1 © 2025 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License The First Record of Fusarium incarnatum as Wilting Agent on Tomato Plants in Iraq Rasha Khalid Hussein AL-Masoudi 1* , Neamat J. AL Judy 2 1,2 Department of Biology , College of Science, University of Baghdad, Baghdad, Iraq. *Corresponding Author. Received: 20 September 2023 Accepted: 9 January 2024 Published: 20 October 2025 doi.org/10.30526/38.4.3746 Abstract Fusarium wilt is one of the most significant diseases that affects tomato plants and is caused by the fungus Fusarium oxysporum, whether in fields or greenhouses. The current research aimed to identify the Fusarium incarnatum (Desm.) Sacc. agent that causes Fusarium wilt on tomato plants for the first time in Iraq by morphological and molecular methods, in addition to detecting the phylogenetic distance and similarity of the Iraqi isolates to other isolates globally. In January of 2023, the wilt signs were apparent on wilted tomato plants in Al-Mahmoudiya fields in Iraq. The isolate was confirmed and molecularly diagnosed according to the internal transcribed spacer (ITS) region. The sequences of ITS region for the isolate have been presented to the database in the NCBI Gen Bank for the first time, and it obtained their accession number (OQ439282.1). F.incarantum was isolated and purified and identified by using morphological and microscopic features, the morphological features of F.incarnatum isolate were observed on PDA media and under a microscope revealed that macroconidia have curved shapes, three to five septate, and basal cells with a foot shape. The pathogenicity of the fungus F.incarnatum showed the fungus's ability to cause wilting disease in healthy tomato plants at 90.33%, this demonstrates that the Fusarium isolate can create strains that can attack the host continually. Additionally, the phylogenetic tree revealed the genetic relationship between the Iraq isolate and other global isolates and it showed the Iraq isolate (OQ439282.1) is in the same clade as the Turkey isolate, India isolates, and China isolate with similarity 100%, and this clade shares 99.60% similarity with another clade, including Thailand, Taiwan, Nigeria, South Korea, Pakistan, Japan, Egypt, USA, and Tunisia isolates. As far as we are aware, this is the first morphological and molecular recording of F.incarantum on an Iraqi tomato plants. Keywords: Fusarium incarnatum, Iraqi isolate, Phylogenetic tree, Tomato Plant, Wilting agent. 1. Introduction It is well known that Fusarium species can infect a wide variety of host plants with a wide range of diseases (1, 2(. Soil-borne filamentous fungi have an important economic role since many of their species are responsible for vascular wilt diseases in ornamental and agricultural crops across the world. )3(. The pathogen can attack the plant because this fungus can remain alive latently for prolonged periods in the soil in the form of spores )4(. One of the crops of https://orcid.org/0000-0003-1008-0625 mailto:rasha.khalid@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-9117-7220 mailto:nemataljudy@gmail.com https://orcid.org/0000-0003-1008-0625 mailto:rasha.khalid@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-9117-7220 mailto:nemataljudy@gmail.com https://orcid.org/0000-0003-1008-0625 mailto:rasha.khalid@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-9117-7220 mailto:nemataljudy@gmail.com https://orcid.org/0000-0003-1008-0625 mailto:rasha.khalid@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-9117-7220 mailto:nemataljudy@gmail.com https://orcid.org/0000-0003-1008-0625 mailto:rasha.khalid@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-9117-7220 mailto:nemataljudy@gmail.com https://orcid.org/0000-0003-1008-0625 mailto:rasha.khalid@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-9117-7220 mailto:nemataljudy@gmail.com IHJPAS. 2025, 38(4) 2 the Solanaceae family, the tomato plant (Solanum Lycopersicon L.), is important due to its great nutritional value to humans )5(. The popular fungus associated with tomato roots is Fusarium oxysporum f. sp. lycopersici, which is the causative factor of tomato wilt; yet, several additional species have been discovered from wilted tomato plants )6(. The Fusarium genus is quite diverse and has more than 1650 species that belong to it; some of these species are registered in the gene bank and others are not, but the number is still vulnerable to rise)7(. F.incarnatum (Desm.) Sacc. is widespread in subtropical and temperate climates and affects crops such as sorghum, corn, and rice )8(. Fumonisin and deoxynivalenol, two toxins produced by F. incarnatum, result in clinical symptoms such as diarrhea and vomiting )9(. Fusarium species are commonly identified by their macroscopic and microscopic characteristics )10(. Over the past 20 years, molecular technologies have been shown to have a great benefit in the detection of plant pathogens)11(. It might be challenging to diagnose Fusarium at the species level only based on morphological features because of the genetic variation of the fungus )12(. PCR-based assays are the most popular way to diagnose and distinguish between species and sub-species and are regarded as a rapid procedure )13(. Since the internal transcribed spacer (ITS) gene is an advantageous genetic area for research on the Fusarium species, it was employed in the Fusarium -ID database as an aid for identification )14(. These sequence data have been extensively employed in the phylogeny and taxonomy of the species of Fusarium )15(. Consequently, The research aims to identify the F. incarnatum agent that caused the wilt of Fusarium on tomato plants for the first time in Iraq. 2. Materials and Methods 2.1. Morphological study of the Fungus In January of the year 2023, symptoms of wilt were observed on tomato plants in many fields in Iraq but F. incaranatum was isolated only from the fields in Al-Mahmoudiya in Iraq. Samples were collected from tomato plants that were exhibiting symptoms. Root and stem samples were cut off with a size of (5 mm) by using a sterilized blade into five pieces, it was taken from the infected plant tissues )16(. Root and stem specimens were sterilized by submersion in a solution of sodium hypochlorite (1% free chlorine) for 2–5 min and washed fully with distilled water, after that, it was cultivated in Petri dishes with sterilized Potato Dextrose Agar (PDA) media, and the plates were incubating for 7 days at a temperature of at 25±2°C. In the purification of fungal growth on PDA medium, the colony shape was observed in PDA media. Fungal mycelium was microscopically examined on a glass slide under a microscope to describe the shape of macroconidia, microconidia, and chlamydospores to confirm the relationship with Fusarium through the morphological characteristics described in )17( .Many species of Fusarium were diagnosed, such as F. oxysporum and F. proliferatum, but F.incarnatum was recorded for the first time as a wilting agent in tomato plants, and its appearance rate was 70٪ while the frequency rate was 48٪. 2.2. Pathogenicity test It was prepared with a mingle of peat moss together with sandy soil at a rate of 2: 1 and disinfected with a formalin. A 6% formalin concentration was added to the soil. The soil was then put into plastic bags, and a suitable volume was added of 45 ml of formalin for each 10 kilograms of the soil. The plastic bags had been properly sealed before being set aside for 5 days. After that, when the bags were opened, the soil was distributed in clean plastic pots., and it was exposed to the outside air for three days, to eliminate the formalin smell. The sterilized soil was dispensed in plastic pots with 1000 g capacity, and it was added the fungal IHJPAS. 2025, 38(4) 3 suspension (1x10 7 conidia per ml) for the pathogenic isolate in three replicates )18(. After that, two seedlings of tomato plants four weeks aged were planted per pot of plastic. The plastic pots remained out for 6 weeks till emerged the symptoms, at which point the damage severity was graded using a scale of damage severity to study and test the pathogenicity of the studied isolate. 1. Rate of Wilting 0-23% 2. Rate of Wilting 24-50%. 3. Rate of Wilting is 51-75% 4 . Rate of Wilting is 76-100%. The calculation of the injury's severity depends on the )17, 18( equation (1) Damage Severity%= ×100 (1) 2.3. Molecular identification 2.3.1.Extraction of genomic DNA Genomic DNA was extracted from F.incarnatum isolated from infected tomato plants according to the protocol of ABIO Pure Extraction: This kit was used to quickly and easily isolate DNA from hard-to-lyse fungus. The isolate utilized in the DNA extraction was initially purified multiple times until any contamination (other fungus spores or bacteria) was removed by culturing the Fusarium isolate on the PDA medium for activation. The activation stage is crucial for DNA extraction to obtain pure cultures and a good product of genomic DNA )19(. 2.3.2.Primers of gene ITS The Macrogen Company provided these primers in lyophilized form. In a stock solution, lyophilized primers were dissolved in nuclease-free water to a final concentration of 100 pmol/μl. To make a workable primer solution containing 10 pmol/μl of these primers, 90μl of nuclease-free water was mixed with 10μ l of primer stock solution (stored at -20 C). The ITS sequences, which are found in all eukaryotes as a conserved region, were amplified using the universal primers (ITS-1 & ITS-4) (Table 1) )20, 21(. Table 1. The specific primer of gene ITS. Primer Name The sequence Temp of annealing ( º C) Size of product ITS 1 5`-TCCGTAGGTGAACCTGCGG-3` 55 570 bp ITS 4 5`-TCCTCCGCTTATTGATATGC-3` 56 570 bp 2.3.3.PCR amplification The PCR amplification was performed in a total volume of 25 µl using a thermal cycler, consisting of 3µl DNA, 12.5 µl of master mix (Promega, USA), 1µl of each primer, and 7.5µl of nucleease-free distilled water. The 35 cycles of amplification were as follows: initial denaturation, denaturation, annealing, and extension at 95°C for 5 min, 95°C for 30 min, 55°C for 30 sec, 72°C for 30 sec, and 72°C for 7 min, respectively. All products of PCR were separated utilizing 1.5% agarose gel electrophoresis, and the results were observed by ultraviolet light. 2.3.4.Phylogenetic analysis of F. incarantum The ITS gene sequences obtained in the current research were compared to other fungal isolate sequences retrieved from the NCBI database by using nucleotide BLAST in Gen Bank .Evolutionary analysis were performed with MEGA6 )22,23(. IHJPAS. 2025, 38(4) 4 3.Results 3.1. Morphological study of the Fungus F.incarnatum isolate was obtained from samples of infected tomato plants. Pathogenic Fusarium isolates were purified and identified by using morphological and microscopic features. The morphological features of F.incarnatum isolate were observed on PDA media, such as a white cottony mycelium colony. Features of F.incarnatum observed under a microscope revealed that macroconidia have curved shapes, three to five septate, and basal cells with a foot shape and their dimension (17.59×3.25) µm. Microconidia are oval, without septa, and their dimension (7.98×3.93) µm in addition to the chlamydospores, as is seen in the culture (Figure 1). Figure 1. Morphological Identification of F. incarnatum, (A) (1) Macroconidia, (2) Microconidia under microscope, (B) Chlamydospore under microscope, (C) Morphology on PDA medium (Front view of the plate), (D) Morphology on PDA medium (back view of the plate). 3.2. Pathogenicity test According to a pathogenicity study of the fungus F. incarnatum, the ability of the fungus to cause wilt disease in a healthy tomato plant was 90.33%. Figure 2 illustrates healthy tomato plants that exhibited wilting symptoms, such as discolored vessels and yellowing leaves. The fungus was also isolated from the infected tomato plant to verify the characteristics of the fungal colony and the shape of the conidia of the fungus, and thus to ensure that the fungus that was isolated was the same one that was used for pathogenicity. Figure 2. The pathogenicity of the fungus F. incarnatum, A. The symptoms of pathogenicity on stem, B. The symptoms of pathogenicity on whole plant. 3.3.Sequencing analysis of ITS region The universal primers that were mentioned previously in the research were used to amplify the internal transcribed spacer region of the isolate to confirm the morphological identity; the amplified product likewise produced a single band in electrophoresis at 550 bp for the sample of Fusarium isolate following the PCR run, as shown in Figure 3. The isolated ITS rDNA B D C A 1 2 A B IHJPAS. 2025, 38(4) 5 sequences have been presented to the database in the NCBI Gen Bank, and it obtained their accession number (OQ439282.1). The Fusarium isolate numbered (OQ439282.1) is similar at 100% to the F.incarnatum (OP006283.1). Figure 3. Result of the ITS region amplification of F. incarantum M: (100 – 1500) bp DNA ladder. Marker . Lanes .1 resemble to 570bp PCR Products. 3.4. Phylogenic analysis of F. incarnatum The phylogenetic tree analysis is represented in Figure 4. The Iraqi isolate of this study (OQ439282.1) is shown in the same clade as the Turkey isolate (OP006283.1), India isolate (OP288161.1), and Chinese isolate (MW534570.1), and this clade shares 99.60% similarity with other clades, including Thai isolate, Taiwanese isolate, Nigerian isolate, South Korean isolate, Pakistani isolate, Japanese isolate, Egypt isolate, American isolate, and Tunisian isolate (MT796350.1, MZ749694.1, MN882828.1, KY508359.1, MT080367.1, AB975304.1, OW983261.1., GQ505680.1, MT312734.1, respectively). Table 2. Shows the global sources of F.incarnatum isolates that were used in comparison with the Iraqi isolate in this research, these sources are from the database of NCBI Source: F. incarnatum Accession Country Isolation source Compatibility ID: OP006283.1 Turkey Pepper 100% ID: OP288161.1 India Cinnamon 100% ID: MW534570.1 China ----- 100% ID: MT796350.1 Thailand ------ 99% ID: MZ749694.1 Taiwan Musk melon 99% ID: MN882828.1 Nigeria Dried food 99% ID: KY508359.1 South Korea sweet potato 99% ID: MT080367.1 Pakistan Pumpkin 99% ID: AB975304.1 Japan ------ 99% ID: OW983261.1 Egypt Lycopersicon esculentum 99% ID: GQ505680.1 USA -------- 99% ID: MT312734.1 Tunisia Soil from industrial area 99% https://www.ncbi.nlm.nih.gov/nucleotide/OP006283.1?report=genbank&log$=nuclalign&blast_rank=1&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/OP288161.1?report=genbank&log$=nuclalign&blast_rank=2&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/MW534570.1?report=genbank&log$=nuclalign&blast_rank=3&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/MT796350.1?report=genbank&log$=nuclalign&blast_rank=12&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/MZ749694.1?report=genbank&log$=nuclalign&blast_rank=21&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/MN882828.1?report=genbank&log$=nuclalign&blast_rank=27&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/KY508359.1?report=genbank&log$=nuclalign&blast_rank=33&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/MT080367.1?report=genbank&log$=nuclalign&blast_rank=39&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/AB975304.1?report=genbank&log$=nuclalign&blast_rank=45&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/OW983261.1?report=genbank&log$=nuclalign&blast_rank=59&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/GQ505680.1?report=genbank&log$=nuclalign&blast_rank=62&RID=Z3UE2RZ101R https://www.ncbi.nlm.nih.gov/nucleotide/MT312734.1?report=genbank&log$=nuclalign&blast_rank=65&RID=Z3UE2RZ101R IHJPAS. 2025, 38(4) Figure 4. The phylogenetic tree was generated using ITS region nucleotide sequence information for F.incaranatum (Iraqi isolates in this study compared with other global isolates). The evolutionary history was inferred using the MEGA6 method. 4.Discussion For the first time, F. incarnatum has been identified as the causal agent of tomato wilt in Iraq. This has significant implications for tomato production in the region. Tomatoes are one of the most important vegetable crops in Iraq, and the emergence of F.incarnatum poses a new challenge for farmers. Early and accurate identification of such pathogens is critical for the development of targeted disease management strategies. The integration of molecular diagnostics with traditional morphological methods can improve monitoring programs and reduce the risk of misdiagnosis, which often leads to ineffective control practices. These morphological characteristics were identical to those of F.incarnatum that were mentioned by )3, 4(. Numerous plants across the world have been documented to be infected by F. incarnatum )7, 8(. However, no official reports of F. incarnatum causing wilt on tomato plants in Iraq have been made )24, 25, 26(. Research and studies have shown that numerous pathogenic fungi, whether pathogenic to plants or existing in the soil, can be revealed and identified by using molecular biology techniques, particularly PCR )15(. The analysis of sequence is the best choice for phylogenetic research in the Fusarium species Since the ITS rDNA is being studied more frequently, it has been demonstrated to provide the optimum resolve at the sub-species level. )27-29(. According to previous investigations, molecular analysis is typically chosen for the characterization and identification of Fusarium species since it makes it simple to identify the differences between them. For an accurate diagnosis of Fusarium, morphological examinations and molecular characteristics must be combined )30(. One of the main pathogenic genera that affect tomatoes is Fusarium. The growth of toxigenic fungal species, the expression of biosynthetic regulatory genes, and the generation of mycotoxin are all impacted by the interaction of environmental stress factors like temperature and water activity )31(. In this study, F. incarantum was identified as the causal agent of wilt disease in the tomato plant in Iraq, which is crucial in reducing the risk of the pathogen spreading to other regions. Additionally, this will provide us with pathogen- related biological management strategies and enable us to use phytosanitary techniques to stop the disease's spread. IHJPAS. 2025, 38(4) 7 5. Conclusion Among the most significant pathogens for plants in the world are Fusarium species. While many Fusarium species are widely known, other pathogenic species are currently undiscovered or characterized. Accurate identification of Fusarium species is achieved by combining molecular techniques and morphological methods. The causative agent of the tomato plant wilting disease was determined to be F. incarnatum based on morphological characters and ITS sequencing data. In this study, F. incarantum was identified as the causal agent of wilt disease in the tomato plant for the first time in Iraq, which is crucial in reducing the risk of the pathogen spreading to other regions. Additionally, this will provide us with pathogen-related biological management strategies and enable us to use phytosanitary techniques to stop the disease's spread. Acknowledgment We are very grateful to the farmers who allowed me to take samples of infected tomato plants from different regions in Iraq. Conflict of Interest The authors declare that they have no conflicts of interest. Funding There is no funding for the article. 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