Inhibitor of stimulant of bacterial burn of fruit crops (erwinia amylovora)

FIELD: biotechnologies.

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

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

1 ex

 

The invention relates to biochemistry and may find application in agricultural biotechnology, Microbiology and plant breeding.

A well-known inhibitor of the herpes virus simple 1st type [Inhibitor of herpes simple 1-St type. RF patent №2022012, 1994. S.B. Alekseev, birch CT, Andjaparidze OG and others] and inhibitor of human immunodeficiency virus [Inhibitor of human immunodeficiency virus. RF patent №2022011, 1994. S.B. Alekseev, Weight B.C., Smirnov I.P., and others], which was used metabolite strain Trichoderma harzianum Rifai - homogeneous enzyme L-lysine-a-oxidase.

In recent years, dramatically increased the import of planting material of ornamental and fruit plants, which is the owner of the causative agent of bacterial blight of fruit crops (Erwinia amylovora). Bacterial blight is one of the most dangerous diseases of fruit crops, has a quarantine status in the Russian Federation. The main and most susceptible to fire blight are fruit and ornamental plants of the family Rosaceae subfamily Pomoideae. Cotoneaster is the most susceptible to fire blight culture, among fruit trees suffer most from the disease of pear. In addition, the pathogen infects hawthorn, quince, chaenomeles, Apple, mountain ash, irgu, medlar, pyracantha, stranvaesia, wild pear, rose [Harmful organisms having Arentino phytosanitary importance for the Russian Federation: a Handbook / edited S.A. Dankvert, M.I. Maslova, US Magomedov, AB Mordkovich. - Voronezh: the Scientific book 2009, 280 - 286].

Bacterial blight of fruit crops causes large yield losses and tree mortality. Harmfulness burn fruit is very high due to the very rapid dissemination. Economic damage is expressed in reduction or complete loss of crops, loss of fruit trees, the cost of vicharchika dead and diseased trees and planting a new garden and others In heavily infected orchards, the causative agent of the disease can strike from 20 to 50%, in some cases up to 90% of the trees, some of which are completely dies [Schneider EJ pest risk Analysis of the causative agent of bacterial blight of fruit Erwinia amylovora (Burrill) Winslow et al. for the territory of the Russian Federation. FGI official group member, 2009, Pp.5-6, 13-14, 24].

In our country, private and industrial gardens occupy large areas, and the applied control measures do not prevent the acclimatization of the pathogen. According to the List of pesticides and agrochemicals permitted for use on the territory of the Russian Federation at the present time, in our country is not registered drugs to fight the causative agent of bacterial blight of fruit crops [List of pesticides and agrochemicals permitted for use on the territory of the Russian Federation. 2010 Spectr-R", Kursk. Reference book, 804 S.] All of this suggests, the penetration and acclimatization of the pathogen E. amylovora brings huge losses to fruit and ornamental horticulture.

The technical result of the invention is to reduce losses of ornamental and fruit crops, caused by the causative agent of bacterial blight of fruit crops E. amylovora.

The technical result is achieved by the fact, as a producer inhibitor of the causative agent of bacterial blight of fruit crops used a strain of the fungus Trichoderma harzianum Rifai VKPM F - 180.

The strain is deposited in Russian national collection of industrial microorganisms under the number F-180 in 1987 (Moscow, Road 1-y passage, 1. Institute of genetics and selection).

Morphological and biochemical characteristics of the strain.

Strain Trichoderma harzianum Rifai on the environment wort agar forms a colony growing. Mycelium hyaline, septate,

outstretched. On the 4th - 5th day of growth appear Mat with conidiophores, cushion-shaped, at first white, yellow or dark green. Fieldy butylate /9-12µ/, verticils of 3 or more. Each felide conidia are formed, glued to the head. Conidia of the fungus rounded, smooth, small /3-5µ/, in transmitted light, pale green, and in the mass of the dark.

On agar medium of čapek colonies producer strain growing, but poorly paronomasia. The strain grows well on among the E. of čapek, however, the best growth was observed on the environment wort-agar. Agar and gelatine not thins, milk is not iphonesimulator mushroom. The temperature optimum of +28 to+29°C. On potato-dextrose agar, the decrease of the growth rate, the formation of aerial mycelium and sporulation. Optimum conditions for growth of the fungus at pH 4-6, but can grow at a pH of from 1.5 to 9.

Trichoderma harziatum Rifai equally internalizes as ammonium and nitrate salts. The best source of nitrogen from organic compounds is peptone. Grows well in a medium with asparagine and glutamic acid. The best sources of carbon are xylose, glucose, sucrose, lactose, galactose, mannitol, starch. In environments with these sugars occur rapidly. Poorly digested alcohols methyl, ethyl, dulcet. Well assimilated as a carbon source, wheat bran. The inoculum of the fungus grown on medium containing wheat bran, gives a positive result when used for fermentation in media of different composition.

Strain Trichoderma harzianum Rifai has L-lysine α-oxidase activity. The activity of L-lysine α-oxidase in culture liquid of Trichoderma harzianum Rifai was calculated as the increase in H2O2, which amount was determined spectrophotometrically orthodeuterium micromethods. The essence of the method concludes the I in the interaction of all formed in the reaction of H 2O2with O-vanishedamericana.com. The incubation mixture contained 20 μg peroxidase, 250 mcg 0-danishdigimida and 0.1-0.5 mg of protein in 1 ml final volume. After 20 minutes incubation in a thermostat at a temperature of +37°C the samples were cooled to +4°C. the Optical density of the colored solutions of the test and control (without substrate) of the samples were measured on the spectrophotometer SF-16 at 540 nm against the second control sample (without peroxidase).

The specific activity of enzyme was expressed by the number of units of activity per 1 mg of protein or 1 ml of culture fluid.

The cultivation conditions of a strain of Trichoderma harzianum Rifai.

Fermentation of the strain was carried out on the equipment Experienced process plant IBPM RAS. G.K. Skryabin (husine).

Used the fermenter type BIOR-01 produced by BLS Bureau, the city of Kirishi, volume 100 l with a fill factor of 0.6. The fermenter equipped with a magnetic stirrer, filters air, temperature and pH.

For the preparation of nutrient medium in the apparatus was filled with 60 l of tap water, contributed 1% wheat bran, stimulant - 0,1%, 1.3% of ammonium sulfate, pH 5.8 to 6.0 has established a 10%HCl solution. Wheat bran and stimulant pre-soaked in 10 litres of water for 4 hours, were sterilized in an autoclave for 1 hour at t°+125°C. Then prepared the fermenter was seeded pic is main material of the flasks. Sowing dose of at least 5%. Cultivation was carried out at a temperature of +26°C, air flow throughout the fermentation of 30 l / minute, the mixer rotation speed of 200 rpm. The pH in the growth process was adjusted to 6.5. Duration of cultivation was 94-98 hours, the final pH from 5.3 to 7.5.

At the end of the fermentation culture liquid was sent to the site pre-treatment, where the fungal mycelium was separated by filtration under vacuum on the suction filter. The weight of the obtained biomass was 3.5 kg Received native solution was subjected to additional separation in the separator of the type RSD at 9000 rpm for one hour at a temperature of +2 to+4°C. Then the native solution in a volume of 55 l, obtained after separation of the biomass was concentrated to 1.5 l (concentrate).

The activity of the obtained extract from the culture of Trichoderma harzianum Rifai was 5.4 U/ml

The causative agent of bacterial blight of fruit crops - bacterium Erwinia amylovora was detected in samples of shoots decorative plants pyracantha (.Pyracantha m.roem.) the polymerase chain reaction and confirmed by inoculation in a nutrient medium.

For the production of experience in determining the inhibitory effect of the extract of strain Trichoderma harzianum Rifai on the bacterium E. amylovora using the method of diffusion in agar. As the nutrient medium used sugar is EIT-peptone agar.

Saharsa-peptone agar, Sucrose Pepton Agar (SPA)

Sucrose20 g
Peptone5 g
K2HPO40.5 g
MgSO4×7H2O0.25 g
Baktagir15 g
Distilled waterto 1 l

pH 7,0-7,2

Sterilized by autoclaving at a temperature of 121°C for 15 minutes.

In Petri dishes were poured nutrient agar medium in the amount of 20-30 ml and allowed it to harden. Seeding cultures on Petri dishes was performed by applying a hinge pure culture of bacteria on a nutrient medium and a uniform distribution by rubbing on the surface of the agar medium using a glass spatula Drygalski.

To accommodate extract of strain Trichoderma harzianum Rifai prepared hole in a frozen environment with surface caused by the culture with a sterile drill bit with a diameter of 10 mm was drilled at the required number of wells depth of about 5 mm, after which the wells were brought extract 1 mm below the level of the medium (200 μl well). In control wells cups who wore sterile distilled water.

Prepared Petri dishes with samples and control Petri dishes were placed in a thermostat (temperature 26±2°C). The samples were kept in thermostat 1-3 days, noting the intensity of bacterial growth on cups, measuring the zone of inhibition of their growth and making the log entry.

Inhibitory effect of the extract of strain Trichoderma harzianum Rifai against Erwinia amylovora was determined by the degree of inhibition of growth of a pure culture of the pathogen in a nutrient medium. Measuring the zone of inhibition of growth of the culture was performed at 24 hours after administration of the experience and then observed changes in the course of 3 days.

Example.

Inhibitory effect of the extract of strain Trichoderma harzianum Rifai determined by the degree of inhibition of growth of a pure culture of the pathogen in a nutrient medium. The experiment was carried out in three replications: experienced cups in the holes made extract of strain Trichoderma harzianum Rifai. In the control plate in holes made with sterile distilled water. 24 hours after surface inoculation of the culture of Erwinia amylovora on nutrient medium and make extracts in the wells in the sample cups were observed zone of inhibition of growth of cultures around the hole. The radius of the zone of suppression was 5-9 mm. On the other surface of the Cup saw the growth of the culture of the pathogen without signs of oppression. On the control plate, the entire surface area of environment demonstrated strong growth of the culture in which the adherents of the disease, no signs of suppression. This indicates that the extract of the strain Trichoderma harzianum Rifai has inhibitory effect on the pathogen of bacterial blight of fruit crops.

The application of strain of the fungus Trichoderma harzianum Rifai VKPM F-180 as a producer inhibitor of the causative agent of bacterial blight of fruit crops.



 

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