, horticulture and forestry;

- to stimulate the development of the root system, which is useful to improve water and mineral nutrition.

They can be used, for example, for seed treatment or add to inoculants containing arbuscular mycorrhizal fungi, or added to the soil or culture substrate plants. Purified or synthetic factors ICC the present invention can be used with any plants, that plants are able to form mycorrhizal fungi, including both legumes and non legume plants, both dicotyledonous and monocotyledonous plants, including grasses. They can also be used for plants grown in growth chamber and in the greenhouse or in the field.

They can also be used to stimulate mycorrhizal colonization in the production of mycorrhizal inoculants (i.e. spores or hyphae of AM fungi, or fragments minorityowned roots), as a Supplement to the culture medium used for the production of these inoculants plants grown in soil or in hydroponic or aeroponic conditions, or by cocultivation mycorrhizal fungi and cut the roots.

The present invention also encompasses compositions comprising purified or synthetic factors ICC or mixtures thereof, and which is acceptable in agriculture media. The composition of the present invention can optionally contain mutant strains of bacteria of the Rhizobiaceae, genetically modified for production factors ICC instead of Nod-factors, as described above. Preferred compositions are those which contain a mixture of sulfated and desulfation factors ICC.

Additionally, the factors of the ICC can be combined with other active elements, such as flavonoids, apocarotenal, such as strigolactone, or jasmonate representing plant substances, which reportedly act as symbiotic signals (Harrison, 2005; Akiyama et al., 2005; Bcsscrer et al., 2006).

The composition of these mixtures depends on the intended method of application (for example, the coating of the seeds, adding to the culture medium for the production of mycorrhizal inoculants, processing plants or soil). They can, for example, be prepared as dispersible in water or water-soluble solids such as powders, granules, coated tablets or film-like liquid aqueous solutions, suspensions, emulsions or gels.

According to a preferred implementation variant, these compounds are associated with fungal and/or plant material, for example with inoculants arbuscular-mycorrhizal fungus or seeds of plants, capable of forming mycorrhiza; preferably, these seeds were coated with this composition.

Preferably, the factors of the ICC are used in the composition in a concentration of from 10-3M to 10-12. Adding to the culture medium for the production of spores of AM fungi can be applied in concentrations from 10-6M to 10-10M, preferably in a concentration of from 10-7up to 10-9M in the environment. When used for seed treatment or to stimulate the development of the root system, they can be used in a concentration of from 10-6M to 10-10M, preferably in a concentration of from 10-7up to 10-9M. using the mixture of sulfated and desulfation factors ICC can be used in concentrations from 10-8up to 10-10M.

The essence of the present invention will become more fully understood in light of the additional description, consisting in the following examples and the attached drawings. It should be understood, however, that these examples and the drawings are illustrative only and do not limit the present invention in any way.

CAPTIONS TO DRAWINGS

Fig.1. Biological tests used for detection

symbiotic signals of AM fungi

a. Test MtENOD11. The roots of transgenic seedlings of M. truncatula Jemalong A17, carrier reporter construct pMtENODl 1-GUS. The GUS activity was determined using histochemical staining with 5-bromo-4-chloro-3-indolyl-β-glucuronide. (1) Roots in control, treated with 2.5% acetonitrile. (2) Fraction after TPV and elution with 50% acetonitrile, resbalon the I 40 times. (3) the same fraction after further tenfold dilution.

b. Test VsHab. Root hairs pea seed (Vicia saliva, ssp. nigra), observed under an optical microscope after staining with methylene blue. (1) Root hairs treated with the inactive fraction, were direct. (2) Root hairs treated with active fractions showed significant branching.

Fig.2. Prepreparatory profile by reversed-phase HPLC on C18 extracts of exudates minorityowned roots.

Initial isocratic phase with 20% acetonitrile lasted 10 minutes, followed by 20-minute 20-100% gradient of acetonitrile. The profile shows a large amount of contaminating material present in the exudates minorityowned roots. The collected fractions was carried out every two minutes, and fractions were tested for biological activity using MtENOD11 and VsHab. Horizontal bars show the retention time of compounds from fraction A, active against MtENOD11, and more hydrophobic compounds from fraction B, which is active towards VsHab.

Fig.3. Prepreparatory profile by reversed-phase HPLC on C18 extracts of exudates germinating spores.

Chromatographic conditions are the same as PA Fig.2. The profile shows that the exudates spores contain much less zagryaznyayushikh the material compared with exudates minorityowned roots. The collected fractions was carried out every two minutes, and fractions were tested for biological activity using MtENOD11 and VsHab. Horizontal bars show the retention time of compounds from fraction A, active against MtENOD11, and more hydrophobic compounds from fraction B, which were active against VsHab.

Fig.4. The influence of soft methanol hydrolysis on the biological activity of fractions of A.

Soft methanol hydrolysis, as reported, removes the sulfate group at the sulfated LHO without changing other structural features of these molecules. Fraction A, collected during prepreparation HPLC exudates germinating spores was subjected to mild hydrolysis and tested for biological activity in tests on MtENOD11 and VsHab. The biological activity represented by vertical bars. While non-hydrolyzed fraction And was active against MtETMODll and inactive against VsHab, hydrolyzed fraction lost activity against MtENOD11 and acquired activity against VsHab. These data indicate that the biological activity of fractions And test MtENOD11 was the result of the presence of the sulfated LHO.

Fig.5. Tetramer sulfated, LHO, N-acylated fatty acids containing 16 carbon atoms.

Mass spectrum WASH/MS mode is otricatelniy ions, fraction 4, isolated after prepreparation HPLC on C18. Shows extracted ion currents corresponding to the sulfated tetramers, and their corresponding spectra. This figure demonstrates that the compounds that gave a signal in the area ratio of the mass-to-charge equal to 1101,5, 1103,5 and 1105,5, were actually present in the samples. These values of the ratio of the mass-to-charge (m/z) correspond to the sulfated tetramer of LHO, N-acylated, respectively, C16:2, C16:1 and C16:0. With regard to the relative intensity of these three signals, 1105,5 (LHA-IV-C1 6:0) was the strongest, followed by 1103,5 (LHA-IV-C16:1).

Fig.6. Tetramer sulfated, LHO, N-acylated fatty acid C18:1.

Mass spectrum WASH/MS in the negative ion mode, fraction 5, isolated after prepreparation HPLC on C18, demonstrating that the compound present in the greatest number (the ratio of mass-to-charge 1135,5), N-acylation of fatty acid C18:1.

This profile also shows that LHO carrying fatty acid C18:0 (the ratio of mass-to-charge 1133,5), was absent in this fraction, since this ion is the only above two relations mass-to-charge LHO carrying chain C18:1. As shown in the second mass spectrum, dimensiony C18-LHO present in very small amounts.

Fig.7. PE is tabernae sulfated, LHO, N-acylated residue of the fatty acid C18:1.

This profile demonstrates that was also attended by lipofectamine, but compared with the corresponding tetramera (see Fig.5), their content was approximately 30 times lower. Was discovered by LHO-V-C18:1.

Fig.8. Check for the presence or absence of a specific connection.

When the estimated weight did not correspond to ions present in the sample in the profile instead of a single peak appeared a very large number of background peaks. Very difficult profile, obtained using the ion current with respect to mass-to-charge 1332.6 demonstrates the absence in the sample of getopenfile C18:2. On the contrary, a distinct single peak observed using the ion current with respect to mass-to-charge 1334.6, clearly indicates the presence of pentamer C18:1.

Fig.9. Comparison of the nature of fragmentation in TMS sulfated primary factor ICC and Nod-factor from S. meliloti.

Demonstration of the presence of compounds having a mass corresponding to the expected retention time in HPLC, insufficient to confirm their structure. For this reason, an analysis was conducted of the main sulfated compounds ICC using TMS. This figure compares, in TMS in the negative ion mode, sulfated tetramer Nod-factor from S. meliloti, N-zillionare C16:2, with the main tetramer "factor of the ICC", present in the sample. Characteristic ions of the reducing end with respect to mass-to-charge 503 (Y2), 605 and 706 (Y3) was clearly detected in both cases, as the characteristic neutral loss fragment mass 101.E. m. (vnutrichechenskie gap), starting at the stage of the molecular ion. An exact match of the nature of the fragmentation points to the structural unity of these two molecules.

Fig.10. Effect of fractions of the extract of the ICC on the formation of lateral roots in M. truncatula.

(A) Signal of the AM fungus, which stimulates FBK is amphiphilic.

Comparison of the aqueous (Aq), butanole (VION) and an ethyl acetate (EA) extract from exudates germinating spores (GSP24) on M. truncatula A17. Butanolic extract stimulated FBK, starting from 5th day (statistically significant at P<0.05), whereas water and an ethyl acetate extracts were inactive.

(B) Stimulation FBK through a symbiotic way of signaling DMI.

Comparison steps butanole extract of the exudate minorityowned roots (MRE1), purified TPV and lirovannomu 50% acetonitrile, M. truncatula wild-type (A17) and the mutant dmil (Y6). Extract the ICC stimulated FBK from plants of the wild type, but not mutant dmil.

(C) As fraction A and fraction B stimulated FBK.

Fractions A and B were collected after preprepared the main HPLC exudates minorityowned roots (MRE-1). Fraction MRE-1A contained sulfated, LHA, and the fraction of MRE-1B - desulfation LHO. Both fractions are statistically significantly stimulated FBK (P<0,05).

Fig.11. The effect of a mixture of sulfated and desulfation factors ICC on memorization Medicago truncatula.

a. Memorysize in axenically conditions. The plants were grown in test tubes on generovanou beveled nutrient medium M containing factors of the ICC at a concentration of 10-8M. 50 sterile spores (Glomus intraradices) were placed near the roots of the seedlings. The degree of minoritatii was measured by counting units infection six weeks after inoculation. The results were analyzed using non-parametric statistical test of Kruskal-Wallis.

b. Memorysize in non-sterile conditions. The plants were grown on a substrate, consisting of baked clay pellets and inoculated with 50 sterile spores of G. intraradices; factors ICC was added to the nutrient solution at a concentration of 10-8M. three weeks after inoculation of root colonization was estimated by the method of intersection of the lattice.

Fig.12. The effect of the factors on the ICC architecture root Medicago truncatula.

a. The effect on the formation of lateral roots. Histogram showing the effect of a mixture of sulfated and desulfation factors ICC (NS+S), sulfated factors ICC (S) and desulfation factors the Directors of the ICC (NS) at a concentration of 10 -8M, 10-9M and 10-10M on the formation of lateral roots of M. truncatula wild-type (A17) eight days after processing.

In the experiment were used forty plants, and statistical analysis was performed using t-test t-test when comparing control and treated plants.

b. The effect on the total length of the root. Histogram showing the effect of a mixture of sulfated and desulfation factors ICC for the total length of the roots of seedlings. Seedlings were grown for eight days, then the roots cut off and perform the scanning and measurement of the root system by using the software WinRhizo. Data were analyzed using a test of Kruskal-Wallis.

(*) and (**) denote, respectively, a significant (P<0,05) or highly significant (P<0.01) difference, and the vertical bar denotes the standard error of the mean (SOS).

Fig.13. Genetic analysis of activated factor ICC path signaling, leading to stimulation of the formation of lateral roots.

Histogram showing the effect desulfuromonas factor ICC 10-8M) on the formation of lateral roots of M. truncatula wild-type (A17) and mutants in a symbiotic way of signaling dmil, dmi2, and dmi3 nsp1. Average values are represented as percentage of control value after eight days of treatment.

For each genotype were volun who inany data of at least two independent experiments with 40 plants in each, and statistical comparisons were made using t-test t-test when comparing the control and each treatment. (**) indicates a highly significant (P<0.01) difference, and the vertical bar denotes the standard error of the mean (SOS).

Fig.14. The effect factors of the ICC on mycorrhizal colonization carved transformed carrot roots in vitro.

a. The effect of a mixture of bacterial sulfated and desulfation

factors ICC. The roots were inoculable sterile spores of G. intraradices (10 spores/ml of culture medium) and treated once a week for three weeks, members of which were present or absent mixture of factors ICC at a concentration of 10-8M. Six weeks later observed the level of mycorrhizal colonization. (**) indicates a highly significant difference with control (t-test t-test, P<0,01). The vertical bar denotes the standard error of the mean (SOS).

b. The effect of a mixture of synthetic sulfated and desulfation factors ICC. The roots were inoculable sterile spores of G. intraradices (100 spores/ml of culture medium) and treated once a week for four weeks, members of which were present or absent mixture of factors ICC at a concentration of 10-8M. Eight weeks later observed the level of mycorrhizal colonization. (*) denotes reliable decomp is the difference with the control (t-test student's t, the value of P=0,0119).

Fig.15. The effect factors of the ICC on memorization Tagetes patula.

a: the Effect of a mixture of sulfated and desulfation factors ICC on the number of units of the infection on the plant (a1), the length of the root (a2) and the density of infection (a3). Plants were inoculable approximately 100 sterile Glomus spores inlraradices and treated twice a week for three weeks, members of which were present or absent factors ICC at a concentration of 10-8M. After four weeks was determined by the number of units of the infection, the root length and density of the units of the infection. (**) indicates a highly significant difference with control (t-test t-test, P value=0,004086).

b: Effect of sulfated (S), desulfation (NS) or a mixture of sulfated and desulfation (NS+S) factors ICC on mycorrhizal colonization of root. Plants were inoculable approximately 100 sterile spores Glomus intraradices and treated twice a week for three weeks, members of which were present or absent factors ICC at a concentration of 10-8M. After four weeks was measured level of colonization.

Fig.16. The effect factors of the ICC on the germination of tomato seed.

a. The effect desulfation (NS), sulfated (S) and a mixture of sulfated and desulfation (NS+S) factors of the ICC on the germination of tomato seed at 14°C. the Factors ICC was added to the Cup for the PoWPA is stania at a concentration of 10 -8M, 10-9M and 10-10M. the Level of germination was assessed daily. The results were analyzed using a test of Kruskal-Wallis. (***) and (**) denote, respectively, very high (P value<0.001) and high (<0,01) no significant difference with the control, and the vertical bar denotes the standard error of the mean (SOS).

b. The effect of a mixture of sulfated and desulfation factors ICC on seed germination at 14°C.

b1. The kinetics of germination. Factors ICC were added at a concentration of 10-10M. the Results were analyzed using the nonparametric test of Kruskal-Wallis. After 6-day differences were highly significant. The vertical bar denotes the standard error of the mean (SOS).

b2. Picture characteristic cups for germination with addition and without added factors ICC ten days after sowing.

MATERIALS AND METHODS

Natural sources of factors ICC

Strain DAOM 197198 AM fungus Glomus inlraradices, which were maintained in co-culture with cut roots for many years (Chabot et al., 1992), well characterized, and its genome is currently sequanorum. This strain is used by PREMIER TECH company for the industrial production of commercial inoculants and for the production of purified spores for research purposes. For example, these purified spores was is used as source DNA for the project to sequence the genome of G. intraradices. The authors of the present invention used two types of exudates, both of which were made from materials purchased at PREMIER TECH BIOTECHNOLOGIES (Rivere-du-Loup, Quebec, Canada):

(i) the Exudates minorityowned roots (EMK). Mycorrhiza was produced by the joint cultivation of G. intraradices with carved transformed roots of carrot. Nutrient medium was overiden with Phytagel. After appropriate growth minorityowned roots gel was razziali by adding sodium citrate as a chelating agent, and a liquid EMK was kept in 4-liter containers, which were stored at 4°C.

(ii) the Exudates germinating spores (XPS). Purified sterile spores of the AM fungus Glomus intraradices was kept in bottles containing approximately one million spores. Bottles were stored at 4°C. Spores were germinated at 30°C in an incubator with 2% CO2within 10 days.

Biotests, used for the purification of factors ICC

To detect the presence of symbiotic signals AM-fungi at different stages of isolation and purification was used three Biotest. (i) it Was shown that the design of M. truncatula ENOD11::GUS is induced in the formation of mycorrhizal fungi and the effects of diffusible compounds of different AM fungi j (Journet ct. al., 2001; Kosuta et al. 2003) (=test MtENOD11). (ii) the Formation of lateral roots in M. truncatula has been shown to be stimulated by defund the dominant compounds of different AM fungi, and for this reaction required a symbiotic way of signaling DMI (Olah et al., 2005) (=test MtLRF). (iii) in Addition, the authors of the present invention used a modified test for branching root hairs in Vicia saliva (pea seed, capable of detecting various desulfation LHO (=test VsHab).

(i) Induction of symbiotic gene MtENODl 1 in transgenic Medicago truncatula.

Previously it was shown through experiments, in which the AM fungus was separated from the root of the plant cellophane membrane that diffusing the connection of the AM fungus can induce the expression of the transgene promoter MtENODl 1-gusA in growing lateral roots of M. truncatula (Kosuta et al., 2003). The authors of the present invention used a previously described Protocol (Andriakaja et al., 2007) with the following modifications: paper disk was not placed on the agar plate was assessed, and the processing was made by adding 40 microlitres to sprout. To check out whether the answer ENOD11 induced via the signal path DMI, compared to the response observed in M. truncatula line wild-type A17 and in the mutant carrying a mutation in the gene DMI1 (mutation Y6).

(ii) the Branching root hairs pea seed

Peas seed (Vicia sativa ssp. nigra) is a small-seeded legume, convenient for microscopic observation of the deformation of root hairs. Deformation root ox is SKOV pea seed are not only called Nod factors specific bacterial symbiont Rhizobium leguminosarum, the biovars viciae, but also many desulfation Nod-factors (Roche et al., 1991; Price ct al., 1992).

Thus, this test is suitable for detecting the presence of desulfation LHO. In previous works, the test was performed in liquid medium. The authors of this invention have developed a test Cup with agar, which is more sensitive and reproducible. The seeds were first sterilized in sulfuric acid for 20 minutes, rinsed twice with sterile water, and then treated for 20 minutes with calcium hypochlorite (5 g/150 ml after filtration through a paper filter) and rinsed five times with sterile water. Seeds kept in water overnight at 4°C, transferred to plates with soft agar and incubated for three days at 4°C to increase the uniformity of germination. Following this, the Cup was passed within 36 hours in the dark at 22°C for germination. Five young seedlings (root length approximately 1 cm) were seeded in Petri dishes with the environment of Pareus, was sealed by Parafilm and incubated for three days in an upright position in a growth chamber at 22°C. When the roots appeared hairs along the roots was carefully placed 40 microliters of the test solution and seedlings grown for 30 hours at 22°C. For observation branching root hairs, roots of prepared slices, which were placed between the objective and patronisation 0.02% th solution of methylene blue and observed under an optical microscope. Watched ten plants for each of the experimental conditions.

Tests memorization

Sources inoculum AM fungi experiments minoritatii were sterile spores of Glomus intraradices acquired in Premier Tech Biotechnologies Ltee (Riviere-du-loup, Quebec, Canada) or received in a cutout transformed roots of carrot, as described Becard and Fortin (1988). Minorityowned the transformed roots of carrot was cultivated as described in Chabot ct al. (1992), and perseval every ten weeks on the medium M (Becard and Fortin, 1988) generovanou with the help of 0.4% Phytagel (Sigma). After liquefaction Phytagel citrate buffer (Doner and Becard, 1991), spores were collected, as described, in sterile conditions and stored at 4°C in Ultrapure water for at least four weeks before using.

Tests memorization were performed on three types of plants, the model legumes M. truncatula and the two are not related to legumes, carrots (Daucus carota, family Umbelliferae) and marigold melkotsvetnye (Tagetes pa tula, family Asteraceae).

Factors ICC was dissolved in water/acetonitrile (50/50) for the preparation of 10-3M basic solution which is then diluted to the desired concentration with water or nutrient medium. An equal number of traces of solvent of acetonitrile was added in the control Cup.

Memorysize carved transformed roots of carrot in vitro

Sterile wire is these transformed roots of carrot was grown on the medium M, generovanou with 0,4% Phytagcl, at 24°C in the dark and perseval every ten weeks (Chabot et al., 1992). Roots were collected by liquefaction Phytagel nitrate buffer (Doner and Becard, 1991) and washed with sterile deionized water. Cups for test memorization were prepared as follows: in Petri dishes (0 to 90 mm) poured the first layer of 20 ml of medium M containing 0.3% Phytagel, and gave it to harden. Then poured the second layer of the same medium containing 20 or 200 spores/ml and the factors of the ICC to the desired concentration. In the control cups instead of the solution factors ICC used equal volume of medium used to prepare the solution of the factors of the ICC. The root fragments were placed on the medium surface is approximately equal to the quantity (number of pieces and length of roots) in different cups. The cups were sealed with Parafilm tape and incubated in the dark in the phytotron at 24°C and 50% humidity for six or eight weeks. Factors ICC was added once a week to the surface of the Cup within the first three or four weeks for experiments lasting six or eight weeks, respectively. For observation of fungal colonization of roots were collected after liquefaction Phytagel citrate buffer, washed and stained with ink-acetic method (Vierheilig et al., 1988). The level of colonization was estimated by the method of intersection of the lattice (Giovanetti and Mosse, 1980).

Memorysize Tagetes patula in vivo

Seeds Tagetes patula, sort Legion d'honneur, were obtained from Caillard (84091 Avignon, France). Seedlings were grown for four weeks in Falcon tubes of 50 ml volume, filled with a substrate made from washed and processed in the autoclave clay (charred granular Montmorillonite; ref "Oil Dry US Special, Brenntag Bretagne, Zl dc Tory, BP41, Avenue des Ferrancins, 71210 Montchanin). To ensure the supply of seedlings, at the base of the tubes were done three small holes, and the tubes were individually placed in plastic boxes (5.5 cm diameter, 7 cm high) with a volume of 120 ml, closed with an opaque lid with holes' where inserted and fixed tubes Falcon.

The boxes were filled with water to 80 ml and wrapped with aluminum foil. Clay substrate in Falcon tubes was moistened with 20 ml nizkoposhibnogo solution long Ashton (Flewitl el al, 1966). In each tube under the surface of the substrate was placed one seed, and a hundred of fungal spores were distributed around the seed in 1 ml of 10-7M solution factors ICC or control solution. Each plant received 1 ml of 10-7M solution factors ICC or 1 ml of control solution twice a week for three weeks. The pots were placed in growth chamber at 25°C, with a light period of 16 h and light intensity of 180 manktelow·m-2·s-1.

We carried out two series of experiments. the first mixture of sulfated and desulfation synthetic factors ICC was tested on 12 seedlings per treatment. In the second sulfated, desulfation factors ICC and their mixture were tested on 20 seedlings per treatment. After 4 weeks the plants were collected. Internal root system was painted in black ink Shaffer (Vicrheilig et al, 1998). Quantification of root colonization by the fungus was performed under a binocular magnifying glass, was used in two ways: (i) in the first experiment the number of infection units (zones containing arbuscules, vesicles and internal network hyphae) was calculated for each plant, and (ii) in the second experiment, the percentage of root length colonized by the fungus, i.e. demonstrating arbuscules, vesicles, or both, was determined by the method of intersection of the lattice (Giovannetti et al, 1980).

Memorysize Medicago truncatula in axenically conditions

The plants were grown in test tubes on generovanou beveled nutrient medium MM (Olah et al, 2005) with a volume of 20 ml, as described in Ben Amor et al. (2003). Factors ICC at a concentration of 10-8M (or control solution) were included directly in a sterile environment. Fifty-sterile spores of G. intraradices were placed at the bottom of the nutrient medium around the root of the seedlings. The tubes were placed in a growing chamber at 25°C with a light period of 16 h and light intensity, 366 microangelo·m-2·s-1. After six weeks, the architecture of the root system was PR is analyzed using the software Winrhizo Scientific Software (Regent Instruments Inc., 2672 Chemin Ste Foy RD, Sainte Foy, Quebec, Canada). Quantification of root colonization was carried out by direct counting units infection under binocular loupe after dyeing root ink-acetic method (Vierheilig et al, 1998).

Memorysize Medicago truncatula on the substrate, in baked clay

Germinated seedlings were grown for three weeks in Falcon tubes of 50 ml volume, as described above for experiments minoritatii Tagetes. Twenty spores of G. intraradices were distributed around the roots of the seedlings in 1 ml of 10-7M solution factors ICC or control solution. Then each plant received 1 ml of 10-7M solution factors ICC or 1 ml of control solution twice a week for two weeks. Conducted two series of experiments using 12 cuttings per treatment. The pots were placed in growth chamber at 25°C with a light period of 16 h and light intensity, 366 manktelow·m-2·s-1.

After 3 weeks the plants were collected. Internal root system was painted in black ink Shaffer (Vierheilig et al, 1998). The percentage of root length colonized by the fungus, i.e. demonstrating arbuscules, vesicles, or both, was determined by the method of intersection of the lattice (Giovannctti et al, 1980).

Biotests for KeepAlive factors ICC development-related

Were developed biotests DL the validation activity of purified or synthetic factors ICC, associated with the development.

(i) Stimulation of root development in model legumes M. truncatula.

Previously, the authors of this invention have shown that the diffusion factor of AM fungi stimulates the formation of lateral roots (FBK) in M. truncatula via the path DMI (Olah et al., 2005). This Biotest used to test the activity of purified factors ICC associated with the development. Used a previously described Protocol except that the vitamins have been added in Wednesday PM

Identification of the genes of the plants involved in the signaling factor ICC, was performed in M. truncatula using the already described genetic analysis of the response FBK (Olah et al., 2005). Answers FBK factors ICC were studied in M. truncatula line wild-type Jcmalong A17, quality control, and defective in symbiosis mutants dmil (Y6), dmi2 (TR25), dmi3 (TRV25) and nspl (JB85).

(ii) seed Germination of tomato

Seeds tomato varieties Heinz 1706 were obtained from the Main collection of tomato seed INRA. They kindly provided by Rene Damidaux from the lab "Genetique et Amelioration des Fruits et Legumes" INRA 84143 Montfavet cedex (France). From this sample the main collection, seeds were multiplied in L1PM (INRA-CNRS, Toulouse). Seeds were stored at 4°C. the Seeds were sterilized for 45 minutes in the filtered solution 0,262 M of calcium hypochlorite (2.5 g l2in 75 ml of water), to which were added two drops of Tween 20. Then the hypochlorite solution was removed,and three times was rinsed seeds sterile distilled water. Cups with agar for germination were prepared by dissolving 9,375 g Difco Granulated Agar (Becton-Dickinson) in one liter of distilled water. 10-3M solution of the factor ICC was prepared in 50/50 water/acetonitrile and then diluted to the desired concentration with water. An equal number of traces of solvent of acetonitrile was added in the control Cup. One Cup had fifteen seeds with six or eight replications per treatment. The cups were incubated in the dark at 14°C, 20°C and 28°C. the germination rate was assessed daily.

Statistical analysis of data

Data biological experiments were statistically analyzed using t-test t-test or analysis of variance for the data corresponding to a normal distribution with uniform variance, or non-parametric tests of Kruskal-Wallis or Wilcoxon for distributions other than normal. It was used the statistical software R system (R Development Core Team, 2009).

Biochemical experiments

Liquid-liquid extraction exudates minorityowned roots:

The first extraction exudates minorityowned roots was performed in a two-liter round the vessel in which 1.6 liters of exudates was mixed with 400 ml (1/4 volume), butanol (1-butanol or 2-methyl-1-propanol), and leave the mixture to assert that in order to teach a clear phase butanol with a thin intermediate phase, that allows a good place to split water and butenolide phase (at least six hours). Then there was a second extraction of the aqueous phase with 350 ml (approximately 1/5 volume) of butanol and left for the night. After this second extraction of the United botanology phase (extraction 1 and extraction 2) was evaporated to a volume of about 0.5 liters, which was washed liquid-liquid extraction with an equal volume of bidistillate. Washed botanology phase was evaporated, was transferred to a small flask and dried using a rotary evaporator. After that, the dry extract was re-dissolved in 5 ml of water/acetonitrile (1/1) and filtered through cotton wool (pre-washed with chloroform) in a glass tube with a volume of 8 ml, and then was dried under a stream of nitrogen.

Liquid-liquid extraction exudates germinating spores:

Exudates from one million germinating spores (approximately 150 ml) was first extracted with 1/3 volume of ethyl acetate. The mixture was left to defend, to get a thin intermediate phase and to achieve good separation of the aqueous phase and ethyl acetate (at least six hours). A second extraction of the aqueous phase was performed using 1/3 of the volume of ethyl acetate during the night. Then there was the extraction of the aqueous phase butanol (1-butanol or 2-methyl-1-propanol) in the same sequence that the COI is lovelas to conduct extraction with ethyl acetate. The volumes of the phases of butanol and ethyl acetate were reduced to a few ml using a rotary evaporator. Each phase was transferred into a test tube with a volume of 5 ml, and dried under a stream of nitrogen.

Cleanup using solid phase extraction (TPV):

Preparation speakers: TPV system consisted of a glass column Chromabond volume of 3 ml, filled with a reverse phase C18 (SUPELCO Discovery DSC-18). The first glass fiber filter was placed at the bottom of the column. The solid phase was added into the column so that it took the amount column of a height of 3.5 see the Second glass fiber filter was placed on top of the solid phase and pushed inside to seal the solid phase. Before use, the column was washed with acetonitrile (LCP) and water, and then balanced 20% solution of acetonitrile (atsn) in the water.

Pre-filtration: the Extract was dissolved in one ml of 20% atsn. The extract was filtered through cotton wool in a Pasteur pipette (pre-washed with chloroform) and was applied to a C18 column. The tube and the filters were rinsed with 1.5 ml of 20% atsn.

Chromatography: Extract pushed through a phase C18 using a syringe. Flow-through fraction of the column was collected in a glass tube with a volume of 8 ml to get rid of is not bound peroxidase substances, phase abundantly washed (20% solution atsn in the water, five times the volume of the solid phase). This amount was collected in the same samples the REC. Then molecules, contacting column, suirable 50% solution atsn in the water. The volume of the eluate equal to 5 volumes of the solid phase was collected in the second glass tube. Finally, strongly bound peroxidase molecules were suirable from the column using 100% atsn. The volume of solvent (approximately 6 ml) was collected in the third test tube. These three solution (20%, 50% and 100% on atsn) was evaporated under a stream of nitrogen to obtain a dry residue. Each residue is subsequently was re-dissolved in a volume suitable for holding prepreparation HPLC. Column TPV used for cleaning approximately 5 liters of exudates minorityowned roots.

Prepreparation HPLC:

Cleanup was performed on the separation module high-performance liquid chromatography Shimadzu LC10 (Shimadzu corporation, Kyoto, Japan) with prepreparation reversed-phase C18 column (8 mm×250 mm; 5 μm, Equisorb, CIL Cluzeau). Volume loop dispenser was 100 µl. Used the following chromatographic procedure: 10 minutes in isocratic mode with solvent a (20% acetonitrile in water), then for 20 minutes followed by a linear gradient from solvent A to solvent B (100% acetonitrile) and another isocratic step at 100%-th acetonitrile for 5 minutes. Two minutes was required to return to initial conditions (20% atsn). The flow rate sostav the La 2 ml·min -1and the UV absorption was measured in the area of 206 nm. The samples were collected during the gradient was performed every minute (2 ml), which amounted to 14 fractions.

Additional analytical HPLC for detection desulfation LHO

Purification was carried out on the separation module high-performance liquid chromatography Shimadzu LC10 (Shimadzu corporation, Kyoto, Japan) with column phase C8 (Bond XDB-C8 HP-Eclipse (Hewlett Packard) 5 μm to 4.6×150 mm) for 5 min in isocratic mode with 30% methanol in water solvent, and then for 20 minutes followed by a linear gradient to 100%-WMD methanol solvent, and then another isocratic step at 100%-th methanol for 5 minutes. 2 minutes was required to return to initial conditions. The flow rate was 1 ml·min-1and the UV absorption was measured in the area of 206 nm. Sampling was performed along the gradient and isocratic phase 100% wage methanol every minute (about 1 ml) from 15 to 23 min, which amounted to 8 fractions.

Experiments on WASH-EAP MS:

Each fraction collected in prepreparation HPLC were subjected to analysis using WASH-MS Acquity UPLC connected to a mass spectrometer Q-Tof Premier (Waters Corporation). As speakers for WASH used column Acquity (2.1 mm×10 cm, 1.7 mm) (Waters, USA), and the flow rate was 0.45 ml/min For more hydrophilic joint is (fractions 1-9 of prepreparation HPLC) used a linear gradient from 10% atsn (1% om aqueous solution of acetic acid) to 100% atsn within 7 minutes then within 2 minutes was followed by isocratic step at 100%-th atsn, and then return to initial conditions (2 minutes), and finally, the stage of regeneration for 1 minute in a 10% ohms atsn (1% om aqueous solution of acetic acid). To improve the resolution of more hydrophobic compounds (fractions 6-11 from prepreparation HPLC) gradient WASH was expanded: used a linear gradient, starting at 25% wage atsn 0.1% th aqueous solution of acetic acid and up to 100% atsn within 7 minutes. The capillary of the mass spectrometer was set at 3.2 kV and the cone - 10 B. Internal adjustment weight was performed by continuous introduction of the source solution leucine-enkefalina. The spectrometer was calibrated before each experiment. More hydrophilic compounds (fractions 1-9 of prepreparation HPLC) was analyzed in the regime of negative ions and positive ions, in order to facilitate detection, respectively anionic (sulfated) and cationic (desulfation) connections.

For fragmentation of molecules of certain ions were selected and were subjected to analysis of TMS using the collision energy 15 B.

Mild hydrolysis:

This method is used to remove sulfate groups at the sulfated LHO without changing the rest of the molecule (Roche et al., 1991b). Fraction A, the elution of which took place between 15 and 16 m is the alike prepreparation HPLC, was transferred to a glass beaker with a screw top lid and dried under a stream of nitrogen. Then it was twice dissolved in anhydrous methanol and again dried to remove residual water. To the dry sample was added to 250 μl of 0.05 M Hcl in methanol. The reaction was carried out at room temperature over night. Then the sample was again dried under a stream of nitrogen and washed twice with anhydrous methanol to remove all acid.

Production milligramme quantities of factors ICC

Purification factors of the ICC exudates germinating spores Glomus intraradices and minorityowned roots gives an extremely low yield. For the production of a large number of these molecules have used two strategies using genetic engineering of bacteria.

(i) the Production factors of the ICC in mutants of rhizobia.

Rhizobia produce Nod factors, which represent substituted LHO, which have similar structural features with the factors of the ICC. The essential difference is that the factors of the ICC are very simple LHO with a very limited number of substitutions, essentially leading to a possible O-sulfation reducing residue N-acetylglucosamine. Our strategy was to use mutants of rhizobia, modified by genes that encode the enzymes responsible for the replacement of the predecessors Nod-facto is s, and because secreting very simple LHO similar to the factors of the ICC. The authors of this invention have opted for mutant strains derived from any species of rhizobia that produce the maximum tetramer of LHO and minimum (approximately 10%) pentamers of LHA, as in the case of fungal factors ICC.

To obtain sulfated factors ICC authors of the present invention used double mutant nodFEnodL Sinorhizobium meliloti. Mutation nodL inhibits O-acetylation nereguliruemaia terminal residue N-acetylglucosamine and mutation nodFE blocks the synthesis of unsaturated fatty acid 16:2, which leads to N-acylation of fatty acids C18:1 (vaccinology) or C16:0 (palmitic) (Ardourel et al., 1994). To boost the output of LHO in the mutant strain was introduced multicopying plasmid RMN, carrying regulatory genes nod. The resulting highly productive strain GMI 6629 cultivated in a liquid nutrient medium containing 5 μg/ml of tetracycline to maintain plasmids RMN and luteolin (10 μm) as an inducer of nod genes (Ardourel et al, 1994). When reaching the bacterial culture density on the order of 109cells per ml, Nod-factors were isolated by liquid-liquid extraction with butanol or ethyl acetate (Roche et al, 1991). Then spent cleaning LHO by HPLC on a reversed-phase C18 column, as it was opisaniya (Dcmont et al., 1993) with the following modification gradient water-acetonitrile: a 10-minute isocratic phase in 20% acetonitrile followed a linear gradient from 20 to 65% acetonitrile over 30 minutes at a flow rate of 2 ml/min Peak fractions containing the sulfated LHO were collected at the site gradient between 32 and 35% acetonitrile and analyzed using mass spectrometry. Most of LHO were tetramer, and lower pentamers, as factors of the ICC. LHO were O-sulfotyrosine at the reducing end and N-etilirovany fatty acids C18:1 and C16:0 in nereguliruemaia the end. O-acetylation was not found.

For the production of desulfation factors ICC used the strain LPR5045 (RMR). It is a derivative of strain RCR5 R. leguminosarum, biovars trifolii, which was eliminated by the Sym plasmid, and then entered multicamera plasmid RMR containing the shared genes nodABCIJ (Lugtenberg et al, 1995). Highly productive strain were grown in culture medium, containing 5 µg/ml tetracycline to maintain plasmids RMR and 10 µm naringenin as inducer of nod-gene (Spaink. et al, 1994). LHO was isolated from the culture medium as described above. The HPLC purification was performed on the same reversed-phase C18 column, as in the case of the sulfated LHO with a 20-minute isocratic phase 26.5% acetonitrile and subsequent linear gra what antom acetonitrile-water from 26.5% to 100% acetonitrile over 40 minutes at a flow rate of 2 ml/min Peak fractions corresponding to desulfuromonas LHO were collected at approximately 50% acetonitrile and analyzed using mass spectrometry. Most LHO were tetramer, and the minority - pentamers, as factors of the ICC. In the N-acylation participated fatty acids C18:1 and C16:0. O-acetylation or O-sulfation was not found.

The structure of the main sulfated and desulfation LHO produced by mutant strains of rhizobia GMI 6629 Sinorhizobium meliloti and LPR5045 (RMR) Rhizobium leguminosarum, biovars trifolii, respectively, below. They with great accuracy imitate factors ICC produced by the AM fungus Glomus intraradices (see Example 2).

These factors ICC prepared from cultures of mutant rhizobia, were tested for biological activity. Example 7 shows that a mixture of these sulfated and desulfation factors ICC greatly stimulates the formation of mycorrhiza (Figure 14a), indicating that these molecules act as genuine signals minoritatii.

(ii) Production factors ICC methodology using a "cell factory".

These synthetic factors ICC were courtesy of Eduardo Andres Martinez and Hugues Driguez from the laboratory CERMAV CNRS in Grenoble, France. The procedure they used was essentially the same as what isana in the literature (Samain and el,. 1999): the result of the cultivation of recombinant strains of E. coli carrying genes nodBC or nodBCH from Sinorhizobium meliloti, in conditions of high density of cells received NI,II,III-triacetyl-itinerate and 6OI-sulfo-NI,II,III-triacetyl-itinerate as the major compounds together with small amounts of the corresponding pentameron.

After extraction and purification of these compounds was carried out selective N-acylation using acid chlorides palmitic or oleic acids in various water-organic solvents, or free acids, and method of N-acylation, developed previously, to obtain lipophilicity factors in the formation of nodules (Ohsten Rasmussen et al, 2004).

We have received the following four lipoperoxide:

LHO IV (contaminated≈10% LHO V) C16:0

LHO IV (contaminated≈10% LHO V) S C16:0

LHO IV (contaminated≈10% LHO V) C18:1

LHO IV (contaminated≈10% LHO V) S C18:1

RESULTS

EXAMPLE 15: PURIFICATION FACTORS OF the ICC EXUDATES MINORITYOWNED ROOTS AND EXUDATES GERMINATING SPORES

A common strategy

Source factors ICC was used strain DAOM 197198 Glomus intraradices, because it has the wide range of hosts and is used for large-scale industrial production of inoculants AM fungi. This strain is well characterized, and its genome is currently sequanorum. Used two complementary source factors of the ICC. The advantage of exudates minorityowned roots is possible to carry out the allocation of a large amount of obtaining large number of factors ICC. The disadvantage of this source is that the exudates contain a mixture of compounds of plant and fungal origin. For this reason, the authors of this invention have used another source, exudates treated germinating spores, which contain only substances of AM fungal origin, but have the disadvantage in obtaining extremely low concentrations of factors ICC.

Biologically active compounds present in the exudates of AM fungi are amphiphilic.

Exudates minorityowned roots were first subjected to liquid-liquid extraction with butanol and ethyl acetate. Water, butanediol and an ethyl acetate phase was tested for biological activity using biotests on MtENOD11 and VsHab: activity was detected in butanole fraction, which indicates that the factors of the ICC are amphiphilic compounds. As shown in Fig.1, an active connection is manifested in the test PA MtENOD1l as blue staining appearing on growing roots, and test VsHab in ka is este visible branching near the top of the root tips of pea seed.

Then butanolic extract was subjected to solid phase extraction (TPV) with reversed-phase C18 column and subsequent elution of 20%, 50% and 100% combined acetonitrile solvent. Biological activity in tests on MlENOD11 and VsHab was detected in fractions, elyuirovaniya 50% acetonitrile, confirming that the active connection (or connections) is amphiphilic. Similar results were obtained with more than five independent samples of exudates minorityowned roots. A small amount of activity in the test for VsHab could also sometimes be observed in the eluate 100% acetonitrile, which indicates that different compounds may be responsible for the activity in tests on MtENOD1l and VsHab, and the connection is operating in test VsHab is slightly more hydrophobic than the connection, the current test MtENOD11.

Exudates germinating spores were isolated using the same liquid-liquid extraction with butanol and ethyl acetate. Activity in tests on MtENOD11 and VsHab attended only butanole phase. Similar results were obtained from five independent samples of germinating spores. Thus, we can conclude that the amphiphilic compound (connection) active in tests MtENOD11 and VsHab has an AM fungal origin.

Two types of active compounds in the exudates minorityowned cor is she

For further separation of compounds exhibiting activity in tests on MtENOD11 and VsHab, and information about their chromatographic properties, it was necessary to analyze botanology fraction by HPLC. However, because the exudates minorityowned roots were heavily contaminated with substances of root and Phytagel, botanology faction before phase HPLC was subjected to TPV, as described in the previous paragraph. Fraction TPV, allerona 50% acetonitrile and active in two biotests was then analyzed using prepreparation HPLC on reversed-phase C18 column in a gradient of acetonitrile-water. With an interval of two minutes were collected fourteen fractions. A typical profile is shown in Fig.2. The activity of each fraction was verified in tests on MtENODl 1 and VsHab. It was found that fractions, erwerbende in 30-48% acetonitrile (atsn) (fraction A), the active test MtENOD11 and faction, erwerbende in 64-72% LCN (fraction B), the active test VsHab. These data show that active in different biogest are different connections. Connection (connection), active in the test PA MtENOD11, is more hydrophilic than the connection (connection), active in the test for VsHab.

It is interesting to note that the characteristics of the elution fractions, active test MtENOD11 well correspond to the elution characteristics, there is Emim the sulfated Nod factors from Sinorhizobium meliloti and Rhizobium tropici (33-45%), which, in turn, can also be active in Biotest on MtENOD11. On the other hand, the characteristics of the elution fractions, active test VsHab well correspond to the elution characteristics observed in desulfuromonas Nod-factor of R. Leguminosarum, biovars viciae (67%), and deacetylating Nod-factors from Rhizobium meliloti nodHnodL (66%), which, in turn, can also be active in Biotest using seed peas. These data do not contradict the hypothesis that the exudates minorityowned roots contain a mixture of sulfated and desulfation LHO.

Two types of active compounds in the exudates germinating spores

Butanolide extracts from exudates germinating spores were analyzed using prepreparation HPLC under conditions similar to those described above. As can be seen in Fig.3, exudates dispute also contain two types of active compounds, more hydrophilic, which is active in Biotest on MtENODl 1 (fraction A), and more hydrophobic, which is active in Biotest on VsHab (fraction B). The elution characteristics of these two types of compounds are identical to the elution characteristics observed in two types of active compounds from exudates minorityowned roots. These results indicate that the two active compounds present in the exudates minoritarianism, have the AM fungal origin. Their chromatographic dynamics and biological properties do not contradict the hypothesis that these compounds correspond sulfated and desulfuromonas LHO.

Change activity associated with desulfuromonas fraction A

It was shown that soft methanol hydrolysis of sulfated, LHO, such as Nod-factors of S. meliloti, removes sulfate groups, without causing other structural modifications (Roche et al., 1991b). To check, could the biological activity of A fraction collected after HPLC butanolic extracts from exudates germinating spores, in relation to MtENODl 1 to be caused by the presence of the sulfated LHO, sample fraction A was subjected to mild hydrolysis. Processed fraction completely lost the activity in relation to the MtENOD11 (see Fig.4). It is interesting to note that while the fraction A was not initially active in Biotest on VsHab treated fraction showed significant activity in this test (Fig.4). The fact that A fraction after mild hydrolysis can acquire a new function - activity in relation to VsHab - demonstrates that soft methanol hydrolysis did not lead to significant degradation of the active compound fractions A and just modified it, probably by removing the sulfate group, which is the PTS is ery movable O-substituent in LHO. These data indicate that the activity of fractions of A with respect to MtENOD11 may be due to the presence of sulfated(s) LHO, and the activity of the more hydrophobic fraction in relation to VsHab is a consequence of the presence desulfuromonas(s) LHO.

EXAMPLE 2: BIOCHEMICAL CHARACTERIZATION FACTORS ICC

LC/MS and WASH/MS

The different fractions obtained after prepreparation reversed-phase HPLC exudates minorityowned roots were subjected to individual chromatography on an analytical reversed-phase column under ultra-high pressure (WASH). The measurement was performed using IER-MS.

The results are shown in Figures 5-8. These figures show the ion currents corresponding to LHO that, as expected on the basis of indicators of retention time in HPLC and WASH and biological activity was present in the samples.

If compounds with calculated mass within their isotopic distribution was present in the analyzed sample, they would have had an effect on the chromatogram as peaks. Because the resulting peaks could represent artifacts (peaks could correspond to a minor component of the isotopic profile), the corresponding spectra are also shown in the lower part of each figure.

The first eight fractions HPLC, for which the chromatographic Dean is Mika and biological properties assumed the presence of the sulfated LHO, were analyzed in negative ion mode. According to data retention time, measured in HPLC using standard tetramer (SP4) and pentamers (SP5) LHO, search exact masses (error less than 10 ppm), corresponding sulfated compounds with SP4 and SP5, conducted in fraction 4, which showed the highest activity in the test for MtENOD11. In this faction, you can easily discover a mass corresponding sulfated compounds with SP4, acylated fatty acids containing 16 carbon atoms (Fig.5). In accordance with the indicators of retention time in HPLC, we found in the previous fraction (fraction 3) appropriate connections with SP5 (Fig.7), while in the subsequent fraction (fraction 5) - sulfated compounds with SP4, with a chain of 18 carbon atoms (Fig.6). In the following fractions (6-8) search for compounds with SP3 no results. Compounds were characterized, first, by using different ion currents (the correspondence between the calculated exact masses and expected retention times), and secondly, in relation to the isotopic profile of the corresponding spectra. Fig.8 illustrates the effectiveness of the method for establishing the presence or absence of specific compounds with a certain mass. Ion current with the value of the ratio of mA the son to the charge, equal 1332,6 corresponding to the alleged LHO (V, C18:2, S), gave only the amplification of background noise (no single well-defined peak), while the ion current with the value of the ratio of mass-to-charge equal to 1334,6 corresponding to the alleged LHO (V, C18:1, S), clearly demonstrated the presence of a peak WASH containing compound with the expected mass (data are confirmed registered mass spectrum). Thus, the extracts of the ICC contain pentamers sulfated, LHO N-acylated C18:1, but not their derivatives, acylated C18:2.

When using this method, it was impossible to detect sulfated compounds with SP4 or SP5, O-substituted such functional groups as acetyl, carbarnoyl or fucosyl, or N-substituted by such groups as methyl, which are very common in lipophilicity Nod factors produced by various strains of rhizobia.

Fractions 7-11 were then analyzed using WASH, but the measurements were made in the positive mode IER-MS. Was applied the same strategy: the choice of the ion with subsequent analysis of the corresponding spectra.

Extracts minorityowned roots were also analyzed using LC/MS. Faction, erwerbende between 20 and 23 minutes prepreparation HPLC, were pooled, dried under a stream of nitrogen and re RA who met in 150 μl of 50% atsn in water and 1% acetic acid. Solutions were made directly in the source IER spectrometer Q-Tof Ultima (Waters, US). The capillary was set at 3 kV, the cone voltage was 70 V, a Rf lens 35 C. the Molecular ions of two minor compounds with respect to mass-to-charge equal to 1045,5 and 1047.5, can be detected in the positive mode, which corresponded cationizing sodium, LHO containing acyl radicals C16:1 C16:2 and do not have O-substitution. Mass and isotopic profiles confirmed the proposed structure.

Since a significant amount of pollution (e.g., PEG), eluruumid together with the wanted desulfation LHO compounds in fractions 9-11, prevented their detection by MS, we performed additional purification by HPLC. Fractions 9 and 10 of prepreparation HPLC were pooled and applied to a C8 analytical column, and then suirable gradient, starting 30% Meon in the water and ending with 100% Meon. Pollution were suirvey from 1st to 15th minute. The alleged LHO connection expected around 20th minute. The fraction collected between 15 and 23 minutes, were separately analyzed using WASH-MS and detection of specific ions was carried out on the basis of relevant observable sulfated species (SP4 and SP5; acyl chain C16:0 and C18:1). The signal calculated ion exact mass with respect to the masses for the poison, equal 1027,56 (DP4, C16:1), was detected in fractions eluting between 18 and 19 minutes. Retention time in comparison with synthetic standards, as well as accurate mass and isotope profile was consistent with the desired compound. The structure was finally confirmed by registered mass spectrum, which showed classic In-fragmentation in the areas of relationship mass-to-charge equal to 400,2, 603,3, 806,4.

TMS

Demonstration of the presence of compounds having a mass corresponding to the expected retention time in HPLC or WASH, insufficient to confirm their structure. For this reason, the authors of the present invention conducted an analysis of one of the alleged LHO compounds using TMS. Fig.9 shows a comparison, in TMS in the negative ion mode, sulfated Nod factor SP4 C16:2 of S. meliloti with possible "factor of the ICC", LHO (IV, C16:0,S), present in the sample. Characteristic ions of the reducing end with respect to mass-to-charge 503 (Y2), 605 and 706 (Y3) was clearly detected in both cases, as the characteristic neutral loss fragment mass 101.E. m. (vnutrichechenskie gap), starting at the stage of the molecular ion. An exact match of the nature of the fragmentation points to the structural unity of these two molecules, and that the sulfate group is located on the reducing balance is glucosamine, while the balance of fatty acids is PA end nereguliruem the glucosamine residue. Because fragmentation is not led to the emergence of ions beta-elimination (ions fatty acids), it is highly likely that the fatty acid acylation occurred at the N atom of the glucosamine residue.

EXAMPLE 3: STIMULATION of the formation of LATERAL ROOTS FACTORS ICC

After further purification using solid phase extraction (TPV) and elution with 50% acetonitrile, butanolic extract exudates minorityowned roots were made into cups with the medium M and tested by growing seedlings A17 M. truncatula. This purified extract ICC significantly stimulated the formation of lateral roots (P=0.05). This extract ICC did not stimulate the formation of lateral roots of a mutant dmil (Y6) M. truncatula, which indicates the presence of this procedendo extract the signal of the ICC, which activates the formation of lateral roots (FBK) through a symbiotic way of signaling DMI (Fig.10A).

Exudates germinating spores were isolated using ethyl acetate and butanol. Three extracts (water, an ethyl acetate and butanolic) were tested for ability to stimulate BCF in seedlings of M. truncatula A17. Butanolic extract significantly stimulated FBK (P=0.05), whereas water and an ethyl acetate extracts showed no activity (Fig.10B). This experiment confirms the AM fungal p is kishorganj amphiphilic compounds (compounds), stimulating FBK.

After TPV, butanolic extract exudates minorityowned roots was further purified using prepreparation HPLC, and the fractions corresponding to the sulfated LHO (fraction A, the active test MtENODl 1) and desulfuromonas LHO (fraction B, the active test VsHab), were separately collected and tested at the seedling A17 M. truncatula. It was found that these two fractions significantly stimulated BCF (Fig.10C). These data indicate that factors ICC consist of a mixture of sulfated and desulfation simple LHO, both of which can stimulate the formation of lateral roots in plants.

EXAMPLE 4: EFFECT of FACTORS ON the ICC FORMATION of AM IN MODEL LEGUMES M. TRUNCATULA

Synthetic factors ICC, obtained using the methodology of the cell of the plant, as described in Materials and Methods, were used to study the possible influence of factors of the ICC on memorization roots of the model legume plant Medicago truncatula AM fungus Glomus intraradices.

In the first series of experiments, seedlings of M. truncatula was grown in axenically conditions in test tubes on generovanou beveled nutrient deficient phosphorus and nitrogen, into which were added factors ICC at a concentration of 10-8M. Each sprout was inoculable 500 sterile spores of the fungus (Olah et al., 2005). Counting the Isla units infection (zone, containing arbuscules, vesicles and internal network hyphae) in terms of one plant was performed under a binocular magnifier six weeks after inoculation. Processing factors ICC increased the number of units of the infection in terms of one plant by 148% (see Fig.11a).

In the second series of experiments, seedlings of M. truncatula was grown on the substrate, consisting of baked clay pellets, in non-sterile conditions, and each sprout was inoculable 50 spores of the fungus. Factors ICC were added to the medium at a concentration of 10-8M. Percentage of root colonization was estimated by the method of intersection of the lattice three weeks after inoculation. The percentage minorityowned roots of plants treated with factors ICC, was 28.5% higher than in the control plants (Fig.11b).

Conclusions: In low concentration (10-8M) synthetic factors ICC stimulate the formation of AM in model legumes M. truncatula, providing further evidence that we found the factors of the ICC are true signals minoritatii.

The capacity factors of the ICC effectively stimulate the formation of AM at bean opens up opportunities for a wide range of applications in horticulture (for example, beans, chickpeas, lentils), agriculture (e.g., soybeans, peas, horse beans, alfalfa, peanuts), forestry (for example, lucali).

p> EXAMPLE 5: EFFECT of MYC FACTORS ON ROOT DEVELOPMENT IN LEGUMES

AM-fungi secrete diffusible compounds that induce the formation of lateral roots (FBK) in model legumes Medicago truncatula (Olah et al., 2005). It was shown (see Example 3) that the HPLC fractions containing sulfated and desulfation LHO fungi, have the same stimulation FBK. To demonstrate that this stimulation FBK actually is a consequence of the presence of LHO and not polluting compounds of the fungus that may be contained in these HPLC fractions, the authors of the present invention used a synthetic sulfated and desulfation LHO having the same structure as that of the compounds detected in mushroom exudates (see Example 4 and Materials and Methods).

At a concentration of 10-8M pure synthetic sulfated factors ICC or clean desulfation factors, as well as a mixture of sulfated and desulfation factors ICC, clearly stimulated FBK (see Fig. 12A), demonstrating that both types of compounds act as plant growth regulators. In contrast, at a concentration of 10-10M a mixture of sulfated and desulfation factors ICC was still extremely active, while net connection, whether it be sulfated or not, were inactive. These data show that the mixture Sul is atroveny and desulfation factors ICC is significantly more active than pure sulfated or desulfation factors ICC.

Thus, the factors of the ICC not only are symbiotic signals, activating the symbiotic program of the host plant in the early stages of minoritatii, but can also act as a genuine regulators of plants, to stimulate the formation of lateral roots and influence on the architecture of the root.

From agricultural point of view it was important to investigate the possible influence of factors of the ICC not only on the branching root, but also on the overall development of the root system. After growing seedlings for 8 days in a nutrient medium, containing or not containing a mixture of sulfated and desulfation factors ICC, roots cut off and perform the scanning and analysis of the root system by using the software WinRhizo. Processing factors, the ICC has led to an increase in the total length of root in 13,16% (Fig.12b). Thus, processing factors, the ICC is able to stimulate the development of the entire root system.

Conclusions: Both sulfated and desulfation factors ICC are active signals operating in low concentrations (10-8M), but a mixture of sulfated and desulfation factors ICC is clearly more active (current concentration is reduced to 10-10M).

They are now the incentive is irout the formation of lateral roots and root system development and are therefore not only symbiotic signals, Cox also a powerful plant growth regulators.

These results open the possibility of using these molecules in horticulture, agriculture and forestry to stimulate the development of plant roots and their growth.

EXAMPLE 6: FACTORS ICC EVOKE RESPONSES of PLANTS in a SYMBIOTIC WAY of SIGNALING DMI

Symbiotic way of signaling was detected in M. truncatula together with the genes encoding the reception Nod-factor (NFP), calcium signaling {DMI1, DMI2 and DMI3) and activator of transcription-specific klubenkoobrazovaniya (NSP1) (Catoira et al., 2000; Smit et al., 2005). Mutations in the genes DMI1, DMI2 and DMI3 result in changes to the formation of nodules, but also the formation of mycorrhiza, pointing PA that these three genes DMI participate in a way of signaling the formation of nodules and minoritatii (Catoira et al., 2000). In contrast, mutations in NSP1 gene lead to a defect in the formation of nodules, but do not affect memorization (Catoira et al., 2000). This discovery has led to the emergence of the hypothesis, according to which the symbiotic signals minoritatii, factors ICC, stimulate mycorrhizal program plants through the path DMI (Catoira et al., 2000). It is impossible to determine whether genes DMI to stimulate the formation of AM factors ICC, because the dmi mutants defective in the formation of mycorrhiza. Therefore, to examine the possible involvement of plant symbiotic genes in otvette factors ICC used the test on the formation of lateral roots (FBK) M truncatula described in Examples 3 and 5.

Sulfated factors ICC exhibit some structural similarities with the Nod-factors Sinorhizobium meliloti. To avoid possible cross-influence between ways of signaling Nod-factors, ICC, used desulfation synthetic factors of the ICC. The authors of this invention have studied the response to stimulation FBK on M. truncatula line wild-type A17, quality control, and mutants dmil (Y6), dmi2 (TR25), dmi3 (TRV25) and nspl (V).

As already described in Example 5, treatment of wild type line 10-8M desulfation factors ICC has led to a clear stimulation of the formation of lateral roots. On the contrary, defective in the mycorrhiza mutants dmil, dmi2 and dmi3 factors ICC did not cause stimulation FBK (see Fig.13). The mutant nspl, which is defective in the formation of nodules, but has normal mycorrhizal phenotype, and whose Nod-factors able to stimulate FBK, factors ICC caused clearly visible stimulation BCF (Fig.13). These data indicate that factors ICC evoke responses of plants at stages following the action DMI genes through specific mycorrhizal signaling path other than the path of the signaling Nod-factor (NSP1).

Conclusions: the ability of factors ICC to stimulate FBK blocked mutants dmi1, dmi2 and dmi3. This fact indicates that development-related responses caused by the actors of the ICC, implemented through a symbiotic way of signaling DMI, additionally confirming that the factors of the ICC are truly symbiotic signals.

Specific to education nodules NSP1 gene is not required for response to stimulation FBK, indicating that factors ICC cause this is associated with the development of the response through a specific path for them signaling, acting independently from the DMI genes and later them and independent of specific signaling pathways education nodules (NSP1). This is additional evidence that we found factors ICC represent true signals minoritatii.

EXAMPLE 7: the STIMULATION of the formation of AM CUT IN TRANSFORMED ROOTS of CARROT AS a SYSTEM FOR PRODUCTION of MYCORRHIZAL INOCULANTS

AM fungi are obligate symbionts: they are not able to proliferate and form spores in pure culture. For growth, they need to colonize the roots of host plants. This hard need an obstacle both for fundamental studies of AM symbiosis, and opportunities for large-scale production of inoculants AM fungi in horticultural and agricultural purposes. An important breakthrough was achieved through the use of crops carved transformed roots for cultivation AM-Gris is s, making possible the production of large quantities of sterile mushroom spores (Becard and Fortin, 1988). The system, which was used for a long time (Chabot et al., 1992), involved a joint cultivation of the strain DAOM 197198 AM fungus Glomus intraradices with clone carved carrot root transformed by Agrobacterium rhizogenes. This co-culture system is used, in particular, a biotechnology company Premier Tech (Quebec) for the production of commercial inoculants AM fungi. The authors of this invention have addressed the question of whether the use of factors of the ICC in low concentrations, as an additive to the medium to stimulate memorization cut the roots.

Was originally used a mixture of sulfated and desulfation factors ICC derived from the corresponding mutants of rhizobia (see Materials and Methods), which was added to the culture medium at a concentration of 10-8M. Exericise cut the carrot roots were inoculated with sterile spores of G. intraradices. The percentage of length of root colonized by the AM fungus was estimated by the method of intersection of the lattice (Giovannetti and Mosse, 1980). Was done five reps. Counting was performed after eight weeks using a binocular double-blind method.

In Fig.14 shows that the addition of a mixture of sulfated and desulfation the factors of the ICC at a concentration of 10 -8M in the culture medium led to a very strong increase in the percentage of colonization (+68,6%).

In the second experiment, a mixture of sulfated and desulfation synthetic factors ICC was added to the culture medium at a concentration of 10-8M. Was conducted fifteen replicates. As shown in Fig.14b, after eight weeks, the effect factors of the ICC to the stimulation of the formation of AM was highly significant (+20,5%.).

Conclusions: the Mixture of sulfated and desulfation factors ICC actively stimulates the formation of AM in the roots of carrots, which does not belong to the legume family. This is further proof that we identified and synthesized factors ICC are true signals minoritatii.

As synthetic factors ICC obtained biochemical methods, and the factors ICC obtained from mutant strains of rhizobia, effectively stimulated the formation of mycorrhiza, suggesting that these two types of strategies are appropriate for large-scale production factors ICC.

These data open the possibility of applying factors of the ICC as supplements to nutritional environments for the production of AM inoculants in the biotechnology industry, using the carved transformed roots.

EXAMPLE 8: EFFECT of FACTORS ON the ICC FO is the FORMATION I AM NEBBIOLO PLANT TAGETES PATULA

Tagetes patula, a plant of the family Asteraceae, was selected as Nebbiolo the host plant. T. patula (marigold melkotsvetnye) is a very popular garden plant. This view is used as a plant-companion for many vegetable crops. Reported that root allocation of this plant is able to kill nematodes in the soil, and it repels harmful insects, such as whiteflies for tomatoes. During flowering this plant can be together and be used to obtain essential oil, which is used in perfumery. T. patula used in tests on memorization, because it is a small plant, easy to handle and showing a quick root colonization of AM fungi. We used a sort of "Legion d'honneur". Tests memorization conducted by growing seedlings on a substrate composed of particles of baked clay. Seedlings were inoculable sterile spores of G. intraradices and added factors ICC at a concentration of 10-8M.

In the first series of experiments used a mixture of sulfated and desulfation synthetic factors of the ICC. The degree of minoritatii was assessed four weeks after inoculation by counting the number of infection units. The treated factor ICC sprouts were observed very significant, 153,5%, increase in the number of units the SIC infection on the plant (Fig.15a1). Factors ICC could increase the number of infectious areas by stimulating the development of the root system, either by increasing the density of infection. Indeed, processing factors ICC led to an increase of 49.1% of the length of the root (Fig.15a2) and to increase by 30.9% of the density of infection (Fig.15a3).

In the second experiment, inoculated plants were treated pure sulfated or desulfation factors ICC, or a mixture of both. Four weeks after inoculation, the level of colonization was estimated by the method of intersection of the lattice. The results are presented in Fig.15b. Treatment with a mixture of sulfated and desulfation factors ICC have caused substantial doubling of the level of root colonization (+104,5%), whereas the processing of pure sulfated and clean desulfation factors ICC has led to an increase in this level, respectively, by 42.3% and 75.4%.

Conclusions:

Factors ICC stimulate the formation I AM not related to leguminous plants, providing additional evidence that the factors ICC identified by us are genuine signals minoritatii.

As sulfated and desulfation factors ICC active, but their mixture of activity is clearly superior to separate groups of factors.

The fact that the factors of the ICC effectively stimulate the formation of the e AM and root development in nabibovich plants, opens up opportunities for their extremely wide use in gardening, agriculture and forestry.

EXAMPLE 9: EFFECT OF MYC FACTORS ON THE GERMINATION OF NEBBIOLO TOMATO PLANTS.

In the previous examples, the authors of this invention have shown that the factors of the ICC are not only symbiotic signals, activating mycorrhizal program plants, but can also act as regulators of plant growth and to stimulate the development of the root system at a very early stage of development of seedlings. Therefore explored the possible influence of factors of the ICC on the germination nabibovich plants. The variety of tomato Heinz 1706 was chosen, because this line is well characterized and has been used in the project to sequence the genome of tomato in the United States. In addition, it created a DNA microarray, which makes it possible to study the expression profile of genes in this line of tomato.

In these experiments we used purified synthetic factors ICC: sulfated, desulfation or their mixture. Factors ICC was added to the agar medium for germination and poured into Petri dishes. Seeds were placed on the surface of the cups with agar and incubated in the dark at 14°C, 20°C and 28°C. the germination Percentage was assessed daily.

In our experiments we used seeds that were Revisionary storage at 4°C during the course the e at least eight weeks. The presence of the factors of the ICC in the environment for germination in the concentration range from 10-8M to 10-10M resulted in a strong stimulation of germination at 14°C and 20°C (see Fig.16). Each type of factors ICC, sulfated or desulfation showed activity, but interestingly, a mixture of both types was clearly more active. At the highest temperature, 28°C, we were unable to detect any significant effect of the factors of the ICC on seed germination (data not shown). These results indicate that the effect of the stimulation is manifested at temperatures corresponding to the range of the normal temperature of the soil. It is possible to hypothesize that the coevolution of plants and their AM fungal symbionts were affected not only the formation of mycorrhiza in the developing roots, but also a very early stage of their interaction - the stage of germination. Both partners could benefit from combining the efficiency of seed germination and early root development with the presence of the fungal partner. Stimulation of germination was associated with subsequent best development of the seedlings, as shown in Fig.16b.

The fact that the seeds react to factors ICC, shows that plant components required for the perception of the factor ICC (receptors and signal transduction, are present and functioning in the seeds. This achiev that opens up the possibility for treatment of seeds of agricultural crops factors of the ICC in terms of agriculture. The observation that the factors of the ICC have an effect on the germination of seeds in extremely low concentrations (10-10M), opens the possibility of creating technology for the treatment of seeds, low-cost (low desired amount of active material) and ecological (the use of very low concentrations of natural compounds).

Conclusions:

As sulfated and desulfation factors ICC stimulate the germination of tomato, Nebbiolo plants, but their mixture is noticeably more active. Therefore, these two types of factors of the ICC are not only symbiotic signals, but also a powerful plant growth regulators, acting as legumes and nebbie plants.

From agricultural point of view, these results open the way to important applications in horticulture, agriculture and forestry: seed treatment factors ICC, preferably a mixture of sulfated and desulfation factors, may improve the percentage and germination rate and stimulate the development of young seedlings at the most cultivated plants, most of which is able to establish such endomycorrhizal symbiosis.

References

Akiyama K, Matsuzaki K, Hayashi H (2005) Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature, 435: 824-827.

Andriakaja A Now-Dernier A, Frances , Sauviac L, Jauncau A, Barker DG, Carvalho-Niebel F (2007) AP2-ERF reduced factors mediate Nod factor dependent Mt ENOD11 activation in root hairs via a novel cis-regulatory motif. Plant Cell 19:2866-2885.

Ardourel M, Demont N, Debelle F, Maillet F, de Billy F, Promc JC, Denarie J, Truchet G (1994) Rhizobium meliloti lipooligosaccharide nodulation factors: Different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses. Plant Cell 6:1357-1374.

Becard G, Fortin JA (1988) Early events of vesicular-arbuscular mycorrhiza formation in Ri T-DNA transformed roots. New Phytol., 108:211-218.

Benamor B, Shaw S, Oldroyd G, Maillet F, Penmetsa RV, Cook D, Long S, Denarie J, Gough C (2003). The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation. Plant.J., 34: 495-506.

Bcsscrer A, Puech-Pages V, Kiefer P, Gomez-Roldan V, Jauneau A, Roy S, Portais JC, Roux C, Becard G, Sejalon-Delmas N (2006) Strigolactones stimulate arbuscular mycorrhizal fungi by activating the mitochondria. PLoS Biol., 4(7):e226.

Catoira R, Galera C, De Billy F, Penmetsa RV, Journet EP, Maillet F, Rosenberg C, Cook D, gough C, Denarie J (2000) Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway. Plant Cell 12: 1647-1666.

Chabot S, Becard G, Piche Y (1992) The life cycle of Glomus intraradices in root organ culture. Mycologia, 84: 315-321.

Demont N, Debelle F, Aurelle H, Denarie J, Prome JC (1993) Role of the Rhizobium meliloti nodF and nodE genes in the biosynthesis of lipo-oligosaccharidic nodulation factors../ Biol. Chem., 268: 20134-20142.

Denarie J, Debelle F, Prome JC (1996) Rhizobium lipo-chilooligosaccharidc nodulalion factors: signaling molecules mediating recognition and morphogenesis. Annu. Rev. Biochem., 65: 503-535.

D Haeze W, holsters division M (2002) Nod factor structures, responses, and perception during initiation of nodule development. Glycobiology, 12: 79R-105R.

Doner LW, Becard G (1991) Solubilization of gellan gels by chelation of cations. Biotechnol Tech., 1991; 5:25-28.

Giovanetti M, Mosse B (1980) An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in rots. New Phytologist, 84:489-500.

Grenouillat N, Vauzeilles B, Bono J. T, Samain E, Beau JM. (2004) Simple synthesis of nodulation-factor analogues exhibiting high affinity towards a specific binding protein. Angew Chem Int Ed Engl 43:4644-4646.

Harrison M (2005) Signaling in the arbuscular mycorrhizal symbiosis. Annu. Rev. Environ, 59: 19 to 42.

Hewitt, E. J (1966) Sand and water culture methods used in the study of plant nutrition. Technical Communication No. 22 (revised 2nd edn). Com. Bur. of Horticul and Plant Crops East Mailing, Maidstore, Kent, UK.

Journet EP, El-Gachtouli N, Vernoud V, de Billy F, Pichon M, Dedieu A, Arnould C, Morandi D, Barker D, Gianinazzi-Pearson V (2001) Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbusculc-containing cells. Mol. Plant Microbe Interact., 14: 737-748.

Kosuta S, Chabaud M, Lougnon G, Go ugh C, Denarie.1, Barker DG, Becard G (2003) A diffusible factor from arbuscular mycorrhizal fungi dosage symbiosis-specific MtENODl 1 expression in roots of Medicago truncatula. Plant Physiol, 131: 952-962.

Navazio L, Moscatiello R, Genre A, Novero M, Baldan B, Bonfante P, Mariani P (2007) A diffusible signal from arbuscular mycorrhizal fungi elicits a transient cytosolic calcium elevation in host plant cells. Plant Physiol. 144: 673-681.

Olah, Briere C, Becard G, Denarie J, Gough C (2005) Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DM11/DMI2 signalling pathway. Plant.!., 44: 195-207.

Ohsten Rasmussen M, Hogg B, Bono JJ, Samain E, Drigucz H (2004) New access to lipo-chitooligosaccharide nodulation factors. Org. Biomol. Chem.,2: 1908-1910.

Price NPJ, Relic B, Talmont F, Lewin A, Prome D, Pueppke SG, Maillet F, Denarie J, Prome JC, Broughton WJ (1992) Broad-host-range Rhizobium species strain NGR234 secretes a family of carbamoylated, and fucosylated, nodulation signals that are O-acctylated or sulphated. Mol. Environ, 23: 3575-3584.

R Development Core Team (2009) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-http://project.org.

Rcmy W, Taylor TN, Hass IT, Kerp H (1994) Four hundred-million-ycar-ld vesicular arbuscular mycorrhizae. Proc. Natl. Acad. Sci. USA, 91:11841-43.

Roche P, Debelle F, Maillet F, Lerouge P, Faucher C, Truchet G,.-Denarie J, Prome JC (1991a) Molecular basis of symbiotic host specificity in Rhizobium meliloti: nodli and nodPQ genes encode the sulfation of lipo-oligosaccharide signals. Cell, 61: 1131-1143.

Roche P, Lerouge P, Ponthus C, Prome JC (1991b) Structural determination of bacterial nodulation factors involved in the Rhizobium meliloti-aiaia symbiosis../. Biol. Chem., 266: 10933-10940.

Samain E, Drouillard S, Heyraud A, Drigucz H, Gram-scale synthesis of recombinant chitooligosaccharides in Escherichia coli. Carbohydrate Research, 302: 35-42.

Samain E, Chazalet V, Geremia RA (1999), Production of (9-acetylated and sulphated chitooligosaccharides by recombinant Escherichia coli strains harboring different combinations of nod genes. Journal of Biotechnology, 72: 33-47.

Smit P, Racdts J, Portyanko V, Debelle F, Gough C, Bisscling T, Geurts R (2005) NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced reduced. Science, 308:1789-91.

Smith SE, Read DJ (2008) Mycorrhizal symbiosis. 787 pp., Academic Press.

Stacey G, Libault M, Brechenmacher L, Wan J, May G (2006) Genetics and functional genomics of legume nodulation. Curr. Opin. Plant Biol., 9:110-121.

Spaink HP, Wijfjes AHM, van der Drift KMGM, llaverkamp.1, Thomas-Oates JE, Lugtcnberg BJJ (1994) Structural identification of metabolites produced by the NodB and NodC proteins of Rhizobium leguminosarum. Mol. Environ., 13:821-831.

Spaink HP, Wijfjes AHM, Lugtenberg BJJ (1995) Rhizobium Nodi and NodJ proteins play a role in the efficiency of secretion of lipochitin oligosaccharides. J. Bacteriol., Ml: 6276 6281.

Vierheilig H, Coughlan AP, Wyss U, Piche Y (1998) Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl. Environment. Environ., 64:5004-5007.

Weidmann S, Sanchez L, Descombin J, Chatagnier O, Gianinazzi S, Gianinazzi-Pcarson V (2004) Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula. Mol. Plant Microbe Interact., 17: 1385-1393.

1. The use of lipoperoxide, which corresponds privedennyynizhe the formula (I):

where n = 2 or 3, R1is a lipid Deputy, which is chain fatty acids containing from 16 to 18 carbon atoms which may be saturated or mono - or dimensional, a R2represents H or SO3H, for the stimulation of minoritatii plants.

2. The use of lipoperoxide formula (I) under item 1 to stimulate the development of the root system of a plant.

3. The use of lipoperoxide formula (I) under item 1 for better germination of plants.

4. The use of lipoperoxide formula (I) under item 1 as an additive in the production of arbuscular mycorrhiza inoculants.

5. Application under item 1, characterized in that the specified lipohyalinosis formula (I) chosen from:
- lipoicacid formula (I), where n=2 or 3, R1represents a saturated or monounsaturated chain fatty acid containing 16 carbon atoms, a R2represents H or SO3H,
- lipoicacid formula (I), where n=2 or 3, R1represents a saturated or monounsaturated chain fatty acid containing 18 carbon atoms, a R2represents H or SO3H.

6. The use according to any one of paragraphs.1-5, characterized in that a mixture of lipoperoxide formula (I), where R2represents H, with what pohitivshem formula (I), where R2represents the SO3H.

7. The use according to any one of paragraphs.1-5, characterized in that the specified lipohyalinosis formula (I) is used in concentrations from 10-5up to 10-12M

8. Application under item 7, characterized in that the specified lipohyalinosis formula (I) is used in concentrations from 10-7up to 10-10M

9. A mixture of lipoperoxidation, which contains an effective amount of lipohyalinosis formula (I), where R2represents H, and lipohyalinosis formula (I), where R2represents the SO3H to stimulate arbuscular-mycorrhizal symbiosis plants, to stimulate germination of seeds of the plants or to stimulate the development of the root system of the plant.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to a combustion resistant mixture. The mixture contains at least one combustible polymer or a copolymer of a styrene monomer and hexa-, hepta- or octa ester of sucrose and a mixture of brominated C16-C18 fatty acids or a mixture of such esters. Also disclosed is a method of endowing the combustible polymer or styrene monomer copolymer with flame retardant properties.

EFFECT: invention provides excellent flame retardation of combustible polymers.

6 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a compound having general structural formula , in which n is equal to 1 or 5 and R6 is COOH or CH2OPO3H2, to a pharmaceutically acceptable salt of the said compound. The invention also pertains to a pharmaceutical composition based on the said compounds or their pharmaceutically acceptable salts, meant for inducing or boosting immunoreaction in a subject.

EFFECT: invention relates to a method of inducing or boosting immunoreaction in a subject, as well as to a method of relieving or essentially preventing an infectious disease, involving administration of an effective amount of the said compound to the subject.

7 cl, 5 ex

FIELD: chemistry.

SUBSTANCE: in method of obtaining compound aminoalkyl glucosaminide 4-phosphate of formula , X represents , Y represents -O- or NH-; R1, R2 and R3, each is independently selected from hydrogen and saturated and unsaturated (C2-C24) aliphatic acyl groups; R8 represents -H or -PO3R11R11a, where R11a and R11a, each is independently -H or (C1-C4) aliphatic groups; R9 represents -H, -CH3 or -PO3R13aR14, where R13a and R14, each is independently selected from -H and (C1-C4) aliphatic groups, and where indices n, m, p, q each independently is a integer from 0 to 6 and r is independently integer from 2 to 10; R4 and R5 are independently selected from H and methyl; R6 and R7 are independently selected from H, OH, (C1-C4) oxyaliphatic groups -PO3H2, -OPO3H2, -SO3H, -OSO3H, -NR15R16, -SR15, -CN, -NO2, -CHO, -CO2R15, -CONR15R16, -PO3R15R16, -OPO3R15R16, -SO3R15 and -OSO3R15, where R15 and R16, each is independently selected from H and (C1-C4) aliphatic groups, where aliphatic groups are optionally substituted with aryl; and Z represents -O- or -S-; on condition that one of R8 and R9 represents phosphorus-containing group, but R8 and R9 cannot be simultaneously phosphorus-containing group, including: (a) selective 6-O- silylation of derivative of 2-amino-2-desoxy-β-D-glucopyranose of formula , where X represents O or S; and PG independently represent protecting group, which forms ester, ether or carbonate with oxygen atom of hydroxy group or which forms amide or carbamate with amino group nitrogen atom, respectively; by means of tri-substituted chlorosilane RaRbRcSi-Cl, where Ra, Rb and Rc are independently selected from group, consisting of C1-C6alkyl C3-C6cycloalkyl and optionally substituted phenyl, in presence of tertiary amin, which gives 6-silylated derivative; (b) selective acylation of 4-OH position of obtained 6-O-silylated derivative with 6-3-alkanoyloxyalcanoic acid or hydroxyl-protected (R)-3-hydroxyalkanoic acid presence of a carbodiimide reagent and catalytic 4-dimethylaminopyridine or 4-pyrrolidinopyridine to give a 4-O-acylated derivative; (c) selectively deprotecting the nitrogen protecting groups, sequentially or simultaneously and N,N-diacylating the resulting diamine with (R)-3-alkanoyloxyalkanoic acid or a hydroxy-protected (R)-3-hydroxyalkanoic acid in presence of peptide condensation reagent; (d) introducing a protecting phosphate group at 3-position with a chlorophosphate or phosphoramidite reagent to give a phosphotriester; and (e) simultaneous or sequential deprotecting phosphate, silyl, and remaining protecting groups.

EFFECT: method improvement.

11 cl, 3 ex

FIELD: organic chemistry, microbiology, medicine, pharmacy.

SUBSTANCE: invention relates to novel derivatives of caloporoside of the formula (I): wherein R1, R2 and R3 mean independently of one another hydrogen atom (H) or acyl residues with 1-10 carbon (C)-atoms; R4 means hydrogen atom (H) or -C(O)(CH2)n-COOH wherein n = 1-7 under condition that not all R1, R2, R3 and R4 mean hydrogen atom (H), and their physiologically acceptable salts also. Also, invention relates to a method for synthesis of compound of the formula (I) or its physiologically acceptable salts. Method involves fermentation of the strain Gloeoporus dichrous (Fr:Fr) Bres. ST001714, DSM 13784 under aerobic conditions at temperature 18-35°C and pH = 5-8, isolation of one or some derivatives of caloporoside that can be conversed, if necessary, to physiologically acceptable salts, and to using compound of the formula (I) as CDK-inhibitor in treatment of cancer or another diseases associated with pathological damage of cells proliferation, and to a medicinal agent based on thereof and to a method for its preparing.

EFFECT: improved preparing method, valuable medicinal properties of derivatives.

14 cl, 5 tbl, 8 ex

The invention relates to(16)the glucosamine disaccharides having the General formula I, where R1, R2, R3, R4, R2', R3', R4', R6' have the meanings indicated in the claims, and method of production thereof and to pharmaceutical compositions comprising as an active ingredient of these disaccharides

-d - glucosaminidase with antitumor activity" target="_blank">

The invention relates to a new connection, specifically to the derivatives of carbohydrates, namely, methyl-2,4-6-tri-O-acetyl-3-deoxy-3,3-With-dicyano- D-glucosaminide with antitumor activity, of the formula

Specified a new connection, its properties and the method of obtaining not described in literature

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used in agriculture. The Trichoderma harzianum Rifai strain is deposited in the Russian National Collection of Industrial Microorganisms under registration number VKPM F-180. The strain is capable of producing L-lysine-alpha-oxidase and can be used particularly as an inhibitor of cucurbit bacterial spot caused by Acidovorax citrulli bacteria.

EFFECT: invention reduces loss of cucurbit crop.

FIELD: agriculture.

SUBSTANCE: method comprises treatment of soil, seed treatment, harrowing the soil, and crop tending. Linseed flax is grown on sod layer. Before sowing, the complex fertiliser is applied in the soil. Crop treatment is carried out in phase of "herring-bone" with the biologically active substance which is used as the feed additive "Floravit"® which is sprayed to the plant. The concentration of the preparation is 2.5·10-4 mg/ml, the flow rate of the aqueous solution is in the volume of 200-400 l/ha.

EFFECT: method enables to increase the yield of linseed flax.

3 tbl

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology. A method of obtaining a granulated product includes growing filamentous fungi of a Monilialeae family, preferably Arthrobotrys conoides Dreschsler in a suitable liquid culture medium. The obtained culture of the filamentous fungus is mixed with at least one type of modified starch and starch flour, with the modified starch and modified flour being present in weight ratios, constituting from 30:70 to 60:40. Filling agents and in case of necessity nutritional substances are added to the obtained mixture with obtaining a paste. The granulation of the paste is carried out. The obtained granules are dried until the moisture level lower than 13% is achieved, preferably to the level constituting from 9 to 10%. The granulated product, obtained by the method described above, is used for the application in a pesticidal composition.

EFFECT: group of inventions provides obtaining the target product with high dispersability and a high percent of survival of the filamentous fungi propagules.

9 cl, 1 dwg, 4 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to the use of the concentrate of the culture liquid of the strain Trichoderma harzianum Rifai, deposited in the Russian National Collection of Industrial Microorganisms under the number of RNCIM F-180 as an inhibitor of Andis virus of potato mottling.

EFFECT: invention enables to reduce losses of potato from the plant infection with the Andis mottling virus.

2 ex

FIELD: biotechnology.

SUBSTANCE: strain of unicellular algae Dunaliella salina IPPAS D-295-producer of biologically active substances having antioxidant activity was deposited in the culture collection of microalgae of the Institute of Plant Physiology n.a. K.A.Timiryazev RAS (IPP RAS (IPPAS)) under registration number IPPAS D-295 and can be used to produce biologically active substances having antioxidant activity and antagonistic activity against opportunistic pathogenic bacteria.

EFFECT: invention enables to increases the yield of antioxidant substances.

3 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: composition contains an agent designed for biocontrol, for reduction of level of pollution of food and fodder with aflatoxin, a binding agent, an agent having osmoprotector and adhesive properties, a carrier and a source of nutrient elements for an agent, designed for biocontrol.

EFFECT: reduced level of food and fodder contamination with aflatoxin in raw materials.

29 cl, 4 tbl, 6 ex

FIELD: biotechnologies.

SUBSTANCE: strain of fungus Trichoderma harzianum Rifai VKPM F-180 is used as a producent of an inhibitor of a stimulant of bacterial burn of fruit cultures.

EFFECT: invention makes it possible to reduce losses of decorative and fruit cultures caused by bacteria Erwinia amylovora.

1 ex

FIELD: chemistry.

SUBSTANCE: entomopathogenic biopreparation for protecting plants from pests contains a dry mixture of a concentrate of a culture fluid based on infection units of entomopathogenic fungi of the type Verticillium lecanii or Beauveria bassiana, or Metarhizium anisopliae, aerosil and an additive which protects the infection units from loss of viability and biological efficiency when drying, with the following ratio of components, wt %: culture fluid concentrate 69.0-77.5, additive - 10.8-13.8, aerosil - 11.6-17.2. The additive is a polyatomic alcohol, carbohydrate, vegetable oil, sodium gluconate and water, with the following ratio of components, wt %: polyatomic alcohol 6.2-13.4, carbohydrate 38.0-41.9, vegetable oil 38.7-41.0, sodium gluconate 0.4-0.5, water 9.3-10.5. The biopreparation is obtained by mixing the culture fluid concentrate with the additive in ratio of 5-7:1, and then adding aerosil in ratio of 4.5-7.6:1 and drying the mixture. Efficiency of the biopreparation on white flies is 95-100% in a working concentration of 1·107 spores/ml; the titre of viable units is (0.35-5.5)·1010 CFU/g.

EFFECT: invention increases biological efficiency of the biopreparation, preserves and increases viability of infection units of fungi during drying and storage.

8 cl, 3 tbl, 15 ex

FIELD: chemistry.

SUBSTANCE: invention relates to plant protection agents. Disclosed is a preparation having fungicidal properties, where the active components used are terbuconazole, imazalil and prochloraz or metalaxyl. The ratio terbuconazole:imazalil:prochloraz/metalaxyl is equal to 1:(0.5-15.0):(0.5-15.0), with consumption rate of active substances of 45-300 g/t seeds.

EFFECT: invention widens the range of biological action against snow mould, increases biological efficiency against root rot.

4 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and use of a Trichoderma harzianum Rifai strain as a producer of an Impatiens necrotic spot tospovirus inhibitor. The Trichoderma harzianum Rifai strain, which is deposited in the Russian National Collection of Industrial Microorganisms under No.F-180, is a producer of the homogeneous enzyme L-lysine-a-oxidase, which exhibits marked antiviral activity with respect to Impatiens necrotic spot tospovirus.

EFFECT: reduced loss of decorative and vegetable crops caused by Impatiens necrotic spot tospovirus.

2 ex

FIELD: agriculture.

SUBSTANCE: invention relates to agriculture and biotechnology. The invention is a method of rapid determining of the parameters of symbiotic interaction of arbuscular mycorhiza and a plant.

EFFECT: proposed method enables to carry out an assessment of mycorhisation of plant roots much more accurate and faster, which can be used in rapid determining of the parameters of symbiosis of arbuscular mycorhiza - symbiotic effectiveness of arbuscular mycorhiza fungi belonging to the biological preparations - growth intensifiers of plants, as well as indices of mycorhisation.

6 cl, 4 dwg, 4 tbl, 4 ex

FIELD: agriculture.

SUBSTANCE: invention relates to the field of agriculture. The invention is a method of determining the size of symbiotic nitrogen fixation in soybean in the field environment, comprising simultaneous sowing the soybean initial variety and the test culture, determining the dry biomass accumulation in the phase of mass flowering and complete ripeness of the plant, where the test culture is used as a mutant of soybean with ineffective tubercles, and a part of the symbiotic nitrogen in the soybean plants is determined by the difference between the dry biomass of the plants of the soybean variety under study with inefficient symbiosis and soybean mutant, not fixing nitrogen of the atmosphere, expressed as a percentage.

EFFECT: invention enables to determine reliably, fast (without determining the nitrogen content in plant samples) the amount of symbiotic nitrogen fixation by the part of biomass growth, to evaluate varietal responsiveness of soybean to inoculation.

3 tbl, 1 ex

The invention relates to Mycology and plant physiology, namely endomycorrhizal fungi, forming mycorrhiza vesicular-arbuscular type plant

FIELD: agriculture.

SUBSTANCE: invention relates to the field of agriculture. The invention is a method of determining the size of symbiotic nitrogen fixation in soybean in the field environment, comprising simultaneous sowing the soybean initial variety and the test culture, determining the dry biomass accumulation in the phase of mass flowering and complete ripeness of the plant, where the test culture is used as a mutant of soybean with ineffective tubercles, and a part of the symbiotic nitrogen in the soybean plants is determined by the difference between the dry biomass of the plants of the soybean variety under study with inefficient symbiosis and soybean mutant, not fixing nitrogen of the atmosphere, expressed as a percentage.

EFFECT: invention enables to determine reliably, fast (without determining the nitrogen content in plant samples) the amount of symbiotic nitrogen fixation by the part of biomass growth, to evaluate varietal responsiveness of soybean to inoculation.

3 tbl, 1 ex

Up!