Producer of cucurbit bacterial spot agent (acidovorax citrulli) inhibitor

FIELD: chemistry.

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

EFFECT: invention reduces loss of cucurbit crop.

 

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 I-th type (Inhibitor of herpes virus simple I-th type. RF patent №2022012, 1994.), inhibitor of tick-borne encephalitis virus (Inhibitor of tick-borne encephalitis virus. Patent for invention No. 2473689, 2013), virus necrotic spots balsam (Producer inhibitor of virus necrotic spots balsam. Patent for invention No. 24813923, 2013). As inhibitor was used metabolite strain Trichoderma harzianum Rifai is the extract of the culture liquids producer of the enzyme L-lysine-and-oxidase.

In recent years, phytosanitary services of the different countries concerned by the emergence and rapid spread of dangerous diseases bacterial spot of pumpkin crops, caused by the bacterium Acidovorax citrulli. Quarantine status of this agent for the Russian Federation is not defined, but due to the risk of importation with the seed, large yield losses in the countries of distribution and a wide range of host crops cultivated on the territory of our country, it is of interest. This pathogen causes significant yield losses. During early infection of plants can lose up to 100% (Feng, J. J., Li, J. Q., Walcott, R. R., Zhang,G. M., Luo, L. X., Kang, L., Zheng, Y. and Schaad, N. W. (2013). Seed Sci. & Technol., 41, 1-15).

Most sensitive to the causative agent of bacterial spot are watermelon (Citrullus lanatus) and melon (Cucumis melo). Bacteriosis also affects cucumbers (Cucumis sativus), different types of pumpkins (Cucurbita pepo, C. moschata), squash (Cucurbita pepo var. patisoniana), zucchini (Cucurbita pepo var. giromontina), betel (Piper betle is a family of pepper).

Artificially could be infected plants of the family Solanaceae: pepper (Capsicum spp.), tomatoes (Lycopersicon esculentum), eggplant (Solanum melongena) (EPPO (2011a). Acidovorax citrulli - Bacterial fruit blotch of cucurbits. EPPO Alert list).

The pathogen enters the field with an infected seeds, which are seedlings with characteristic lesions on cotyledon leaves. In the case of latent infection of a plant can remain undetected in the field. Wet and warm weather, sprinkler irrigation, protecting plants, failure to comply with phytosanitary conditions in the field or greenhouse are conducive to disease development and transfer agent on adjacent healthy plants. Bacteria Acidovorax citrulli with leaves fall on green fruits, which can be affected at an early stage of development. The fruit is most susceptible to the pathogen at the age of 2-3 weeks, before the formation of the wax layer. After a few days the damage on fruits become visible and appear on their surface cracks. Infected fruit rot in the field, in financial p is, which brought them to the infected seeds, which are a source of diseases that fall into the soil (E. Karimov Century, a New bacterial disease of pumpkin crop Protection and plant Quarantine, 2012 No. 6, S. 35-36).

The technical result of the invention is to reduce losses pumpkin crops caused by the causative agent of bacterial spot of pumpkin crops Acidovorax citrulli.

The technical result is achieved by the fact that, as a producer inhibitor of the causative agent of bacterial spot of pumpkin crops Acidovorax citrulli using a strain of Trichoderma harzianum Rifai. The strain is deposited in Russian national collection of industrial microorganisms under the number F-180 (Moscow, Road 1-y passage, D. 1 Institute of genetics and breeding, 1987).

Morphological and biochemical characteristics of the strain

Strain Trichoderma harzianum Rifai on the environment wort agar forms a colony growing. Mycelium hyaline, septate, open. 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 sporanox the E. The strain grows well in the environment of čapek, but best growth occurs on the environment wort-agar. Agar and gelatine not thins, milk is not peptonized. Aerobic fungus. 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, however, can grow and at a pH of from 1.5 to 9.

Trichoderma harzianum 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 Mick what metodom. The essence of the method lies in the interaction of all formed in the reaction of N2About2with O-vanishedamericana.com. The incubation mixture contained 20 μg peroxidase, 250 μg O-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 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 (, busine).

Used the fermenter type BIOR-01 produced by BLS Bureau, 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.

Nutrient medium was prepared directly in the apparatus according to the technology developed previously (Smirnov, I. P., and other Technology separation and purification of L-lysine α-oxidase, Biotechnology, 2010, No. 6, S. 52-60). For this purpose, 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 with seed material from 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 spot of pumpkin cultures of the bacterium Acidovorax citrulli was obtained in pure culture from bacteriological collection of the Institute of agricultural research (Ivia), Spain, Valencia. Then d is I the identification of the obtained culture was performed sequencing of the 16S rRNA gene length 500 p. N., amplifierarava universal primers 8UA_519B.

For the production of experience in determining the inhibitory effect of the extract of strain Trichoderma harzianum Rifai on the bacterium Acidovorax citrulli using the method of diffusion in agar. As the nutrient medium used environment king Century

Wednesday king (according to King et al., 1954)

Peptone20 g
Glycerin10 ml
K2HPO41.5 g
MgSO4×7H2O1.5 g
Agar15-20 g
Distilled waterTo 1 l
pH 7.0 to 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 rubbing on the surface of the agar medium using a glass spatula Drygalski.

To accommodate extract of strain Trichoderma harzianum Rifai th is has Ofili wells, why in the solidified medium 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 he made extracts in the wells at 1 mm below the level of the medium (200 μl well). In the wells of the control cups were made with sterile distilled water.

Prepared Petri dishes with samples and control Petri dishes were placed in a thermostat (temperature +37°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 Acidovorax citrulli 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.

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 repetitions: 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 culture Acidovorax citrulli on nutrient medium and making extract moons in the experimental cups observed zone of inhibition of growth of cultures around the hole. The radius of the zone of suppression was 5-8 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, on the entire surface of the medium was observed actively growing culture of the causative agent of the disease, without evidence of suppression. This indicates that the extract of the strain Trichoderma harzianum Rifai has inhibitory effect on the causative agent of bacterial spot of pumpkin crops Acidovorax citrulli.

The application of strain Trichoderma harzianum Rifai, deposited in Russian national collection of industrial microorganisms under the number F-180, as producer inhibitor of the causative agent of bacterial spot of pumpkin crops (Acidovorax citrulli).



 

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