Biogenic catalyst for delaying plant development processes

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of delaying plant development processes, associated with ethylene biosynthesis involving exposing a plant or plant part to one or more bacteria which produce one or more enzymes selected from a group consisting of nitrile hydratase, amidase, asparaginase, and mixtures thereof and said one or more bacteria are selected from a group consisting of Rhodococcus, Brevibacterium ketoglutamicum, and mixtures thereof, and wherein said one or more bacteria act on the plant or plant part in an amount sufficient to delay the plant development process. Disclosed is an apparatus for delaying the plant development process associated with ethylene biosynthesis having several layers. At least one of the layers contains a catalyst which contains one or more bacteria selected from a group consisting of Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum and mixtures thereof, wherein the one or more bacteria produce one or more enzymes selected from a group consisting of nitrile hydratase, amidase, asparaginase and mixtures thereof.

EFFECT: invention increases efficiency of delaying plant development process associated with ethylene biosynthesis.

59 cl, 4 dwg, 9 tbl, 5 ex

 

The technical FIELD

The present invention relates to a method of delaying development of plants, including the impact on the plant or plant part of one or more bacteria or enzymes. Additionally, a device to delay the process of development of plants.

The LEVEL of TECHNOLOGY

The formation of ethylene in plants and parts of plants caused by a variety of external factors and stressors, including damage, the use of hormones (e.g. auxin), anaerobic conditions, cooling, heat, dryness and infection by pathogens. Increased formation of ethylene is also observed in many processes of plant development, including maturation of fruits or vegetables, sprouting seeds, falling leaves and withering flowers.

The biosynthesis of ethylene in plants is traditionally portrayed as a biocatalytic scheme, including three enzyme, commonly called “the cycle of leadership, in which synthase S-adenosyl-L-methionine (SAM) catalyzes the conversion of methionine to S-adenosyl-L-methionine (AdoMet); synthase 1-aminocyclopropane-1-carboxylic acid (ACC) catalyzes the conversion of AdoMet to ACC; ACC oxidase catalyzes the conversion of ACC into ethylene and by-products carbon dioxide and hydrogen cyanide. General description of the biosynthesis of ethylene in plants and processes of plant development, regulated by ethylene, can be found, for example, Srivastava (201) Plant Growth and Development: Hormones and Environment (Academic Press, New York).

Earlier studies have shown that the ripening of climacteric fruits is caused, at least partially, a sudden and significant increase of ethylene biosynthesis. Although the sharp increase in the formation of ethylene is involved in the process of ripening of climacteric fruits, the exact mechanism of this process, especially for climacterics fruit, not fully understood. Although climacterical the fetus there is no sudden increase in the production of ethylene, climacteridae fruit also responds to ethylene. Moreover, fruits, vegetables and other plant foods vary in the amount of synthesized ethylene, as well as the sensitivity of a particular product to ethylene. For example, for apples characterized by a high level of ethylene synthesis and sensitivity to ethylene, whereas the artichokes detected a low level of ethylene biosynthesis and sensitivity to ethylene. See, for example, Cantwell (2001) “Properties and Recommended Conditions for Storage of Fresh Fruit and Vegetables” on the website postharvest.ucdavis.edu/Produce/Storage/index.shtml (date last accessed: March 6, 2007), which is fully incorporated into this application by reference. Maturation of the fetus usually leads to discoloration, softening of the pericarp and the change of sugar content and taste of the fruit. While maturation in the early stages makes the fruit more nutritious and attractive is for human consumption, this process ultimately leads to the degradation and deterioration of the quality of the fruit, making it unacceptable for use and leading to significant commercial loss in monetary terms. Control of the maturation process is desirable to improve retention and increase the time available for transportation, storage and sale of fruit and other agricultural products that are subject to maturation.

In addition to the sharp increase in the biosynthesis of ethylene in climacteric fruits, changes during maturation is also associated with increased intensity of breathing. The result of respiration of fruits, vegetables and other plant products produces heat and, consequently, it has an impact on retention, and the required storage conditions (for example, storage in the refrigerator) of these products. Herbal products with more intense breathing (e.g., artichokes, cut flowers, asparagus, broccoli, spinach, etc) have a shorter shelf life than products with less intense breathing (e.g., nuts, dates, apples, citrus, grapes, etc). Breathing is influenced by several environmental factors, including temperature, atmospheric composition, physical stress, daylight, chemical stress, radiation stress, caused by lack of water, growth regulators and defeat what toynami. In particular, the temperature plays a significant role in the intensity of breathing. For a General overview of respiratory gas exchange and recommended controlled atmospheric conditions for fruits, vegetables and other plant products, see, for example, Kader (2001) Postharvest Horticulture Series No. 22A:29-70 (University of California, Davis); Saltveit (University of California, Davis) “Respiratory metabolism” usna.usda.gov/hb66/019respiration.pdf (date last accessed: March 6, 2007); and Cantwell (2001) “Properties and Recommended Conditions for Storage of Fresh Fruit and Vegetables” postharvest.ucdavis.edu/Produce/Storage/index.shtml (date last accessed: March 6, 2007), each of which is fully incorporated into this application by reference.

Methods and compositions for delaying ripening fruit include, for example, the use of silver salts (such as silver thiosulfate), 2,5-norbornadiene, potassium permanganate, 1-methylcyclopropene (1-MCP), cyclopropene (CP) and their derivatives. These compounds have significant disadvantages, such as the presence of heavy metals, bad smells and explosive properties when compressed, which makes them objectionable or which restricts their applicability for use in the food industry. Transgenic methods of controlling formation of ethylene to delay processes of plant development (e.g., fruit ripening) by introducing sequences nuclein the o acids, restrict the formation of ethylene, especially by reducing the expression of the enzyme ACC synthase or ACC oxidase, also are under study. Public reaction to genetically modified agricultural products, however unflattering.

Accordingly, the industry remains a significant need for safe methods and devices for delaying processes of plant development. Such methods and devices could provide better control over the ripening, ripening vegetables, fading colors, falling leaves and germination, and extend the shelf life of various agricultural products (e.g. fruits, vegetables and cut flowers), thereby allowing the transport of these products over long distances without the need for cooling, increasing the attractiveness of the product for consumers and reducing cash costs associated with loss of product due to premature ripening and decay.

BRIEF description of the INVENTION

Sure, you can delay the process of development of plants, including but not limited to the above: ripening, ripening vegetables, wilting flowers and falling leaves. The methods according to the present invention typically include effects on plants or part of plants is Oia, one or more bacteria in a quantity sufficient to delay the development of interesting plants. In some aspects of the present invention, the bacteria are selected from the group consisting of: species Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and mixtures thereof. Bacteria used for the practical application of the methods according to the present invention, can be further processed stimulating agent, including, for example, asparagine, glutamine, cobalt, urea, and mixtures thereof, to induce the bacteria's ability to delay the development of interesting plants.

The present invention additionally provides a device delays the process of plant development, including the catalyst containing one or more bacteria, especially species of Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, or their mixture. Any device that allows the effect of the catalyst on the plant or part of plant and delays the development of interesting plants included in the scope of the present invention. Typical devices include those in which the catalyst is immobilized on the matrix and placed inside, is placed over or otherwise attached to any physical structure. Various configurations of the described devices are provided and described in more detail below in this application. The methods and devices according to the present invention to delay the process of development of plants can be used, in particular, to increase shelf life and make it easier to transport long-distance products of plant origin, such as fruits, vegetables and flowers, improve customer satisfaction in the product and reduce product loss due to premature ripening or decay.

BRIEF DESCRIPTION of DRAWINGS

Thus, after the invention described in General terms, this section presents the accompanying figures, which are not necessarily made to scale, and in which:

In FIG. 1 shows a non-limiting scope of the invention the image is a three-layer device to delay fruit ripening. The outer layers (marked A and B) ensure the structural integrity of the device. The catalytic layer, as defined below in this application includes one or more of the enzymes according to the present invention, and it is located between the outer layers.

In FIG. 2A-C presents a non-limiting scope of the invention images of different devices to delay fruit ripening. These devices include catalytic layer, one or more layers that are designed to ensure structural integrity, and one or more layers that must be removed before using the device. Removing one or more of these layers may, for example, the TCD is set adhesive for attaching the device to a different physical structure.

In FIGURES 3A-3B presents a non-limiting scope of the invention, the image device to delay fruit ripening. The device includes a catalyst immobilized on the layer of film and fixed on the physical structure (for example, mailbox, suitable for storage/transportation of the fruit).

In FIG. 4 shows a non-limiting scope of the invention, the image device to delay fruit ripening. The device includes a segmented abdominal structure, which allows the insertion and replacement of one or more catalytic modular sections, as described below. The outer layers of the physical structure can consist of a material that allows air to penetrate into the catalyst.

DETAILED description of the INVENTION

The present invention will be described hereinafter in more detail with reference to specific implementations of the present invention and, in particular, on the various figures presented in the Appendix. In fact, the present invention can be implemented in many different forms and the scope of the invention is not limited to variants of the implementation described in this application; rather, these implementation options are presented so that the present description of the invention satisfy the relevant legal requirements. In the present description and in prilog the emnd claims, the singular includes reference to the plural, unless the context clearly indicated otherwise.

Everywhere in this description, the word “including”or its grammatical forms, it should be understood in the sense that implies the inclusion of a specified item, number, or step, or group of elements, integers or steps but not the exclusion of the possibility of the presence of any other element, number, or step, or group of elements, integers or steps.

The present invention provides methods of delay interest of the process of plant development, including the impact on the plant or plant part of one or more bacteria. In specific embodiments of the methods described delay the process of plant development, including the impact on the plant or plant part of one or more bacteria selected from the group consisting of the species Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and mixtures thereof, the said one or more bacteria affect plant or plant part in a quantity sufficient to delay the process of plant development. Additionally, a device for delaying the interesting process of plant development and for the implementation of the methods described in this application. The methods and devices according to the present invention can be applied, for example, to delay the ripening process of fruit is/vegetables or process of withering flowers and to increase shelf life of fruits, vegetables or flowers, which thus facilitates the transportation, distribution and sale of such products of plant origin.

In this application, the term “plant” or “plant part” is widely defined and includes the whole plant or any part of the plant, including, but not limited to the above-fruits, vegetables, flowers, seeds, leaves, nuts, embryos, pollen, ovules, branches, bones, ears, cobs, peels, stems, roots, root tips, anthers, and the like. In specific implementations, the part of the plant is a fruit, vegetable or flower. In some embodiments of the present invention, the part of the plant is a fruit, specifically climacteric fruit, described in more detail below.

The methods and devices according to the present invention is directed to a delay in the development process plants, for example, the process of development of plants, usually associated with increased biosynthesis of ethylene. The term “process development plant” means any process of growth or development of plants or parts of plants, including, but not limited to the above: ripening, ripening vegetables, wilting flowers, falling leaves, seed germination, and the like. In particular embodiments of interest, the process of plant development is the maturation plaguily vegetables, wilting flowers or fall leaves, in particular the ripening of fruit or vegetables. Defined in this application, the term “delay of the development process plants”and its grammatical forms, refers to any slowdown, interruption, suppression or inhibition of the development process are interested in plants or phenotypic or genotypic changes in plants or parts of plants, usually related to a specific process of plant development. For example, when interested in the process of plant development is a ripening, delayed fruit ripening may include inhibition of the changes normally associated with the maturation process (for example, change the color, softening of the pericarp (i.e. the wall of the ovary), increased sugar content, taste change, total destruction/deterioration of the fetus and the potential decrease in the attractiveness of the fruit for consumers, as described above). For the person skilled in the art it is obvious that the period of time required for ripening, will vary, for example, depending on the type of fruit, and specific applicable storage conditions (e.g. temperature, humidity, ventilation, etc). Accordingly, delay of fruit ripening can be from 1 to 90 days, specifically from 1 to 30 days, more specifically from 5 to 30 days. Methods of estimation of the delay process is and development of plants, such as ripening, ripening vegetables, wilting flowers and falling leaves, known to specialists in this field and can be based, for example, in comparison with the processes of plant development for raw plants or parts of plants. In some aspects of the present invention, delays in the development process plants, due to the use in practice of the methods according to the present invention can be evaluated by comparison with untreated plants or parts of plants, or plants or parts of plants that have been processed by one or more agents interested in delaying the process of plant development. For example, a delay in the maturation of the fruit, due to the implementation of the method according to the present invention, can be compared with the time of fruit maturity for raw fruit or fruit that has been processed by the agent against maturation, such as described above in this application.

Ways to delay the process of development of plants according to the present invention typically include the implementation of impact on the plant or plant part to one or more of the following bacteria: species Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, or a mixture containing any combination of these bacteria. In some implementations, one or more bacteria include species Rhodococcus, and the hour of the particular strain DAP 96253 Rhodococcus rhodochrous, strain DAP 96622 species Rhodococcus, Rhodococcus erythropolis, or mixtures thereof. In this application, the impact on the plant or plant part to one or more of the above-mentioned bacteria include, for example, exposure of intact bacterial cells, lysates of bacterial cells and bacterial extracts that possess enzymatic activity (i.e. the “enzyme extracts”). Methods of obtaining lysates and enzyme extracts from cells, including bacterial cells are well known in this field. One or more of the bacteria used in the methods and devices according to the present invention, can more generally be referred to in this application as “catalyst”.

In accordance with the methods according to the present invention, carry out impact on the plant or plant part of one or more bacteria in a quantity sufficient to delay the process of plant development. The implementation of the “impact” on the plant or plant part of one or more bacteria according to the present invention includes any method of exposure of the bacterium to the plant or plant part. Indirect methods include, for example, placing bacteria or a mixture of bacteria at an arbitrary distance from plants or parts of plants (i.e. indirect effects). In other implementations, the bacteria can without ystavat on the plant or plant part by close or direct contact. Moreover, used in this application, the term “sufficient” quantity of one or more bacteria according to the present invention depends on many factors, including, but not limited to the above: concrete used in the way that bacteria form in which bacteria affect plant or plant part (e.g., in the form of intact bacterial cells, cell lysates or enzyme extracts, as described above), ways of influence of bacteria on the plant or part of plant, and the time period of exposure. The person skilled in the art using conventional experiments will be able to define “sufficient” quantity of one or more bacteria necessary to delay the development of interesting plants.

Although specific embodiments of the present invention, one or more bacteria selected from the group consisting of: species Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, any bacterium, which delays the process of plant development with its impact on the plant or plant part can be used in the methods and devices according to the present invention. For example, bacteria belonging to the genus Nocardia [see patent application Japan room 54-129190], Rhodococcus [see patent application Japan room 2-470], Rhizobium [see patent application Japan room 5-236977], Klebsiella [patent application Japan room 5-30982], Aeroonas [patent application Japan room 5-30983], Agrobacterium [patent application Japan room 8-154691], Bacillus [patent application Japan room 8-187092], Pseudonocardia [patent application Japan room 8-56684], Pseudomonas and Mycobacterium represent non-limiting examples of microorganisms that can be applied according to the present invention. Not all the species belonging to this genus can exhibit the same properties. Thus, it is possible that the genus of which it is known that it includes strains that are able to show the desired activity (e.g., the ability to withhold a certain process of development of plants, such as fruit ripening, may also include one or more species that normally do not exhibit the desired activity. In light of the description of the present invention described in this application and the prior art in this field nevertheless, the specialist in this area will require routine experiments, to analyze and to determine whether certain types of one or more of the desired activities.

In addition, specific examples of bacteria used according to the present invention, include, but are not limited to the above: view of Nocardia, Rhodococcus species, Rhodococcus rhodochrous, Klebsiella species, Aeromonas species, Citrobacter freundii, Agrobacterium rhizogenes, Agrobacterium tumefaciens, Xanthobacter flavas, Erwinia nigrifluens, Enterobacter species, species of Streptomyces, Rhizobium species, Rhizobium loti, Rhizobium legminosarum, Rhizobium merioti, Candida guilliermondii yeast, Pantoea agglomerans, Klbsiella pneumoniae subspecies pneumoniae, Agrobacterium radiobacter, Bacillus smithii, Pseudonocardia thermophila, Pseudomonas chloroaphis, Pseudomonas erythropolis, Brevibacterium ketoglutamicum, Rhodococcus erythropolis, Nocardia farcinica, Pseudomonas aeruginosa and Heliobacter pylori. In specific implementations, the bacteria of the genus Rhodococcus, namely strain DAP 96253 Rhodococcus rhodochrous (number ATCC Deposit in the 55899; deposited in the ATCC on December 11, 1996), strain DAP 96622 species Rhodococcus (number ATCC Deposit in the 55898; deposited in the ATCC on December 11, 1996), Rhodococcus erythropolis, or mixtures thereof, used in the methods and devices according to the present invention.

In some embodiments of the present invention, the specified one or more bacteria “stimulated”in order to give them the desired properties (for example, the ability to delay the process of plant development, such as ripening), by exposure to or treatment with a suitable stimulating agent. Stimulating agents include, but are not limited to the above: asparagine, glutamine, cobalt, urea or any mixture. In specific implementations, the bacteria is exposed to or treated with a stimulating agent asparagine, in particular, a mixture of stimulatory agents, including asparagine, cobalt and urea. A stimulating agent can be added at any point in the cultivation of specified cells. For example, with regard to bacteria in the culture medium can be made stimulating agent before Naalakkersuisut bacteria. Alternatively, bacteria can be grown in the environment within a predetermined amount of time to grow bacteria, and a stimulating agent can be added to one or more predefined points in time, in order to induce the desired enzymatic activity of the bacteria. Moreover, the stimulatory agent can be added to the nutrient medium (or in a separate mixture, including previously grown bacteria)in order to induce the desired activity of the bacteria after growth of bacteria.

Not limited to a particular mechanism, “stimulation” of bacteria according to the present invention can lead to the formation (or enhanced the formation of one or more enzymes, such as nitrilimines, amidase and/or asparaginase, and promote one or more of these enzymes may play a role in the delay of development of interesting plants. “Nitrilimines”, “amidase” and “asparaginase” includes a family of enzymes present in the cells of various organisms, including, but not limited to the above: bacteria, fungi, plants and animals. Such enzymes are well known to experts in the field, and every class of enzymes has a well-known enzymatic activities. “Enzymatic activity” in this application generally relates to the sposobnosti enzyme to act as a catalyst in the process, such as the conversion of one compound to another. In particular, nitrilimines catalyzes the hydrolysis of the nitrile (or tangerine) into the corresponding amide (or hydroxy (Aha). Amidase catalyzes the hydrolysis of the amide to the corresponding acid or gidrokshikislotu. Similarly, the enzyme asparaginase, such as asparaginase I, catalyzes the hydrolysis of asparagine to aspartic acid.

In some embodiments of the present invention, fermenta activity may be specified in the “units” on the mass of the enzyme or cells (usually based on the dry weight of the cells, for example, units/mg ICS). “Unit”generally refers to the ability to convert a certain amount of connection in another connection under certain conditions, as a function of time. In certain implementations, one “unit” of activity nitrilimines can refer to the ability to convert one micromole of Acrylonitrile in the corresponding amide in a minute, milligram of cells (dry weight) at pH 7.0 and a temperature equal to 30ºC. Similarly, one unit of activity of amidase can refer to the ability to convert one micromole of acrylamide in the appropriate acid per minute at milligram of cells (dry weight) at pH 7.0 and a temperature equal to 30ºC. Additionally, one unit of activity of asparaginase can refer to spasopreobrazensky one micromole of asparagine in the appropriate acid in a minute, on milligram of cells (dry weight) at pH 7.0 and a temperature equal to 30ºC. Tests for measuring the activity of nitrilimines, amidase, or activity of asparaginase is known in this field and include, for example, detection of free ammonia. Cm. Fawcett and Scott (1960) J. Clin. Pathol. 13:156-159, which is fully incorporated into this application by reference.

Ways to delay the process of plant development, including the impact on the plant or plant part of one or more enzymes selected from the group consisting of nitrilimines, amidase, asparaginase, or mixtures thereof, with one or more enzymes affect the plant or part of plant in the amount or level of enzyme activity, sufficient to delay the development process plants, additionally included in the scope of the present invention. For example, whole cells, which produce stimulated to produce, or genetically modified to produce one or more of the above enzymes (i.e. nitrilimines, amidase and/or asparaginase), can be used in ways to delay the process of plant development. Alternatively, nitrilimines, amidase and/or asparaginase, you can select to clear or partially clear of any of the above cells and subjecting them to the action of a plant or plant part in more isolated forms of the. See, for example, Goda and others (2001) J. Biol. Chem. 276:23480-23485; Nagasawa and others (2000) Eur. J. Biochem. 267:138-144; Soong and others (2000) Appl. Environ. Environ. 66:1947-1952 consolidation; Kato and others (1999) Eur. J. Biochem. 263:662-670, each of which is fully incorporated into this application by reference. In addition, for the person skilled in the art it is obvious that one type of cell may be capable of producing (or it can be stimulated or genetically modified to produce) more than one of the enzymes according to the present invention. These cells are suitable for use in the described methods and devices.

Sequences of nucleotides and amino acids for several nitrilimines, amides and asparaginase from different organisms are described in publicly available databases of sequences. A non-limiting list of typical nitrilimines and aliphatic amides known in this field, are shown in Tables 1 and 2 and in the list of sequences. “The protein pokazal”referred to in Tables 1 and 2, provides a General idea of the percentage confidence intervals (% int. of truth.) identification of selected proteins on the basis of the results of mass spectroscopy.

Table 1
Information about the amino acid sequence for a typical nitrilimines
The body-sourceThe access numberID sequenceProtein index
(% int. of truth.)
View Rhodococcus806580SEQ ID NO:1100%
View Nocardia27261874SEQ ID NO:2100%
Rhodococcus rhodochrous49058SEQ ID NO:3100%
Rekultivirovana bacterium (BD2); beta-subunit of nitrilimines27657379SEQ ID NO:4100%
View Rhodococcus806581SEQ ID NO:5100%
Rhodococcus rhodochrous581528SEQ ID NO:6100%
Rekultivirovana bacterium (SP1); the alpha-subunit of nitrilimines7657369SEQ ID NO:7100%

Table 2
Information about the amino acid sequence for a typical aliphatic amides
The body-sourceThe access numberID sequenceProtein soon
(% int. of truth.)
Rhodococcus rhodochrous62461692SEQ ID NO:8100%
Nocardia farcinica IFM 1015254022723SEQ ID NO:9100%
Pseudomonas aeruginosa PAO115598562SEQ ID NO:1098,3%
Helicobacter pylori J9915611349SEQ ID NO:1199,6%
Helicobacter pylori 266952313392SEQ ID NO:1297,7%
Pseudomonas aeruginosa150980SEQ ID NO:1394%

As a rule, any bacterial, fungal, plant or animal cell, which is one to make or which can be stimulated to produce nitrilimines, amidase, asparaginase, or any combination thereof, can be used in implementing the present invention. Nitrilimines, amidase and/or asparaginase can be produced constitutively in the cell, taken from a specific organism (e.g., in the cell, bacterium, fungus, plant or animal) or, alternatively, the cell can produce the desired enzyme or enzymes only after “induction” appropriate stimulating agent. The term “constitutive” is intended to indicate that at least one enzyme according to the present invention is continuously formed or expressed in a specific cell type. Other types of cells, however, may need to “stimulate”as described above, to Express nitrilimines, amidase and/or asparaginase in sufficient numbers or with sufficient enzyme activity to delay the development of interesting plants. That is, the enzyme according to the present invention can be produced (or produced at a sufficient level) only after exposure to or treatment with a suitable stimulating agent. Such stimulatory agents known in the field and above. For example, in some embodiments of the present invention, one or more bacteria is treated with a stimulating agent, such as spurgin, glutamine, cobalt, urea, or any mixtures thereof, more specifically, a mixture of asparagine, cobalt and urea. Moreover, as described in application for U.S. patent, pending, room 11/669,011, entitled “Induction and Stabilization of Enzymatic Activity in Microorganisms”, filed January 30, 2007, the activity of asparaginase I can be induced in DAP 96622 Rhodococcus rhodochrous (gram-positive) or DAP 96253 species Rhodococcus (gram-positive), in medium supplemented amesterdam amino acids, or their derivatives. Other Rhodococcus strains can also preferably similarly to induce that they showed enzymatic activity of asparaginase I, applying amesterdam amino acids, or their derivatives.

In other embodiments of the present invention, P. chloroaphis (number ATCC Deposit in the 43051), which produces active asparaginase I, in the presence of asparagine, and B. kletoglutamicum (number ATCC Deposit in the 21533), gram-positive bacterium, for which it is also known that it produces active asparaginase, used in the described methods. Fungal cells, such as cells of the genus Fusarium, plant cells and animal cells that Express nitrilimines, amidase and/or asparaginase, can also be used in the methods and devices described in this application, either in the form of whole cells, either in the form of the source from which you can select one or boleas the above-mentioned enzymes.

In additional implementations, the cells of the hosts that have been modified by the methods of genetic engineering, for ekspressirovali nitrilimines, amidase and/or asparaginase, can be applied, subjecting them to the action of a plant or plant part, in accordance with the methods according to the present invention and devices for delaying the process of plant development. In particular, polynucleotide, which encodes nitrilimines, amidase or asparaginase (or many polynucleotides, each of which encodes nitrilimines, amidase or asparaginase), you can enter using standard techniques of molecular biology of the cell host to obtain transgenic cell that expresses one or more enzymes according to the present invention. The use of the terms “polynucleotide”, “polynucleotide structure, nucleotide or nucleotide structure” does not mean limiting the present invention polynucleotide or nucleotides comprising DNA. For medium to specialists in this field will be obvious that polynucleotide and nucleotides may include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules, and synthetic analogs. In polynucleate the s according to the present invention also includes all forms of sequences, including, but not limited to the above: single-stranded forms, double-stranded forms, and the like.

Variants and fragments of polynucleotides that encode polypeptides that retain the desired enzymatic activity (i.e. nitrilimines, imidazol or asparaginase activity), can also be used in implementing the present invention. The term “fragment” means a portion of polynucleotide and, therefore, it also encodes part of the corresponding protein. Polynucleotide, which represent fragments of the nucleotide sequence of the enzyme, typically include at least 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 consecutive nucleotides, or up to the number of nucleotides present in a full-sized polynucleotide sequence of the enzyme. The movie polynucleotide encodes a polypeptide with the desired enzymatic activity and, as a rule, encodes at least 15, 25, 30, 50, 100, 150, 200 or 250 consecutive amino acids, or up to the total number of amino acids present in a full-sized amino acid sequence of the enzyme according to the present invention. The term “variant” is intended to refer to essentially the same sequences. Generally, options are determined by what sledovatelnot enzyme according to the present invention at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a reference sequence of the enzyme, which can be determined using standard sequence alignment. Options polynucleotide included in the scope of the present invention encode polypeptides with the desired enzymatic activity.

In the context of producing transgenic cells, the term “introduction” is intended to mean providing a host cell, more specifically, a microorganism such as Escherichia coli, polynucleotide, which encodes nitrilimines, amidase and/or asparaginase. In some implementations, polynucleotide is provided so that the sequence gains access to the internal space of the host cell, including its possible integration into the genome of the host cell. The methods according to the present invention do not depend on a particular method of introducing the sequence into a cell of the owner, except that polynucleotide gets access to the internal space of the at least one host cell. Methods of introduction of polynucleotides in the cells of the host are well-known in this field, including, but not limited to the above: how stable transfection, ways of temporary transfection and methods-mediated virus. The term “stable transfect the means, what polynucleotide structure introduced into the cell host, integrate into the host genome and can be inherited by its offspring. The term “transient transfection” or “transient expression” means that polynucleotide introduced into the cell of the host, but does not integrate into the host genome.

Moreover, the nucleotide sequence of nitrilimines, amidase or asparaginase may contain, for example, in plasmid for introduction into a cell of the host. Typical interest plasmids include vectors with certain sites of the cloning, the starting point of replication and breeding markers. The plasmid may further include sequences to initiate transcription and translation and transcription terminators and broadcast. The plasmids may also include a typical expression cassette containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both (e.g., Shuttle vectors)and selection markers for both prokaryotic and eukaryotic systems. Vectors suitable for replication and integration in prokaryotes, eukaryotes, or, ideally, both. For General descriptions of the systems and methods cloning, packaging and expression, see Giliman and Smith (1979) Gene 8:81-97; Roberts and others (1987) Nature 328:731-734; Berger and Kimmel (1989) Guide to Molecular Cloning Tecniques, Methods in Enzymology, volume 152 (Academic Press, Inc., San Diego, Calif.); Sambrook and others (1989) Molecular Cloning: A Laboratory Manual, volumes 1-3 (2nd ed.; Cold Spring Harbor Laboratory Press, Plainview, new York); and Ausubel and others, as amended (1994) Current Protocols in Molecular Biology, Current Protocols (Greene Publishing Associates, Inc., and John Wiley & Sons, Inc., New York; 1994 Supplement). Transgenic cells of the hosts that Express one or more enzymes according to the present invention can be used in the described methods and devices as whole cells or as a biological source from which you can select one or more of the enzymes according to the present invention.

Additionally, a device to delay the process of development of plants and for the implementation of the methods according to the present invention. In specific implementations, a device for delaying the development of plants, more specifically, to delay fruit ripening, including the catalyst, which contains one or more bacteria selected from the group consisting of: species Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and mixtures thereof, is included in the scope of the present invention. Strain DAP 96253 Rhodococcus rhodochrous strain DAP 96622 species Rhodococcus, Rhodococcus erythropolis, or mixtures thereof, can be used in certain embodiments of the present invention. One or more bacteria of the device according to the present invention is provided in a quantity sufficient to delay the development process who are interested in plants, specified in the application above. In other embodiments of the present invention, the catalyst comprises one or more enzymes (i.e. nitrilimines, amidase and/or asparaginase) or at the level of enzyme activity, sufficient to delay the process of plant development. Sources of enzymes are preferred for use as a catalyst in the device according to the present invention are also described in detail above. For example, the catalyst can be used in the form of whole cells, which produce (or induced or genetically modified to produce one or more enzymes according to the present invention, or may include the enzyme(s) selected, purified or partially purified form.

Device for delaying development of plants included in the scope of the present invention, can be provided in many suitable forms and can be designed for a single application or multiple applications (e.g., “rechargeable”). Moreover, the device according to the present invention find application in both domestic and commercial environments. For example, such devices can be built into domestic or commercial refrigerators included in trains, trucks, etc. for transportation over long distances fruits, vegetables ilicito, or used as free-standing cabinets for storage or transportation of such products of plant origin. Typical non-limiting device according to the present invention is described below in this application and shown in Figures 1-4.

In specific implementations, the catalyst is provided in an immobilized form. Any process or matrix for immobilization of the catalyst can be applied provided that the ability of one or more bacteria or enzymes) to delay the process of development of plants is maintained. For example, the catalyst may be immobilized on a matrix comprising alginate (e.g., calcium alginate), carrageen, diethylaminoethylcellulose (DEAE-cellulose) or polyacrylamide. Other such matrices are well known in this field and can be optionally cross-linked with any suitable cross-linking agent, including, but not limited to the above: glutaraldehyde or polyethylenimine, to increase mechanical strength of the catalyst matrix. In one embodiment of the present invention, the catalyst is immobilized on DEAE-cellulose matrix, cross-linked with glutaraldehyde. The catalyst, more specifically, an immobilized catalyst, may also be named in this application “catalytic who aulnoy section. Catalytic modular section includes a catalyst, such as an immobilized catalyst, within a secondary structure, which, for example, to reduce possible contact with the catalyst, facilitates the replacement of the catalyst or allows air to pass through the catalyst.

In one implementation, the matrix comprises alginate, or its salt. Alginate is a linear copolymer with homopolymer blocks of (1-4)-linked ß-D-mannuronate (M) and its epimerase C-5 residues of α-L-guluronate (G), respectively, covalently linked together in different sequences or blocks. The monomers can be present in the homopolymer blocks of consecutive G residues (G-blocks), sequential M-residues (M-blocks), alternating M and G residues (MG-blocks), or randomly arranged blocks. In one implementation, the calcium alginate is used as the substrate, specifically, calcium alginate, which was associated cross-connections, as, for example, polyethylenimine, with the formation of thickened calcium alginate. Additional description of such immobilization techniques can be found in Bucke (1987) “Cell Immobilization in Calcium Alginate” in Methods in Enzymology, vol. 135(B) (Academic Press, Inc., San Diego, California; Mosbach, as amended), which is incorporated in this application by reference. A typical method of immobilization used with the tion of calcium alginate, the associated cross-connections using polyethylenimine, also described below in Example 5. In another implementation, the matrix comprises nesoderzhaschii polymer. Any polymer comprising one or more amide groups, you can apply according to the present invention. In one implementation, the substrate includes a polyacrylamide polymer.

The increased mechanical strength of the matrix with immobilized catalyst can be achieved by cross-linking relations. For example, cells can chemically bind transverse relationship with education agglutinative clusters of cells. In one implementation, collected from cell culture were associated transverse relationship with the use of glutaraldehyde. For example, cells can be suspended in a mixture of deionized water and glutaraldehyde, and then add polyethylenimine until then, until it reaches the maximum flocculation. Related cross-connected cells (usually in the form of particles formed by several cells) can be distinguished by conventional filtration. Additional description of such techniques is given in Lopez-Gallego and others (2005) J. Biotechnol. 119:70-75, which is hereby fully incorporated by reference. Traditional methods of immobilization of cells, in particular cells of the species Rhodococcus, DEAE-cellulose, cross-linked with p the power of glutaraldehyde, also described below in Example 4.

In some embodiments of the present invention, the immobilized catalyst or one or more catalytic modular sections placed inside, placed on top of or attached to the “physical structure”. The physical structure includes, but is not limited to the above: a film, a sheet, a covering layer, carton, bag, package, or segmented chamber capable of holding one or more catalytic modular sections. In some implementations, the physical structure includes a container suitable for transport or storage of fruits, vegetables or flowers. The physical structure may optionally include more than one separate structure, by means of which all individual structures connected to the Central catalyst or catalytic modular section. The physical structure described in the above application, may possibly be cooled by external means or include a cooling module in the physical structure.

Items to monitor the effectiveness of the catalyst for the delay in the development process of the interesting plants (for example, for assessing when a catalyst or catalytic module should be replaced), or for measuring or regulating ventilation, moisture/humidity, and levels of carbon dioxide can be enabled is enabled in the device according to the present invention. Any delay device development process plants may optionally include one or more elements to ensure penetration of air to the catalyst or catalytic modular sections or through them. The person skilled in the art will easily be able to anticipate other possible modifications of the devices described in this application, for the control and regulation of atmospheric conditions (e.g., ventilation, humidity, and levels of carbon dioxide) for catalyst, catalytic modular partition or physical structure. Conditions such as temperature, atmospheric composition (e.g., relative humidity, levels of O2and CO2), physical stress, daylight, chemical stress, radiation, stress caused by lack of water, growth regulators and damage by pathogens play an important role in breathing intensity and significantly affect the shelf life of fruits, vegetables, flowers and other plant products. Although temperature and atmospheric storage conditions vary depending on the specific fruit, vegetable or other important product of vegetable origin, recommended storage temperatures usually lie in the range from approximately 0° to approximately 20°C at levels of O2and CO2lying in the approximate ranges of 1-10% and 0-20%, respectively. Regarding the additional humidity from about 50% to about 100%, specifically, from 85% to about 95%, more specifically, from about 90% to about 95%, usually recommended for storage of fruits, vegetables and similar products of vegetable origin. Given the significant correlation between the intensity of respiration and shelf life of foods of plant origin, control of the above factors are important to delay the destruction of such products. Accordingly, the device may be a sink of carbon dioxide to reduce the content of carbon dioxide.

In specific embodiments of the present invention provides a device with a breathable catalyst to delay the process of development of plants, including several layers. For example, Figure 1 shows the catalytic device 10, which may include outer layers 12 and 14 and intermediate catalytic layer 16 located between the outer layers 12 and 14. The catalytic layer 16 includes one or more bacteria (e.g., species Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and mixtures thereof) or enzymes (nitrilimines, amidase, asparaginase, and their mixtures), the one or more bacteria or enzymes are provided in a quantity sufficient to delay the development of interesting plants, and the third layer. In this implementation, the Dean or more of the outer layers 12 and 14 provides structural integrity of the catalytic device 10. The outer layers 12 and 14 normally allow air to penetrate to the catalytic layer 16, although, in some implementations, it may be useful to have an outer layer that is not permeable to air, for example, if the device forms a wall of the box and there is a requirement not to allow the outer layer of the box to expose the catalytic layer of the environment. The catalytic device 10 may be provided in the form of packets or bags, allow or not reusable, in accordance with the present invention. In one implementation, the catalytic layer 16 includes cells of Rhodococcus species, specifically, the strain DAP 96253 Rhodococcus rhodochrous strain DAP 96622 species Rhodococcus, Rhodococcus erythropolis, or mixtures thereof. Bacterial cells used as catalyst in the device according to the present invention, it is possible to induce one or more stimulating agents (e.g., asparagine, glutamine, cobalt, urea, or mixtures thereof)that described in detail above.

In Figures 2A-2C depicts an alternative device delays the process of plant development, in accordance with the present invention. These devices include multiple layers, one or more layers are removed. Figure 2A shows a device that includes a breathable structural SL is th 22 and the catalytic layer 24. Remove layers 26 and/or 28 may be located along the structural layer 22 and/or the catalytic layer 24, and, as a rule, they should be removed before use or activation of the catalyst. In some embodiments of the present invention, removal of the removed layers 26 and 28 opens the adhesive material, which facilitates the placing or attaching the catalytic structure to a separate physical structure. Figure 2B shows an alternative implementation in which the device 30 includes two air-permeable structural layer 32 and 34, intermediate catalytic layer 36 and remove the layer 38. Figure 2C depicts another embodiment in which the device 40 includes two air-permeable structural layer 42 and 44, intermediate catalytic layer 46 and two deleted layer 48 and 50.

In Figures 3A-3B depict an alternative embodiment 60, in which the catalyst is attached to the inner side of the container such as a cardboard box. Figure 3A shows the side 62 of the container, which includes a catalytic layer 64 attached thereto using adhesive layer 66. Peelable film 68 can be made adjacent to the catalytic layer 64 to protect the catalytic layer from exposure to the environment. Peelable film 68 can be removed to activate the catalyst in analiticheskoy layer 64, exposing the catalyst part of the plant located in the container, to thereby delay the undesirable process of plant development.

Figure 3B depicts the catalytic structure 70 before attaching the catalytic structure to the inner side of the container, as shown in Figure 3A. In addition to the catalytic layer 64, the layer of adhesive 66 and peelable film 68, the catalytic structure 70 includes additional peelable film 72. Peelable film 72, like the peelable film 68, protects the catalytic structure 70 during packaging, transportation or storage. Peelable film 72 can be removed to open the layer of adhesive 66 to provide attachment of the catalytic structure 70 to the inner side of the container by the method depicted in Figure 3A.

The Figure 4 shows the catalytic structure 80, which includes two slots 82 and 84 for receiving a catalytic cartridge (for example, cassette 86). Catalytic cartridge 86 is breathable, and it can be easily inserted or removed from the slot 84. Thus, the catalytic cartridge 86 can be easily replaced, if needed a new catalytic cartridge for use in catalytic structure 80. Catalytic cartridge 86 includes a catalyst, such as described in this application, and which is preferred is entrusted immobilized on the matrix. The catalytic structure 80 may include being opposite each other permeable surfaces 88 and 90, such as lattice partitions, to allow air to pass through the catalytic cartridge 86. The catalytic structure 80, in alternative implementations may include only one air-permeable surface, the two are not opposite each other permeable surface or more than two air-permeable surfaces, which should be obvious to a person skilled in this field. Although Figure 4 shows the catalytic structure comprising two slots 82 and 84 for receiving a catalytic cartridge (for example, cassette 86), for a specialist in this field should be obvious that the catalytic structure 80 may include one or more slots for receiving the cartridge. The catalytic structure 80 may be provided inside the container used to transport parts of plants, such as fruit or flowers, or can be attached to the container, for example, using a layer of adhesive substance described in this application.

The methods and devices according to the present invention can be used to delay the development process of any interesting plants or parts of plants. In specific embodiments of, methods and devices according to the present image the structure aims to delay ripening, and part of the plant is a fruit (climacteric menopausal or not), vegetable or other plant part that is subjected to maturation. For professionals in this field should be obvious that the “climacteric fruit” are characterized by a sudden burst of formation of ethylene during ripening, whereas it is believed that “climacterics fruit” uncharacteristically significant increase of ethylene biosynthesis during ripening process. Typical fruits, vegetables and other interesting products of plant origin include, but are not limited to the above: apples, apricots, biriba, breadfruit, cherimoya, feijoa, figs, guava, jackfruit, kiwi, bananas, peaches, avocados, apples, cantaloupe, mangoes, cantaloupes, nectarines, persimmons, sapota, anon needle, olives, papaya, passion fruit, pears, plums, tomatoes, peppers, blueberries, cocoa beans, cashew nuts, cucumber, grapefruit, lemons, limes, peppers, cherries, oranges, grapes, pineapple, strawberries, watermelons, tamarillo and nuts.

In other embodiments of the present invention, methods and devices directed to delay wilting flower, topple, falling or collapse of the petals. This invention is applicable to any flower. Typical interesting colors include, but are not limited to the above: roses, carnations, orchids, Portola is, mallow and begonias. Cut flowers, more specifically, cut flowers, important from a commercial point of view, such as roses and carnations, are of particular interest. In some embodiments of the invention use the flowers that are sensitive to ethylene. Ethylene-sensitive flowers include, but are not limited to the above: flowers delivery Alstroemeria, Aneomone, Anthurium, Antirrhinum, Aster, Astilbe, Cattleya. Cymbidium, Dahlia, Dendrobium, Dianthus, Eustoma, Freesia, Gerbera, Gypsophila, Iris, Lathyrus, Lilium, Limonium, Nerine, Rosa, Syringa, Tulipa, and Zinnia. Typical ethylene-sensitive flowers also include flowers from families Amaryllises, Onion, Landyshovich, Hemerocallis, Giacintova, Liliaceae, Orchidaceae, the), Cactus, Bell, Clove, Weariness, Gorchakova, Malvaceae, Plumbago, Portulacaceae, Solanaceae, Aganovich, Asphodeline, Asparagus, Begonia, Honeysuckle, Vorsanova, Euphorbiaceae, Legumes, Cereals, Myrtle, Cipriani, Rosales and Verbena. See, for example, Van Doorn (2002) Annals of Botany 89:375-383; Van Doorn (2002) Annals of Botany 89:689-693; and Elgar (1998) “Cut Flowers and Foliage - Cooling Requirements and Temperature Management” hortnet.co.nz/publications/hortfacts/hf305004.htm (date last accessed: March 20, 2007), each of which is fully incorporated into this application by reference. Methods and devices for delaying the falling leaves also included in the scope of the present invention. In the industrial plant, the grower is TBE, horticulture and floriculture there is considerable commercial interest to methods and devices for regulating processes of plant development, such as ripening, wilting and falling.

In addition, for the person skilled in the art it is obvious that any of the methods or devices described in this application can be combined with other known methods and devices for delaying the process of development of plants, particularly those processes that are typically associated with increased biosynthesis of ethylene (for example, ripening of fruits/vegetables, fading colors and falling leaves). Moreover, as described above, increased the formation of ethylene was also observed in case of damage by pathogenic organisms of plants or parts of plants. Accordingly, the methods and devices according to the present invention can be further used to improve the response of plants to pathogens.

The following examples are offered to illustrate but not to limit:

EXPERIMENTAL PART

The present invention is described with specific reference to various examples. The following examples are not intended to limit the present invention and, rather, intended as typical implementation options.

Example 1: Delayed ripening after exposure induced species Rhodococcus

<> Cells of Rhodococcus species, induced asparagine, Acrylonitrile or acetonitrile, were immobilized on the matrix of DEAE-cellulose, cross-connected by means of glutaraldehyde. Methods of inducing cells and the receipt of the aforementioned matrix is described in more detail in this application below.

The catalytic matrix of the connected cross-DEAE-cellulose were placed in three separate paper bag (approximately 1-2 grams of compacted wet weight of cells per package, each package contained unripe bananas, peaches or avocado. As negative controls, the same fruit were placed in separate paper bags in the absence of catalytic matrix. Paper bags were kept at room temperature, and the products were examined daily for signs of maturation and destruction of the fruit.

All products are subjected to a catalytic matrix, showed a significant delay in the ripening of fruit. In particular, the rigidity and integrity of the peel peaches remained longer in the presence of catalytic matrix. Similarly, for bananas, the appearance of brown spots, was delayed, and the rigidity was maintained longer in comparison with negative control.

Example 2: General methods of fermentation and induction

The fermentation process

Following the traditional met the Dickey and culture medium were used for fermentation of Rhodococcus species: strains DAP 96622 species of Rhodococcus and DAP 96523 Rhodococcus rhodochrous for use in other experiments.

Fermentation tanks were equipped with contact sensors for measuring dissolved oxygen (DO) and pH, as well as samplers for measuring the concentration of glucose (offline). Additional inlets used to adjust (e.g., acid, base or antifoam), making inductors, nutrients and additives. Pre-cleaned jars were sterilized in place. Used a suitable basic medium (1 or 1.5X) R2A or R3A. Specific components of these cultural environments below. In some experiments, the medium composition was changed. For example, Proflo®(Trader's Protein, Memphis, Tennessee) is sometimes used instead of proteinopathy and/or Kazimirovich acids. Moreover, in some experiments, Hy-Cotton 7803®(Quest International, the estate of Hoffman, Illinois), Cottonseed Hydrolysate (hydrolyzed seed cotton, Cottonseed Hydrolysate-Ultrafiltered (hydrolyzed cottonseed subjected to ultrafiltration) (Marcor Devolpment Corp., Karlstad, new Jersey) was used in place Proflo®(Trader's Protein, Memphis, Tennessee).

Profile recharge to add nutrients was installed so that gradually replace the basic environment R2A or R3A on a nutrient medium, namely 2X YEMEA, the components of which are also described in more detail below. Other possible additives nutrients included Malte the zu 50% (weight/volume) and dextrose 50% (weight/volume). Commercial products containing the equivalent of dextrose (glucose, maltose and higher polysaccharides), sometimes used instead of maltose and dextrose.

The inoculum (crops) were obtained from cultures of strains DAP 96622 species of Rhodococcus and DAP 96523 Rhodococcus rhodochrous on a suitable solid medium and incubated at a suitable temperature (e.g. 30°C). In specific implementations, the cells were grown on plates with agar medium YEMEA within 4-14 days, preferably 7 days. Alternatively, the inoculum was obtained from frozen concentrate cells from previous cycles of fermentation. The concentrated cells were usually received in concentrations exceeding 20 times present in the fermenter concentration. In addition, the inoculum was sometimes obtained from a suitable two-phase medium (i.e., the combination of a liquid medium, covering the solid medium with the same or different composition). When used two-phase environment, the environment, usually contained YEMEA both in liquid and in solid layers.

For induction nitrilimines, at t=0 hours, sterile CoCl2·6H2O and urea were added to achieve concentrations equal 5-200 ppm CoCl2and 750 mg/l - 10 g/l of urea, preferably, generally 10-50 ppm CoCl2and 7500 mg/l 7.5 g/l of urea. In a specific implementation, urea and/or cobalt was again added and twisting during the fermentation. For example, an equivalent amount of urea and 150 ppm CoCl2added after 4-6 hours 24-30 hours. In addition to urea, was added Acrylonitrile/acetonitrile to a final concentration of 300 to 500 ppm or 0.1 M-0.2 M asparagine, incrementally or at constant speed, starting at different times. Cycles of fermentation was stopped when the cell mass and concentrations of enzymes were suitable, usually through another 24-96 hours.

The cells were then collected by any suitable means, including, but not limited to the above: periodic or continuous centrifugation, decanting or filtering. The collected cells resuspendable up to 20X concentrated volume in a suitable buffer such as 50 mm phosphate saline buffer solution (FBI), supplemented with the inducer used during the fermentation process. The concentrated cells were then frozen, more specifically, by rapid freezing. The frozen cells were stored at -20°C -80°C or in liquid nitrogen for further use.

Description of cultural media

The R2A medium (see Reasoner and Geldreich (1985) Appl. Environ. Environ. 49:1-7)

Yeast extract 0.5 g

Protectorate #3 0.5 g

Kasuminome acid 0.5 g

Glucose 0.5 g

Soluble starch 0.5 g

K2HPO40.3 g

MgSO4.7H2O 0.05 g

Pyruvate sodium 0.3 g

Deionizer. or remote. H2O 1.0 liter

Wednesday R3A (see Reasoner and Geldreich, above.)

Yeast extract 1.0 g

Protectorate #3 1.0 g

Kasuminome acid 1.0 g

Glucose 1.0 g

Soluble starch 1.0 g

K2HPO40.6 g

MgSO4.7H2O 0.1 g

The sodium pyruvate, 0.5 g

Deionizer. or remote. H2O 1.0 liter

Wednesday YEMEA

1X 2X

Yeast extract 4.0 g 8.0 g

The malt extract 10.0 g 20,0 g

Glucose 4.0 g 8.0 g

Deionizer. or remote. H2O 1.0 liter1.0 liter.

Induction

Following the traditional method used for induction of Rhodococcus species: strains DAP 96622 species of Rhodococcus and DAP 96523 Rhodococcus rhodochrous.

Volatile inducing fluid (for example, Acrylonitrile/acetonitrile) was added to the volumetric in the form of sterilized by filtering the liquid inductors, depending on the specific density of the liquid inductor. In the case of solid inducers (e.g., asparagine/glutamine), solids were weighed and added directly to the culture medium. The resulting environment autoclaved. When used sterilized by filtering the liquid inductors, culture medium separately autoclaved and cooled to 40°C before adding liquid inductor. Typical concentrations are interested inductors were:500 ppm Acrylonitrile/acetonitrile; 500 ppm asparagine/glutamine; and 50 ppm of succinonitrile. The cells were then grown in these environments and additionally analyzed for specific enzyme activity and biomass.

Example 3: Analysis nitrilgidrataznoi, amidases and asparaginase activity and biomass in induced asparagine cells of the species Rhodococcus

Nitrilimines, imidazol and asparaginase activity and biomass were assessed in induced asparagine cells of Rhodococcus species: strains DAP 96622 species of Rhodococcus and DAP 96523 Rhodococcus rhodochrous. Analyzed various components of culture media, techniques, velocities and concentrations of asparagine, which is supplied to the cells, and cell source in relation to their impact on the activity of the above enzymes and biomass. In sections A through G in this example, the described features of each set of test conditions and provides a brief description of enzyme activity and biomass obtained when each of the specific conditions.

A. essentially as described above in Example 2, 20-liter fermenter, inoculated with cell DAP 96253 Rhodococcus rhodochrous collected from a solid medium, was continuously applied to the inductor asparagine (120 µl/min 0.2 M solution). Hy-Cotton 7803® was used in place of proteinopathy #3 in the environment R3A described above. By the end of the fermentation cycle, Acrylonitrile-specification is ing nitrilimines activity imidazol activity and biomass was measured in accordance with standard techniques known in this field.

Results for nitrilgidrataznoi activity, amidase activity and biomass are presented below in Table 3, when this activity is provided in units/mg SVK (dry weight of cells). One nitrilgidrataznoi activity refers to the ability to convert 1 µmol of Acrylonitrile in the corresponding amide in a minute, milligram of cells (dry weight) at pH 7.0 and a temperature of 30ºC. One amidase activity refers to the ability to convert 1 µmol of acrylamide in the appropriate acid per minute at milligram of cells (dry weight) at pH 7.0 and a temperature of 30ºC. Biomass is presented in the form of cells, measured in g/l QMS (wet weight of cells).

Table 3
Enzyme activity and biomass of cells DAP 96523 Rhodococcus rhodochrous after induction by asparagine
Nitrilgidrataznoi activity
(units/mg SVK)
Amidala activity (units/mg SVK)Biomass
(g/l QMS)
168236

B. Evaluated the enzyme activity and biomass of cells DAP 96523 Rhoococcus rhodochrous essentially as described above in Example 3A, with changes in the composition of the medium, noted below. In particular, YEMEA, dextrose or maltose was added to a modified environment R3A, optionally containing Hy-Cotton 7803®, is used instead of proteinopathy #3. 0.2 M solution of asparagine was added at a constant flow rate of 120 µl/min, starting at time t=8 hours. By the end of the fermentation cycle, measured Acrylonitrile-specific nitrilimines activity, imidazol activity and biomass. The results are presented in Table 4. Increased biomass yield was observed when added to the environment YEMEA, dextrose or maltose.

Table 4
Enzyme activity and biomass of cells DAP 96523 Rhodococcus rhodochrous after continuous induction-asparagine
Nitrilgidrataznoi activity (units/mg SVK)Amidala activity (units/mg SVK)Biomass
(g/l QMS)
155652

C. Cells DAP 96622 species Rhodococcus of solid media was used as a source of inoculum for cycle 20 liter fermentation (see Example 2 for a more detailed description of the fermentation process). 0,2 the solution of asparagine was added semi-continuous every 6 hours, starting at time t=24 hours, within 50-70 minutes at a flow rate of 2 ml/minute. Hy-Cotton 7803® was used in place of proteinopathy #3 in a modified environment R3A. By the end of the fermentation cycle, measured Acrylonitrile-specific nitrilimines activity, imidazol activity and biomass. The results are shown in Table 5.

Table 5
Enzyme activity and biomass of cells DAP 96622 species Rhodococcus after semi-continuous induction-asparagine
Nitrilgidrataznoi activity (units/mg SVK)Amidala activity (units/mg SVK)Biomass
(g/l QMS)
172244

D. Cells DAP 96622 species Rhodococcus of solid media was used as a source of inoculum for cycle 20-liter fermentation. 0.2 M solution of asparagine was added semi-continuous every 6 hours, starting at time t=12 hours 12-85 minutes at a flow rate of 2.5 ml/minute. Cottonseed Hydrolysate was used in place of proteinopathy #3 in a modified environment R3A. By the end of the fermentation cycle, measured Acrylonitrile-specific nitrilimines activity, imidazol AK is Yunosti and biomass, and the results are presented in Table 6.

Table 6
Enzyme activity and biomass of cells DAP 96622 species Rhodococcus after semi-continuous induction-asparagine
Nitrilgidrataznoi activity (units/mg SVK)Amidala activity (units/mg SVK)Biomass
(g/l QMS)
165257

E. Pre-frozen cells DAP 96253 Rhodococcus rhodochrous was used as a source of inoculum for cycle 20-liter fermentation. YEMEA, dextrose or maltose was added to a modified environment R3A, which additionally contained Hy-Cotton 7803® as a substitute proteinopathy #3. 0.15 M solution of asparagine was added at a constant flow rate of 120 µl/min, starting at time t=8 hours. By the end of the fermentation cycle, measured Acrylonitrile-specific nitrilimines activity, imidazol activity and biomass. The results are presented in Table 7.

Table 7
Enzyme activity and biomass of cells DAP 96523 Rhodococcus rhodochrous after continuous induction Aspar what Gino
Nitrilgidrataznoi activity (units/mg SVK)Amidala activity (units/mg SVK)Biomass
(g/l QMS)
171474

F. Cells DAP 96253 Rhodococcus rhodochrous grown on two-phase environment, was used as a source of inoculum for cycle 20-liter fermentation. Used a modified environment R3A, which was supplemented by the addition of carbohydrate (i.e. YEMEA, dextrose or maltose) and additionally contained Cottonseed Hydrolysate instead of proteinopathy #3. 0.15 M solution of asparagine was added at a constant flow rate equal to 1000 ál/min, starting at time t=10 hours. By the end of the fermentation cycle, measured Acrylonitrile-specific nitrilimines activity, imidazol activity, the activity of asparaginase I, and biomass. The results are shown in Table 8.

Table 8
Enzyme activity and biomass of cells DAP 96523 Rhodococcus rhodochrous after continuous induction-asparagine
Nitrilgidrataznoi activity (units/mg SVK)Amidala activity (units/mg SVK)AK is Yunosti asparaginase I (units/mg SVK) Biomass
(g/l QMS)
159221616

G. Cells DAP 96253 Rhodococcus rhodochrous grown on two-phase environment, was used as a source of inoculum for cycle 20-liter fermentation. Used a modified environment R3A, which contained maltose (instead of dextrose) and Hy-Cotton 7803® as a substitute proteinopathy #3. 0.15 M solution of asparagine was added at a constant flow rate equal to 476 μl/min, starting at time t=8 hours. By the end of the fermentation cycle, measured Acrylonitrile-specific nitrilimines activity, imidazol activity and biomass, and the results are shown in Table 9.

Table 9
Enzyme activity and biomass of cells DAP 96523 Rhodococcus rhodochrous after continuous induction-asparagine
Nitrilgidrataznoi activity
(units/mg SVK)
Amidala activity (units/mg SVK)Biomass
(g/l QMS)
137635

Example 4: Immobilization of cells of Rhodococcus species on DEAE-cellulose, the knitted cross links via glutaraldehyde

The modified process, obtained from the methods described in U.S. patent number 4,229,536 and Lopez-Gallego and others (2005) J. Biotechnol. 119:70-75, was used for cell immobilization of Rhodococcus species in the matrix, including DEAE-cellulose, cross-linked by cross-linking via glutaraldehyde.

Obtaining cells

Cells of Rhodococcus were grown in a suitable culture medium (e.g., YEMEA-maltose + inductors, two-phase culture, etc) and separated by centrifugation at 8,000 rpm for 10 minutes. The precipitate cells resuspendable in 100 ml of 50 mm phosphate buffer (pH of 7.2) and centrifuged at 8,000 rpm for 10 minutes. This process resuspendable sediment cells and centrifugation at 8,000 rpm for 10 minutes was repeated twice. Recorded compacted wet mass (cm) of the final sample of cells. Performed assessment nitrilgidrataznoi activity of a small sample of cells to assess the enzymatic activity of whole cells.

Immobilization of cells

Received number of DEAE-cellulose equivalent to that of the collected cells of Rhodococcus species, and cells, and DEAE-cellulose resuspendable in 100 ml of deionized H2O. the Amount of 25% solution of glutaraldehyde sufficient to achieve a final concentration of 0.5%, was added with stirring to the mixture of cells/DEAE-cellulose. The mixture was stirred for 1 hour, on the East the treatment which was added 400 ml of deionized H 2O with additional stirring. With stirring, was added 50% (by weight solution) polyethylenimine (PEI; molecular weight 750,000). Stirring is continued until, until it was ended by flocculation. Flocculated mixture was filtered and punching through the syringe of appropriate size. Extruded from a syringe mass of immobilized cells was divided into small pieces, dried overnight, and crushed to obtain granules with a size of approximately 2-3 mm

Example 5: Immobilization of cells of Rhodococcus species on the alginate calcium and hardening of the granules of calcium alginate

Process, adapted from the method described by Bucke (1987) “Cell Immobilization in Calcium Alginate” in Methods in Enzymology, volume 135(B) (Academic Press, Inc., San Diego, California; Mosbach, as amended), was used for immobilization of cells of the species Rhodococcus in calcium alginate.

Obtaining cells

Cells of Rhodococcus species were obtained as described above in Example 4.

Immobilization of cells

25 g of 4% solution of sodium alginate was obtained by dissolving 1 g of sodium alginate in 24 ml of 50 mm Tris-HCl (pH of 7.2). 25 mg metaperiodate sodium was added to the alginate solution and stirred at 25°C for 1 hour or until until the alginate is not completely dissolved. Cells obtained by the method described above, resuspendable to a final volume of 50 ml in 50 mm Tris-HCl (pH of 7.2), and then added to a solution of sodium alginate when PE is emisiuni. The obtained granules were passed through a 27G needle diameter in 500 ml of 0.1 M solution of CaCl2. The needle typically placed approximately two inches above the solution in order to prevent penetration of air into pellets and to prevent sticking of the granules. The pellets were treated for 1 hour in a solution of CaCl2and then the granules were washed with water and kept at 4°C in 0.1 M solution of CaCl2before applying.

Making the hardness of the granules of calcium alginate, including cells of Rhodococcus species

Granules of calcium alginate, obtained as described above, can be further strengthened by the formation of cross-links with PEI. The pellets were incubated in 2 l of 0.5% PEI in 0.1 M solution of CaCl2(20 g 50% PEI in 0.1 M solution of CaCl2). the pH of the final solution was brought to 7.0 with HCl or NaOH, if necessary, and the pellets were incubated for 24 hours. The granules are then washed with water and kept at 4°C in 0.1 M solution of CaCl2before applying.

Many modifications and other options to implement the inventions described in this application will come in the head of the person skilled in the art to which the present invention belongs to the useful ideas presented in the foregoing descriptions. Therefore, it should be apparent that the present invention should not be limited to the specific described variants of implementation and that modifications and other is e implementations are assumed to be included in the scope of the attached claims. Although in this application are used in concrete terms, they apply only in a generic and descriptive sense, but not to limit.

1. The method of delays in the development process plants associated with the biosynthesis of ethylene, including the impact on the plant or plant part of one or more bacteria, characterized in that one or more bacteria produce one or more enzymes selected from the group consisting of nitrilimines, amides, asparagines and mixtures thereof, and the specified one or more bacteria selected from the group consisting of the species Rhodococcus, Brevibacterium ketoglutamicum, and mixtures thereof, and the specified one or more bacteria affect plant or plant part in a quantity sufficient to delay the process of development of plants.

2. The method according to claim 1, characterized in that the specified one or more bacteria include species of Rhodococcus.

3. The method according to claim 2, characterized in that these species include Rhodococcus strain Rhodococcus rhodochrous ATCC 55899, the strain of the species Rhodococcus ATCC 55898, Rhodococcus erythropolis, or mixtures thereof.

4. The method according to claim 1, characterized in that the specified one or more bacteria induce the influence of a stimulating agent selected from the group consisting of asparagine, glutamine, cobalt, urea and mixtures thereof.

5. The method according to claim 4, characterized in that the specified one or more bacteria induce ondastan asparagine.

6. The method according to claim 4, characterized in that the specified one or more bacteria induce the influence of asparagine, cobalt and urea.

7. The method according to claim 1, characterized in that the specified plant or part of plant is subjected to the indirect action of one or more bacteria.

8. The method according to claim 1, characterized in that the specified plant or part of plant is subjected to the direct action of one or more bacteria.

9. The method according to claim 1, characterized in that the process of plant development is the maturation of fruits or vegetables.

10. The method according to claim 9, characterized in that the specified part of the plant is a fruit or a vegetable.

11. The method according to claim 10, characterized in that the fetus is a climacteric fruit.

12. The method according to claim 11, characterized in that the specified climacteric fruit selected from the group consisting of bananas, peaches, plums, nectarines, apples, tomatoes, pears and avocados.

13. The method according to claim 10, characterized in that the fetus is climactically fruit.

14. The method according to claim 9, characterized in that said plant is a cucumber.

15. The method according to claim 1, characterized in that the specified part of the plant is the flower and the process of plant development is a fading flower, topple, dropping petals Il the folding of the petals.

16. The method according to item 15, characterized in that the flower is a carnation, rose, Orchid, purslane, mallow or begonia.

17. The method according to claim 1, characterized in that the process of plant development is the falling leaves.

18. The method according to claim 1, characterized in that the specified part of the plant is a cut flower.

19. The method according to claim 1, characterized in that the specified one or more bacteria immobilized and placed in the physical structure, placed on the physical structure or attached to the physical structure, suitable for transportation or storage plant or plant part.

20. Device for delaying the development of plants associated with the biosynthesis of ethylene, comprising multiple layers, wherein at least one layer contains a catalyst which contains one or more bacteria selected from the group consisting of the species Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and mixtures thereof, and one or more bacteria produce one or more enzymes selected from the group consisting of nitrilimines, amides, asparagines and mixtures thereof, and the catalyst may be placed in physical structure, placed on the physical structure, or attached to the physical structure, such as film, sheet, covering the layer box, bag, package, or segmentina these Luggage, and the specified one or more bacteria is contained in a quantity sufficient to delay the process of development of plants.

21. The device according to claim 20, characterized in that the specified one or more bacteria include species of Rhodococcus.

22. The device according to item 21, wherein the types include Rhodococcus strain Rhodococcus rhodochrous ATCC 55899, the strain of the species Rhodococcus ADS 55898, Rhodococcus erythropolis, or mixtures thereof.

23. The device according to claim 20, characterized in that the specified one or more bacteria induce the influence of a stimulating agent selected from the group consisting of asparagine, glutamine, cobalt, urea, and mixtures thereof.

24. The device according to claim 20, characterized in that the process of development of plants selected from the group consisting of ripening of fruits or vegetables, fading colors, topple, folding petals and leaves falling.

25. The device according to claim 20, characterized in that the specified one or more bacteria immobilized on a matrix comprising cross-linked bonds DEAE-cellulose, the matrix comprising alginate, matrix, including carrageenan, the matrix comprising cross-linked bonds alginate, the matrix comprising cross-linked bonds carrageenan, matrix, including polyacrylamide, or granules of calcium alginate.

26. The device according A.25, characterized in that the said matrix comprises connected the second transverse relationship DEAE-cellulose, while DEAE-cellulose cross-linked bonds using glutaraldehyde.

27. The device according to claim 20, characterized in that the catalyst is placed in catalytic module.

28. The device according to claim 20, further comprising adjustment means for regulating the exposure of the catalyst to the plant or plant part.

29. The device according to claim 20, further comprising monitoring means for monitoring catalyst efficiency is to delay the process of development of plants.

30. The device according to item 27, characterized in that the catalytic module is removable and replaceable with a second catalytic module.

31. The device according to item 27, wherein more than one catalytic module is placed in physical structure, placed on the physical structure or attached to the physical structure.

32. The device according to item 27, wherein the specified physical structure allows air to penetrate into the catalytic module.

33. The device according to p, further comprising an element for controlling the flow of air to the catalytic module.

34. The device according to item 27, wherein the specified physical structure provided in the form of a cooled structure.

35. The device according to item 27, further comprising an element for regulating the moisture content in the physical is the structure.

36. The device according to item 27, further comprising an element for regulating the level of carbon dioxide in the physical structure.

37. The device according to claim 20, characterized in that it is a breathable catalytic device for delaying the process of plant development, including:
the first layer; and
a second layer that includes a catalyst containing one or more bacteria selected from the group consisting of the species Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and mixtures thereof, and one or more bacteria produce one or more enzymes selected from the group consisting of nitrilimines, amides, asparagines and mixtures thereof, and one or more bacteria is provided in a quantity sufficient to delay the process of plant development;
the first layer provides structural integrity to the device.

38. The device according to clause 37, wherein the specified one or more bacteria include species of Rhodococcus.

39. The device according to clause 37, wherein the specified types include Rhodococcus strain Rhodococcus rhodochrous ATCC 55899, the strain of the species Rhodococcus ATCC 55898, Rhodococcus erythropolis, or mixtures thereof.

40. The device according to clause 37, wherein the specified one or more bacteria induce the influence of a stimulating agent selected from the group consisting of asparagine, glutamine, cobalt, urea, and mixtures thereof.

41. The device p is clause 37, characterized in that the process of development of plants selected from the group consisting of ripening of fruits or vegetables, the withering of the flower and the leaves falling.

42. The catalytic device according to clause 37, further comprising a third layer, characterized in that the third layer can be removed from the specified second layer to open the layer of sticky substance that can be used to attach the catalytic device to a separate structure.

43. The catalytic device according to § 42, characterized in that the second layer represents the layer of sticky substance.

44. The catalytic device according to § 42, further comprising a fourth layer adjacent to the specified third layer, which can be removed from the specified third layer to open the layer of sticky substance that can be used to attach the catalytic structure to separate the structure.

45. The catalytic device according to item 44, characterized in that the third layer represents the layer of sticky substance.

46. Breathable package or bag for delays in the development process plants associated with the biosynthesis of ethylene, comprising a catalytic device according to claim 20.

47. The method of delays in the development process plants associated with the biosynthesis of ethylene, including the impact on the RA shall buy or part of the plant enzyme extract from one or more bacteria, selected from the group consisting of the species Rhodococcus, Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and mixtures thereof, and one or more bacteria produce one or more enzymes selected from the group consisting of nitrilimines, amides, asparagines and their mixtures, and these bacteria induce a stimulating agent selected from the group consisting of asparagine, glutamine, cobalt, urea, and mixtures thereof, and the plant or plant part is subjected to the action of the specified enzyme extract in a quantity sufficient to delay the process of development of plants.

48. The method of delays in the development process plants associated with the biosynthesis of ethylene, including the impact on the plant or plant part of one or more bacteria, characterized in that one or more bacteria produce one or more enzymes selected from the group consisting of nitrilimines, amides, asparagines and mixtures, and one or more bacteria are Pseudomonas chloroaphis, which were induced by exposure to a stimulating agent selected from the group consisting of asparagine, glutamine, cobalt, urea, and mixtures thereof, and the specified one or more bacteria affect plant or part plants in a quantity sufficient to delay the process of development of plants.

49. The method according to p, wherein the specified one or blueberi induce the influence of asparagine, or asparagine, cobalt and urea.

50. The method according to p, characterized in that the specified plant or part of plant is subjected to direct or indirect action of one or more bacteria.

51. The method according to p, characterized in that the process of plant development is the maturation of fruits or vegetables.

52. The method according to p, characterized in that the specified part of the plant is a fruit, vegetable or flower.

53. The method according to paragraph 52, characterized in that the fetus is climactically or climacteric fruit.

54. The method according to item 53, wherein the specified climacteric fruit selected from the group consisting of bananas, pears, plums, nectarines, apples, tomatoes, peaches and avocado.

55. The method according to p, characterized in that the specified part of the plant is the flower and the process of plant development is a fading flower, topple, dropping petals or folding of the petals.

56. The method according to § 55, characterized in that the flower is a carnation, rose, Orchid, purslane, mallow or begonia.

57. The method according to p, characterized in that the process of plant development is the falling leaves.

58. The method according to p, characterized in that the specified part of the plant is a cut off the branches.

59. The method according to p, characterized in that the specified one or more bacteria immobilized and placed in the physical structure, placed on the physical structure or attached to the physical structure, suitable for transportation or storage of plants or parts of plants.



 

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10 ex

FIELD: medicine, pharmaceutics.

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7 cl, 9 dwg, 12 tbl, 10 ex

FIELD: agriculture.

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1 tbl

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1 ex

FIELD: agriculture.

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4 tbl, 1 ex

FIELD: medicine, pharmaceutics.

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7 cl, 9 dwg, 12 tbl, 10 ex

FIELD: chemistry.

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4 ex

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SUBSTANCE: method of plant cultivation comprises includes treatment of plant seeds prior to their sowing and of plant in the growing season with a biological product in liquid form. As a biological product, a mixture of strains of bacteria of B. subtilis of RNCIM B-10641, B. amyloliquefaciens RNCIM B-10642 and B. amyloliquefaciens RNCIM B-10643 is used, with a titer of not less than 106 CFU/ml in the form of an aqueous suspension.

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3 cl, 4 dwg, 5 tbl, 9 ex

FIELD: chemistry.

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

2 ex

FIELD: agriculture.

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EFFECT: biological preparation enables to protect plants from fungal and bacterial diseases, to improve the phytosanitary condition of soil and improve its fertility, to increase crop yields and improve the quality of agricultural products.

5 tbl, 8 ex

FIELD: agriculture.

SUBSTANCE: method to stimulate growth and to protect small-fruit crops against diseases caused fungic pathogens includes treatment of plants with a biopreparation in a liquid form by soaking roots of seedlings prior to planting, or their sprinkling after planting, or sprinkling of plants in the period of vegetation and fruiting, or sprinkling of soil around plants with its further soaking with water. The biopreparation is a mixture of strains of bacteria B. subtilis VKPM B-10641, B. amyloliquefaciens VLPM B-10642, B. licheniformis VKPM B-10561 and B. licheniformis VKPM B-10562 or a mixture of strains of bacteria B. subtilis VKPM B-10641, B. amyloliquefaciens VKPM B-10642 and B. licheniformis VKPM B-10562 with a titre of each strain of not less than 105 CFU/ml in the form of a water suspension.

EFFECT: invention provides for higher protection of small-fruit crops against diseases caused by fungic pathogens, due to use of a mixture of strains of different types of bacteria of Bacillus type, having higher antagonistic activity, and also provision of the possibility of stimulation of growth of small-fruit crops.

5 cl, 10 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: strain Trichoderma harzianum Rifai, having L-lysine-alpha-oxidase activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number RNCIM F-180 and may be used in agricultural biotechnology and plant growing.

EFFECT: invention makes it possible to reduce losses of decorative and vegetable crops.

2 ex

Antibiotic peptides // 2472805

FIELD: chemistry.

SUBSTANCE: peptides and peptide derivatives have general formula Sub1-X1NX2X3PVYIPX4X5RPPHP-Sub2, where Sub1, X1, X2, X3, X5, Sub2 are given in the claim. The disclosed peptides or peptide derivatives have at least one of the following advantages compared to natural apidecine peptides: (i) longer half-life in mammal serum owing to higher protease stability, (ii) high antimicrobial activity with respect to one or more bacterial strains, particularly human pathogens or fungi, or other microbial infections, (iii) demonstrate a wide range of antimicrobial activity, (iv) cause slower development of resistance in microorganisms and (v) are not toxic for human cells, including erythrocytes.

EFFECT: improved properties of peptides.

21 cl, 8 dwg, 7 tbl, 6 ex

FIELD: agriculture.

SUBSTANCE: agent comprises salicylic acid, lipids from mycelium of fungus Ascichyta pinodes and the source of magnesium - magnesium sulfate MgSO4 or magnesium chloride MgCl2 at the following ratio of components: wt %: salicylic acid - 0.001, lipids from mycelium of fungus Ascichyta pinodes - 0.0001; the source of magnesium (magnesium sulfate MgSO4; or magnesium chloride MgCl2) - 0.001; water - 99.9979.

EFFECT: invention enables to increase the growth indices of pea, yield, disease resistance.

7 tbl, 7 ex

FIELD: plant production.

SUBSTANCE: method includes spraying of vegetative solanaceous plants with Steinermena feltiae suspension in combination as antidesiccant with agent obtained from biomass of Mortierella jenkinii micromycete according to claimed technology.

EFFECT: insect pest control with improved effect.

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