RU2548199C1 - METHOD OF PRODUCTION OF BIOLOGICAL PREPARATION FOR PROTECTION OF WINTER GRAIN CROPS FROM PINK SNOW MOULD Microdochium nivale - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: in the method the potato-dextrose agar (PDA) is prepared, the fungus is isolated in pure culture, the inoculum is prepared for infectious background, the nematodes (A. saprophillus) are isolated from the infected plants. The mycohelminths (A. saprophillus) are massively propagated on the fungus M. nivale at a temperature of +5°C. The suspension of mycohelminths A. saprophillus is prepared and applied in autumn on the fields affected by pink snow mould of winter grain crops, the suspension with the mycohelminths (A. saprophillus) at a dose of 160 thousand pcs/m².

EFFECT: method enables to reduce the number of fungicides used, destroys fungal mycelium of M. nivale of the pathogen PSM, promotes the plants to resist affected by PSM in the autumn-winter-spring period, provides obtaining the environmentally clean grain, and improves its quality.

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Description

A method of obtaining a biological preparation for protecting winter crops from pink snow mold belongs to the field of agriculture and can be used in biotechnology for biological protection of cereal crops.

Pink snow mold (RSP) is caused by the phytopathogenic low-temperature fungus Microdochium (Fusarium) nivale (Fr.) Samuels & I.C. Hallet Infection of plants occurs primarily through the soil, since the pathogen can persist on crop residues, but infection with seed is also possible. In heavily opened crops, the spread of infection is particularly promoted by high air humidity and low temperatures close to zero. If crops on unfrozen soil are covered with snow for a long time, especially favorable conditions are created for the development of this fungus. The disease becomes noticeable only in the spring after the snow melts. Immediately after the snow melts, the diseased plants are usually covered with filamentous mycelium, from dirty white to light pink, which disappears in sunny and windy weather. Abundant plaque formation leads to gluing of leaves; as a result, the affected leaves die off. With severe damage, the death of the tillering node, leaf sheaths, roots and the death of the whole plant is observed. Surviving plants may lag behind in their development, and inferior grains often form in their ears. The harmfulness of the CPD is the thinning of crops, and often their partial or complete death. With severe damage to plants, sometimes you have to resort to reseeding spring. The yield loss in this case can be 20-50%. To suppress CPD, chemical fungicides are used in preplant seed dressing and spraying as close as possible to the establishment of snow cover, the timing of which is difficult to predict. In addition, the use of fungicides has many negative aspects (pesticides contamination of soil and natural waters, accumulation of toxic substances in food, violation of the ecological integrity of soil biota, etc.). Currently, work is underway in many countries around the world to green the cultivation of crops, for which the biological method is of great importance.

The developed biological preparation for protecting winter wheat from CPD consists in the use of low-temperature mycohelminths as a biological agent. These are nematode-specific nematodes armed with a stylet, with which they pierce the walls of mycelium hyphae and suck out its contents. As a result, the mycelium of the fungus M. nivale dies.

We are not aware of the use of these mycohelminths in the fight against pink snow mold of winter wheat.

The purpose of the patent is to develop a technology for the biological protection of cereal crops from pink snow mold using specially selected high-performance low-temperature mycohelminths, as well as to develop optimal conditions for their use.

Examples of specific performance

To obtain a biological preparation, special techniques have been developed:

1. The method of preparation of potato dextrose agar (PDA)

To prepare a nutrient medium for mushrooms, we used a ready-made PDA from HIMEDIA (India).

Stir 39 g of powder in 1000 ml of distilled water. The flask with the solution was heated in a water bath until the particles were completely dissolved. Autoclaved at 1.1 atm (121 ° C) for 15 minutes.

2. The method of isolation of the fungus in a pure culture

The M. nivale fungus mycelium isolates taken from the affected plants were rinsed in sterile water and plated on PDA in Petri dishes and grown at room temperature. After 5 days, the mycelium of the fungus completely spread over the surface of the nutrient medium.

3. Methods of preparing the innoculum for an infectious background

Sterilized in an autoclave (at a pressure of 1.5 atmospheres, 60 min) and then cooled wheat grain was seeded with M nivale fungus. The ratio of wheat to water is 1: 1 (by weight). Then the flasks were placed in a menopause chamber (SanyoMPR-311D (H), Japan) for 30 days, maintaining t = 10 ° C.

4. The method of isolation of nematodes from affected plants

The materials for the research were collections of apparently healthy and affected CPF winter wheat plants and basal soil. To extract nematodes from different parts of plants and soil, a modified Berman method was used. Samples, cleaned of excess impurities, were laid out with a layer of 3-5 mm on a cotton filter, in a sieve. The sieve was inserted into a funnel with a diameter of 12-15 cm, on the socket of which a piece of rubber hose 10-15 cm long was put on. An entomological tube was inserted into the lower end of the tube to collect the isolated nematodes. A funnel with a rubber tube and a test tube was installed in a wooden tripod with specially made holes for the funnels. The funnel was filled with fresh tap water so that the liquid covered the mass sprinkled on the sieve.

The duration of plant exposure is 24 hours, for the soil - 48 hours. When determining nematodes, the number of females, males and larvae is taken into account. When analyzing the material under a microscope, a suspension of nematodes from an entomological tube was applied to a glass slide, viewed and the species was determined. In determining the species composition of nematodes, work was done on the systematics of nematodes (Paramonov, 1962).

5. The method of mass reproduction of A. saprophillus mycohelminth on the fungus Alternaria tenius Nees

The cultivation and reproduction of nematodes was carried out according to the method of A. Shesteperov. (1995). The results of Shesteperov's studies show that mycotrophic nematodes reproduce best on the fungus Alternaria tenius.

When propagating fungi, PDA was used. It was poured into 200 ml two-liter mattresses and covered with cotton-gauze plugs. Then autoclaved at an atmosphere of 0.5 for 0.5 hours. After sterilization, the mattresses were folded onto a flat surface until the medium was cooled and solidified. The fungus was sown on a completely cooled nutrient substrate and grown for 3-10 days in an incubator at a temperature of 26-27 ° C. Two-liter mattresses populated by the fungus were infected with an aqueous suspension of nematodes in an amount of 500 ind. and left at room temperature. After 30-40 days, the number of nematodes increased 80-100 times. The mattresses were stored in a climacteric chamber at a temperature of + 5 ° C.

6. The method of making inoculum on experimental plots

Grains overgrown with mycelium of the fungus were weighed from flasks with the inoculum of the fungus M. nivale, which was weighed on a torsion balance (100 g) and distributed in packets. Suspended and distributed by the inoculum of the fungus, it was evenly scattered over the surface of the experimental plot (1 × 1 m ² ).

7. Methodology for the preparation of an aqueous suspension of A. saprophillus mycohelminth

A. saprophillus was cultured in the laboratory on M. nivale fungus at t + 5 ° C. Mycohelminths were propagated in two-liter flasks. When the fungus mycelium was eaten in most flasks, the nematodes were washed off the walls of the flasks. The resulting suspension (in a volume of 800 ml) was brought to a volume of 1 l (for ease of calculation). The resulting solution was thoroughly mixed (using a magnetic stirrer) and the number of nematodes in 1 ml was calculated (1 ml = 100 copies).

The resulting suspension was shaken and poured into a sprayer. The nematode was sprayed onto the affected plants in the fall. In the spring, the biological effectiveness of the use of the A. saprophillus mycohelminth in the fight against CPD was determined, and in the autumn, economic efficiency was determined.

In laboratory experiments, mycohelminths Aphelenchoides saprophillus Franklin, Paraphelenchus tritici Baranovskaja, Aphelenchus avenae Bastian, were introduced in an amount of ± 100-200 ind. / tube (20 × 200 mm) on the mycelium of M. nivale fungus at temperatures of + 5 ° C, + 15 ° C and + 27 ° C. The temperature of + 5 ° C turned out to be the most favorable for the cultivation of mycohelminths of P. tritici, A. avenae and A. saprophillus on the mycelium of the fungus M. nivale. The breeding coefficient at this temperature in all species ranged from 4.8 to 7.1, while the nematodes used all the food resources presented (M. mycelium nivale mycelium) for 50-75 days. However, at a temperature of + 15 ° C, the breeding rate of mycogelminthes decreased (breeding coefficient - 4.65-6.09), which naturally affected the population size (Table 1). At the end of the experiment, in test tubes stored at a temperature of + 15 ° C, single dead individuals were noted, which was not observed in test tubes at a temperature of + 5 ° C. At the same time, in species P. tritici, A.avenae, and A. saprophillus, no breeding was recorded at a temperature of + 27 ° C (the breeding coefficient is 1.14–1.48).

Table 1 The effect of temperature on the number of mycohelminths on the mycelium of the fungus Microdochium nivale during cultivation Types of mycohelminths t = + 5 ° C t = + 15 ° C t = + 27 ° C Hcp ₀₁ Aphelenchoides saprophillus 661 570 146 23 Aphelenchus avenae 627 536 116 95 Paraphelenchus tritici 536 444 93 31 Hcp ₀₁ 7 16 91

An analysis of the obtained data on the cultivation of three types of mycohelminths on the fungus M. nivale showed that the temperature influenced the reproduction of nematodes and the terms of use of the presented food resources. It was found that the temperature + 5 ° C (close to the temperature under the snow cover) is favorable for the propagation of three species of nematodes, and the total number of nematodes at this temperature was quite high. Since A. saprophillus showed the most aggressive qualities for the fungus M. nivale (complete suppression of the growth of the fungus population in test tubes, while mycelium of the fungus was preserved with other types of mycohelminths) and it reproduced better at the three studied temperatures, compared with other types of mycohelminths significant difference (HCP ₀₁ -23). Further studies were conducted with this particular type of mycohelminth (Schukovskaya, A.G. 2012).

Conducted small-scale field experiments showed that the introduction of mycogelminthes in autumn on crops of winter wheat affected by CPN reduced the degree of disease damage in the spring of next year.

table 2 The influence of the number of mycohelminths on the development of winter wheat CPF Experience Options Qty Plants, pcs / m ² Number of productive stems, pcs / m ² Plant height, cm Spike Length, cm No. of grains in Kolos, pcs. The mass of grain in the ear, g Mass of 1000 grains, g Productivity, t / ha Control (M nivale fungus without introducing nematodes) 273.5 270,2 74.5 7.95 23.7 2.7 47.45 68 Mushroom M nivale + A. saprophillus (38,000 thousand specimens) 321 326 100.7 7.87 23.75 2.2 47.7 71.1 Fungus M nivale + A. saprophillus (80,000 thousand specimens) 338.75 333.75 101.7 8.5 25.5 2.19 44.75 78,2 Fungus M nivale + A. saprophillus (160,000 thousand specimens) 345.2 344.5 104 8.37 23.75 2.28 48.8 79,4 Hcp ₀₅ 10.1

In the experiments, the introduced nematodes significantly reduced the degree of development of the disease and improved the indicators of the productive qualities of winter wheat plants, which, in turn, affected the increase in yield.

In the variant where 160,000 thousand copies were introduced. Nematodes, the percentage of development of RSP was only 20.5%, while in the control the development of the disease was 2.5 times greater (Table 3). In options with the number of applications 80,000 thousand copies. and 38,000 thousand copies. nematodes, the development of the disease was (26% and 31.25%, respectively).

In the control, the affected plants had fewer productive stems (270.2) compared with the variant M nivale + A. saprophillus (160,000 thousand specimens) - 344.5 pcs. There was a decrease in the mass of grain in the ear, 1000 grains (47.45 g - control and 48.8 g in the variant M. nivale + A. saprophillus 160,000 thousand copies).

The calculation of biological effectiveness showed (Table 3) that in the variant where the maximum concentration of mycohelminths was added to 160,000 pcs / m ^{2, the} biological efficiency is 62.7%, in the variant with a concentration of 80,000 pcs / m ² - 52.7%, and with a concentration of 38000 pcs / m ² - 43.1%. In the variant where the nematodes were introduced in the maximum concentration, the economic efficiency was higher by 6.7%, in comparison with the variant where the average concentration was added and 45.3%, where the minimum concentration of mycohelminths was introduced.

Table 3 Biological and economic efficiency of the use of A. saprophillus mycohelminth suspension on winter wheat plants affected by CPN Option Biological efficiency,% The defeat of the CPD,% Economic efficiency,% Productivity, t / ha M. nivale (control - without introducing nematodes) 0 55 0 68 M. nivale + min. 43.1 31.25 4.3 71.1 concentration (38000 ind./ plot) M. nivale + environments concentration (60,000 ind / plot) 52.7 26 13.04 78,2 M. nivale + max. concentration (160,000 ind / plot) 62.7 20.5 14.45 79,4 19 19.6 10.5 10

The presented research results were statistically processed in Microsoft Excel (Statstica).

The list of essential features of the invention

When using a suspension of mycohelminth A. saprophillus in the fight against mycelium of RSP, the following significant signs were noted:

- Reduces the degree of development of CPD in the autumn-winter-spring period.

- Helps plants withstand CPD damage in winter.

- Provides environmentally friendly grain and improves its quality.

- Does not enter phytopathogenic organisms on crops.

Literature

1. Shesteperov A.A., Savotikov Yu.F. “Quarantine phytohelminthoses” // Book 1. - M .: Kolos, 1995 .-- 463 p.

2. Schukovskaya A.G., Shesteperov A.A., Tkachenko O. B. “Winter wheat mycohelminths are potential bioagents of the Microdochium nivale fungus” // Mat. scientific conf. "Theory and practice of controlling parasitic diseases." M. 2012. Issue 13. S.466-468.

3. Hoshino T., Xiao N., Yajima Yu., Tkachenko O.B. Fungi in Cryosphere: Their Adaptations to Environments. Part 1. Biodiversity in Cold Ecosystems. Chapter.

4. Cold-adapted Microorganisms. Edited by Isao Yumoto. Norfolk, UK: Caister Academic Press, 2013 .-- P.51-68.

Claims (1)

The method of introducing a biological preparation for protecting winter crops from pink snow mold (Microdochium nivale), which consists in preparing potato-dextrose agar (PDA), isolating the fungus in a pure culture, preparing an inoculum for an infectious background, isolating nematodes (A. saprophillus) from affected plants, mass propagation of mycohelminth (A. saprophillus) on M. nivale fungus at a temperature of + 5 ° C, preparation of a suspension of A. saprophillus mycohelminth and application of winter crops to the fields affected by pink snow mold in autumn sii with mycohelminths (A. saprophillus) at a dose of 160 thousand pcs./m ² .