DE112006000480B4 - Method for evaluating the vitality of chlorophyll-bearing biological samples - Google Patents

Abstract

Method for evaluating the vitality of chlorophyll-bearing biological samples with a time course measurement of an oxygen concentration (c) in an immediate spatial environment of the biological sample with changing illumination by measuring a reaction time (D) between the time of an abrupt change of illumination and the time of a local extreme in the time-dependent oxygen concentration for determining the adaptation behavior of the photosynthetic power, wherein the time course measurement of the oxygen concentration (c) in an immediate spatial environment of the biological sample is carried out with changing illumination with the following method steps: - a measurement of a temporal change in the oxygen concentration (c) during a dark time (tD) a switched off illumination, - a measurement of the reaction time (D) between a switch-on time (ton) of the lighting and a time (tmin) of a switch-on time following local minimum of the oxygen concentration, - a measurement of a temporal change in the oxygen concentration during a bright time when the lighting is switched on, - a determination of a set when the lighting illumination saturation value (M) of the oxygen concentration, characterized in that from the temporal change of the oxygen concentration (c ) during the dark time (tD), this temporal change descriptive Respirationsparameter (R) and from the temporal change of the oxygen concentration (c) during the bright time, this time change descriptive slope parameter (S) is determined, wherein from the negative ratio of the slope Parameters "S" and the respiratory parameter "R" a coefficient of photosynthetic activity KphA = -S / R is determined.

Description

The invention relates to a method for evaluating the vitality (life energy) of chlorophyll-carrying biological samples according to the preamble of claim 1.

Green plants are autotrophic organisms. This means that they absorb inorganic substances with the aid of light energy, in particular from the radiation spectrum of sunlight, and convert them into organic substances, in particular carbohydrates, fats and proteins. The plants are known to take carbon dioxide from the air and water and mineral salts from the soil and release molecular oxygen into the environment. This process of autotrophic assimilation is called photosynthesis and occurs within the chloroplasts of plant cells. In the following, the term "plants or plant parts" is understood to mean all autotrophic organisms or parts of such organisms.

The basic reactions in the cycle of the assembly and disassembly of organic material can be formulated as follows: Photosynthesis (assimilation) 6CO ₂ + 6H ₂ O → C ₆ H ₁₂ O ₆ + 6O ₂ CO _{2 is} taken up and oxygen is released (exchange of gaseous products). Cellular respiration (dissimilation) C ₆ H ₁₂ O ₆ + 6O ₂ → 6CO ₂ + 6H ₂ O whereby oxygen is taken up and CO _{2 is} liberated (exchange of gaseous products).

First of all, the evidence of oxygen or carbon dioxide in these processes appears to be equivalent and equally meaningful in these processes. The above-mentioned reaction equations are greatly simplified.

In assessing the effects of certain agrochemical active ingredients or fertilizers, for example herbicides or insecticides and various artificial fertilizer preparations, it is necessary to assess the so-called vitality of the plants under the effect of the substances mentioned. This assessment criterion is currently not clearly defined and so far results from a subjective rating by farmers, gardeners, foresters or other experts who can estimate on the basis of many years of experience, whether a respective tested plant on the treatment with the appropriate drug "good", "Less good" or "detrimental" responds or behaves sufficiently resistant to the drug. For this purpose, the focus is primarily on visual impressions, which naturally provide an exclusively qualitative and very highly subjective evaluation basis. Moreover, in this type of evaluation almost exclusively optical impressions of the plants are observed and taken over into the evaluation calculation. Furthermore, assessments by "eye-sighting" only lead to the result if the plant is usually already succumbing to the influences positively or negatively.

As a criterion of effect z. B. the yield, but a premature indication is not possible; another criterion is already present disease symptoms, but early detection is not possible here. It is understood that such an approach in terms of accuracy and objectivity is extremely unsatisfactory and can only provide results that are to be compared and generalized with caution. The assessments (scores) lead to the labeling "plant vitality". Vitality ratings (eg number and quality of leaves and growth in comparison to the norm state) are ultimately qualitative statements of the energy supply in the plant cells and their use for the development of reserve resources, biomass and resistance. In this respect, it is more than natural for quantitative statements on plant vitality to favor the detection of the reaction products, which are a direct indicator of the energy supply in the plant cells. In the above comparison of CO ₂ and O ₂ , therefore, the choice turns out clearly in favor of O ₂ , since this product is formed in the release of protons that provide the energy storage. Therefore methods are sought, which measure the energy supply (production and consumption).

To measure the photosynthetic activity of plants as a hallmark of vitality, a number of methods and devices are known from the prior art. For example, in the DE 43 32 290 C2 or the DE 10 2004 058 402 A1 proposed a number of suitable devices in which the oxygen balance of a trapped in a transparent measuring cell plant or a plant part under the light incidence of a high-power light emitting diode by means of an oxygen measuring probe is detected. The cited documents propose for this purpose a number of devices which are suitable in the one case for normal leaves or Laubblattabschnitte and in other cases for needles derived from conifers or even for algae.

From the DE 43 32 163 A1 a generic method is previously known, where it is about to implement a parallel determination of different variables, namely on the one hand the fluorescence and on the other hand, the evolution of oxygen to ultimately assess the physiological state of an algae-water mixture can. Depending on a continuous light irradiation, the course of the evolution of oxygen and the fluorescence signals emitted by the mixture are detected. The measured values of fluorescence and oxygen evolution are then compared as the basis for a pollutant analysis.

According to the method for detecting and evaluating light-induced changes in chlorophyll fluorescence DD 300 049 A7 It is known to generate a cycle with defined pre-exposure and defined dark break by means of an electronic control and then to process the detected characteristic values. Specifically, not the dark adapted state of the plant to be examined, but a defined by pre-exposure and short dark break intermediate state is used.

In the method for characterizing the photosynthetic system of plants DE 44 27 438 A1 After a dark break, an irradiation with rectangular light pulses is performed, whereby the induced fluorescence radiation is detected. According to the teaching there, the dark phase is shorter than one second and the rectangular light pulse is generated by a laser, the pulse duration being between one and 30 msec.

It is therefore the object underlying the invention to provide a quantitative method for determining the vitality of plants or parts of plants, which ensures an objective assessment of plant vitality under well-defined conditions.

The solution is carried out with a method for assessing the vitality of chlorophyll-carrying biological samples according to the features of claim 1, wherein the claim 2 describes a convenient and advantageous embodiment of the method.

In these methods, a time course measurement of an oxygen concentration in an immediate spatial environment of the biological sample with changing illumination in conjunction with the measurement of a reaction time between the time of a change of illumination and the time of passing through an extreme value in the time-dependent oxygen concentration.

The method is based on the experimentally confirmed finding that the vitality of plants is particularly evident in their adaptive behavior of photosynthesis performance. Appropriately, a system description is hereby transferred to reaction kinetic parameters, whereby system properties such as elasticity in the reaction of the plant, potential overloadability (stress), current system status of the plant, etc. are determined. In this connection, reference should again be made to the above-mentioned context of CO ₂ or O ₂ detection. Comparisons of the behavior of CO ₂ and O ₂ show that the solubility of CO _{2 is} 23 times greater than that of O ₂ , resulting in a kind of buffer in the case of CO ₂ , which can lead to delayed light-change dynamics , And since CO _{2 has} a higher molecular weight, CO ₂ diffuses about 20 times better than O ₂ at the same concentration gradient. These properties clearly indicate a detection of O ₂ over CO ₂ when comparing reaction kinetic parameters. In the situation underlying the method, the photosynthetic performance in the form of oxygen concentration under both different lighting conditions, as well as an abrupt, rarely occurring in the natural environment lighting changes studied and recorded in the form of objectivable parameters that are independent of the purely visual assessment of the state of the plant ,

The time course measurement of the oxygen concentration in an immediate spatial environment of the biological sample with changing illumination takes place with the following method steps. In a first step, a measurement of a temporal change in the oxygen concentration during a dark time is performed when the lighting is switched off. Then, a delay time between a switch-on time of the illumination and a time of an extreme value following the switch-on time is measured over the course of time of the oxygen concentration. This is followed by a measurement of a change over time in the oxygen concentration during a bright time with the lighting switched on. The saturation value which then occurs when the lighting is switched on is also determined. Finally, the measured changes in the oxygen concentrations are analyzed in a data-processing evaluation.

Such a procedure can be transferred and implemented in a very simple manner into a measurement program for the automated assessment of vitality, wherein the measured variables to be determined are clearly defined.

According to the invention, a temporal change of the oxygen concentration during the dark time is used to determine a respiration parameter describing this temporal change. Furthermore, a temporal change of the oxygen concentration during the clearing time is used to determine a slope parameter describing this temporal change.

Both parameters describe the manner of changing the concentration of molecular oxygen in the environment of the sample during the respective periods in a clearly defined manner.

From the ratio of the slope parameter and the respiration parameter, a photosynthetic activity coefficient is determined. This coefficient describes the ratio of the photosynthesis performance during the light-time to the respiration process of the plant sample during the dark period and thus provides an indication of the relationship between the two states of activity of the plant in the light and in the dark.

The respiration parameter and / or the slope parameter are readily determined by fitting a linear function to the measured time-varying oxygen concentration values during the dark and light periods, respectively. Linear adjustments can be programmed and automated in a particularly simple way by the mathematical method of linear regression.

The inventive method will be described below with reference to embodiments and the 1 to 12 be explained in more detail. The same reference numbers are used for identical or equivalent parts and method steps.

It shows:

1 an exemplary arrangement for carrying out the method,

2 an exemplary time-dependent oxygen concentration measured during the process,

3 a diagram for the reproducibility of the results obtained by the method using the example of the Slope parameter, measured on wheat leaves of plants that are under the influence of various external influences.

4 a diagram of an exemplary correlation analysis of individual measurement series of the slope parameter "S" using the example of wheat leaves,

5 an exemplary diagram of a series of time-dependent oxygen concentrations, measured on wheat leaves with different inoculation variants with a number of fungi at the end of a first trial week,

6 an exemplary diagram of a dynamics of the photosynthetic activity of wheat leaves of individual variants in the course of ontogenesis,

7 an exemplary diagram of the photosynthetic activity of wheat leaves of various inoculation variants in a trial period between the fourth and ninth trial week,

8th a diagram comparing the vitality dynamics of the individual inoculation variants over 14 days,

9 a diagram for a comparison of a vitality dynamics of the individual inoculation variants over the course of 39 days,

10 a diagram of the effect of humic substances of different concentrations on algae growth within a period of 21 days,

11 Course of the oxygen balance as a function of the entry of organically bound carbon (see box) on the humic substances. The decisive parameter used here is the vitality parameter "S" for further evaluation.

12 a correlation diagram between a conventional biological state parameter (here the presence of hydrogen peroxide with concentration) and the inventively determined photosynthesis performance (parameter "S") of the unicellular algae.

1 shows an exemplary arrangement for carrying out the method. In a protected against environmental influences, especially light and temperature fluctuations measuring station 10 there is a measuring cell 20 with a biological sample enclosed therein 15 , As biological sample both aqueous solutions with phytoplankton, for example green algae in fresh or salt water, sections of shoot plants, for example parts of leaves or needles or microcultures in a suitable plant substrate, in particular seedlings from a germ husk, can be used. For meaningful measurement results, in general, one area of the chlorophyll-carrying biological sample, for example a leaf section or an algae solution, with a surface area between 5 and 20 mm ^{2 is} sufficient. Furthermore, the temperature is recorded both at the sensor and in the sample holder. Furthermore, the light source is arranged to illuminate the underside of the sample.

The measuring cell 20 consists of a translucent biologically neutral glass or plastic material, which has a particularly high transparency especially in the visible red light spectrum in the wavelength range of about 620 nm to 700 nm. The measuring cell may cover the sample either completely or partially to ensure a microenvironment above the sample. As a measuring cell, in particular a cuvette can be used. Into the interior of the measuring cell protrudes directly into the environment of the biological sample, an electrochemical sensor 25 , Preference is given here to a sensor of the Clark type.

The light source 30 preferably consists of a high-power LED that emits light in the predominantly red spectral range between 620 nm and 700 nm. Conveniently, the LED is immediately adjacent to the measuring cell 20 arranged in such a way that the light source, the sample and the detector are on a common axis. An arrangement for uniform irradiation, which is at least the sample 15 Homogeneous illumination on one side is particularly advantageous.

The named components are assigned a series of control or evaluation units. A timer 35 , In particular, a timer or a time signal generating software program, both gives a timing to a control electronics 36 for the light source 30 , as well as to a protocol unit 45 out. Furthermore, a measured value acquisition unit 40 for the electrochemical sensor 25 provided that the sensor signals according to an internal calibration function in measured values for the oxygen concentration and the measurement temperature at the target within the measuring cell 20 transferred. In the protocol unit 45 are those of the data acquisition unit 40 supplied oxygen concentration and temperature readings together with the timing (t ₁ , t ₂ , ..., t _n ) of the timer 35 as a series of measurements 50 stored in the form of a tabular value store c (t ₁ ), c (t ₂ ), ..., c (t _n ) and T (t ₁ ), T (t ₂ ), ..., T (t _n ) and are available for a subsequent evaluation, in particular for a graphical representation or a data processing.

The components 35 . 36 . 40 and 45 can also be implemented in the form of appropriate software routines and driver programs, which are executed in a PC with a typical device configuration.

The measuring cell 20 is due to a thermal stabilization in the form of a control loop 60 with a Peltier element in direct thermal contact with the measuring cell 65 , a temperature sensor located in the measuring cell 66 and an electronic control system 67 kept at a constant temperature. As a result, the measurement conditions falsifying heating of the sample 15 prevented and on the other hand can be selected by the operator a desired measurement temperature, different from the ambient temperature.

2 shows an example, with the arrangement 1 recorded measurement result. After a measurement of the time-dependent oxygen concentration using the electrochemical sensor 25 , the data logger 40 and the protocol unit 45 and signal processing in a corresponding, provided for this system software appears on the screen of a connected computer shown in the figure exemplary curve. The oxygen concentration "c" in this diagram is given in mg / l or as partial pressure in mbar on the ordinate, the time forms the abscissa with a division into seconds or minutes.

The measurement curve represents the time course of the oxygen concentration measured at the electrochemical sensor. In this case, the part of the measurement curve marked by the parameter "R" designates a time segment in which the oxygen concentration in the surroundings of the sample decreases due to the breathing of the plant material occurring in the dark , This part of the trace is called the respiratory phase. It provides initial information about the vitality of the sample or plant from which the sample was taken.

The parameter "R" is usually given in the units mg / l · s or in mbar / s, ie either in units of the change in concentration per unit time or a partial pressure change per unit time. It describes the linear decrease in the time-dependent oxygen concentration by respiratory or respiratory processes of the plant sample during a switched off light source 30 , This parameter is always negative.

At a time t _on , which can be chosen freely by the operator and is thus known by the experimental design or is determined by the measured value detection system upon reaching a test parameter to be selected in advance, the light source becomes 30 switched on. At this moment, the sample is subject to strong exposure to light, causing the plant sample to undergo adaptation processes. Photosynthesis now takes place within the cells over a certain period of time and respiration is continued with the same or altered parameters. As a result of these overlapping processes, the oxygen concentration passes through a local minimum, which is traversed with a certain time delay after the light source is turned on. This delay is described by the time parameter "D" (delay). It is defined as the time between the known switch-on time t _{on of} the light and the time t _min of passing through the curve minimum and is usually measured in seconds or minutes.

After the adaptation of the vegetable sample material, the oxygen concentration in the now switched-on light source increases 30 initially continuously. This increase in concentration is caused by the now proceeding photosynthesis in the sample material, ie by the thereby occurring photolysis of the water. The rise of the measurement curve is linear in a first approximation. The linear increase in oxygen concentration is described by a slope parameter "S". The physical unit of "S" is measured as well as that of the parameter "R" in mg / l · s or mbar / s. The value for "S" is always positive.

The dimensions listed here for "R" and "S" are device-dependent. For "R" and "S" optionally the dimension mmol / m ² · s or derived therefrom dimensions such. B. μmol / m ² · s, can be used. These are generally device-unspecific, since the conversion takes into account device parameters, for example the volume within the measuring cell, and can therefore be very advantageous for comparing the values with other parameters measured with other devices and / or with other methods.

In the further course of the curve goes into a saturation region of the oxygen concentration and has a substantially constant temporal saturation value. This results from a balance between oxygen production and oxygen uptake by the plant sample as well as shrinkage and a resulting, almost constant oxygen concentration in the vicinity of the sensor head. The saturation value provides a parameter "M". It describes the maximum time of the trace that is constant in the bright time and is given in mg / l or mbar.

From a combination of "R" and "S", the coefficient of photosynthetic activity can be formed by a quotient K _phA = -S / R. It turns out that in certain cases this coefficient still more clearly describes the vitality / photosynthetic activity of the examined sample as "R" and "S".

The aforementioned parameters "R", "S", "D" and "M" or the coefficient of the photosynthetic activity "K _phA " are objectively determinable indicators for the description of qualitative and / or quantitative states or prognostic statements about the others vegetative and / or generative development of the examined plant. For example, developments in green matter and root mass or reproductive or generative development processes, such as the development of fruiting processes of the chlorophyll-bearing system, can be assessed and assessed solely on the basis of subjective factors visual assessments are not recognizable or can only be determined by incomparably higher metrological effort. This concerns, for example, the optimal determination of the harvest time of certain market crops, in which the method of many years of experience already deals with the above-mentioned disproportionate dominance of the subjective factor. In the following some embodiments are given for this purpose.

Embodiment I: Interaction of winter wheat and soil fungi

In recent years, extensive research has been carried out on the interaction of pathogenic and soil fungi especially in agricultural crops such as grain and oil crops, but also in soft fruit. Here, a reduction of chemical pest control and its replacement by a biological variant as a research goal in the foreground. This is particularly necessary, since in the case of an infestation with Verticillium a chemical soil disinfection is prohibited by new legal provisions.

Of particular importance in wheat production is the common "Partial Taubährigkeit" disease, which is mainly caused by Fusarium graminearum and leads to high quality and harvest losses and mycotoxin contamination of the harvested products. These losses can be further increased by the infestation with Alternaria alternata, which is also a potential mycotoxin producer.

Alternative methods of chemical control of these pathogens are needed. The arbuscular mycorrhiza (AM) plays a special role. Mycorrhiza, the association of higher plant roots with fungi, is a symbiotic relationship that allows plants to absorb minerals (phosphate, bound nitrogen) from the soil in the most effective way and provides the fungus access to plant assimilation products.

• 1. Winterweizen (WW) als Kontrollvariante;
• 2. WW + Glomus intraradices (WW + Gl);

In several experiments, the influence and the interaction of these fungi on the development of winter wheat were investigated. Traditional plant physiological as well as microbiological and biochemical investigations were carried out and compared with the results of plant vitality studies according to the method of the invention. The above-mentioned parameter "S" was considered particularly significant. The following variants were analyzed:

• 1. Winter wheat (WW) as a control variant;
• 2. WW + Glomus intraradices (WW + Gl);

• 3. WW + Fusarium graminearum (WW + Fus);
• 4. WW + Alternaria alternata (WW + Alt);
• 5. WW + Glomus intrarad. + Fusarium gramin. (WW + Gl + Fus)
• 6. WW + Glomus intrarad. + Alternaria altern. (WW + Gl + Alt);
• 7. WW + Fusarium gramin. + Alternaria altern. (WW + Fus + Alt);
• 8. WW + Glomus intrarad. + Fusarium gramin. + Altern. altern. (WW + Gl + Fus + Alt).

After about four weeks both variants were inokoliert with the pathogenic fungi in the following variants:

• 3. WW + Fusarium graminearum (WW + Fus);
• 4. WW + Alternaria alternata (WW + Old);
• 5. WW + Glomus intrarad. + Fusarium gramin. (WW + Gl + Fus)
• 6. WW + Glomus intrarad. + Alternaria aging. (WW + GI + ALT);
• 7. WW + Fusarium gramin. + Alternaria aging. (WW + Fus + Alt);
• 8. WW + Glomus intrarad. + Fusarium gramin. + Aging. aging. (WW + Gl + Fus + Alt).

The reproducibility of the results is in 3 shown. It is generally higher than 90%. Using a suitable software program, a statistical evaluation of the test results, both of the measurement results with the method according to the invention using the parameter "S", and of the microscopic, plant physiological and biochemical examinations was carried out.

The result of the correlation analysis of individual measurement series on wheat leaves shows 4 , Tables 1a to 1c show the mean values of the photosynthetic activity of the wheat leaves over a period of 9 weeks and changes Δ compared to the control variant (O ₂ measurement, "S" in μmol / m ² · s). Table 1a Mean values of photosynthetic activity using slope parameter "S" in wheat leaves from week 1 to week 3 and changes Δ versus the control variant Inoculation variant 1 week. Δ% 2 weeks. Δ% 3 weeks. Δ% WW 10.93 12.22 14.83 WW + Eq 14.78 35.2 14.15 15.8 15.27 3.0 WW + foot 11.27 3.1 2.17 -82.3 0 -100.0 WW + Alt 8.81 -19.4 11.12 -9.0 13.64 -8.0 WW + Gl + Fus 10.30 -5.8 9.67 -20.8 6.81 -54.1 WW + GI + ALT 8.49 -22.4 11.93 -2.4 14.85 0.2 WW + Fus + Alt 7.03 -35.7 8.31 -31.9 5.12 -65.5 WW + Gl + Fus + Alt 9.54 -12.8 10.24 -16.2 5.82 -60.7 Table 1b Mean values of photosynthetic activity using the slope parameter "S" in wheat leaves from week 4 to week 6 and changes Δ to the control variant Inoculation variant 4 weeks. Δ% 5 weeks. Δ% 6 weeks. Δ% WW 13.63 11.77 10.11 WW + Eq 14.34 5.2 14.15 20.2 12.84 27.0 WW + foot 0 -100.0 0 -100.0 0 -100.0 WW + Alt 15.20 11.6 12.64 7.4 9.89 -2.1 WW + Gl + Fus 2.30 -83.1 0 -100.0 0 -100.0 WW + GI + ALT 11.36 -16.6 14.67 24.6 14.85 46.9 WW + Fus + Alt 0 -100.0 0 -100.0 0 -100.0 WW + Gl + Fus + Alt 0 -100.0 0 -100.0 0 -100.0 Table 1c Average values of the photosynthetic activity using the slope parameter "S" in wheat leaves from week 7 to week 9 and changes Δ compared to the control variant Inoculation variant 7 weeks. Δ% 8 weeks. Δ% 9 weeks. Δ% WW 10.94 10.43 8.87 WW + Eq 13,09 19.6 13.54 29.8 11.05 24.5 WW + foot 0 -100.0 0 -100.0 0 -100.0 WW + Alt 8.53 -22.0 9.44 -9.5 8.51 -4.1 WW + Gl + Fus 0 -100.0 0 -100.0 0 -100.0 WW + GI + ALT 12.89 17.9 10.85 4.0 9.82 10.6 WW + Fus + Alt 0 -100.0 0 -100.0 0 -100.0 WW + Gl + Fus + Alt 0 -100.0 0 -100.0 0 -100.0

The diagram 5 shows, by way of example, the time-dependent oxygen concentration in the case of the different inoculation variants investigated at the end of the first week. The plants inoculated with Fusarium graminearium showed decaying vitality within the second week and died completely at the end of the second week.

The results obtained were compared with plant physiological and microbiological measurements. The following parameters were measured:
The green matter (scion FM) and the dry mass of the shoot (shoot TM), the fresh mass of the roots (root FM) and their dry matter (root TM) and the degree of mycorrhization of the roots for each variant depending on their ontogenesis. The individual values are not listed here, only the results of the correlation of the parameters of the statistical evaluation with those of the photosynthetic activity at the respective time are shown and put into relation. The results of the Correlation analysis is presented in Table 2 for the three different series of measurements in which 4 the relevance of averaging could be demonstrated: Table 2 Correlations. Marked correlations significant for p <0.05; N = 126 (case-by-case exclusion of MD) O_1 O_2 O_3 Sprout FM 0.1291 0.0802 0.0325 p = 0.150 p = 0.372 p = 0.718 Sprout TM 0.1190 0.0837 0.0265 p = 0.184 p = 0.351 p = 0.769 Root FM -0.0977 -0.1645 -0.1446 p = 0.276 p = 0.066 p = 0.106 Root TM 0.1093 0.0203 0.0426 p = 0.223 p = 0.822 p = 0.636 Mycorrhizierung 0.3748 0.4217 0.4217 p = 0.000 P = 0.000 p = 0.000

Table 3 shows experimental correlations: 100 pieces of 1 g FM root, independent of root mass: Table 3. Correlations. Marked corr. Significant for p <0.05 N = 127 (case-by-case exclusion of MD) Sprout FM Sprout TM Root FM Root TM Mycorrhizierung -0.0750 -0.0915 -0.3513 -0.1114 P = 0.402 P = 0.306 p = 0.000 p = 0.212

Table 4 presents the correlations between photosynthetic activity and mycorrhization compared to the correlation to the total amount of mycorrhizae: Table 4 Correlations. Marked corr. Significant for p <, 05000 N = 127 (case-by-case exclusion of MD) Mycorrhizierung O_1 O_2 O_3 0.3852 0.4351 0.4351 p = 0.000 p = 0.000 p = 0.000 0.3145 0.3080 0.2993 p = 0.000 p = 0.000 p = 0.001

Result of the evaluation:

• 1. There are no significant correlations between the harvest parameters and the photosynthetic activity of the wheat leaves in the individual inoculation variants.
• 2. There is a significant correlation between the photosynthetic activity of the wheat leaves in the individual inoculation variants and the mycorrhization.
• 3. The correlation is greater in considering mycorrhization than in the same total root mass consideration,
• 4. The vitality parameter in the present case describes a qualitative relationship to the examined plant, the mycorrhization.

• 1. Durch Inokulation mit Glomus wird die Sauerstoffproduktion der Pflanze erhöht.
• 2. Durch den Einfluss von Fusarium nimmt die Vitalität stark ab, während gemeinsam mit Glomus das Kontrollniveau wieder erreicht wird.
• 3. Alternaria hat gegenüber der Kontrollvariante keinen wesentlichen Einfluss, gemeinsam mit Glomus wird ein stabilisiertes Kontrollniveau erreicht.
• 4. Folgende Antagonismen sind zu erkennen: Gl gegen Fus; Gl gegen Alt, Alternaria steht in Konkurrenz zu Fusarium (kein additiver Effekt!); Gl + Fus + Alt erreicht etwa das Kontrollniveau.

With this knowledge, the correlations of the individual variants and the microbiological correlations contained therein are considered. The following basic statements can be made for the second trial week:

• 1. Inoculation with Glomus increases the oxygen production of the plant.
• 2. Due to the influence of Fusarium, the vitality decreases strongly, while together with Glomus the control level is reached again.
• 3. Alternaria has no significant influence on the control variant; together with Glomus a stabilized control level is achieved.
• 4. The following antagonisms can be recognized: Gl against Fus; Gl vs Alt, Alternaria competes with Fusarium (no additive effect!); Gl + Fus + Alt reaches about the level of control.

• 1. Es zeigt sich eine parallele Dynamik der Kontrollvariante WW mit den Varianten WW + Glomus und WW + Alternaria.
• 2. Andererseits liegt eine geänderte Dynamik bei der Kombination WW + Glomus + Alternaria vor.
• 3. Die Anfangswirkung von Alternaria ist sehr hoch.
• 4. Die Wirkung von Glomus setzt etwa ab dem 8. Tag ein und erstreckt sich etwa bis zum 30. Tag.
• 5. Ab dem 30. Tag ist keine Wirkung mehr zu verzeichnen.

Finally, reference should be made to the dynamics of correlations with the results of the correlation analysis presented in Table 4. This is in the diagrams in 8th and 9 shown. 8th shows a comparison of the dynamics of the individual inoculation variants over a period of 14 days, while the diagram in 9 represents a comparison of the dynamics of the individual Inokulationsvarianten over a period of 39 days. From the diagrams, the following conclusions can be drawn:

• 1. It shows a parallel dynamics of the control variant WW with the variants WW + Glomus and WW + Alternaria.
• 2. On the other hand, there is a changed dynamic in the combination WW + Glomus + Alternaria.
• 3. The initial effect of Alternaria is very high.
• 4. The effect of Glomus begins around the 8th day and extends until about the 30th day.
• 5. From the 30th day is no longer noticeable.

Exemplary embodiment II: growth of algae and humic substances

Investigation of the effect of humic substances (HS) on the growth of green algae of the type Monoraphidium convolutum

With the increasing use of mineral fertilizers in crop production worldwide, there is an increasing input of nutrients into surface waters everywhere. This, in turn, has a lasting impact on the growth of algae and higher plants in this eco-area, which, when reached a certain limit, has a strong negative impact on this ecosystem. It is therefore important to know whether and to what extent this growth can be stopped by biological means, instead of chemical growth inhibitors. These investigations lead to very specific quantitative conclusions for each individual ecosystem and therefore have to be carried out separately for each individual system.

It is shown an example of using the method according to the invention can be in the clarified on the vitality parameters "S", as measured by the PlantVital ^® 5000, whether and in what order in the case of concrete algae humic substances serve as an additional nutrient or inhibitory effect on the Algae growth act. The investigations were carried out by establishing a correlation between the measured vitality parameter "S" of the photosynthetic activity of the algae and the oxidative products produced in the algae by the action of humic substances of different concentration. In 10 the growth of algae over a period of 21 days is shown as a function of the concentration of the organically bound carbon (see box in the picture), introduced via the humic substances.

11 shows the measured with the inventive method time course of the oxygen concentration (see. 2 ) depending on the entry of the organically bound carbon over the humic substances.

Using biochemical methods (determination of stress enzymes, such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), glutathione-S-transferase (GST) and hydrogen peroxide (H ₂ O ₂ )) has now been tried, the to achieve the same result in order to determine the primary causes for the decrease in photosynthetic activity. The latter methods have shown in many experiments a good correlation to the experiments with humic substances. It is known that the biological investigations are very time and material consuming and the availability of several expensive equipment is required. Furthermore, a great deal of experience of the investigating scientist is required to achieve good results.

The result of the comparative investigations is in 12 shown. This figure shows the correlation between the photosynthetic activity of algae expressed by the vitality parameter "S" and the hydrogen peroxide concentration in the algal material as determined by a variety of experiments.

The correlation coefficient r ² = 0.90 has a quite satisfactory value. r ² = 1 would be the ideal value.

The example shows that routine measurements can be carried out very simply and expediently with the method according to the invention, which otherwise requires a multiple of time and enormous experimental effort. It also shows that, with the method according to the invention, the vitality parameter "S", determined as the objective variable from the photosynthetic activity of the examined species, is a parameter which describes the system objectively and quantitatively.

Embodiment III: Vitality of road trees

Examination of the location influence on the vitality of street trees in Berlin-Charlottenburg and in Strausberg (Green City on the lake) on the same day

With the method according to the invention, three varieties of road trees, chestnuts, oaks and limes were examined on the same days both in a busy area in Berlin-Charlottenburg and in a less traffic-strained street in Strausberg (Green City on the lake) in Berlin. It was taken care that the trees were about the same age and the samples were taken from about the same height and the same direction. Even otherwise, the lighting conditions were comparable for the samples compared.

From each sample 5 specimens were measured, from which then averages were formed.

The parameters of the respiration "R" and the parameter of the assimilation "S" were determined as measured values (as indicated in 2 ).

The mean values determined in this way according to the method according to the invention are contained in Table 5. The table also shows the coefficients of the photosynthetic activity K _phA = -S / R. From this table it can be seen that, in relevant cases, this coefficient according to the invention is a useful quantitative value which expresses the vitality ratios even more clearly for the purpose of describing the condition of the chlorophyll-bearing examination objects. Table 5: Vitality parameters in μmol / m ² · s of road trees in Strausberg (SRB) and in Berlin (B) as well as their quotients. The table only shows the measures parameter chestnut chestnut chestnut Oak Oak Oak lime lime lime Location SRB B SRB / B SRB B SRB / B SRB B SRB / B "+ S" 1.03 0.70 1,471 4.56 4.06 1,123 6.80 4.91 1.385 "-R" 2.58 3.68 0.701 3.50 3.55 0.986 3.91 7.92 0.494 "K _phA " 0,399 0,190 2,098 1,303 1,144 1,139 1,739 0,620 2,805

The numerical values clearly show that the coefficient of the photosynthetic activity K _phA = -S / R can represent existing differences in comparable objects more clearly than the primary vitality parameters "S" and "R".

LIST OF REFERENCE NUMBERS

10

shielded measuring station

15

biological sample

20

cell

25

electrochemical sensor

30

light source

35

timer

36

control electronics

40

Data acquisition unit

45

protocol unit

50

measurement series

60

thermal control circuit

65

Peltier element

66

temperature sensor

67

control electronics

c

oxygen concentration

D

time delay parameter

M

maximum saturation value

R

Respirationsparameter

S

Slope parameter

t _on

switch-on

_min

Time of the local minimum

K _phA

= -S / R coefficient of photosynthetic activity

Claims (2)

Method for evaluating the vitality of chlorophyll-bearing biological samples with a time course measurement of an oxygen concentration (c) in an immediate spatial environment of the biological sample with changing illumination by measuring a reaction time (D) between the time of an abrupt change of illumination and the time of a local extreme in the time-dependent oxygen concentration for determining the adaptation behavior of the photosynthesis power, wherein the time course measurement of the oxygen concentration (c) in an immediate spatial environment of the biological sample is carried out with changing illumination with the following method steps: - a measurement of a temporal change in the oxygen concentration (c) during a dark time (t _D ) with a switched off illumination, - a measurement of the reaction time (D) between a switch-on (t _on ) of the lighting and a time (t _min ) of a on the on switching time following local minimum of the oxygen concentration, - a measurement of a temporal change in the oxygen concentration during a bright time when the lighting is switched on, - a determination of a set when the lighting illumination saturation value (M) of the oxygen concentration, characterized in that from the temporal change of the oxygen concentration (c ) during the dark time (t _D ), this temporal change descriptive Respirationsparameter (R) and from the temporal change of the oxygen concentration (c) during the light time, a time change describing slope parameter (S) is determined, wherein from the negative ratio of Slope parameter "S" and the respiratory parameter "R" a coefficient of photosynthetic activity K _phA = -S / R is determined. A method according to claim 1, characterized in that the respiration parameter (R) and the slope parameter (S) is determined by fitting a linear function to the measured time-varying oxygen concentration values during the dark and the light time respectively.

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