Researches prove: Scarlet, Benefis and Polaris are irreplaceable

In the conditions of intensive maintaining agricultural production dependence of a yield on optimization of agricultural crop protection methods from weed plants and other harmful organisms increases. It is very important that this protection will be based on the regulation of number of harmful organisms to the economically reasonable and environment-friendly level. This approach is impossible without using in the industry the most progressive technologies and innovations of modern science. Nowadays polymeraze chain reaction (PCR) is the most powerful tool for finding and qualitative analysis of wide spectrum of phytopathogens in agricultural crops.

PCR diagnostics is based on finding the area of genome specific for every concrete pathogen. In biological laboratory «Schyolkovo Agrochim» the comparative analysis of efficiency of three seeds herbicides Scarlet, Benefis, Polaris was carried out by means of PCR. The essence of experiment consisted in the following: dressed seeds of wheat variety «Moskovskaya 56» were grown in the conditions aggressive infection background, created with fungus mycelium Fusarium culmorum, during 2 weeks. At the end of this period the assessment of quantity of pathogen organism in the grown plant and in the substrate round its seeds was done. Due to the degree of leaves infection we concluded the system influence of pesticide on plant in general. Dynamics of accumulation of fungus DNA in the substrate testified to impact of an herbicide on the infectious background surrounding seeds. As test plants were use d plants grown from undressed seeds: positive test group were infected with Fusarium culmorum, negative test group remained uninfected.

The principle of PCR operation consists in copying specific for every concrete pathogen area of deoxyribonucleic acid (DNA) by means of special enzyme (polymerase) with artificial way in a glass-tube. During reaction the number of copies of a diagnostic fragment of DNA increases in million times, to easily revealed quantities.

PCR diagnostics includes three main procedures: sample preparation (DNA allocation), actually reaction and detection (visualization) of its products.

First of all, it is necessary to receive object which it is necessary to analyze - DNA. Cells of plants contain a huge amount of organic chemicals. Polysaccharides (cellulose of cellular walls, starch, protopectin, hemicellulose), proteins, lipids and lipoproteins, nucleic acids, secondary metabolites (polyphenols, alkaloids, terpenes, etc.) are referred to them. Elements of these multicomponent mixtures can physically connect and chemically degrade molecules of nucleic acid that significantly complicates its isolation and can lead to inhibition (stop) of polymerize chain reaction. Therefore the choice of a technique of allocation of DNA has huge value for receiving adequate result in PCR. As a whole, procedure of allocation of DNA represents serial processing of vegetable fabric by various chemicals with the subsequent centrifugation for division of the fractions containing different intracellular components, including nucleic acids. For DNA isolation from plants the set of techniques is offered but among them there is no universal, equally suitable for work with any vegetable fabrics. It is connected with features of their chemical composition.

Concentration of DNA is defined by a way of spectrophotometric measurement of quantity of the ultra-violet light absorbed by the nucleobases. The method is based on ability of DNA in solution as much as possible to absorb ultra-violet waves of 260 nanometers long. This photometric absorption directly correlates with concentration of DNA.

Indicators of purity of the emitted nucleic acids are so-called «the relations of invading quantities». So, proteins absorb with a wave length of 280 nanometers therefore the relation of 260/280 nm is used for identification of protein impurity. Pure DNA has to have the relation value of 260/280 nearly 1,8. Decrease in this indicator speaks about availability of protein impurity, phenol or other contaminating agents. At the RNA presence in preparation the value 260/280 increases. Other indicator of preparation DNA or RNA purity is the characteristic value dependences of absorption of 260/230 nm. In case of a pure preparation it is usually 2.2. Smaller values testify to preparation pollution with salts and other components of the solutions used for procedure of allocation of DNA.

Table 1 presents the results of DNA allocation with methods of cetitrimethylammonium bromide (CTAB) (Murray, Thompson, 1980). Spectral characteristics of preparations testify that the received samples contain DNA, in different degree contaminated with impurities. Degree of contamination is low and they can be used for carrying out PCR.

Table 1 - Concentration of total DNA, allocated from different plant organs

The obtained data illustrate the dependency of allocation quality of DNA from chemical content of plant fabric. For example, from 500 mkg of leaves, we obtained DNA in the concentration of 500-800 ng/mcl, from grains rich in carbohydrates and proteins – in a total 100-280 ng/mcl. The fungus parasitizing on a plant does not strongly influence quality of DNA allocation. So, the quantity of total (cumulative) DNA from infected Fusarium culmorum wheat is lower than concentration of DNA from a healthy plant less than twice (490 ng/mcl and 740 ng/mcl respectively). Also the presence in a sample of small amount of active substance of herbicides does not influence allocation process. Also we can stress that the obtained DNA is more that enough for doing the number of amplifications with sensitivity that positive result can be obtained having at the beginning of the reaction one molecule of the detective DNA (determined). The obtained nucleic acid serves as a matrix for replication (doubling) of new chains in the process of PCR. In diagnostic practice several reaction varieties of PCR are used: PCR with reverse transcription (RT-PCR), inserted PCR (Nested PCR), PCR with usage of hot start (Hot-start PCR), PCR with detection by the terminal point (FLASH PCR), quantitative PCR ( PCR in real time) , etc. The presented work was done on the equipment and test-systems, designed for setting PCR in real time.

Method PCR in real time (RT-PCR) significantly exceeds precision, peculiarities, efficiency and safety of PCR diagnostics. Unique peculiarities of this method are possibility of direct observation of amplification of DNA-target by means of monitoring of the formed product. The reaction mixture composition except enzyme Tag-polymerase and defined by us DNA-matrix includes deoxynucleoside triphosphates (dNTP) – building material of future amplicons, two synthetical oligonucleotides – direct and reverse primers, terminating the succession-target from two sides and hybridization probe, additional oligonucleotide, on different ends of which there are fluorophor-colourant and fluorescence-stabilizator of this colourant.

Design of oligonucleotides for test-system with which research associates of «Schyolkovo Agrochim», is developed with LLC «Agro Diagnostics». Test-system is intended for finding fungus Fusarium culmorum. Fluorometric detection of amplification products in RT-PCR is done in the course of their accumulation. During amplification at the account of 5-exonuclease activity of Taq- polymerase fluorescent tag transfers into the solution, releasing from neighborhood of stabilizator and generates fluorescent signal increasing in real time proportionally accumulation of amplificat (Figure 1).

Figure 1 - Scheme of fluorescent signal formation

PCR results can be expressed in the form of graphs and in relative digital quantities. One channel of device DT-96 after every amplification cycle records the signal, testifying accumulation of the DNA fragment F. сulmorum. By these data the kinetic curve PCR is built. If fluorescence of this investigated sample definitely exceeds the value of background fluorescence (threshold value), reaction result is positive. The more the initial number of copies of specific DNA in the analyzed sample (star concentration of DNA-target), the less amplification cycles are necessary to get the threshold value.

Table 2 - Report on the results of the PCR analysis:

Determination of relative concentration of DNA Fusarium culmorum in different wheat fibers

* Manual (threshold) method of analysis (B,F) Threshold_FAM = 10,0 Threshold_HEX = 0,0

Algorithm of definition of amount of target-oriented DNA in the sample is based on simultaneous analysis of the researched samples and compared test sample having the DNA-target in the known concentration. As the standard we used DNA F. culmorum in the concentration 10 ng/mcl (D9). The fungus DNA in concentration of 2,9 ng/mcl (С8) appeared in the leaves of plants from the positive test group. In the leaves of plants grown from the dressed seeds fungi are not detected (С5, С6, С7). The remarkable changes occurred in the substrate. In untreated grain considerable number of DNA fusarium – 24,0 ng/mcl (D7) is accumulated. Infection background round dressed seeds is significantly lower. So, the processing with Scarlet decreases it in 10 times (D4), with Polaris – in 25 times (D6), with Benefis – in 50 times (D5) (Table 2).

Thus, the results of the carried out experiment prove the presence of the system effect from herbicides application and their control influence on the infection background round seeds. At that, innovative three-components Benefis and Polaris exceed two-components Scarlet by these parameters.

Researches on the DNA level have become already routine practice in medicine, veterinary, selection, taxonomia, bioengineering, etc. Modern level of PCR-diagnostics application in plant breeding is not high. Implementation of molecular methods into this branch allow analyzing great amount of plant organisms to find genetically modified plants, precise and fast identification of phytopathogens, to control the efficiency of anti pests stoppers, sorting out unfavorable seeding material, monitoring of infection background of sown areas, etc.