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DETECTION AND IDENTIFICATION OF SOME BIOLOGICAL CHARACTERISTICS OF POTATO L-VIRUS USING REAL-TIME PCR METHOD
M.M.Kholmatova1, V.B.Fayziyev2
1Samarkand State University of Veterinary Medicine, Animal Husbandry and Biotechnology Tashkent
city, Chilanzarsky district, microdistrict 20, building 35 A 2Chirchik State Pedagogical University, Tashkent, Uzbekistan region, 100074, Universitetskaya street,
4, ChDPU
*Corresponding author email: Ivmona.92(a>mai1.ru.
E-mail addresses of co-authors: v. fayziyev@cspi.uz
Abstract. Nowadays, several phytopathogenic viruses infect the potato plant and cause great economic damage. Early detection of the virus from sowing material and natural reservoir plants is the main reason to prevent its spread, so that measures can be taken to control these viruses. This article presents the results of research on detection and identification of potato L-virus (PLV) from natural host plants and different disease symptoms, using real-time polymerase chain reaction (PCR-real-time) method.
Keywords: PCR, RNA, virus, identification, potato, amplification, variety, reservoir. ОПРЕДЕЛЕНИЕ НЕКОТОРЫХ БИОЛОГИЧЕСКИХ СВОЙСТВ И ИДЕНТИФИКАЦИЯ Л ВИРУСА КАРТОФЕЛЯ С ПОМОЩЬЮ МЕТОДА ПЦР
М.М.Холматова1, В.Б.Файзиев2
Самаркандская государственная ветеринарная медицина,Университет животноводства и биотехнологии.Ташкент, Чиланзарский р-н, микрорайон 20, дом 35 А 2Чирчикского Государственного Педагогического Университет, Узбекистан, Ташкент обл, 100074,ул.Университетская 4, ЧГПУ "■'Соответствующий автор email: lvmona.92(5>mai1.ru.
Адреса электронной почты соавторов; v, fayziyev@cspi.uz
Аннотация. В настоящее время некоторые фитопатогенные вирусы поражают растения картофеля и наносят большой экономический ущерб. Раннее обнаружение вируса из эквивалентного материала и природных растений-резервуаров является основной причиной предотвращения его распространения, чтобы можно было принять меры против этих вирусов. В этой статье проведены исследования по обнаружению и идентификации L-вируса картофеля (KLV) из природных растений-хозяев и различных симптомов заболевания с использованием метода полимеразной цепной реакции в реальном времени (ПЦР в реальном времени], а результаты представлены эти исследования.
Ключевые слова: ПЦР, РНК, вирус, идентификация, картофель, амплификация, сорт, резервуар.
Agricultural products are of great importance in human life. Potatoes, one of these products, play an important role in the diet. In developed countries, potatoes are used as raw materials for food, fodder, starch and alcohol production. As in other
plants, there are phytopathogenic diseases in potatoes, which have a serious negative impact on its growth, yield and product quality. In most cases, during the growth period of the plant, under the influence of various infectious diseases,
most of the crop is lost and its quality decreases sharply. As a result of the lack of regular control measures and other preventive measures, diseases are often found in crop sown fields and private farms and cause great damage.
Potatoes are the fourth major food crop in the world and feed a large part of the world's population. In recent years, many diseases and pests have negatively affected the productivity of agricultural crops. Among agricultural crops, potato is one of the main crops, and the productivity of this plant is also decreasing due to various diseases. Viruses are the most dangerous pathogens that damage potato plants in agriculture. More than 50 viruses naturally infect the potato plant worldwide. These viruses can reduce the yield of potato plants by up to 80%. Of the 400 phytoviruses known to date, 52 cause damage to the potato plant, 36 of which are widespread, and the rest are found only in South American countries. Of these, 16 species are very common, and 6 species are found in the Republic of Uzbekistan. They are: PVX, PVY, PVM, PVS, PLV, PVA [6,7].
Despite the improvement of breeding efforts to introduce disease-resistant and high-yielding varieties, potato production in cultivated fields is low due to various biotic and abiotic factors, including viruses. Viral diseases play a major role in the drastic reduction of plant productivity, insects (beetles, leafworms and whiteflies), fungi and tubers serve as a source of virus infection and a vector of virus, as well as, a reservoir of infection. Low phytopathogenic isolates of potato L-virus alone can reduce yield by 15-35%, strong pathogenic isolates by 65-80%. Studies have shown that infected potato tubers
replanted over many years reduce yield by almost 50% [6,10,12].
L- virus belongs to the Luteovirus family, and its virions are spherical in shape, 23-25 nm in size. The protein (m.m) of the virus is 28,3x106 D, virion consists of 28% RNA and 72% protein. The RNA m.m. 2,0x106 D, protein m.m. 26,3x103 D, sedimentation coefficient 127 C, E260 0,1% lcm«5. The degree of the temperature resistance of the virus is 80°C, the virus is stored at 42-43°C for 1-5 days, and at 20°C for more than a month.
PLRV is not mechanically transmitted, but is very easily transmitted by stem and tuber-core grafting. In natural conditions, it is transmitted by a number of aphids and plant lice, such as Myzus persicae Sulz., Myzus ascalonium, Myzus circum flexus BucktMyzus ornofus, Myzus convolvuli Kalt., Macrosiphum solanifolii Ashm.
PLRV is transmitted by sap in a circulation way: from the phloem they move to the hemocoel and through the hemolymph to the salivary glands. Its localization in plants is in the phloem, it causes tissue necrosis, stunted growth, loss of chlorophyll and yellowing of leaves. Myzus persicae is its main vector. Studies have shown that PLRV can be transmitted to infected plants by aphids and exert pathogenicity on infected plants. However, the molecular mechanisms mediating these
relationships are still poorly understood. A plant infected with the PLRV virus through aphids will retain the virus for the next year. The seeds of an infected potato plant in the first year are a reservoir for the virus, resulting in unhealthy plants. Infected potato seeds serve as a systemic translocation of PLRV to all plant tissues. The degree of
temperature resistance of this virus is 80%, and the virus can survive at 42-43°C for 1-5 days, at 20°C for more than a month [6, 7, 9].
I.T. Ergashev detected natural reservoirs of potato L - virus in several wild plants and weeds: including, field ivy [Convolvulus arvensis], sorrel (Rumex confertus), plantain (Plantago lanceolata), jimsonweed (D. stramonium), black nightshade (Solanum nigrum), etc.. In the research work of V.B. Fayziev it was reported that the plants, such as eggplant (S. melongena L.J, rough cocklebur [Xanthium strumarium L], wormwood
The cryptogram of this virus is R/l:2/28:S/S:S/Ap; S/Ap. The protein of the virus (m.m) is 26,3x103 D, the diameter of the virion is 25-30 nm, each nucleocapsid contains 32 capsomeres. About 28% of the virion is RNA, 78% is protein. According to the classification of luteoviruses, it belongs to IV group of viruses. Each virion contains a single strand of (+) RNA. The genome consists of a sequence of 5300 to 5900 nucleotides [2, 3, 4, 9, 14].
Potato L-virus causes disease symptoms such as yellowing and coarsening of the leaf color and crinkled or wavy leaf plate in the plant. The abovementioned disease symptoms are differentiated from other potato virus diseases.
(Artemisia annua L.J, black nightshade (Solanum nigrum L.), thornapple (Datura metel L.), water foxtail (Alopecurus geniculatus L.J, mugwort (Artemisia vulgaris LJ, alfalfa (Medicago sativa LJ, tomato (Lycopersicum esculentum Mill), goosefoot (Ch, amaranticolor), cucumber (Cucumis sativus LJ, dodder (Cuscuta approximata Babing.J, common marshmallow (Althaea officinalis LJ, common mallow (Malva neglecta Wall], velvetleaf (Abutilon theophrasti Medic.), curly dock (Rumex crispus L.) are natural reservoirs of L-virus [7, 9].
The PCR method was chosen for accurate and precise diagnosis of the disease and identification of the virus.
1. Polymerase chain reaction (PCR) is considered the most acute, accurate and fast method of molecular diagnosis of phytopathogenic viruses. When analyzed by real-time PCR method in a biological sample taken from the stem, leaf or tuber of a potato plant, it allows to determine the genetic molecule (DNA/RNA) of the pathogen. PCR diagnostics can detect even the lowest stage of viral infection in plant samples. The difference between the molecular diagnostic method and other methods is that the causative agent (pathogen) of the disease is diagnosed not by the connection between the antigen and the antibody, but by the direct recognition of the genetic molecule of the infection [10,11,16].
MATERIALS AND METHODS Conditions for research
The research work was carried out in the "Molecular Biology and Bioinformatics" scientific research laboratory of the "Biology" department of the faculty of Chirchik State Pedagogical University and in the IFT and PCR laboratory of the State Center for the Diagnosis of Animal Diseases and Food
Safety of the Republic, as well as a potato growing farms in the Tashkent and Qibray districts of the Tashkent region.
The methods of research
Sampling for PCR analysis. For conducting analysis, potato leaves by 4 pieces from each site of the potato planting area, a total of 80 plant leaves from 20 sites, including plant stems and leaves of weeds growing among the
potato plants, were collected and placed (+4°C) and brought to the laboratory for in separate polyethylene bags and then PCR analysis
put in a thermos with special ice packs
O o o o
o o o o
1-HyKia o o o o 4-nyKTa
2- iiVKTa O o 3- nyicra 5-HyK'ra O o
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[Fig.!).
Figure-1. Scheme of envelope method
The infestation level of plants was determined by using PCR based on the collected samples. During scientific research, monitoring was carried out by RV-PCR method for symptomatic differentiation of PVL with other viruses [1,2].
Sequencing was performed according to the following algorithm to determine PVL by polymerase chain reaction method:
1. Isolation of RNA of PVL from the sample;
The set of reagents "PhytoSorb" RN-520 manufactured by the "SINTOL" scientific production company (Russia) was used for the isolation of RNA of virus from the plant sample.
2. Mixing isolated RNA with specific primers and enzymes (TAQ-polymerase, revertase, etc.);
"Reagent set for detection of RNA of PVL by real-time-polymerase chain reaction-repetitive transcription (RV-PCR-QT) method" (SINTOL PV-002) manufactured by "SINTOL" scientific production company (Russia) was used for the research. This test set can detect the RNA of the pathogen of PVL in any vegetation period of plants.
3. Amplification of the RNA storage mixture placed in microtubes in PCR amplifiers and interpretation of the result.
amplmg from field crop area
The amplification process was carried out in qT0WER3/G amplifier equipment. The first 15 minutes of amplification were reverse transcribed at 45°C. After that, 50 cycles of denaturation, annealing and elongation processes were carried out sequentially.
RESULTS AND THEIR DICUSSION
4 samples of potato plant leaves of 4 varieties from 5 sites were taken from the fields of potato farms in Kibray district. During the sampling, plants with typical PVL symptoms shown in the literature were selected. Selected samples were sorted to differentiate PVL from other viral diseases of potato (PVY, PVX). Also, in order to study the preservation of the virus from one season to another in the fields planted with potatoes, samples of wild plants were taken and monitored.
Samples were homogenized using extracting solution in autoclaved sterile porcelain flasks. RNA of PVL was isolated from the homogenized sample using sequencing nucleic acid isolation reagents. Using a special PCR mixture, the isolated RNA of PVL was mixed with primers. The prepared mixture was placed in the equipment for amplification. At the end of the amplification, the following result was obtained (Table 1).
1-Figure. A) healthy leaf В) PVL infected leaf
Table-1
Expertise conclusion on PVL detection by PCR amplifier.
Tested samples Sample name Dye Ct mean Ct
A1 Santa variety 1 site FAM 38,83 38,83
B1 Santa variety 2 sites FAM 35,54 35,54
CI Santa variety 3 sites FAM 32,55 32,55
D1 Santa variety 4 sites FAM 33,35 33,35
El Azara variety lsite FAM 42,17 42,17
F1 Azara variety 2 sites FAM 37,78 37,78
G1 Azara variety 3 sites FAM 31,15 31,15
HI Azara variety 4 sites FAM 39,56 39,56
A2 Pikasso variety 1 site FAM 32,66 32,66
B2 Pikasso variety 2 sites FAM 30,56 30,56
C2 Pikasso variety 3 sites FAM 21,65 21,65
D2 Pikasso variety 4 sites FAM 26,36 26,36
E2 Kizil kuz variety 1 site FAM 15,53 15,53
F2 Kizil kuz variety 2 sites FAM 17,59 17,59
G2 Kizil kuz variety 3 sites FAM 19,56 19,56
H2 Kizil kuz variety 4 sites FAM 21,20 21,20
A3 PKO (K+) FAM 30,74 30,74
B3 ОКО (K-) FAM no Ct
According to the results of the research, Santa, Azara, Picasso and Kizil kuz potatoes were infected with PVL. From all 4 samples, it was determined from the results of PGR that the infestation of Kizil kuz variety potato with the virus was higher.
The samples provided for the identification of potato L-virus reservoirs were examined by PCR method and the following results were obtained (Table 2).
2-Figure. PVL reservoirs: a- Malva L, b- Brossica campestris L.
Table-2
Expertise conclusion on the identification of PVL reservoirs carried out by the PCR
amplifier
Tested samples Sample name Dye Ct mean Ct
Al Fizalis fPhysalis) FAM no Ct
B1 Potato (S.tuberosum] Kayroki variety tuber FAM 34,1 34,1
CI Goosefoot Chenopodiumalbum FAM no Ct
D1 Henbane (Hyoscyamus L.) FAM no Ct
El Field mustard (Brossicacampestris L.) FAM no Ct
Fl Purslane (Portulacaoleracea L.) FAM no Ct
Gl Clover (Trifolium) FAM no Ct
Hl Hedge bindweed fConvolvulussepium) FAM no Ct
A2 Black nightshade (Solanumnigrum] FAM no Ct
B2 Potato (S.tuberosum] tuber of red potato FAM 29,79 29,79
С2 Tomato (Solanumlycopersicum) FAM no Ct
D2 Malva (Malva L.) mallow FAM no Ct
Е2 Sweet pepper (Capsicum annuum L] FAM no Ct
F2 Thistle (Cirsiumarvense) FAM 30,37 30,37
G2 Potato (S. tuberosum] local Gala variety tuber FAM no Ct
H 2 PKO (K+) FAM no Ct
A3 ОКО (K-) FAM 28,86 28,86
According to the results of the body of the wild and cultivated potato
analysis conducted to identify the plants shown in the literature has a high
reservoirs of PVL in the weeds grown in titer of the virus. (Fig. 1. a, b). the potato fields: it was found that the
3-Figure. PVL reservoir: a-(S.tuberosum) Kayroki variety tuber, b-(S.tuberosum) local Gala variety tuber.
Taking into account that wild and cultivated potato plants grow in autumn, their tubers overwinter under the soil, and continue to grow in spring, the storage of PVL in reservoir plants causes the periodic circulation of the virus and its spread to a wider area.
CONCLUSION
One of the main conditions for ensuring a high and quality harvest from plants is timely detection of the disease, correct and prompt diagnosis, and proper protection of crops from diseases. Therefore, it is necessary to have information about the identification of the causative agent, its development, spread, and how it is preserved from one season to another.
In this study, disease symptoms such as leaf discoloration or yellowing and coarsening as well as crinkling of the leaf plate were studied by PCR monitoring.
From the studied literature and conducted experiments, it was found that: PVL is found mainly in representatives of the nightshade family. It was also found that these plants, which are found in potato fields as weeds, keep PVL in their bodies and participate in the periodic cycle.
Identifying natural reservoir plants and avoiding their growth in fields is very important in preventing the spread of this virus.
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