Molecular characterization by PCR-ITS technique of Fusarium oxysporum isolated from tomato in Baghdad city

Keywords: Fusarium oxysporum, PCR, gene sequence

The present study aimed to estimate the pathogenesis of Fusarium oxysporum isolates and identify them by molecular technique Polymerase Chain Reaction-Internal Transcript Spacer (PCR-ITS). A total of thirty Fusarium oxysporum isolates were isolated from infected tomato plants and diagnosed depending on 'morphological characteristics. The pathogenicity test was performed for thirty Fusarium oxysporum isolates. So it was procedure steps that Genomic DNA was extracted from seven F. oxysporum isolates according to pathogenicity test by using'' ZR Fungal/Bacterial DNA'' MiniPrep™ kit. Seven F. oxysporum isolates showed high pathogenicity. The concentration and purity of the DNA extracted from the seven F. oxysporum isolates was (150 -201) ng/µl and (1.4 -1.9) respectively. ITS gene was amplified by using PCR-ITS The sequencing results of amplified product of ITS gene from Fusarium oxysporum isolates indicated that all the seventh sequenced isolates where from one Formae speciales of Fusarium oxysporum as Fusarium oxysporum f. sp. Lycopersici. These results have been devoted to study genetic variability among the species involved in study. Molecular markers are highly important'' to focus on F. oxysporum isolates below'' the species level therefore, the DNA array containing "genus, species'' and forma specialis specific" detector'' for the detection and identification ''of F. oxysporum. Also, Cluster analysis and phylogenetic tree depending on genetic distance revealed the genetic relationship between the thirteen F. oxysporum isolates and confirmed sequencing analysis. 

Introduction

Growth and productivity of tomato plants (Lycopersicon esculentum Mill.) are facing different challenges due to the outbreak of pest and diseases [1] Wilt disease caused by Fusarium oxysporum f. sp. lycopersici is highly destructive in both greenhouse and field causes economically loss for this crop [2,3]. Controlling of tomato wilt disease give significant defies because of the ability of this fungus to remain dormant in the soil in the form of spores [4]. In Iraq, fungal diseases are the main reason for the reduction of tomato production, fungal diseases cause significant economic losses, especially wilt diseases, root rot and dapping off caused by Fusarium oxysporium [5,6]. Fusarium oxysorum is one of the most important pathogenic fungi that infects plants through roots in all the plant's growth stages. Pathogen causes the symptoms of necrosis and vascular wilt diseases in a wide variety of economically important crops [7]. Fusarium classified under the Fungi kingdom, Division of Ascomycota, Class of Sordariomycetes, Hypocreales Order, Family of Nectriaceae, Genus of Fusarium and Species of Fusarium oxysporum [8]. Fusarium wilt pathogens show a high grade of host specificity and based on the plant species and plant cultivars that infect by this fungus. F. oxysporum are classified into more than 100 formae specials and strains [9,10]. F. oxysporum that attack Tomato is F. oxysorum f.sp. Lycoperesici, which is one of the most important diseases of Tomato in Iraq [1] and it is an economically important disease that exists where a crop is grown causing the destruction of plants that are cultivated in the fields and protected houses, especially in warm areas of the world, where the rate of loss in the result of the incidence of this fungi is between 10 -50% per year [11,12]. F. oxysporum that attack Solanum melongena is F. oxysporum f.sp. melongenae, and it's considered the most pathogenic fungus on eggplant. F. oxysporum f.sp. melongenae was initially described in 1958 from S. melongena plant suffering from a vascular wilt disease [13] and other cases of financially imperative formae speciales incorporate melonis, tulipae (on tulips), cubense (on banana) and asparagi (on asparagus plants). In general, morphological characterization of plant pathogenic fungi is achieved by traditional pathogenicity bioassays under greenhouse conditions and this method takes a long time and high costs, also this identification does not give the specific classification of the isolates because it depends only on the morphological characters under microscope and this is not enough to identify the under-species levels because of the high similarity of this species isolates. So, wilt disease in the fields of tomato, middle of Iraq was identified using molecular approaches [14]. Also, characterization and molecular identification of F. oxysporum isolates were investigated [15]. At present, molecular biology technologies are being used to determine the specific formae specilas and strains of F. oxysporum, since these technologies are usually quick, accurate, and low-cost [16] The aim of the study was to concentrate on the molecular Identification of Fusarium oxysporum which are isolated by PCR-ITSgene. 

Materials and Methods


Isolation, Purification and Identification of Pathogens

The infected tomato plants were collected during the month of December 2017 from two greenhouses in Al-Rashidiya's farm (Baghdad province) and a number of plants in each field was 750. The twenty infected tomato plants were collected by crossing the diameters from each greenhouse, then they were put in polyethylene bags individually and the name of the area and the date of collection were written on each bag. After that the samples were taken to the laboratory to isolate and identify the pathogens. The infected plants were identified based on the symptoms that appeared on the shoot and root, which included yellowing and drooping of leaves, death of some branches, reddish-brown streaks were visible in the vascular tissues when cut with a knife and finally death of plants. The stems of infected plants were cut at a height of 15 cm above the crown area. After that, the roots were washed by tap water for two hours to remove of the mud suspended. The stems and roots were cut into small pieces (0.5-1 cm) and sterile by immersing them for 3 minutes in the sodium hypochlorite solution (1% free chlorine), then washed by immersing them in sterilized distilled water for three times, after that dried with sterile filter paper. Four pieces (one for the root and three for the stem) were cultured for each infected plant in petri dishes containing autoclaved PDA and incubated for 4 days at 28±2 ˚C [1] The isolates fungi were purified by taking mycelial plugs with a five mm diameter from the growing margin and put overhead onto fresh PDA in the center of the petri dish and incubated for five days at 27±2 ˚C. F. oxysporum were identified based on the morphology of the colony, conidiophores and spores shapes according to [17,18] by Assistant Professor Dr. Alaa Mohsen Al-Aaraji. All fungal isolates were cultured and maintained on autoclaved PDA and incubated for 5 days at 28±2 ˚C. Slant cultures were also prepared, and all stored in a refrigerator at 4 ˚C [19]. 

Fusarium oxysporum pathogenicity test:

This test was carried out to detect the pathogenicity of F. oxysporum isolates. Local cress seeds untreated with fungicide were surface sterilized by immersion in sodium hypochlorite (1 % free chlorine) for two min, rinsed three times with sterile distilled water, dried by sterile filter paper. Ten seeds for three replicates were placed on the sterile filter paper inside a Petri dish containing moisture by putting drops of sterile distilled water, then leaves for 7 days, then the percentage of seeds germination account was counted according to the following formula. 

P = Number of non germinating seeds

Total number of seeds X 100 P = the percentage of non-germination seeds Genomic DNA extraction Genomic DNA was extracted from seven Fusarium oxysporum isolates infected tomato plants were selected according to pathogenicity test (named Fox1-7) by using ZR Fungal/Bacterial DNA MiniPrep™ (USA, the epigenetic company). This kit was designed for the simple and rapid isolation of DNA from tough-to-lyse fungi and bacteria in minutes. In the beginning all the isolates used in the DNA extraction were purified several times till getting rid of any contamination (other fungus spores or bacteria) by culturing the isolates on the PDA medium for activation. The step of activation is very necessary for extraction to get pure culture and good yield of genomic DNA. 

Agarose gel electrophoresis of DNA:

Agarose gel Electrophoresis was done to determine DNA pieces after the process of extraction PCR reaction to detect bands by using standard DNA to distinguish the band size on the agarose gel. 

Primers of gene ITS

The primer pair of ITS gene that shown in the table (1) was purchased at lyophilized form, they dissolved in the free H 2 O (dd) to give a final concentration of 100 pmol/µl as stock solution and keep a stock at -20 to prepare 10 pmol/µl concentration as work primer suspended, 10 µl of the stock solution in 90 µl of the free H2O (dd) water to reach a final volume 100µl, was investigated by IDT (Integrated DNA Technologies company, USA). 

Table 1. The specific primer of gene ITS Primer Sequence


Amplification of ITS region:

The universal primers (ITS-1& ITS-4) were used for the amplification of rRNA gene and ITS regions which is found in all eukaryote as a conserved region [23]. The thermal cycling conditions were done as follows: Initial Denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 40s, 56°C for 35sec and 72 °C for 35sec with final extension 2 at 72 °C for 10 min using a thermal Cycler (Gene Amp, PCR system 9700; Applied Biosystem). All PCR products were separated by 1.5% agarose gel electrophoresis and visualized by exposure to ultraviolet light (302nm) after staining by red safe stain (Intron Korea). 

Sequencing and sequence alignment

All PCR product isolates were sent for Sanger sequencing using ABI3730XL, automated DNA sequences, by Macrogen Corporation -Korea. Homology search was leaded using Basic Local Alignment Search Tool (BLAST) program which is available at the National Center Biotechnology Information (NCBI) online at (http://www.ncbi.nlm.nih.gov) and BioEdit program to detect polymorphism. The results are received by email then analyzed using genious software. 

Results and Discussion

Isolation and identification of Fusarium oxysporum isolates Thirty F. oxysporum isolates were obtained from infected tomato plants from two greenhouses in Al-Rashidiya's farm (Baghdad Governorate). The pathogenic fungus F. oxysporum isolates were purified and identified depending on morphological and microscopically characters. The morphological characteristics on PDA medium were observed (Figure 1) cottony mycelium and variable color according to age, from cream-colored colony to pinkish violet. Microscopically characters of F. oxysporum showed that macroconidia have three-to five-septate, sickle-shaped, with foot-shaped basal cells. Microconidia without septa, oval and borne in false heads, and chlamydospores were visible in culture [17,18,21,22] In Iraq, many researchers have pointed to the isolation of F. oxysporum from tomato plants infected with it and root rot disease [14,15] Also in study of [23] seven Fusarium isolates isolated from Serbia were examined macroscopically and microscopically. 
During morphological observation, aerial mycelia of all isolates were white at the initial stage, while the underside of colonies became pale pink, violet, purple and brown in the later stages on PDA. Based on their morphological characteristics (shape, form, separation, size, microconidia, macroconidia, phialides, chlamidospores, sclerotia), the isolates were identified as Fusarium oxysporum. 

Pathogenicity Test of Fusarium oxysporum

Results of pathogenicity test showed that all F. oxysporum isolates were high pathogenic for local cress seeds, and seven F. oxysporum isolates (1, 11, 12, 13, 19, 29 and 30) showed high pathogenicity more than other isolates, which recorded 100 % inhibition growth of local cress seeds (table 2). These seven F. oxysporum isolates were selected to complete this study and named (Fox1-7). These results were consistent with [24], who noted that all isolates of F. oxysporum were highly capable of infecting eggplant plants. The results of this study were in agreement with a study done by [23] who demonstrated that the results of the pathogenicity test revealed that all seven isolates of Fusarium species were pathogenic to the seedlings. They showed symptoms like crown rot and root necrosis or root rot. These symptoms were observed within 30 days after inoculation as necrosis and brown discoloration of the root, lower part of the stem necrosis, and a complete rottenness and decomposition. 

Table 2. Pathogenicity test of Fusarium oxysporum on local cress seeds plants.

*each number represents three replicates. 

DNA Extraction:

The DNA was extracted efficiently from seven Fusarium oxysporum isolates by using wizard ZR Fungal/Bacterial DNA MiniPrep™ Kit. Purity and concentration of DNA were measured using Nanodrop [25]. The yield of the DNA extracted from the F.oxysorum isolates was in range of (150 -201) ng/µl with purity ranged from (1.4 -1.9). Then the product of DNA was confirmed by agarose gel electrophores (Figure 2). Genomic DNA was successfully extracted from F. oxysporum isolates using ZR fungal DNA kit for the specific PCR and ITS-PCR analysis [26] Fusarium oxysporum identification by gene sequence: 
The identification of the seven F. oxysporum isolates was based on the ITS region. The intergenic spacer region was successfully amplified from all F. oxysporum isolates. All isolates yielded a unique product size of approximately 550-600bp as shown in (Figure 3). This result is similar to those obtained in previous studies on identification, characterization and genetic variability of the Fusarium isolates where the amplification of the rDNA region of Fusarium isolates yielded amplicons in the size range of 600pb. 
The Fusarium species specific PCR primers, ITS1 and ITS4 can provide a fast and accurate tool for the identification and characterization of Fusarium species. 

Gene Bank identification of F. oxysporum isolates:

Seven DNA samples amplified PCR-products for ITS gene from Fusarium species were sequenced for the detecting SNPs within these sequences. Our sequences were compared with reference sequence of National Center Biotechnology Information's (NCBI) [27] who studied sixteen isolates of Fusarium oxysporum which were collected from the root and stem fragments of guava plants growing in six districts of Bangladesh. Species identity was based on the colony character, nature of conidiogenous cell, morphology of microconidia, macroconidia and chlamydospores. Eleven isolates were confirmed as F. oxysporum through polymerase chain reaction (PCR) using species specific primers designed from the conserved regions of 18S rRNA gene. 

Phylogenetic tree structuring

The phylogenetic tree diagrammatic by Molecular Evolutionary Genetics Analysis (MEGA) software version 6.0. Sequences that showed the highest identity (>95%) and maximum coverage (100%) as shown in (Figure 4). The genetic distance between Iraq and the isolates of the world is detailed according to the Phylogenetic tree and the comparison. 
Cluster analysis and phylogenetic tree depending on genetic distance in order to reveal the genetic relationship between the thirteen F. oxysporum isolates. Analysis of this dendrogram, the overall result indicated that all F. oxysporum isolates were classified into two groups. The first major group was further divided into two subgroups. The first subgroup is divided into two divisions, the first division include the first two isolates which are (F1 and F2), while the second division include the second two isolates which are (F6 and F7). The second subgroup is separated into two divisions, the first division include the first isolate which is (F5), while the second division include the second two isolates which are (F3 and F4). 

Conclusion

Plant pathogenic fungi Fusarium oxysporum ''which causes wilt is a serious pathogen ''which economically important ''because many members are considered'' the causal agents of vascular wilt'' or root rot diseases in agricultural'' of Iraq. The DNA array containing "genus, species'' and forma specialis specific" detector'' for the detection and identification ''of F. oxysporum. And The sequence of ITS gene product (565bp) were appeared presence of one forma specialis ''F. oxysporum (7 Fusarium oxysporum'' f. sp. Lycopersici). Also, Pathogenicity tests cannot determine'' the specific species and subspecies of F. oxysporum. 

Figure (appendix1). Sequence analysis of ITS gene. Appendix 2. Alignment of nucleotide sequencing of isolate (F2) gene bank of NCBI, and appeared compatibility of gene under sequence ID: MG136705.1

After alignment the product amplification of ITS gene for the (F2) isolate that shown to have one transition A>G, and two transversion T>A, and G>C from the Gene Bank, found that part of ITS gene having 99% compatibility with standard in Gene Bank ITS gene as it shown in Figure (appendix 2 After alignment the product amplification of ITS gene for the (F4) isolate that shown to have one transition C>T from the Gene Bank, found that part of ITS gene having 99% compatibility with standard in Gene Bank ITS gene as it shown in Figure (appendix4) 



  premix kit (Intron/Korea) was used for PCR amplification, which contained 2.5U I-Taq DNA Polymerase, 2.5mM DNTPs, 1X Reaction buffer (10X), and 1X Gel loading buffer. The PCR amplification mixture of the specific reaction for diagnosis gene was performed in a total volume of 20µl containing 1.5µl DNA, 1 µl Taq PCRPreMix, 1µl of each primer (10 pmol) then 16.5 µl of distilled water was added into tube to a total volume of 20µl. 


Figure 1 .

 1 Figure 1. Fusarium oxysporum (A) under microscope (B) morphology on PDA medium. 


Figure 2 . 1 %

 21 Figure 2. 1 % Agarose gel Electrophoresis of total genomic DNA for Fusarium oxysporum isolates, (70 Volt/30 minute). 


Figure 3 .

 3 Figure 3. shown PCR product of ITS gene band size 565bp that was electrophoresis on 2% agarose gel ITS gene. 


Figure 4 .

 4 Figure 4. Dendrogram showing genetic diversity among seven of Fusarium oxysporum isolates 


Appendix 4 .

 4 Alignment of nucleotide sequencing of isolate (F4) gene bank of NCBI, and appeared compatibility of gene under sequence ID: MG136705.1 




  Gene Bank.The samples were treated with AB13730XL APPLIED BIOSYSTEMS machine in National Instrumentation Center for Environmental Management NICM/USA company online at (http://nicem.snu.ac.kr /main/?en_skin=index.html).The result of the sequence analysis has become clear in terms of BLAST search in the National Centre Biotechnology Information (NCBI) online at (http:// www.ncbi.nlm.nih.gov) and BioEdit program to detect polymorphism.After sequencing analysis on blast website, the acquired results allowed to determine Fusarium species level.The sequencing results of amplified product of ITS gene from Fusarium oxysporum isolates indicated that all the seventh sequenced isolates where from one Formae speciales of Fusarium oxysporum as Fusarium oxysporum f. sp.Lycopersici as shown in Appendix.The result of this research is consistent with the research of Hussien et al.,(2013) 



appendix 2). Sequence analysis of ITS gene. Appendix 3. Alignment of nucleotide sequencing of isolate (F3) gene bank of NCBI, and appeared compatibility of gene under sequence ID: KF914451.1

  ). Amplified product of ITS gene from Fusarium species of isolate (F3) appeared 100% compatibility for ITS gene as it shown in Figure(appendix 3). 


	Query CCAACCCCTGTGACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACG 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 17 CCAACCCCTGTGACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACG 1 76 Query 61 GGACGGCCCGCCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAA 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 77 GGACGGCCCGCCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAA 136 Query 121 CAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAG 180 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 137 CAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAG 196 Query 181 CAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCA 240 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 197 CAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCA 256 Query 241 CATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAG 300 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 257 CATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAG 316 Query 301 CTCAGCTTGGTGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGT 360 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 317 CTCAGCTTGGTGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGT 376 Query 361 CGAGCTTCCATAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCG 420 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 377 CGAGCTTCCATAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCG 436 Query 421 TAAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAG 480 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 437 TAAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAG 496 Query 481 CATATCAAAAGGCCGGAGGAA 501 ||||||||||||||||||| Sbjct 497 CATATCAATAAGCCGGAGGAA 517 17 CCAACCCCTGTGACATACCAATTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGG 76 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 3 CCAACCCCTGTGACATACCAATTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGG 62 Query 77 AACGGCCCGCCAGAGGACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCAT 136 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 63 AACGGCCCGCCAGAGGACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCAT 122 Query 137 AAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGC 196 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 123 AAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGC 182 Query 197 AAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCAC 256 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 183 AAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCAC 242 Query 257 ATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGC 316 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 243 ATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGC 302 Query 317 CCCCGGGTTTGGTGTTGGGGATCGGCGAGCCTCACGGCAAGCCGGCCCCGAAATACAGTG 376 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 303 CCCCGGGTTTGGTGTTGGGGATCGGCGAGCCTCACGGCAAGCCGGCCCCGAAATACAGTG 362 Query 377 GCGGTCTCGCTGCAGCTTCCATTGCGTAGTAGTAAAACCCTCGCAACTGGTACGCGGCGC 436 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 363 GCGGTCTCGCTGCAGCTTCCATTGCGTAGTAGTAAAACCCTCGCAACTGGTACGCGGCGC 422 Query 437 GGCCAAGCCGTTAAACCCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCG 496 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 423 GGCCAAGCCGTTAAACCCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCG 482 Query 497 CTGAACTTAAGC 508 |||||||||||| Sbjct 483 CTGAACTTAAGC 494 Figure (Query Figure (

appendix 3). Sequence analysis of ITS gene.

  

. Sequence analysis of ITS gene. Appendix 5. Alignment of nucleotide sequencing of isolate (F5) gene bank of NCBI, and appeared compatibility of gene under sequence ID: MG136705.1

  . After alignment the product amplification of ITS gene for the (F5) isolate that shown to have three transversion A>T, T>A and C>G from the Gene Bank, found that part of ITS gene having 99% compatibility with standard in Gene Bank ITS gene as it shown in Figure(appendix 5). 


	Query AACCCCTGTGACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCTGTAAAACGGG 60 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 19 AACCCCTGTGACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACGGG 1 78 Query 61 ACGGCCCGCCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAACA 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 79 ACGGCCCGCCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAACA 138 Query 121 AATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCA 180 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 139 AATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCA 198 Query 181 AAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACA 240 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 199 AAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACA 258 Query 241 TTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCT 300 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 259 TTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCT 318 Query 301 CAGCTTGGTGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGTCG 360 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 319 CAGCTTGGTGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGTCG 378 Query 361 AGCTTCCATAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCGTA 420 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 379 AGCTTCCATAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCGTA 438 Query 421 AAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCA 480 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 439 AAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCA 498 Query 481 TATC 484 |||| Sbjct 499 TATC 502 3 ACCCCTGTGACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACGGGA 62 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 20 ACCCCTGTGACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACGGGA 79 Query 63 CGGCCCGCCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAACAA 122 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 80 CGGCCCGCCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAACAA 139 Query 123 ATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAA 182 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 140 ATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAA 199 Query 183 AATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACAT 242 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 200 AATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACAT 259 Query 243 TGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCTC 302 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 260 TGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCTC 319 Query 303 AGCTTGGTGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGTCGA 362 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 320 AGCTTGGTGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGTCGA 379 Query 363 GCTTCCATAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCGTAA 422 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 380 GCTTCCATAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCGTAA 439 Query 423 AACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCAT 482 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 440 AACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCAT 499 Query 483 ATCATAAAGGCGGAGGAA 500 ||||||||||||||| Figure (appendix 4)Query Sbjct 500 ATCAATAAGCCGGAGGAA 517

Figure (appendix 5). Sequence analysis of ITS gene. Appendix 7. Alignment of nucleotide sequencing of isolate (F7) gene bank of NCBI, and appeared compatibility of gene under sequence ID: MG136705.1

  Amplified product of ITS gene from Fusarium species of isolate (F7) appeared 100% compatibility for ITS gene as it shown in Figure(appendix 7). 


	Query	1
	TGACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACGGGACGGCCCG 60	
	||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||	
	Sbjct	27
	TGACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACGGGACGGCCCG 86	
	Query	61
	CCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAACAAATAAATC 120	
	||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||	
	Sbjct 87 CCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAACAAATAAATC
	146	
	Query	121
	AAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGA 180	
	||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||	
	Sbjct	147
	AAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGA 206	
	Query	181
	TAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCC 240	
	||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||	
	Sbjct 207 TAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCC
	266	
	Query	241
	GCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCTCAGCTTGG 300	
	||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||	
	Sbjct 267 GCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCTCAGCTTGG
	326	
	Query	301
	TGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGTCGAGCTTCCA 360	
	||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||	
	Sbjct 327 TGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGTCGAGCTTCCA
	386	
	Query	361
	TAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCGTAAAACCCCA 420	
	||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||	
	Sbjct	387
	TAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCGTAAAACCCCA 446	
	Query	421
	ACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATA 480	
	||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||	
	Sbjct	447
	ACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATA 506	
	Query 481 AG 482	
	||	
	Sbjct 507 AG 508	

Figure (appendix 7). Sequence analysis of ITS gene. 
Appendix

Appendix 1. Alignment of nucleotide sequencing of isolate (F1) gene bank of NCBI, and appeared compatibility of gene under sequence ID: MG136705. 1 After alignment the product amplification of ITS gene for the (F1) isolate that shown to have one transition A>G, one transversion T>A from the Gene Bank, found that part of ITS gene having 99% compatibility with standard in Gene Bank ITS gene as it shown in Figure (appendix1). 

Appendix 6. Alignment of nucleotide sequencing of isolate (F6) gene bank of NCBI, and appeared compatibility of gene under sequence ID: MG136706.1

After alignment the product amplification of ITS gene for the (F6) isolate that shown to have one transversion T>A and one transition A>G from the Gene Bank, found that part of ITS gene having 99% compatibility with standard in Gene Bank ITS gene as it shown in Figure (appendix 6). 

Figure (appendix 6). Sequence analysis of ITS gene.
Noor Saad, Nahi Al-Adhami, Alaa Mohsen Al-Araji
Molecular characterization by PCR-ITS technique of Fusarium oxysporum isolated from tomato in Baghdad city