RU2798766C1 - Method of determining microbial-plant interaction in the process of development of microorganisms by measuring heat release from it and a device for its implementation - Google Patents

Abstract

FIELD: biotechnology, experimental veterinary medicine, medicine and pharmacology.

SUBSTANCE: invention relates to a method and device for determining microbial-plant interaction. The method of determining the microbial-plant interaction in the process of development of microorganisms by measuring the heat release from it includes the following stages: a plant test sample is placed in the measuring cell; placing the test microbial sample in a passive state on the plant test sample or at a distance at which they can exert their influence on the development of the plant test sample when they enter the active state; create conditions for the development of plant test and microbial samples under study; monitoring the heat release of the plant test sample with the microbial sample under study at the first stage, when the microbial sample under study is still in a passive state in the lag phase, and at subsequent stages i, when the test sample enters the active log phase; determine the activity coefficient of the plant test sample from the following equation: G2=(dQ/dt)_i/(dQ/dt)_l-1, where dQ is the heat released; dt is a unit of time, while the activity coefficient at the first stage must coincide with the previously measured activity coefficient of the plant test sample under similar conditions without the studied microbial sample, calculated from the equation: G1=(dQ/dt)_l; the activity coefficients of the selected time intervals i are calculated, according to which the influence of the microbial sample under study on the plant test sample is judged.

EFFECT: ability to study the activity of interaction with plants mold fungi.

4 cl, 5 dwg, 1 tbl

Description

The invention relates to agricultural biotechnology. It can be used in veterinary medicine and medicine, as well as in pharmacology to control technological parameters in the production of medicines. In addition, to develop express methods and equipment for in-depth study of the influence of various factors on the development of biological objects, including microbial-plant interaction; for accelerated breeding of new plant varieties resistant to the action of harmful microorganisms.

Known methods [Calve E., Prat A. Microcalorimetry. Devices and methods. Application in physical chemistry and biology, Moscow, Izd-vo IL, 1963. - 478 p.] and devices [Boyko B.N. Applied microcalorimetry "Nauka", Moscow, 2006.] to determine the activity (viability) of plants and microorganisms, based on the registration of heat release from the samples under study. They are successfully used to study the biochemical reactions that occur in a living organism to maintain life, to study the processes that allow organisms to grow and reproduce, maintain their structures and respond to environmental influences. Existing methods and devices, however, make it possible to determine the physiological state (active or passive) of only the same type of plant or microorganism populations. The authors are not aware of instrumental methods that would make it possible to determine the activity and strength of the interaction of two different types of biological objects in the process of development.

It is known that the role of microbial-plant interaction in microbial-plant symbioses is of great importance in agrobiotechnologies [Kovalevskaya N.P., Sharavin D.Yu., Bessonova L.V., The role of microbial-plant symbioses in increasing the stress resistance of crops in the Cis-Urals . /https://cyberleninka.ru> article> rol-mikrobno-rastiteln…, 2018].

The closest method and device - the prototype - for determining the activity (viability) of biological objects is the method of differential thermograms based on microthermistors for an accelerated assessment of the viability of the E. Coli bacterial population [A.I. Drapeza, N.V. Pleshko, V.A. Loban, G.A. Skorokhod, E.I. Gudkova The method of differential thermograms based on microthermistors for an accelerated assessment of the viability of the bacterial population of E. Coli.].

The disadvantage of the prototype is:

1. A relatively large spread in the measured differential temperature on the given thermograms up to 0.005 ° C, which requires the use of “methods of statistical analysis” for their processing over a time interval of up to 80 minutes. This is probably due to the insufficiently high sensitivity of the temperature sensors in the measuring cell, which does not allow obtaining thermograms with a more stable (more even) change in the value of the measured differential temperature. A possible explanation for the chaotic fluctuations of thermograms within +/- 0.005 degrees may be insufficiently good thermal protection of the measuring cell from external heat fluxes.

2. The inability to determine the microbial-plant interaction in the process of development of microorganisms.

The objective of the invention is the possibility of determining the microbial-plant interaction in the process of development of microorganisms.

The essence of the invention lies in the fact that in the method for determining the microbial-plant interaction in the process of development of microorganisms by measuring the heat release from it, characterized in that

- place the plant test sample in the measuring cell;

- placing the tested microbial sample in a passive state on the plant test sample or at a distance at which they can exert their influence on the development of the plant test sample when they enter the active state;

- create conditions for the development of plant test and microbial samples under study;

- monitor the heat release of the plant test sample with the microbial sample under study at the first stage, when the microbial sample under study is still in a passive state in the lag phase, and at subsequent stages i, when the test sample enters the active log phase;

- determine the activity coefficient of the plant test sample from the equation:

G2=(dQ/dt) _i /(dQ/dt) _l -1,

where dQ is the heat released;

dt - unit of time,

at the same time, the activity coefficient at the first stage should coincide with the previously measured activity coefficient of the plant test sample under similar conditions without the studied microbial sample, calculated from the equation:

-G1=(dQ/dt) _l ;

- calculate the activity coefficients of the selected time intervals i, which are used to judge the effect of the studied microbial sample on the plant test sample.

The essence of the invention also lies in the fact that in the method for determining the microbial-plant interaction in the process of development of microorganisms by measuring heat release from it, characterized in that at least three measuring cells are used with simultaneous monitoring of heat release from each cell in real time.

The essence of the invention also lies in the fact that a device for determining microbial-plant interaction in the process of development of microorganisms, including an installation with a microcalorimetric chamber, consisting of a heat-insulated block, the outer cylindrical frame of which is made of aluminum, and the inner one is made of copper, the frame cylinders are placed in a thermostat, in which the temperature is maintained with an accuracy of 0.1 degrees, between the aluminum and copper cylinders there is a thermostatic shell with a heater and an electronic unit for stabilizing the temperature, a thin-walled tube is brought out to humidify and introduce samples into the measuring cells, inside the chamber there are measuring and comparative cells, identical according to its design, the cell diameter is 25 mm, the height is 40 mm, at the bottom of the cells there are semiconductor thermosensitive elements with a sensitivity of at least 50 mV/degree, the unit is designed to monitor thermograms on a PC in real time.

The essence of the invention also lies in the fact that a device for determining the microbial-plant interaction in the process of development of microorganisms, characterized in that at least three measuring cells are used.

At the same time, according to the invention, the activity of a biological object, for example, seeds of cereals, then the first test biological object, is proposed to be determined by measuring the heat release from it, for example, by the method of differential thermal analysis (DTA).

Distinctive features of the proposed method is that in order to detect the presence of a second biological object with a very small, insufficient for measurement, heat release, to assess its biological activity (viability), for example, mold fungi, to determine the microbial-plant interaction with the first test bio-object, the first test bio-object placed in the measuring cell near a high-resolution temperature-sensitive element, create conditions for it and for the second biological object for development over a sufficiently long time interval 1-iN, in which the first does not change its active state, and the second changes its activity leaving the passive state in the process of development , where 1 is the initial and N is the final boundary of this interval. Next, the heat release from the first test bioobject is measured, the activity coefficient is determined for it according to the formula G1=(dQ/dt) _l , and the presence of the second one, its activity (viability) is determined at subsequent time intervals i according to the activity coefficient from the formula G2=(dQ/dt ) _i /(dQ/dt) _l -1, in which G2 at the first time interval takes on the value zero when the second biological object is in a passive state, positive - in the case of a stimulating effect of the second biological object on the development of the first test, and negative - with the overwhelming influence of the second bioobject for the development of the first test. In this case, the value minus one corresponds to the complete suppression of the development of the first test bioobject by the second.

The essential features of the invention include:

1. The use of highly sensitive semiconductor elements with a sensitivity of at least 50 mV / degree, which can significantly increase the reliability of measurements and simplify the registration of thermograms in real time by improving the signal-to-noise ratio.

2. Use as a test sample (as an example) of seeds of cereals and vegetable crops, which provide a sufficiently high heat release during germination when creating known conditions for exiting dormancy and subsequent development. At the same time, the seeds of grain and vegetable crops are subject to the influence of microorganisms, in particular mold fungi, which the authors propose to use to determine the biological activity and, at the same time, the viability of microorganisms, as well as to study the microbial-plant interaction.

3. The use of the first test sample, for example, wheat seeds over the entire time interval under study in the active state, and the second sample, for example, mold fungi, with changing activity, which makes it possible, when interacting with the change in the activity of the first test sample, to judge the change in the activity of the second ( microorganisms).

4. Using the obtained mathematical expression to determine the parameters characterizing the physiological state of the studied biological objects, including their biological activity and microbial-plant interaction.

To determine the microbial-plant interaction in the process of development of microorganisms placed in the measuring cell during the test period, showing their activity in the event of their viability, is determined by the activity coefficient (growth intensity) according to the formula G1=(dQ/dt) _l , where dQ is the isolated heat from the biological object per unit time dt. Index - the unit in the above expression indicates the initial time interval. Microbial-plant interaction in the process of development of microorganisms of a biological object should be expressed in units of mikelvin (mK) per unit of time equal to an hour - mK/h. One mikelvin is equal to 0.001°C.

The minimum values in the study of germination activity in the installation (device) used by us are at the level of unit mK/h. Typical values for the studied various samples of seeds of wheat, amaranth and vegetable crops for the selected modes in the installation used are, when determining the activity (viability) of biological objects, from units to 80 mC/h (80 mC/h - under the action of growth accelerators).

When several biological objects are located in the measuring cell, the intensity of heat flows from them is summed up in accordance with the activity of the studied biological objects and the conditions of heat transfer from the studied sample to the temperature-sensitive element.

It should be noted here that the heat fluxes that must be supplied to the temperature-sensitive elements and characterize the activity of mold fungi are extremely low due to their very low density and high friability. This makes it extremely difficult to measure heat fluxes from them, especially at the stage of their exit from the dormant state (lag phase), and it is practically impossible to simultaneously measure the heat flux from germinating plant seeds and the heat flux from mold fungi and, at the same time, determine accordingly their activity in the process of development in terms of heat release.

As an example of a specific implementation of technical solutions, the algorithms of the proposed method for determining the microbial-plant interaction in the process of microorganism development are given below.

same type bioobjects. The algorithm for determining activity and evaluating viability for the same type of biological object is based on measuring heat release directly from the test sample and includes the following operations:

1. The biological object is placed in the measuring cell close to the temperature-sensitive element.

2. Create conditions for germination.

3. Monitor the heat release from the test sample.

4. The activity of the biological object is determined by calculating the activity coefficient from the expression G1=(dQ/dt) _l .

5. The value of G1 is used to judge the activity and, accordingly, the viability of the biological object. The minimum values for standard germination conditions in the plant used are at the level of one mK/h. Typical values for the studied test samples with wheat grains for the installation used are from 1 to 80 mK/h.

6. To study the reaction of a biological object to external influences, repeat steps 1-5, registering the features of changes on the thermogram depending on the impact.

Diverse bioobjects

An algorithm for determining the microbial-plant interaction during the development of microorganisms and assessing the viability of two types of biological objects based on the analysis of thermograms, one of which is constantly in an active state and is a test sample, and the second is in a passive lag phase for the period of placement in the measuring cell.

Method and device for its implementation for determining the physiological state (activity) and viability, for example, of Mucor-type molds by their effect on the activity of a test sample.

1. Place the test sample in the measuring cell.

2. The biological object under study is placed in a passive state - for example, mold spores in a measuring cell on a test sample or at a distance at which they can exert their positive or negative influence on the development of a test sample when they switch to an active state.

3. Create conditions for the development of test and test samples.

4. The heat release of the test sample with the test sample is monitored at the first stage, when the biological object under study (for example, mold fungi) is still in a passive state in the lag phase and then at subsequent stages i, when the test sample passes into the active log phase.

5. Determine the activity coefficient of the test sample from the expression G2=(dQ/dt) _i /(dQ/dt) _l -1 at the first time interval 1 dQ/dt) _l when the test sample is still in a passive state. Previously, when choosing a test sample, this value must be measured under similar conditions, but without a second sample. Values G1=(dQ/dt) _l previously measured without the second bioobject and the value measured according to paragraph 5 with the second bioobject under test must coincide in the first interval within the limits of the error of the experiments.

6. Calculate the activity coefficients for the test sample (for example, molds of the Mucor type) according to the formula G2=(dQ/dt) _i /(dQ/dt) _l -1 at the selected time intervals i and judge the physiological state of the biological object under study.

The activity of the bioobject G2 takes the value minus one for the test sample, when by its activity it completely suppresses the development of the first test sample in the presence of nutrition and favorable conditions for development (growth), zero - when it practically does not have any effect on the development of the test sample.

The claimed technical solutions differ from the prototype in new functionality, including the ability to determine the biological activity (viability) of fungi and, at the same time, microbial-vegetative interaction with seedlings of grain and vegetable crops.

The proposed method for determining the activity of mold fungi and assessing the interaction with the first test biological object in the form of plant seeds is based on monitoring the biological activity of the test bioobject, which has a sufficiently high heat release in the active state, and its activity significantly depends on the activity and strength of interaction with mold fungi, in particular Mucor type.

Technical solutions are implemented on a setup with a microcalorimetric chamber (Fig. 5). The studied biological objects are placed in a measuring cell with a diameter of 25 mm and a height of 40 mm. At the bottom of the measuring and comparative cell, high-resolution semiconductor thermosensitive elements are fixed (Fig. 5, position 1).

The measurement and comparison cells are identical. Thermal sensing elements are also identical, included in the bridge circuit. To register heat releases from the studied biological objects, differential thermal analysis was used - the signal from the measuring bridge is measured. The input stages of electronic units made it possible to measure signals from 1 microvolt with the registration of signals on a PC. The device allows you to monitor thermograms on a PC online with a sufficiently high resolution (Fig. 1-3).

The measuring and comparison cells are placed in a carefully thermally insulated block, which includes several cylindrical frames made of aluminum (external) and copper (internal). Frame blocks are placed in a thermostat, in which the temperature is maintained with an accuracy of 0.1 degrees. Between the aluminum and copper cylinders there is a thermostatic shell with a heater (Fig. 5, position 3) and with an electronic unit for temperature stabilization. A thin-walled tube 5 was introduced into the measuring cell to humidify and introduce the substance.

The temperature resolution in the measuring cell is up to 0.0003 degrees. The parameters of temperature-sensitive elements with electronic blocks allow, if necessary, to increase the temperature resolution up to 0.0001 degrees. The presented technical solutions make it possible to measure heat release with a significant excess of the useful signal relative to noise, which is clearly seen in the thermograms (Fig. 1-3).

To expand the presented functionality for determining the activity of biological objects, it is proposed to use at least three measuring cells, which will expand the possibilities of studying biological objects with different properties under identical conditions (different varieties, different storage times, different pre-treatment, interaction of biological objects located in neighboring measuring cells). ). A comparison cell with identical characteristics is included with each measuring cell according to the DTA method.

The use of 3 channels when testing identical samples makes it possible to increase the reliability of the results of precision measurements, and when studying (monitoring) samples with slightly different properties, revealing more subtle features of the development of biological objects and their interaction.

Table -1 shows the results of the analysis of the intensity of development of the test object (germination of wheat seeds) at various time intervals and the activity indicators of Mucor molds in the process of development.

In the initial section from the beginning of germination to the time mark of 19.1 hours, an almost constant slope of 34.7 mK/h is observed. This corresponds to the germination of the test sample under the selected conditions in the absence of any side effects on it. In this area, the mold fungi were still in the passive lag phase and did not have a detrimental effect on the test sample. Therefore, this value corresponds to the values for the intensity of germination of test samples, measured without the presence of any second biological objects, equal to 34.7 mK/h.- (Table 1). In subsequent sections 2 and 3, the activity coefficient of the test sample decreases markedly, since the activity of mold fungi increases in these intervals.

During the transition of molds of the Mucor type from the passive phase to the active phase in section 2 in the interval from 19.1 hours to 68.7 hours, the value for the intensity of growth of the test image is 22.3 mK/h.

Further, in section 3, in the interval from 68.7 hours to 93.6 hours, the growth rate of the test sample during the active phase of molds drops to a value of 8.7 mK/hour. The value for the activity of the test sample G2 in this time period is (-) 0.75, which, with a maximum value of one, indicates a significant influence of the Mucor mold on the test sample. The minus sign indicates its inhibitory effect on the test sample in the form of grains of wheat seeds.

The proposed method and device make it possible to monitor on a PC online the change in the physiological state of microorganisms, in particular molds of the Mucor type, already when they pass from the passive state of the lag phase to the active log phase, to determine their viability and microbial-plant interaction with wheat seeds.

The figure 1 shows the dependencies of the output signals from the three measuring cells in the absence of the studied biological objects in them. It is clearly seen that the zero lines are quite stable, and the output signal from the temperature-sensitive elements changes from the zero line by no more than +/- 25 μV at a sensitivity of 50 mV/degree.

The figure 2 shows the thermogram of wheat seeds in the process of germination. The sufficiently high sensitivity of the device for recording heat release from the studied wheat seeds weighing 1 gram, moistened with one milliliter of distilled water, is clearly visible. In figure 2, one division along the X axis is 1000 seconds or 16.666 minutes, one division along the Y axis is 100 μV or 0.002 degrees. The left section of the thermogram along the X axis (40 minutes from the start) displays the process of moisture absorption. The next 90 minutes on the graph correspond to the active state of the studied wheat seeds. There is a release of thermal energy, leading to an increase in temperature in the measuring cell with an intensity of 55.8 millidegrees per hour. A full increment in the graph shown in 1.5 hours caused a temperature change of 84 millidegrees. The selected degree of moisture is sufficient for seed germination with a constant activity coefficient G1=(dQ/dt) _l for 1.5 hours.

The figure 3 shows the thermogram of the germination of wheat seeds with molds of the Mucor type. One division along the X axis is 50000 seconds or 13.888 hours, one division along the Y axis is 200 millidegrees or 0.004 degrees.

The decrease in the intensity of heat release from the studied samples approximately from the middle of the thermogram is explained by the increase in the intensity of growth of mold fungi in this period. Since the Mucor type molds have an overwhelming effect on seed germination, at this stage, due to the increase in mold activity, we observe a decrease in the activity coefficient for the test sample.

To photograph the presence of mold fungi in the measuring cell, the experiment was interrupted after 100 hours from the start. On the thermogram, respectively, there is a sharp peak and some deviation of the curve from the steady state, caused by the opening of the measuring cell.

In figure 4, in the photograph of the measuring cell, wheat seedlings are clearly visible, covered with loose growths of mold fungi, which is consistent with changes in the thermogram in figure 3.

Figure 5 shows a microcalorimeter chamber, measuring and comparison cell with temperature sensitive elements.

The results of the experiments to confirm the performance of the proposed technical solutions are summarized in Table 1.

The proposed method allows you to study the microbial-plant interaction at a qualitatively new level.

The invention can be used in experimental veterinary medicine and medicine, in pharmacology to control technological parameters in the production of drugs from molds.

In addition, it can be useful in the field of agricultural technologies for the development of express methods and equipment for assessing the physiological state of biological objects, studying the influence of various factors on the development of biological objects, for accelerating the selection of new plant varieties, and express determination of seed viability.

Claims (15)

1. A method for determining the microbial-plant interaction in the process of development of microorganisms by measuring heat release from it, characterized in that - place the plant test sample in the measuring cell; - placing the tested microbial sample in a passive state on the plant test sample or at a distance at which they can exert their influence on the development of the plant test sample when they enter the active state; - create conditions for the development of plant test and microbial samples under study; - monitor the heat release of the plant test sample with the microbial sample under study at the first stage, when the microbial sample under study is still in a passive state in the lag phase, and at subsequent stages i, when the test sample enters the active log phase; - determine the activity coefficient of the plant test sample from the equation: G2=(dQ/dt) _i /(dQ/dt) _l -1, where dQ is the heat released; dt - unit of time, at the same time, the activity coefficient at the first stage should coincide with the previously measured activity coefficient of the plant test sample under similar conditions without the studied microbial sample, calculated from the equation: - G1=(dQ/dt) _l ; - calculate the activity coefficients of the selected time intervals i, which are used to judge the effect of the studied microbial sample on the plant test sample. 2. The method according to claim 1, characterized in that at least three measuring cells are used with simultaneous monitoring of heat release from each cell in real time. 3. A device for determining microbial-plant interaction during the development of microorganisms, including an installation with a microcalorimetric chamber, consisting of a heat-insulated block, the outer cylindrical frame of which is made of aluminum, and the inner one is made of copper, the frame cylinders are placed in a thermostat, in which the temperature is maintained at with an accuracy of 0.1 degrees, a thermostatic shell with a heater and an electronic unit for temperature stabilization is located between the aluminum and copper cylinders, a thin-walled tube is brought out to moisten and introduce samples into the measuring cells, inside the chamber there are measuring and comparative cells, identical in their design, diameter cells is 25 mm, height - 40 mm, at the bottom of the cells there are semiconductor thermosensitive elements with a sensitivity of at least 50 mV/degree, the installation is made with the possibility of monitoring thermograms on a PC in real time. 4. Device according to claim 3, characterized in that at least three measuring cells are used.

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