RU2634390C1 - Strains of nematophage fungus arthrobotrys oligospora infecting eggs and larvae of cyst-forming golden nematode globodera rostochiensis in cysts - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of the nematophage fungus Arthrobotrys oligospora T-15, which has the ability to germinate inside cysts and infect the eggs, as well as the nematode larvae in them, destroying their contents, is deposited in the All-Russian Collection of Industrial Microorganisms under the registration number VKPM F-1303. The strain of nematophage fungus Arthrobotrys oligospora can be used to produce biological preparations for plant protection.

EFFECT: increased potato yield.

4 dwg, 3 ex

Description

The invention relates to a strain of the nematophagous fungus Arthrobotrys oligospora, which affects the eggs and larvae of the cyst-forming golden potato nematode Globodera rostochiensis, and can be used in biotechnology and the microbiological industry to obtain biological preparations for plant protection.

Problems associated with soil contamination with phytoparasitic nematodes are relevant both for Russia and other developed and developing countries. Golden Potato Nematode Globodera rostochiensis is a dangerous pest of potatoes. Loss of yield due to severe infection of the soil can be 70-90%. The nematode causes the disease, glauberosis, and is subject to the external and internal quarantine of the Russian Federation. There are no chemical preparations for glauberosis, and only foreign drugs Bionemat, manufactured by Biotech International, based on the fungus Paecilomyces lilacinus, which parasitizes nematode eggs, can be used as biological ones. The fungus P. lilacinus secrets a complex of enzymes, including chitinolytic enzymes that allow them to penetrate the integument of eggs. The effectiveness of the fungus depends on the number of conidia of the fungus that have fallen on the stationary stages of the nematodes. However, numerous mobile nematode larvae that infect the roots of potato plants remain outside the access of the fungus.

Against the mobile stages of nematodes - invasive larvae - the most effective are nematophagous (carnivorous fungi), which are widespread in the soil and play an important ecological role in utilizing the huge biomass of microscopic roundworms - nematodes.

Known strains of nematophagous fungi forming various trapping devices for nematode larvae (Teplyakova T.V. Bioecological aspects of the study and use of predatory fungus hyphomycetes. - Novosibirsk, 1999. - 252 p .; Teplyakova T.V., Ananko G.G. hyphomycete fungi against parasitic nematodes // Plant protection and quarantine. - 2009. - No. 6. - S. 22-25). Preparations introduced under potatoes do not have time to cope with the larvae of nematodes, and at this time the females form new cysts with eggs on the roots of the potato, which are well preserved in the soil until new potato plantings. In this state, cysts without a host plant can persist up to 8 years. In the spring, after planting potatoes, invasive larvae under the influence of root secretions exit cysts and infect roots. The exit of larvae from cysts takes place within 3-6 weeks. The larva penetrates the root through the root cap and moves along the root conduction system for several days. Then it stops and forms a food zone of several giant cells. Depending on the temperature of the soil, males and females form from larvae within one and a half to two months. A few days after copulation, the female begins to accumulate eggs in the body cavity. Young females of white color in the fall acquire a golden color and in this state go to winter. By the end of life, the female’s body is completely filled with eggs, turning into a cyst.

Known strains of nematophagous fungi: Arthrobotrys oligospora BKMF-2461D (USSR Author's Certificate No. 1264393, IPC C12N 1/14, publ. 1986), Arthrobotrys oligospora (US Patent No. 4975105, IPC C05F 11/08, publ. 1990), showing a high nematophagus Effect. Preparations for the control of gall nematodes were obtained on various organic substrates. The active principle is conidia and mycelium of the fungus.

The closest analogue (prototype) is the Arthrobotrys oligospora 3062D strain (USSR Author's Certificate No. 1688818, IPC A01N 63/00, publ. 1991), which, in comparison with the above strain, exhibits a higher nematophage effect against gall nematode larvae and stability of properties. The active principle is also conidia and mycelium. This strain is the basis of the developed nematophagin-BL biological product, approved in 1990 by the VASKhNIL Commission for Microbiological Means of Protection, Soyuzselkhozkhimiya, and is recommended for implementation. Nematofagin-BL is included in the list of biological products approved for use in Russia.

However, in all the above analogues and prototype, strains are used that exhibit an effect only with respect to the mobile stages of nematodes - larvae.

The technical result of the claimed invention is to obtain a strain of the predatory fungus Arthrobotrys oligospora, which affects not only the larvae of the cyst-forming potato nematode located in the soil, but also the larvae and eggs of the potato cyst-forming nematode located inside the cysts.

The specified technical result is achieved by isolating from the natural conditions a new strain of the predatory fungus Arthrobotrys oligospora T-15 (the author's name), which produces in the soil trapping devices for nematode larvae and is able to germinate inside cysts and infect eggs, as well as nematode larvae in them, destroying their contents. The strain Arthrobotrys oligospora was isolated from soil contaminated with potato golden nematode in the Kolos garden community, p.p. Koltsovo, Novosibirsk region in May 2915 and deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) under the number F-1303 (a certificate of deposit is attached).

The inventive strain is characterized by the following features.

Cultural and morphological features of the strain on an agarized nutrient medium. Peptone-corn agar (PKA), g: glucose - 10.0; sodium chloride - 5.0; calcium carbonate - 0.5; corn extract - 5.0; peptone - 5.0; agar-agar - 20.0; tap water - up to 1 liter. On this medium, colonies with a dense white cotton-like mycelium are formed, colonies with a smooth edge. The mycelium is slightly rising, the size of the colonies is 30 mm - 3 days, 55 mm - 5 days, 90 mm - 10 days, sporulation by conidia, the reverse side of the colony (reverse) is from light sand to light brown in color. The color of the colony is slightly sandy as it ages.

Micromorphological signs. Mycelium hyphae are even, 4-8 microns in size, conidia are formed on simple straight conidiophores, 300 to 500 microns long, at the base 7-12 microns wide, at the distal end 4-6 microns.

The conidia are bicellular, elongated pear-shaped, the distal cell is almost two times larger than the proximal, 25-27 × 10-14 microns in size, collected in the form of heads of 8-12 pieces on the tops of the conidiophores, as well as in the form of individual clusters on the conidiophores, the number of which can reach 5-10. In the soil and in the culture on nematodes thick-walled chlamydospores are formed, oval or round in size 25-35 microns. In the presence of nematodes, round loops and their plexuses in the form of networks form on hyphae. The internal size of the catching devices is on average from 25 to 30 microns, but there are rounded rings up to 40 microns in diameter.

The product synthesized by the strain is a complex of compounds (attractants, toxins, enzymes) involved in the mechanism of predation and produced by the fungus actively in the process of formation of hunting devices. The nematophage effect is determined in relation to larvae and eggs of nematodes by the ability to destroy them under laboratory conditions, indicators of predation (the number of catching devices) and activity (the number of sacrificed larvae of nematodes, non-viable eggs in%) are estimated.

To determine the activity, a suspension with larvae, eggs, nematode cysts is placed in Petri dishes with a diameter of 40 mm or on glass slides on which paraffin rings are applied. A fungal culture is introduced into the nematode suspension and viewed daily under a stereoscopic microscope (MBS-10).

The strain is stored in refrigerators (+ 2-6 ° C) in the form of cultures grown on a slanted medium (PKA) in test tubes, reseeding is carried out once a year.

Optimum media for reseeding:

- liquid based on molasses and corn extract, g: corn extract - 7.5; beet molasses - 30.0; KH ₂ PO ₄ - 5.0; MgSO ₄ × 7H ₂ O - 2.0; NaNO ₃ - 5.0; tap water - up to 1 liter;

- agar based on molasses and corn extract to the liquid medium indicated above, agar-agar is added at the rate of 20 g per 1 liter of prepared medium;

- PKA: glucose - 10.0; sodium chloride - 5.0; calcium carbonate - 0.5; corn extract - 5.0; peptone - 5.0; agar-agar - 20.0; tap water - up to 1 liter.

As mentioned above, in the section on the characterization of analogues, strains of the fungus Arthrobotrys oligospora are widely used to protect plants and animals from nematodes. The USSR State Chemical Commission in 1990, based on another strain 3062D (USSR author's certificate No. 1688818), allowed the production and use of Nematofagin BL for biological plant protection (Soprunov F.F. Predatory hyphomycetes and their use in the fight against pathogenic nematodes. - Ashgabat, 1958, 394 p .; Teplyakova T.V. Bioecological aspects of the study and use of predatory fungal hyphomycetes. - Novosibirsk, 1999, 252 p.). The strain is not genetically modified and does not contain the genes of other organisms; transferred resistance genes; genetic changes associated with the use of genetic engineering techniques.

An advantage of the claimed strain is its ability to affect not only the motile larvae of the potato nematode in the soil, but also the immobile ones - eggs surrounded by a cyst membrane that is resistant to external influences. This will allow the use of a drug based on this strain not only during planting of potato tubers, but also after harvest, when numerous cysts of a dangerous pest remain in the soil.

In FIG. Figure 1 shows conidiophores with conidia of the fungus Arthrobotrys oligospora on the surface of a potato nematode cyst. In FIG. Figure 2 shows eggs in a cyst of a potato nematode, the structure is broken, hyphae are visible, an increase of 400 times. In FIG. 3 presents hyphae of a carnivorous fungus, filled the eggs located in the cyst, magnification 1000 times. In FIG. Figure 4 shows eggs and larvae not affected by a predatory fungus in the cyst of potato nematode, magnification 400 times.

Example 1. The cultivation of biomass of the fungus based on the inventive strain Arthrobotrys oligospora F-1303 surface method.

Obtaining biomass can be carried out on agarized and granular media. Peptone-corn agar (PKA) is used as an agarized medium, g: glucose - 10.0; sodium chloride - 5.0; calcium carbonate - 0.5; corn extract - 5.0; peptone - 5.0; agar-agar - 20.0; tap water - up to 1 l).

After placing all the components of the medium in the dishes with a capacity of at least one and a half liters, the mixture is heated to melt the agar-agar, with constant stirring. For the same purpose, a water bath can be used. Then the medium is poured into flasks or vials of 200 ml and sterilized in an autoclave for 40 minutes at a temperature of 120 C (1 ATM).

Then the medium is poured into sterile Petri dishes and after cooling, they are seeded with pieces of sterile culture of the fungus. The growth time of the colony until the cup is completely overgrown and sporulation appears at a temperature of + 28 + - C is 12-14 days.

As a granular medium, wheat grain is used. 1500 ml of water is added to 1 kg of grain, and grain is boiled for 15 minutes to saturate the grains with water. After that, the grain is thrown into a sieve or colander to remove moisture, add 5 g of chalk to it, pack 750 ml flasks, filling them in 1/3 of the volume, and sterilize in an autoclave at a temperature of 120 ° C (1 atm) for 40 minutes . After cooling, the flasks with the grain are shaken and inoculated with pieces of the mycelium of the carnivorous fungus. Mycelium grows on grain within two to three weeks.

Example 2. The cultivation of biomass of the fungus based on the inventive strain Arthrobotrys oligospora F-1303 deep method.

Deep cultivation is carried out on a liquid medium: corn extract - 7.5; beet molasses - 30.0; KH ₂ PO ₄ - 5.0; MgSO ₄ × 7H ₂ O - 2.0; NaNO ₃ - 5.0; tap water - up to 1 liter. The medium is poured into 100 ml flasks; inoculation is performed on the spore-mycelial suspension of the fungus at the rate of 5 ml per 100 ml of medium. The suspension is obtained from an agarized culture of the fungus grown in a biological tube on mowed agar. 20 ml of sterile water is poured into the test tube, the mycelium with conidia is removed with a sterile glass pipette or spatula and shaken to obtain a more uniform distribution of conidia and scraps of hyphae in the water. The flasks are placed in a temperature-controlled rocking chair for 5-6 days, biomass is grown at a temperature of 28 ° C and a speed of 180 revolutions / min. When viewing culture samples under a microscope, the presence of spiral branches at the ends of growing hyphae is a sign of biomass maturity. The finished biomass has a brownish color and a gel-like consistency, and also contains at least 15 g / l of dry matter.

Example 3. Test biomass of surface and deep cultivation of the inventive strain Arthrobotrys oligospora F-1303 on cysts of the potato nematode Globodera rostochiensis in laboratory conditions. To test the effectiveness of the Arthrobotrys oligospora F-1303 strain against eggs and larvae of potato nematodes enclosed inside cysts, an aqueous agar-agar solution (20 g per 1 liter of water without adding nutrients, agar melting and sterilization according to Example 1) and nematode cysts were used collected on potato roots and in soil at an infected site.

A sterile agar-agar solution was poured in a thin layer into Petri dishes and after cooling, nematode cysts were placed in the form of a circle at a distance of 3 cm from the center in an amount of 10 pieces. In the center of the Petri dish, a piece of agarized culture 0.5 cm in diameter was placed (die-cutting with a metal tube from a colony) or a biomass of liquid culture occupying the same area. In the control, no mushrooms were added. A carnivorous fungus from the center grew toward cysts, forming at first barely noticeable hyphae. After 2-3 days, the mycelium reaches the cysts and envelops them, conidiophores with conidia heads move away from the cysts in all directions. Under the stereoscopic microscope Stemi 2000-ZEISS, sporulation was seen in the form of conidia heads and whole garlands of them. On hyphae near the cysts, active formation of traps in the form of loops and their plexuses took place. The study of the contents of cysts is carried out on a glass slide after the destruction of their integrity using dissecting needles in a drop of water. From above, the destroyed cyst is covered with a coverslip and a temporary preparation is viewed under an Axsioskop microscope with an increase of 100 to 1000 times (see Fig. 1-4). The contents of the affected eggs differs from those normally developing by large granularity. In the affected eggs and larvae inside the cysts using a × 40 and × 100 microscope objective, fungal hyphae are clearly detected. After 5 days, the fungus forms a mass of chlamydospores on hyphae within the cysts, which are the life form of predatory fungi in the soil, their natural habitat. From the shells of the eggs, almost nothing remains. In the control on cysts, predatory bends were not found, on some cysts fluffy deposits of microscopic fungi from species living in the soil were formed. When viewing the contents of cysts, only live eggs were found in them, and sometimes larvae were not found, carnivorous fungi were found.

Example 3. Determination of nematophage activity of the inventive strain Arthrobotrys oligospora F-1303 in soil conditions.

The soil with cysts of potato nematode was placed in plastic vessels weighing 300 g; they added the biomass of the fungus obtained on grain at a dose of 1.0% by weight of the soil. This dose was previously determined in experiments as the most effective for fungi of the genus Arthrobotrys.

We used as control soil without introducing fungus, as well as soil with the introduction of another strain of carnivorous fungus, Daddingtonia flagrans F-882, which does not have the ability to penetrate the cysts of the potato nematode and infect the eggs and larvae inside them. However, this strain is active against motile larvae of nematodes located in the soil. The experiment was carried out in July 2015, potatoes of the Svitanok Kiev variety were used, pieces of tubers along with green sprouts were introduced into the soil. The task was to assess the total number of nematodes in the soil 1 month after laying the experiment. Nematodes were counted according to the Berman method. The average number of nematode larvae in 10 g of soil was: in the variant with Arthrobotrys oligospora F-1303-98, in the variant with Daddingtonia flagrans-192, in the control - 317. Thus, under the influence of Arthrobotrys oligospora F-1303, 1 month later, the soil remained in the soil almost 2 times less nematode larvae than under the influence of another strain, taken by us for comparison. This indicates that the Arthrobotrys oligospora F-1303 strain infects the eggs of the potato nematode at the cyst stage, preventing them from turning into larvae, thereby preventing their entry into the soil and re-infection of the roots.

Thus, the above examples 1-3 confirm the achievement of the claimed technical result, namely obtaining a strain of the predatory fungus Arthrobotrys oligospora, which affects not only the larvae of the cyst forming potato nematode located in the soil, but also the larvae and eggs of the potato cyst forming nematode inside the cysts.

Claims (1)

The strain of the nematophagous fungus Arthrobotrys oligospora T-15, which has the ability to infect the larvae of the potato nematode Heterodera rostochiensis in the soil, as well as penetrate the cysts, infecting eggs and larvae in them, and deposited in the All-Russian collection of industrial microorganisms (VKPM) under the number F-1303.

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