Diagnosis of Olive Leaf Flay Daisenura oleae by PCR Technique in Nineveh Governorate

Through the PCR diagnostic technique, it found the Daisenura oleae, from the Cecidomyiidae family and the order Diptera. Olive leaf flay were characterized by their activity and continued presence during the activity season. The results of the electrophoresis of flys DNA, which was extracted from olive leaf adults. Obtained pure DNA, and the purity of the parasite DNA was confirmed by a successful operation. Electrophoresis using Agarose gel and showing the nucleic acid bundles clearly. The sequence of the nitrogenous bases of the samples (Cyanobacteria) that were under investigation was determined, as products were sent for PCR representation, and the sequence reading of the genes was completed by depending on the 3130 genetic analyzer that was equipped from the Japanese company Hitachi. BLAST software was used to compare the gene sequences that were sequenced with the gene sequences that were documented in the National Center for Biotechnology Information (NCBI). The findings of this comparison were then analysed. The products of the amplified packages resulting from the PCR reaction were sent outside Iraq to study the sequence and sequence of the nitrogenous bases and compare them with the original cytochrome c oxidase subunit I (cox1) gene stored in the Global Genome Bank with specific sites of the gene to be studied, and it was found that there are sites of variation or variation in the nucleotide sequence compared to with the original gene at the NCBI website, and the isolate match was 100%. The isolate was recorded in the Gen Bank with accession number LC757225.1, and the genetic tree was drawn to find out the relationship between the Iraqi insect isolate and the rest of the insect isolates, and the closest was the American isolate.


Introduction
The olive tree is exposed to many types of insects, which cause great economic damage and at the same time affect the quality of the crop and thus lead to its deterioration in terms of quantity and quality.unionalis Hubner, Euphlura olivine Costa olive, and Phloeotribuss carabeoidse (Bren) olive neron.The olive leaf fly, Dasineura oleae F. Loew, is one of the olive insects that appeared in recent years in olive groves in some parts of the world.It recorded many infections in the Mediterranean regions, where it was recorded as one of the most dangerous insect pests on olive trees in coastal areas in Greece [1].The olive leaf fly is one of the most dangerous pests that infect olive trees.Infestations 1259 (2023) 012089 IOP Publishing doi:10.1088/1755-1315/1259/1/012089 2 were recorded for the first time in 2011 in many orchards in the rural area around and central Macedonia, where the olive leaf fly caused severe deformities and tumors evident on the leaves and tender branches [2].In a previous study [3], showed that this insect is a major pest in Italy, as well as it could be in other countries.The olive leaf fly also spreads in Palestine, Jordan, Turkey, Syria and Cyprus [4,5].The olive leaf fly is one of the dangerous insects that infect olive trees in Iraq, which prompted farmers to use many control methods in an attempt to control this insect and reduce its damage, especially the use of chemical pesticides, which is well known for its side effects on the environment and public health.As well as the possibility of the emergence of strains tolerant or resistant of this type of insecticides used in control operations.Therefore, integrated control programs today seek to find plant species that are tolerant or resistant to pest infection, and breeding plants that are resistant to insect pests is one of the most important goals that plant breeding programs seek to achieve.The importance and position of pest-resistant varieties in agricultural pest control programs has increased, and resistant varieties have succeeded in varying degrees in saving millions of dollars that were spent on control as well as the economic loss in the crop due to pest infestations, which made the process of using varieties resistant to insect pests an issue that receives more acceptance by farmers in developed countries, In developing countries, where the use of resistant varieties is one of the modern trends to protect the environment, especially since the use of pesticides in these countries is a modern practice compared to developed countries, and investment in the production of pestresistant varieties has today become a profitable investment for many companies, but it is noted that This investment focused in the field of production of pest-resistant vegetables and field crops, while the interest and investment was less in the field of breeding pest-resistant olive tree varieties [6].And due to the lack of sufficient research confirming the diagnosis of the insect using the PCR technique in Iraq, especially in Nineveh Governorate, the idea of diagnosing this insect with this modern technique was crystallized.

Materials and Methods
Samples were collected from flies during the 2022 season from the Nineveh governorate station of olive varieties, Bakowal, Nepali, Khastawi and Syrian.The samples were placed in 70% alcohol and preserved until the diagnosis process.

Extraction of the DNA of the Insects
The analysis kit prepared by (Monarch® Genomic DNA Purification Kit) was relied upon to extract DNA from samples of flies, according to the steps proven in the attached file.Insects were collected from the olive leaf fly, one adult insect was weighed and placed in an Eppendorf tube, the weighed insects were placed in the freezer at 80 ° C for 24 hours, after that the insects were ground using a glass rod and some small grains of sand were added to ensure that the insect was well ground [7].
 A homogenous powder should be created by weighing 0.2 milligrammes of insect material, adding liquid nitrogen to it in a ceramic mortar, and grinding the combination for three seconds until it is completely pulverised. After transferring the powder to a tube with a capacity of 1.5 milliliters, 400 microliters of GP1 and 5 microliters of RNase are introduced to the tube before being mixed using a mixing device.(Vortex). The test container should be kept in an incubator with a temperature of 60 degrees Celsius for ten minutes, during which time it should be turned once every three minutes. After adding 100 µl of the GP2 Buffer solution, mix everything together using a device called a Vortex, and then incubate it on cold for three minutes. After transferring the combination into a Filter Column tube, it is then subjected to a centrifugation that lasts for one minute and has an acceleration of one thousand g.After that, the filter tube is thrown away, and the leachate is poured into a 1.5 ml container.Add 1.5 volts of GP3 solution and mix directly for 5 seconds. Place 700 microliters of the solution inside of a GD Column container, then centrifuge the mixture for two minutes at the highest possible speed.
 After discarding the filtrate, the GD Column is reinserted into the collection container.This step is repeated. After adding 400 L of solution W1, centrifuge for a full 30 seconds at the highest possible speed. The filtrate should be discarded, and the GD Column should then be reattached to the collection tube. After adding 600 µl of the Wash Buffer solution and centrifuging at maximum speed for thirty seconds, discarding the supernatant and continuing to centrifuge at maximum speed for three minutes. After placing the GD column in a tube with a capacity of 1.5 milliliters, 100 microliters of elution solution are added to the column, and the tube is allowed to sit undisturbed for three minutes so that the solution can bond with the DNA. Perform the centrifuge for a full thirty seconds at the highest speed.Throw away the GD column, and store the precipitate at a temperature of -20 degrees Celsius until it is needed.

DNA Extraction from the Gel
In order to perform a nucleotide sequence sequencing test, the bundles that resulted from the PCR reaction were extracted from the gel in order to purify them and send them on their way.This procedure was carried out in accordance with the analysis kit that was prepared by (Gene aid) and according to the steps that were as follows:  A sterile scalpel is used to cut the packet out of the agarose gel.This is done while simultaneously removing the largest possible quantity of gel that surrounds the packet. After placing approximately 300 milligrammes of the gel component in a 1.5 millilitre Eppendorf tube, add 500 microliters of the DF buffer, and then mix using the Vorteky device. To completely ensure the formation of the gel component, the tube is incubated for 15 minutes at a temperature of 55-60 degrees Celsius, with the tube being rotated once every three minutes during the incubation period.After the incubation period has concluded, the tube is allowed to cool at room temperature. After transferring 800 microliters of the sample combination to the DF column that has been installed in the collection tube, centrifuge the mixture at 16000g for thirty seconds, and then discard the filtrate. After reattaching the DF column to the collection tube, 600 microliters of cleaning solution are poured in, and the mixture is allowed to sit for one minute. Remove the supernatant from the mixture after centrifuging it at the same speed of 16,000 g for thirty seconds. The action from before is carried out once more. To guarantee that the DF column is completely dry, perform a centrifugation for three minutes. The tube that was used for collecting the sample is thrown away, and the DF column is placed in a fresh tube that has 1.5 ml. Twenty to fifty microliters of extraction buffer should be added to the middle of the column. Wait for two minutes in order to guarantee that the dissolving solution has been absorbed. In order to acquire the DNA that has been dissolved, centrifugation at a speed of 16000g for two minutes.

Electrophoresis Gel Electrophoresis Agarose Gel Electrophoresis
Agarose gel is prepared at a concentration (1.5%).by dissolving (1.5) gm.Of Agarose powder in (100) ml of Tris-Borate EDTA Buffer (TBE).At a concentration of (1X), using a beaker capacity (250) ml.well mixes the Agarose powder with the buffer solution (TBE) in the beaker.Placed the beaker in the microwave oven for (1.(15-20) minutes at room temperature, after that the comb is pulled from the hardened agar with extreme caution, as it remains the location of the comb teeth is empty holes known as Wells.These holes represent the injection sites for DNA samples in preparation for the electrophoresis process.The Agarose gel is immersed in the entire electrophoresis basin, with (700) ml of TBE buffer solution at a concentration of (1X) added to it, after which (1X) placed ( 7) microliter of nucleic acid amplification product The PCR products resulting from the polymerase chain reaction are collected in the specified pits.The DNA Ladder gradients are (100 bp) for each gradient.In addition, the power supply is connected to (80) volts and (300) mA for one hour.After completing the electrophoresis process (chromatography), the Agarose gel was extracted and placed into Gel Doc EZ Gel Documentation System.Where it shines ultraviolet light on the Agarose gel to detect amplification products, and the images of the results are saved for later analysis.

PCR Reactions
In order to achieve the necessary concentration for carrying out the PCR reactions, the concentration of DNA in all of the study samples had to be adjusted by diluting them with TE Buffer solution.The final concentration of DNA in each sample was (50) ng / microliter.Since the master reaction mixture was produced for each PCR reaction by mixing the DNA sample and the special initiator for each gene with the components of the pre-mix inside of a 0.2 ml epipendruff tube that was prepared by the English company Biolaps, we can say that each PCR reaction had its own unique master reaction mixture.The volume of the reaction was brought down to 20 microliters by adding some distilled water, and then the combination was thrown away. in a Microfuge for a time period of (3-5) seconds to ensure mixing of the reaction components, then the reaction tubes were inserted into the thermocycler for the purpose of conducting the multiplication reaction using the special programme for each reaction, after that the sample was loaded into the pits of the agarose gel prepared in advance at a concentration of 2% with the addition of evidence Volumetric DNA Ladder prepared by Biolaps in one of the pits, after that t, then the sample was transferred.

Molecular Diagnosis of Olive Leaf Flay Region-Dependent Cytochrome C Oxidase Subunit I (cox1) Gene
When the contents of the master blend were supplemented with 4 microliters (100 nanograms) of template DNA and 1 microliter (10 picompl) of each gene-specific primer, it became possible to determine whether or not the amplified region was present.This was accomplished.Table 1.The specific gene primer were added to the contents of cytochrome c oxidase subunit I (cox1) gene.

Sequence Primer
5`-ATT CAACCA ATC ATA AAG ATA TTG G-3` Forward SSU 5`-TAA ACT TCT GGA TGT CCA AAA AATCA -3` Revers rDNA After that, the reaction tubes were inserted into the thermocycler to conduct the double reaction using the special program for the reaction as shown table 2.
Table 2. Steps of the double reaction mechanism in the thermo cycler.

DNA Sequencing Analysis of Nucleotides
The sequencing of DNA is the method that is used to discover and locate genetic mutations, as well as SNP variations and the highly conserved region.In most cases, the results of the PCR reaction are used to identify the sequences of amplified portions that must contain genetic differences.In recent years, the results that DNA technology has delivered have been: The sequencing process is extremely effective when it comes to locating genetic mutations [8,9].On the other hand, if the product of the PCR reaction includes more than one package, then the necessary portion of the gel must be purified and isolated.On the other hand, if the product of the reaction is specialised for only one package, then it may be used directly in the process of determining the sequences.Fig. 1 [2].

Determine the Nucleotide Sequence of the Amplified Pieces Based on the DNA Sequencing Technique
Because the PCR reaction products of the aforementioned samples were sent with the primers of the resulting package, and the sequence of the genes was read based on the 3130 Genetic Analyzer equipped from the Japanese company Hitachi, the sequence of the nitrogenous bases of the samples (Cyanobacteria) that were being studied was determined.The gene sequences were compared to gene sequences that were documented in the National Center for Biotechnology Information (NCBI), and the BLAST software was used to analyse the findings of this comparison.

DNA Extraction from Insect
DNA was extracted from the insect and gave a very good genomic DNA after transferring it on an agarose gel at a concentration of 1.5%, and detecting it using the Red Safe dye and after examining it with an ultraviolet light source, a single non-dispersive beam, as in Figure (2), and this is evidence of the purity of the acid Nuclear DNA.The insect isolate was diagnosed using polymerase chain reaction (PCR) to detect the cytochrome c oxidase I (cox1) gene, and using a pair of specialized primers, which are tandem and reverse reaction initiators.The amplification products, after electrophoresis on a 1.5% agarose gel, showed that the resulting bands They were 700 base pairs in size, as shown in   oxidase subunit I (cox1) gene, with a reaction product of 700 bp using a 1.5% agarose gel at 5 V / cm / 1:30 hours.The size of the bundle for the two isolates is 700 base pairs.M volumetric guide.

Results of the Sequensing of Nitrogenous Bases Study
The products of the amplified bundles resulting from the PCR reaction were sent outside Iraq to study the sequence of nitrogenous bases and compare them with the original cytochrome c oxidase subunit I (cox1) gene stored in the Global Genbank with specific sites of the gene to be studied.It was found that there were sites of variation or heterogeneity in the nucleotide sequence compared to the original gene at the NCBI site, and the isolate match rate was 100%, as shown in the table Figure (4).The isolate was recorded in the Gen Bank with accession number LC757225.1, and the genetic tree was drawn to find out the relationship between the Iraqi insect isolate and the rest of the insect isolates, and the closest was the American isolate, as shown in Figure (5).

Conclusion
In this study, the olive leaf fly was diagnosed for the first time by PCR technique, by detection with the original cytochrome c oxidase subunit I (cox1) gene stored in the Global Gen bank with specific sites of the gene to be studied.It was found that there were sites of variation or heterogeneity in the nucleotide sequence compared to the original gene at the NCBI site, and the isolate match rate was 100%.The isolate was recorded in the Gen Bank with accession number LC757225.1, and the genetic tree was drawn to find out the relationship between the Iraqi insect isolate and the rest of the insect isolates, and the closest was the American isolate

Figure 3 .
Figure 3.The product of the PCR reaction for samples of flies based on the primer cytochrome c oxidase subunit I (cox1) gene, with a reaction product of 700 bp using a 1.5% agarose gel at 5 V / cm / 1:30 hours.The size of the bundle for the two isolates is 700 base pairs.M volumetric guide.

7 Figure 4 .
Figure 4.The percentage of the correspondence of the insect isolate under study with the closest isolate recorded in the Gen Bank.

Figure 5 .
Figure 5.The genetic tree to show the genetic affinity between the Iraqi isolate and the isolates registered in the Gene bank.