Bioscience/5. Molecular biology

Candidate of Agriculture Beyshova I.S.

Postgraduate student of natural sciences Kusymbaeva S.K.

Kostanay State University named after A.Baytursynov, Republic of Kazakhstan

The importance of PCR in R-time regime in diagnosis septoria of cereals

Cereals are considered to be one of the main of all agricultural plants in Central Asia, including Kazakhstan. According to statistics FAO in 2013 this region is characterized by high level in using of wheat – more than 200 kg per year. That’s why cereals play an important role in providing the country with provision. More than that Kazakhstan is the main wheat provider that increases the requirements to the corn quality. In wheat production in regions the farmers run into many difficulties, the most dangerous one is spreading of diseases that cause damage to the harvest [1].

According to Kazakhstan Republic Government Decree from 10 December 2002 №1295 “About confirmation of the list of quarantine objects and particularly dangerous harmful organisms” into “The list of dangerous vermin and the diseases of agricultural plants” the septoria was included [2].

This disease is considered to be not only widespread practically in all countries, which grow cereals and also are the main reason of the huge decrease of crop capacity. Sharp continental climate of Kazakhstan, especially in its north regions is favorable for development and spreading of septoria, decreasing profitability of corn production. Decreasing of biological production of plants happens when грибковое disease damage upper leaves of cereals, and also their ears in a period of milk and milk-waxen ripen. Damaging of ears and upper tier of leaves грибами septoria cause the loss of harvest (it’s about 50% during epiphytotics) [3].

For organization of rational harvest protection system, making prognosis for developing of disease, the proper choose of fungicides, time and doses of their application, in taking decision about necessity of pre-sowing processing of seeds, and also in producing of stable sorts we need the exact kind identification agents of septoria. However, the first signs of fungi of the genus septoria on cereal leaves appear after a sufficiently long period of time until the pathogen Septoria runs its incubation period, and thus to diagnose the presence of the disease cannot be visually.

Visual diagnosis of the disease is complicated by the presence within the same field, and even a few plants of this kind septoria. To define by morphology of fruiting bodies conidia complicated by the presence of other similar entities fungi, and after formation of pycnidia fungicide application will not produce the desired effect. Traditional methods of complex diagnostics, including the selection of species in pure culture, selection of media and culture conditions obtaining monospore isolates and microscopy, durable, time-consuming and not effective enough. Therefore, the most optimal methods required for the timely diagnosis of the disease in the early stages of its development.

Adopted in the Republic of Kazakhstan monitoring system for protecting plants from disease particularly dangerous technique uses field surveys visual method of diagnosing, which covers a small part of the acreage, which leads to misdiagnosis and reduced profitability of grain production. There are a number of biotic and abiotic diseases with identical signs. For improving the quality of examination is necessary to introduce modern methods of diagnosis septoria.

Using of polymerase chain reaction in Real-time for laboratory diagnosis modifications led to the revolutionary development of the group of diagnostic methods based on the detection of genetic material. The general principle is based on multiplication of the number of copies of specific DNA fragments of the target enzyme, the DNA-synthesis of new complementary polymerase.

Detection of accumulation of amplicons occurs using an oligonucleotide probe hybridizes to the target site between the primers. Reaction is carried out under conditions of multiple repetition of the temperature cycle that includes three main steps: denaturation, annealing of primers or hybridization and elongation.

If no constraint to effective amplification per cycle number of copies of a specific target DNA region is increased twice, and after 30 cycles more than 109 times. Due to this polymerase chain reaction has been applied in laboratory diagnostics to determine such small amounts of pathogens, which reliably detected without amplification impossible. Theoretically with the help of polymerase chain reaction it can be detected using a single - molecule DNA target. In practice for reliable analysis must be present in the test tube with the reaction mixture several dozen copies of a specific DNA.

Detection target task DNA-target in the test sample using the polymerase chain reaction, thereby, formed is found to detect the end of amplification products, are used for this purpose amplicons gel electrophoresis and hybridization enzyme-linked immunosorbent assay. In the early 1990s was created a new method for detection of amplicons in real time, which has given the opportunity to correctly determine the amount of the original DNA-target in the sample.

In modern laboratory diagnostics Real-Time PCR is being promoted as a method of identifying specific fragments of genetic material of pathogens and is gradually replacing the options with the detection of the amplification products at the end of reaction. The principal advantage is the possibility of detecting the accumulation of amplicons without opening the tube, which minimizes the risk of false-positive results due to contamination of samples reagents and amplification products. The significant decrease in the amount of manipulation of the test sample reduces the time, simplifies the analysis and reduces the likelihood of errors. Application along with primers hybridization probes enhances the specificity analysis. The possibility of independent simultaneous recording of the fluorescent signal from several hybridization of DNA probes allows identification of one study of several different parts of one or different DNA-targets. But the main feature of this methodology allowed to occupy a special place among the methods of laboratory diagnosis, the ability to determine the initial number of copies of a specific DNA target in a clinical trial.

In recent years the principle of specific DNA amplification began actively to use in developing methods for reliable determination of the species of plant raw materials. In comparison with conventional methods for detecting species, establishing the species of plant materials using highly versatile, deeper level of species differentiation, high reproducibility and the ability to quantitative analysis. In spite of using of polymerase chain reaction for species identification of tissues of plant origin was highly appreciated by foreign experts, in our country this area, unfortunately, in practice, is not widely used in the examination of cereal septoria cultures.

Thus, in Kazakhstan the diagnosis septoria of cereals actively investigated by the method and its implementation will be offered in production.

Literature:

1. Koishibaev M., Ismailova E.T. Peculiarities of development and harmful effect of septoria of cereals in Kazakhstan. / Herald of agricultural sciences of Kazakhstan. 1991, №11 p. 31-36.

2. The list of introduced changes by decree of Kazakhstan Parliament from 23.11.2005 №1157.

3. I.M.Polyakov, E.M.Shumakov, the article “Protection of plants” The Big Soviet encyclopedia. – M: The Soviet encyclopedia. 1969 – 1978.

4. Ivanov M.K., Poryvaev V.D., Kandrushin E.V. Peculiarities of quantity analysis by the polymerase chain reaction method in Real-Time/News “Vector-Best”, 2008, №4(50).