Fungi strain duddingtonia flagrans with activity against plant gall nematode and animal parasitic nematode, and stimulating of plant growth and development

FIELD: biotechnology.

SUBSTANCE: invention relates to nematophage fungi strain Duddingtonia flagrans useful as base in biological preparations producing. Strain Duddingtonia flagrans F-882 has activity against plant gall nematode and animal parasitic nematode, and stimulates plant growth and development.

EFFECT: strain with high plant growth and development stimulating activity and nematophage effect.

6 tbl, 2 dwg, 7 ex

 

The invention relates to the microbiological industry, and represents nematophagous strain of the fungus Duddingtonia flagrans, which can be used as a producer to obtain a biologics to combat parasitic nematodes of plants and animals.

Known strains nematophagous mushrooms: Arthrobotrys oligospora BKMF-2461D (USSR Author's certificate, IPC C 12 N 1/14, No. 1264393, publ. 1986), Arthrobotrys oligospora (U.S. patent No. 4975105, IPC 05 F 11/08, publ. 1990), showing high nematophagous effect. Preparative form to fight with root-knot nematodes were obtained on different organic substrates. Current start - conidia and mycelium of the fungus.

However, these strains are characterized by instability of morphological characteristics nematophagous properties. This is manifested in the emergence of sectors and spots in the colonies cultures on agar nutrient medium. Cytochromoxidase analysis detected the anastomosis between hyphae and the difference nuclei size and shape in multi-core basal cells of the conidiophores. Preparation on the basis of conidia can be stored for not more than 1 year.

Know of any other similar strains of Arthrobotrys oligospora 3062D (USSR Author's certificate No. 1688818, IPC a 01 N 63/00, publ. 1991), manifesting as compared with the above strain higher nematophagous effect and stability properties. Current top I is comprised of conidia and mycelium. This strain is the basis of the developed biological product “hematophagy-NL”, approved in 1990 by the Commission on microbiological remedies of agricultural Sciences, Soussaline, and recommended for implementation. “Hematophagy-BL” is included in the list of biological products, authorized for use on the territory of Russia.

However, strains of the genus Arthrobotrys form in terms of surface culture conidia and mycelium, which are not adapted for long-term storage, for use against parasitic infection of animals and need additional funds to protect conidia from exposure to the digestive processes of animals [Pryadko AI, Osipov Per substances as protection conidia predatory fungi from exposure to the digestive processes of animals. Mushrooms-hyphomycetes controls the number of parasitic nematodes. - Alma-ATA: Nauka, 1990. - S-112], and when the soil against phytoparasitic nematodes takes a long time (2-3 weeks) for the implementation of the adaptation process and the formation of chlamydospores and traps that provide predatory fungi exist in this ecological niche.

A more detailed study of the behavior of nematophagous fungi in soil conditions allowed us to establish that the conidia are not a form of existence of the fungus in the soil. Using direct microscopic examination and the method m is brunnich cameras installed, that predatory fungi of the genus Arthrobotrys exist in the soil as chlamydospores.

Part of the conidia and mycelium, soil, subjected to lysis, and some cells transformed into chlamydospores. They have a powerful mehanizirovannoju shell, characterized by increased activity against nematodes, as when seeds germinate immediately form a catching device.

The closest analogue (prototype) is the strain Duddmgtonia flagrans CI 3 DSM 6703 (U.S. Patent No. 5643568, IPC a 01 N 63/00, publ. 1997), with nematophagous properties and used to combat parasitic on animals nematodes.

However, this strain was not tested for activity against root-knot nematodes of plants and does not possess the ability to stimulate plant growth, which narrows the scope of its use.

The technical result of the present invention to provide a new strain nematophagous mushroom, with high efficiency in the destruction of Gallic damaged plants, pathogens helminths of animals, the ability to maintain viability without loss of properties, as when passing through the gastro-intestinal tract of animals, and the condition of the soil, and also has the property to stimulate the growth and development of plants.

This result is achieved in that, as a producer to obtain bioprep the preparations for combating parasitic nematodes of plants and animals are encouraged to use the strain Duddingtonia flagrans T-89, registered in the collection of cultures of microorganisms SRC VB “Vector” number F-S82 (certificate of Deposit of the strain attached).

The inventive strain is characterized by:

- education in surface culture on various solid (agar and dry) environments are produced mostly in the form of chlamydospores, with a thick shell;

the chlamydospores are stored in a dry condition without losing the original properties of the fungus and the viability of not less than two years, easily restored even after 10 years of storage at room culture in suspension of nematodes, which stimulate the germination of chlamydospores;

- has a high efficiency with respect to different nematodes, shows the duration of;

comparative characteristics by the method of genomic fingerprinting showed significant differences of this strain from the prototype.

The strain of the fungus Duddmgtonia flagrans T-89 isolated from soil of a greenhouse complex “Kirovets” , Novosibirsk in 1989 and deposited in the collection of microorganisms of the Institute of culture collections of microorganisms SRC VB “Vector” (named Koltsovo, Novosibirsk region) number F-882.

The strain has the following cultural-morphological and physiological-biochemical characteristics.

On wort-agar and peptone corn agar forms a colony, having fluffy mycelium brown Otten is and. The conidiophores are formed small conidia, two-celled, straight or slightly curved. On top of conidiophores formed from 4 to 12 conidia, an average size of 40×10 μm. On mycelium abundantly formed round chlamydospores, the diameter of which ranges from 25 to 55 μm. They have a dark brown color and a thick rubberductape shell. In the presence of nematodes, the fungus produces adhesive loop and plexus involved in the act of predation.

The fungus absorbs different sources of carbon and nitrogen. Grows well on media containing wort, molasses, corn extract.

Optimum temperature for growth of +(25-26)°C, lower limit +(5-6)°C, top +(29-30)°C.

Stored strain on agar medium - peptone corn agar (PKA) in test tubes at a temperature of +(4-5)°within 2 years. For long-term storage can be used lyophilization, cryopreservation, and storage in sterile soil.

The strain F-882 marker signs is not. There is a characteristic DNA of strain obtained by the method of PCR genomic fingerprinting.

Strains of Duddingtonia flagrans widely applied in various countries (USA, Denmark, Argentina, and others) in experiments on animals (horses, sheep, and others) to combat widespread and dangerous diseases C.-H. and domestic animals - worms.

In the only experimental study of the proposed strain in mice toxicity is not detected. There is the opinion of the Scientific research Institute of occupational medicine and human ecology of the Russian Academy of medical Sciences (Siberian branch of the East-Siberian scientific center), accredited as a testing center in the certification System GOST R (ROSS RU. 0001.510164) on virulence, no toxicity, no oxygenate strain.

Figure 1 shows electrophoregrams RAPD-PCR analysis of genomic DNA of fungi primer W-19. Strains: lane 1 - Phoma exigua 13, 2 - Phoma exigua 3; 3 - Pleurotus ostreatus A, 4 - Rhizoctonia solani var. faviata S, 5 - Rhizoctonia solani var. faviata SC, 6 - Rhizoctonia solani var. faviata P; 7 - Arthrobotrys sp. V-02, 8 - Duddingtonia flagrans T-89, 9 - Arthrobotrys oligospora BKMF-3062D.

Characterization of strains by the method PP-PCR genomic fingerprinting

For studying the genomic polymorphism of special interest hypervariable minisatellite sequence. Method PP-PCR-fingerprinting (RAPD-PCR analysis) [Shahinian I.A., Gunzburg A.L. PCR genetic typing of pathogenic micro-organisms // Genetics 1995, volume 31, No. 5, s-610] using arbitrary primers length 6-20 nucleotides allows to get the number of amplicons of different length with a molecular mass of from 200 to 1,500 base pairs, with a range of amplicons varies for each combination of primer DNA target, but characteristic for each of the combinations. Upon registration of amplification products by electrophoresis in a floor is acrylamide or agarose gels each range of allowed profiles for amplification, or fingerprinting.

Below is a comparative analysis of the genomic DNA helminthophobia fungi of the genus Arthrobotrys and Duddingtonia flagrans method PP-PCR genomic fingerprinting using arbitrary primers.

Genomic DNA of fungi were isolated from biomass according to the method Ahman and others [Ahman J., Ek Century, Rask L., Tunlid A. Sequence analysis and regulation of a gene encoding a cuticle-degrading serine protease from the nematophagouse fungus Arthrobotrys oligospora. // Environ. 1996. V.142, N7. P.1605-1616] using proteinase K and subsequent purification of DNA by phenol and chloroform. The quality of DNA was assessed by electrophoresis in 1%agarose gel ( Maniatis R.A., Fritsch E., Sambrook D. Methods of genetic engineering. Molecular cloning. // M: Mir, 1984. S).

Polymerase chain reaction with arbitrary primers was carried out as follows. The reaction mixture (volume 25 ál) contained: 1.2 ál of the mixture TM deoxy-nucleosidase, 2.5 µl of MPCR buffer (0.5M Tris-Hcl pH 8.8, TM (NH4)2SO4, 20mM MgCl2, 0,TM tween-20), 0.5 μl of Taq polymerase, 2.5 µl M MgCl2, 1 μl of oligonucleotide primer, 1 ng of genomic DNA. Conditions of amplification (30 cycles): 94°s - 1 min (denaturation step); 42°s - 1 min (stage annealing); 72°C - 1.5 min (stage of completion of the chain); 72°With 8 minutes (the final stage of synthesis).

Analysis of amplification products was performed by electrophoresis in 4%polyacrylamide gel followed by staining of b is Emistim by ethidium. The most important step when using PCR-fingerprinting genetic typing of microorganisms is an experimental search and selection primer or their combination for the most effective differentiation of closely related representatives of one or another species of organisms.

For this purpose have been tested a few random primers with a length of 10-20 nucleotides (table 1) on the DNA of fungi Arthrobotrys oligospora (BKMF-3062D) and Duddingtonia flagrans T-89. As negative controls were taken DNA Mycobacterium Mycobacterium bovis and DNA Bacillus Bacillus subtilis 652. The most specific and informative primers for differentiating strains were oligonucleotides GDI8 and sh-19, which further analysis was conducted of the newly selected chromosomal DNA of fungi.

Table 1
Primers for carrying out PP-PCR genomic fingerprinting
Name primersThe composition of the primers
R295'-CCGGCCTTAC
W-145'-AATCGGGCTG
W-165'-GTGATCGCAG
W-195'-AGTCAGCCAC
GD15'-AGGACGAGCTCGCGGATATA
GD135'-CTCGGCGGCCCGGGCAGAGAC
GD185'-AGCCCGCGGGGCCGTCGT

For the analysis in addition to fungi of the genus Arthrobotrys (Arthrobotrys oligospora BKMF-3062D, Duddingtonia flagrans T-89 and Arthrobotrys sp. V-02) were taken by an additional 6 test drugs other fungi of various genera: Phoma exigua 3 and Phoma exigua 13; Rhizoctonia solani var. faviata S, Rhizoctonia solani var. faviata P and Rhizoctonia solani var. faviata SC; Pleurotus ostreatus A. After extraction of chromosomal DNA of fungi spent their characterization method PP-PCR-genotyping using one of the selected primers sh-19 (table 1).

The following results were obtained (Figure 1): all the paintings genomic fingerprints of the control DNA differed from those for the genus Arthrobotrys. For fungi Arthrobotrys like to have pictures of the bands in strains of Arthrobotrys oligospora BKMF-3062D and Arthrobotrys sp. V-02 (tracks 7 and 9). They probably both are in the form of Arthrobotrys oligospora. Strain Duddingtonia flagrans T-89 (lane 8) was significantly different from them, in particular, on electrophoregram in the length range from 400 to 1300 base pairs (mo) strip completely coincide with those on tracks 7 and 9, and in the range of 300-340 mo instead of double bands on track 8 there is only one, because Duddingtonia flagrans T-89 is the representative of another genus of fungi that indicate data and morphological studies of this strain.

Example 1. Comparative nematophagous effect Duddingtonia flagrans and Arthrobotrys oligospora BKMF-3062D in suspension hookworms-alprazolaj Panagrellus rediviras. (table 2).

Table 2.
Nematophagous the effect of different strains of predatory fungi
OptionNematophagous effect in %
1 day2 day3 day4 days
A. oligospora BKMF-3062D9799100100
D flagrans F-882738898100

Duddingtonia flagrans as Arthrobotrys oligospora exhibits good attractive effect, which is detected by the accumulated number of nematodes in blocks from a fungal culture grown on peptone corn agar. Like many sleeping patterns, having a thick shell, chlamydospores Duddingtonia flagrans require for germination a little more time than the prototype, with a culture that presents conidia and mycelium.

Example 2. A large number of chlamydospores, requiring adaptation to soil conditions, allows the use of Duddingtonia flagrans F-882 not only for 2-3 weeks before transplanting plants, smitten parasitic nematodes, but also with plants. In the experiment, where the biological product on the basis of a strain of D. flagrans F-882 was incorporated into the hole together with the cucumber seedlings were observed its prolonged action, which affected the Gauls who's namitah and productivity of the next turnover of tomato (table 3).

Table 3.
The results of the field experience for introduction of a biological product on the basis of D. flagrans against root-knot nematodes
OptionThe yield of cucumberThe yield of tomato
The plan FactThe plan Fact
Hundredweight 1 greenhouseHundredweight 1 greenhouse
Preparation on the basis of Duddingtonia flagrans F-8821602303034
Steaming soil (control)2321433026

Example 3. Feeding within 7 days of the grain of a biological product on the basis of strains of D. flagrans F-882 dose of 3 g per day for 7 days laboratory lines of white mice infected by helminths (of cifali, aspiculuris)showed that the fungus passes through the digestive system of animals, maintains its vitality and nematophagous efficiency. The excrement of the test and control lines were placed in Petri dishes on an empty agar (HA) and was carried out by direct microscopy. The presence of trapping loops and chlamydospores of the fungus on the excrement is about all samples with a wild mushroom, since 2 days. Also noted was the reduction of colonies of fungi of the genus Aspergillium, Penicillium and actinomycetes, as on the surface of the agar, and the excrement experienced option after using the drug for 3 days. After the end of the experiment the helminthological analysis showed the complete absence of nematodes in the faeces of mice in the experiment with mushroom-nematophagous, while in the control version number 1 g averaged 8 pieces.

Example 4. The experience delivered using faeces of horses, spontaneously infested strangulate the gastrointestinal tract. Preliminary studies of ovoscope on Kotelnikovo-Damned showed high contamination of animals. The intensity of infection was 35-50 eggs in one drop. From this sample taken sample of the manure mass 36, and 50, and evenly distributed in Petri dishes. In one Cup with a sample of faeces weight of 50 g was added 78 grains of rye, which was grown fungus Duddingtonia flagrans. Samples were placed in a thermostat at a temperature of 26.7°C. After 13 days of larval nematodes were isolated according to the method of Berman-Orlov. The results are presented in table 4.

Table 4
The influence of the fungus D. flagrans on the number of parasitic nematodes of horses
OptionWeight, gThe number of larvae strongest, copies
On the 13th dayIn 1 g of feces
Congrol36.0822.3
D. flagrans50.0100.2

Analysis of the obtained data showed that on the 13th day after sampling in the experimental sample (D. flagrans) the number of larvae of parasitic nematodes was 11.5 times less than in the control sample.

Example 5. Data on the claimed strain that foster growth and development of plants.

Tests of the fungus Duddingtonia flagrans in experiments on various crops has allowed to establish a stimulating effect on the growth and development of plants. It is marked on the pine seedlings, cyclamen, cucumbers and tomatoes.

Introduction biomass of submerged culture Duddingtonia flagrans in pots under seedlings of cucumber during altercation at a dose of 1% by weight of the substrate, consisting of sawdust, peat and soil, has led to a significant advance plant growth, the formation of leaves. After 4 weeks of the growing season was the analysis of cucumber variety “Crystal”, the results are presented in table 5.

Table 5
Data on the stimulating effect on the growth and development of the s plant biomass Duddingtonia flagrans T-89
OptionPlant height, cmThe number of leavesWeight of above-ground biomass, gThe root weight, g
The biomass of the fungus D. flagrans12,867,73,2
Control7,1442,22,1

Example 6.

In December 2002, a hothouse of “Industrial” , Barnaul cucumbers varieties “Sapphire” in the greenhouse No. 36 was made the drug on the basis of D. flagrans in a dose of 45 g in the hole under the seedlings (450 plants). Surveys conducted after 5 weeks, showed that the average height of the experimental plants was 180 cm, the plants had a lot of ovaries and fruits weighing up to 100 g In a control area, where there were no drug plants had an average height of 150 cm, and the ovaries have just started to develop.

Experiments on different species of nematodes carried out in laboratory and field tests indicate the manifestation of predatory fungus Duddingtonia flagrans nematophagous properties in relation to alprazolaj nematode Panagrellus redivivus, phytoparasitic nematodes of the genus Meloidogyne (M. hapla, M. incognita, M. javanica, M. arenaria), entomopathogenic nematodes Steinernema carpocasae and S. feltiae, and strongylata the gastrointestinal tract of the horse - Trichonema, Strongylus, Alfortia, Delafondia and larvae of Elaphostrongylus sp. - excite is ELU of helminthiasis brain of the deer.

Example 7. To assess the stimulating effect of the fungus Duddingtonia flagrans T-89 (room F-882) on the growth and development of plants and the simultaneous manifestation of nematophagous efficiency was carried out on a laboratory experience in which he used the culture of predatory fungi D. flagrans T-89 and Arthrobotrys oligospora BKMF-3062D (working title T-82). On the basis of these cultures was previously received biologic by growing mushrooms on rye grain.

As a test object used cucumber varieties Zozulya. The experiment was carried out in plastic cups, and as the substrate used calcined river sand. To create infectious background used crushed root galls cucumber from greenhouse complex “Industrial” (Barnaul).

The preparation is made simultaneously with the sowing of sprouted seeds of cucumbers in a dose of 1% by weight of the substrate in the glass, which was g. Each variant was 5 plants. Watering of the plants was carried out at the rate of 20 ml per 1 plant in 2-3 days as the soil dries. Once in 10 days the plants were watered with a solution of Gold Flora”. The control was represented by the blood vessels in which the biological product is not made. The experiment lasted 4 weeks.

The results of experience has enabled us to establish differences in the growth and development of plants, as well as the manifestation of nematophagous efficiency depending on the piece is MMA predatory fungus.

The best results were recorded in the variant with Duddingtonia flagrans T-89 (claimed)strain. By the end of the experience none of the plants in this embodiment, not killed.

In the variant with A. oligospora (T-82) 2 plants died, 2 had dried yellow cotyledon leaves from 5 plants marked chlorosis of leaves. In addition to differences in plant height, root length, number of leaves, the variants differed in the number of galls per plant. As can be seen from table 6, the best growth, plant development and manifestations nematophagous effect were in the variant strain Duddingtonia flagrans (see figure 2)

Table 6
The influence of different strains of predatory fungi on the growth and development of plants of cucumber and manifestation nematophagous efficiency
OptionIndicators
Plant height, cmLength of roots, cmThe number of leaves, piecesThe number of galls on 1 plant, units
Arthrobotrys oligospora T-8213,75.82,343,3
Duddingtonia flagrans T-8915,56,42,528,3
Control10,22,21,35,0

Industrial applicability. The invention can be used in microbiological industry and agriculture as a producer for the production of biological products.

Strain Duddingtonia flagrans T-89, registered in the Collection of cultures of microorganisms SRC VB “Vector” number F-882, showing properties against root-knot nematodes and plant parasitic nematodes of animals and stimulate the growth and development of plants.



 

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EFFECT: higher efficiency.

FIELD: food industry.

SUBSTANCE: the present innovation deals with obtaining gelling concentrate containing structure-forming agents of vegetable and microbial origin. Prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide in the field of ultrasound fluctuations, juice pressing should be carried out under aseptic conditions followed by cultivation upon residues of mycellar fungi of Trichoderma and Aspergillus species of acetic acid fermentation. Then comes separation of culture liquid, its blending with juice, addition of liquid ammonia and supercritical CO2-extract into the blend out of Mortierella alliaceae micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with treated solid residue of Mortierella alliaceae micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

FIELD: food industry.

SUBSTANCE: the present innovation deals with obtaining gelling concentrate containing structure-forming agents of vegetable and microbial origin. Prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide in the field of ultrasound fluctuations, juice pressing should be carried out under aseptic conditions followed by cultivation upon residues of mycellar fungi of Trichoderma and Aspergillus species of acetic acid fermentation. Then comes separation of culture liquid, its blending with juice, addition of liquid ammonia and supercritical CO2-extract into the blend out of Mortierella lignicola micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with treated solid residue of Mortierella lignicola micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

FIELD: food industry.

SUBSTANCE: prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide in the field of ultrasound fluctuations, juice pressing should be carried out under aseptic conditions followed by cultivation upon residues of mycellar fungi of Trichoderma and Aspergillus species of acetic acid fermentation. Then comes separation of culture liquid, its blending with juice, addition of ammonia and supercritical CO2-extract into the blend out of Mortierella verticillata micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with solid residue-treated Mortierella verticillata micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

FIELD: food industry.

SUBSTANCE: prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide in the field of ultrasound fluctuations, juice pressing should be carried out under aseptic conditions followed by cultivation upon residues of mycellar fungi of Trichoderma and Aspergillus species of acetic acid fermentation. Then comes separation of culture liquid, its blending with juice, addition of ammonia and supercritical CO2-extract into the blend out of Mortierella marburgensis micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with solid residue-treated Mortierella marburgensis micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

FIELD: biotechnology, in particular reagent for structural protein hydrolysis.

SUBSTANCE: method for production of protheolytic reagent includes cultivation of producer strain selected from dermatophyte of species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Microsporum canis, Microsporum gypseum, preferably from fungus strains capable to produce complex of structural protein proteinases (scleroproteases) with total protheolytic activity at least 0.7 U/mg including collagenase 0.1-4.5 U/mg; keratinase 0.1-0.5- U/mg; elastase 0.5-1.9 U/mg. Cultivation is carried out, for example, in wort agar-agar or in wort broth for 20-24 days.

EFFECT: scleroproteases with improved specific activity; method for protheolytic cleavage of specific substrates (scleroproteins) with increased rate and depth.

2 dwg, 12 ex

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