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Genetic Diversity of Alternaria Species Associated with Black Point Disease in Wheat (Triticum aestivum) in Karbala, Iraq

Z
Zainab L. Hameed1,*
0000-0003-4891-8331
B
Ban T. Mohammed2
1Department of Field Crops, Agriculture College, University of Kerbala, Karbala, Iraq.
2College of Education for Pure Sciences, University of Kerbala, Karbala, Iraq.

ABSTRACT

Background: Species of the genus Alternaria are widely recognized as important plant pathogens. Several species are frequently associated with cereal crops, causing diseases such as black point, which significantly reduce grain yield and quality.

Methods: A comprehensive survey was conducted in the principal wheat-growing regions of Karbala Province, Iraq, to determine the etiology and genetic relationships of Alternaria species associated with wheat black point disease using morphological characteristics and ITS-based molecular analysis.

Result: Field surveys in major wheat-growing regions of Karbala Province, Iraq, revealed infected grains across all surveyed areas and commonly cultivated genotypes. A total of 172 fungal isolates were recovered, with the highest proportion in Ain Al-Tamr (56%), followed by Al-Khairat and Al-Jadwal Al-Gharbi (18% each), while the lowest incidence (4%) occurred in Al-Hurr and Sahrawiya. Alternaria species predominated, representing 74% of all isolates and eight species were identified. A. alternata was the most prevalent (19.32% incidence; 28% frequency), followed by A. chlamydosporigena (13.8%; 10%) and A. sorghi (7%; 10%). ITS sequence analysis showed high similarity (>99%) among A. alternata and A. chlamydosporigena isolates, whereas the A. sorghi isolate from Sahrawiya exhibited substantial divergence (>60%), suggesting localized genetic differentiation. These results confirm that combined morphological and ITS analyses effectively characterize Alternaria diversity associated with wheat black point disease.

INTRODUCTION

Bread wheat (Triticum aestivum L.) is one of the most important cereal crops worldwide and a principal member of the family Poaceae. Wheat plays a fundamental role in human nutrition. Accordingly, it is extensively cultivated in many countries, including Iraq, where it represents a cornerstone of national agricultural production (Lahuf et al., 2018a; Hameed and Mohammed, 2024). However, Iraq ranks 23rd globally in wheat production, with a total production was estimated at 5,234,171 tons, average productivity remains relatively low compared with other wheat-producing countries (FAOSTAT, 2024). This considerable yield gap underscores the importance of identifying and mitigating the major constraints on wheat productivity in Iraq (Jaber and Lahuf, 2020).
       
The genus Alternaria is widely distributed in agricultural ecosystems and includes species that can survive as saprophytes and act as destructive pathogens (Lahuf, 2019a; Sharma et al., 2024). In wheat-based production systems, Alternaria spp. are commonly associated with black point and leaf blight diseases, which negatively affect grain yield and quality (Woudenberg et al., 2015).
       
In Iraq, black point symptoms have been increasingly observed in major wheat-growing regions; however, comprehensive information regarding the diversity and population structure of associated Alternaria species remains scarce. Accurate species identification within the genus Alternaria is challenging due to substantial morphological and cultural variability, necessitating the use of molecular approaches (Lahuf et al., 2018b). Thus, sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA has become a standard and reliable method for assessing genetic relationships within fungi, including Alternaria (Lahuf et al., 2020a; Hameed et al., 2021).
       
Despite advances in DNA-based research on fungal diversity worldwide (Ozer and Bayraktar, 2018; Lahuf et al., 2019a). However, in Iraq such investigations remain limited. To date, no comprehensive survey has addressed the distribution and genetic diversity of predominant Alternaria species associated with wheat black point disease in Karbala Province. Therefore, the present study aimed to elucidate the etiology and genetic relationships of Alternaria species infecting wheat grains in Karbala, Iraq, through combined morphological characterization and ITS-based molecular profiling.

MATERIALS AND METHODS

Isolation of fungi associated with wheat grains
 
A total of 69 wheat grain field samples were randomly collected from 1-5-2024 to 20-6-2024 from wheat fields distributed across the main five districts and sub-districts (Al-Hurr, Al-Jadwal Al-Gharbi, Sahrawiya, Al-Khairat and Ain Al-Tamr) of Karbala Province. Approximately 0.5 kg of grains of various common wheat cultivars was collected from each field. The samples were placed in tightly sealed plastic bags and properly labeled with relevant information. All samples were transported to the Postgraduate Laboratory, Department of Plant Protection, College of Agriculture, University of Kerbala for further processing.
       
The wheat grain samples were initially washed under running tap water to remove surface debris. Surface sterilization was then performed using 2% sodium hypochlorite solution for 3 minutes. Subsequently, the grains were thoroughly rinsed with sterile distilled water to remove residual disinfectant, then immersed in 70% ethanol for 30 seconds (Lahuf et al., 2019b; Abdulmoohsin et al., 2019). Disinfected grains were plated on water agar (WA) at five grains per plate and incubated at 25±2°C for three days. To purify the emergent fungi, 5 mm marginal mycelial discs were subcultured onto potato dextrose agar (PDA) and incubated under identical conditions (Watanabe, 2010; Alamry et al., 2024).
 
Pathogenicity test of fungi associated with wheat grains
 
To evaluate pathogenic potential of the isolated fungi using the plate method (Christensen et al., 1988; Dakheel et al., 2024), 5 mm marginal agar discs from 7-day-old PDA cultures were centrally inoculated onto WA plates and incubated at 25±2°C for three days. Ten surface-sterilized wheat grains (cv. Aba 99) were placed around the periphery of each fungal colony, with three replicates per isolate. Uninoculated WA plates with sterile grains served as controls. All plates were incubated at 25±2°C until complete germination was observed in the controls. Subsequently, the percentage of seed germination was calculated according to Jaber and Lahuf (2020) using the following formula:

 
The percentage of growth inhibition was also calculated according to Abbott’s formula (Abbott, 1925), using the following equation:


The percentage of occurrence and frequency of fungal isolates were calculated using the following formulas (Sharma and Sumbali, 2021):.



 
Morphological identification
 
Pathogenic isolates were identified based on cultural characteristics (colony appearance, pigmentation, texture) and microscopic features of hyphae, conidiophores and conidia using light microscopy. Species were determined using established taxonomic keys (Simmons, 2007; Watanabe, 2010).
 
Molecular identification
 
Polymerase chain reaction (PCR) and internal transcribed spacer region of the rRNA (ITS)-based molecular analysis were employed to confirm the morphological identification, following the methodology described by Lahuf et al., (2019b) and Ali et al., (2024). For each isolate, a commercially available ready-to-use PCR master mix was utilized to facilitate reaction setup. Two µL of extracted genomic DNA and the universal primer set ITS1/ITS4 (White et al., 1990) were used in this PCR assay (Lahuf et al., 2022; Jasim et al., 2019). PCR amplicons underwent bidirectional sequencing at Macrogen Inc. (Seoul, South Korea). Raw chromatograms were evaluated and trimmed of low-quality bases using ApE software (Lahuf et al., 2020b). Edited sequences were then analyzed using BLAST against the NCBI GenBank database to confirm species-level identification through phylogenetic matching with global reference isolates (Lahuf, 2019b; Mohammed et al., 2025). Aligned sequences were used to construct phylogenetic trees comparing local isolates with global reference strains using MEGA 11 (Tamura et al., 2021).
 
Assessment of genetic variability among fungal isolates associated with wheat grains in Karbala province
 
Genetic variability among the prevalent fungal isolates from Karbala wheat grains was evaluated using ITS rRNA sequence analysis, a reliable marker for species-level phylogenetic discrimination (Lahuf et al., 2023). ITS sequences were aligned and analyzed for DNA polymorphism using ClustalX 2.1. Nucleotide conservation and variability were subsequently visualized using the Sequence Demarcation Tool. All obtained fungal ITS sequences were deposited in the NCBI GenBank database (Shehan et al., 2023).

RESULTS AND DISCUSSION

Isolation of fungi associated with black point of wheat grain
 
Fungal isolation from randomly collected Karbala wheat grains revealed distinct pathological symptoms despite surface sterilisation (Fig 1). Infected grains exhibited dark brown to black discolouration, particularly at the embryo, alongside dark spots on the seed coat. Kernels were frequently shrivelled, malformed and brittle, with rough, chalky, or powdery surfaces. Advanced infections caused tissue softening and disintegration, which severely reduced germination rates and yielded abnormal seedlings with distorted radicles and shoots.

Fig 1: Representative black point disease symptoms observed on infected wheat grains.


       
Symptomatic grains and fungal isolates were widespread across all surveyed regions in Karbala Province. Infection was consistently observed in all 13 surveyed wheat cultivars (Adana, Baghdad, Jad, French, Aba 99, Jehan, Mexipak, Rasheed, Buhuth 22, Sham, Dijla 1, Dijla 2 and Wafiya), indicating broad pathogen adaptability and a lack of strong cultivar-specific resistance under local conditions. A total of 172 fungal isolates were recovered (Fig 2). The highest proportion originated from Ain Al-Tamr (56%), followed by Al-Khairat and Al-Jadwal Al-Gharbi (18% each), while Al-Hurr and the Desert (Sahrawiya) areas yielded the lowest (4%). The high prevalence in Ain Al-Tamr likely reflects its history of extensive, continuous wheat cultivation, which promotes inoculum accumulation in soil and crop residues, unlike the more recently cultivated Al-Hurr and Desert regions (Karbala Directorate of Agriculture, 2022, personal communication).

Fig 2: Percentage distribution of fungal isolates associated with wheat grains across the surveyed regions of karbala province.


       
This observation is consistent with findings reported by Bithell et al., (2015), who demonstrated that repeated cultivation of wheat in monocropping systems significantly elevates the levels of soil-borne fungal pathogens. Such practices promote the build-up of pathogens, such as Gaeumannomyces graminis, the cause of take-all disease, which can accumulate during the first year of wheat cultivation and subsequently infect the root system of seedlings when wheat is replanted in the same field.
 
Morphological and molecular identification and pathogenicity testing of fungal isolates associated with wheat grains
 
Morphological identification
 
Of the 172 isolates recovered from symptomatic grains, 120 were morphologically identified as Alternaria spp. based on Simmons (2007). On PDA, these formed fast-growing, aerial colonies (averaging 4.9 cm radial growth) that transitioned from dark gray or olive-brown with white margins to deep brown over two weeks. Microscopically, they produced light brown conidiophores bearing oval to ellipsoidal conidia (24-28 × 9-12 µm) with 1-4 transverse septa. The remaining isolates belonged to Stemphylium (17) and Cladosporium (13).
 
Pathogenicity test of the isolated fungi associated with wheat grains
 
Pathogenicity assays confirmed all 120 Alternaria isolates were pathogenic, significantly reducing seed germination by 77.77% to 100% compared to the 100% germination in uninoculated controls (Fig 3). This varying virulence-including complete germination suppression by some isolates-substantiates their role in grain rot, black point and seedling damping-off.

Fig 3: Pathogenicity test of selected fungal isolate belonging to Alternaria sp. on wheat grains.


  
The findings of the present pathogenicity test are consistent with previous studies, these Alternaria isolates significantly impaired wheat seed germination (Soomro et al., 2020). This inhibition is largely attributed to the production of germination-suppressing phytotoxins, such as deoxyradicinin and maculosin (Terna et al., 2020). As well as, it is aligning with Greeshma (2023), Alternaria colonization compromises seed viability and seedling establishment by depleting essential energy reserves and inducing tissue damage via degradative enzymes and toxic metabolite.
 
Molecular identification
 
Successful rDNA-ITS amplification yielded 500-600 bp PCR products for all samples. BLAST sequence analysis confirmed morphological identifications, showing 98-100% similarity with GenBank reference strains, where these nucleotide sequences were subsequently deposited (Table 1). Molecular profiling identified eight Alternaria species comprising 74% of total isolates across all surveyed regions in Karbala Province. Alternaria alternata was the most prevalent (19.32% occurrence; 28% frequency), followed by A. chlamydosporigena (13.8%; 10%) and A. sorghi (7%; 10%), alongside five less frequent Alternaria species.

Table 1: Molecularly identified fungal isolates deposited in the genbank database with their pathogenicity.


       
Accurate identification of seed-borne fungi is critical, as these pathogens reduce germination, weaken seedlings and initiate field epidemics that threaten crop productivity. Consequently, molecular detection using the highly discriminatory rDNA-ITS region (comprising ITS1, 5.8S and ITS2) has become a reliable cornerstone for precise fungal identification (Al-Tememe et al., 2019). Offering greater specificity and sensitivity than traditional morphology, ITS sequencing substantially improves diagnostic resolution in fungal taxonomy (Nguyen et al., 2018), as demonstrated by its successful application in recent plant pathology studies (Jaber and Lahuf, 2020; Shehan et al., 2022). Notably, an extensive literature review indicates this is the first documented report of Alternaria chlamydosporigena, A. cumini and A. sorghi on wheat in Iraq. Detecting these previously unreported species expands the known Iraqi wheat mycobiota and underscores the efficacy of molecular tools in revealing hidden pathogen diversity.
 
Assessment of genetic variability among the most prevalent Alternaria spp. isolates in Karbala province
 
ITS sequence analysis (Fig 4) revealed >99% genetic homology among all Alternaria alternata isolates across the surveyed regions of Karbala (Ain Al-Tamr, Al-Khairat, Al-Hurr, Al-Jadwal Al-Gharbi and the Desert/Sahrawiya). Aligned sequences exhibited near-complete identity across the entire ITS region. Phylogenetically, all A. alternata isolates clustered into a single clade, distinctly separated from the Spongospora subterranea outgroup (Fig 5). This pronounced genetic uniformity suggests a stable population structure facing limited evolutionary pressure, likely stemming from a single regional introduction source.                

Fig 4: Percentage similarity among rDNA-ITS nucleotide sequences of Alternaria alternata isolates recovered from wheat fields in Karbala province.



Fig 5: Phylogenetic relationships among Alternaria alternata isolates recovered from wheat fields in Karbala province.

 

Furthermore, the rDNA-ITS sequence analysis (Fig 6) revealed >99% genetic homology among all Alternaria chlamydosporigena isolates from the Ain Al-Tamr and Desert (Sahrawiya) regions. Aligned sequences exhibited near-complete identity across the entire ITS region. Phylogenetically, all A. chlamydosporigena isolates clustered into a single clade with minimal genetic divergence, distinctly separated from the Spongospora subterranea outgroup (Fig 7). This pronounced genetic cohesion strongly suggests a common origin for this local population.

Fig 6: Percentage similarity among rDNA-ITS nucleotide sequences of Alternaria chlamydosporigena isolates recovered from wheat fields in Karbala province.



Fig 7: Phylogenetic relationships among Alternaria chlamydosporigena isolates recovered from wheat fields in Karbala province.


       
In contrast to the previously studied species, rDNA-ITS sequence analysis of Alternaria sorghi (Fig 8) revealed significant population divergence. While isolates from Ain Al-Tamr and Al-Khairat exhibited >99% genetic homology, the desert (Sahrawiya) isolate showed over 60% sequence divergence. Aligned sequences displayed multiple mismatches throughout even the typically conserved central regions. Phylogenetically, the Ain Al-Tamr and Al-Khairat isolates clustered into a single clade, whereas the highly divergent Sahrawiya isolate formed a separate branch, indicating long-term evolutionary separation (Fig 9).

Fig 8: Percentage similarity among rDNA-ITS nucleotide sequences of Alternaria sorghi isolates recovered from wheat fields in Karbala province.



Fig 9: Phylogenetic relationships among Alternaria sorghi isolates recovered from wheat fields in Karbala province.


       
Mainly various members of Alternaria genus were found to cause different diseases on multiple hosts such as leaf spot (Lahuf et al., 2020b; Jangid et al., 2025; Singh et al., 2025) and leaf blight (Chavhan et al., 2015; Jain et al., 2025). However, limited studies were accomplished in regard of other Alternaria diseases. Therefore, the primary objective of this analysis was to assess genetic diversity among the most prevalent Alternaria species isolated from wheat fields across different regions of Karbala Province. The integration of morphological and molecular diagnostic approaches provides a highly reliable framework for identifying taxonomically complex and economically important fungal species, including many members of the genus Alternaria, which are known to cause substantial yield and quality losses (Lahuf et al., 2020a).
       
Genetic relatedness analysis based on molecular markers is widely recognized as a standard and effective approach for characterizing and differentiating fungal species and strains (Chehri et al., 2015). In particular, the rDNA-ITS region has been extensively used in phylogenetic studies to clarify evolutionary relationships among Alternaria species and to provide insights into their genetic diversity and evolutionary history. For example, Pryor and Gilbertson (2000) conducted a comprehensive phylogenetic analysis of Alternaria species using nuclear rDNA sequences in combination with mitochondrial small subunit (mtSSU) markers. Their findings confirmed the utility of rDNA sequences in resolving species-level relationships within the genus. Similarly, Park et al., (2008) re-evaluated phylogenetic relationships among Alternaria species using ITS sequences alongside additional genetic markers, further emphasizing the importance of this region for species delimitation.
       
More recently, Iturrieta-González and Gené (2023) highlighted that the rDNA-ITS region remains a cornerstone in fungal taxonomy, including within the genus Alternaria. Their study demonstrated that combining ITS data with additional genetic markers enhances the accuracy and robustness of species identification. Likewise, Woudenberg et al., (2015) noted that although ITS sequencing is effective for preliminary identification, supplementary genetic markers-or even whole-genome approaches-may be required to achieve a comprehensive understanding of interspecific relationships and to obtain a more detailed and precise genomic resolution within this complex genus.

CONCLUSION

This study provides the first comprehensive characterization of Alternaria spp. causing wheat black point disease in Karbala, Iraq. Morpho-molecular analyses identified eight species-predominantly A. alternata-and represent the first regional report of A. chlamydosporigena, A. cumini and A. sorghi on Iraqi wheat. Pathogenicity assays confirmed these isolates severely impair seed germination, threatening local agriculture. Furthermore, high genetic homology (>99%) among A. alternata and A. chlamydosporigena populations indicates a stable, single-source inoculum across districts. These findings provide a critical foundation for ongoing pathogen surveillance, targeted resistance breeding and sustainable management strategies to safeguard Iraqi wheat productivity.

ACKNOWLEDGEMENT

The current study was supported by the Iraqi Ministry of Higher Education and Scientific Research and University of Kerbala.
 
Disclaimers
 
The views expressed in this article are those of the authors and do not necessarily reflect those of their affiliated institutions. The authors assume no responsibility for any loss arising from the use of this information.

CONFLICT OF INTEREST

The authors declare no conflicts of interest regarding this publication. No funding or sponsorship influenced the study design, data collection, analysis, decision to publish, or manuscript preparation.

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    Full Research Article

    Genetic Diversity of Alternaria Species Associated with Black Point Disease in Wheat (Triticum aestivum) in Karbala, Iraq

    Z
    Zainab L. Hameed1,*
    0000-0003-4891-8331
    B
    Ban T. Mohammed2
    1Department of Field Crops, Agriculture College, University of Kerbala, Karbala, Iraq.
    2College of Education for Pure Sciences, University of Kerbala, Karbala, Iraq.

    ABSTRACT

    Background: Species of the genus Alternaria are widely recognized as important plant pathogens. Several species are frequently associated with cereal crops, causing diseases such as black point, which significantly reduce grain yield and quality.

    Methods: A comprehensive survey was conducted in the principal wheat-growing regions of Karbala Province, Iraq, to determine the etiology and genetic relationships of Alternaria species associated with wheat black point disease using morphological characteristics and ITS-based molecular analysis.

    Result: Field surveys in major wheat-growing regions of Karbala Province, Iraq, revealed infected grains across all surveyed areas and commonly cultivated genotypes. A total of 172 fungal isolates were recovered, with the highest proportion in Ain Al-Tamr (56%), followed by Al-Khairat and Al-Jadwal Al-Gharbi (18% each), while the lowest incidence (4%) occurred in Al-Hurr and Sahrawiya. Alternaria species predominated, representing 74% of all isolates and eight species were identified. A. alternata was the most prevalent (19.32% incidence; 28% frequency), followed by A. chlamydosporigena (13.8%; 10%) and A. sorghi (7%; 10%). ITS sequence analysis showed high similarity (>99%) among A. alternata and A. chlamydosporigena isolates, whereas the A. sorghi isolate from Sahrawiya exhibited substantial divergence (>60%), suggesting localized genetic differentiation. These results confirm that combined morphological and ITS analyses effectively characterize Alternaria diversity associated with wheat black point disease.

    INTRODUCTION

    Bread wheat (Triticum aestivum L.) is one of the most important cereal crops worldwide and a principal member of the family Poaceae. Wheat plays a fundamental role in human nutrition. Accordingly, it is extensively cultivated in many countries, including Iraq, where it represents a cornerstone of national agricultural production (Lahuf et al., 2018a; Hameed and Mohammed, 2024). However, Iraq ranks 23rd globally in wheat production, with a total production was estimated at 5,234,171 tons, average productivity remains relatively low compared with other wheat-producing countries (FAOSTAT, 2024). This considerable yield gap underscores the importance of identifying and mitigating the major constraints on wheat productivity in Iraq (Jaber and Lahuf, 2020).
           
    The genus Alternaria is widely distributed in agricultural ecosystems and includes species that can survive as saprophytes and act as destructive pathogens (Lahuf, 2019a; Sharma et al., 2024). In wheat-based production systems, Alternaria spp. are commonly associated with black point and leaf blight diseases, which negatively affect grain yield and quality (Woudenberg et al., 2015).
           
    In Iraq, black point symptoms have been increasingly observed in major wheat-growing regions; however, comprehensive information regarding the diversity and population structure of associated Alternaria species remains scarce. Accurate species identification within the genus Alternaria is challenging due to substantial morphological and cultural variability, necessitating the use of molecular approaches (Lahuf et al., 2018b). Thus, sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA has become a standard and reliable method for assessing genetic relationships within fungi, including Alternaria (Lahuf et al., 2020a; Hameed et al., 2021).
           
    Despite advances in DNA-based research on fungal diversity worldwide (Ozer and Bayraktar, 2018; Lahuf et al., 2019a). However, in Iraq such investigations remain limited. To date, no comprehensive survey has addressed the distribution and genetic diversity of predominant Alternaria species associated with wheat black point disease in Karbala Province. Therefore, the present study aimed to elucidate the etiology and genetic relationships of Alternaria species infecting wheat grains in Karbala, Iraq, through combined morphological characterization and ITS-based molecular profiling.

    MATERIALS AND METHODS

    Isolation of fungi associated with wheat grains
     
    A total of 69 wheat grain field samples were randomly collected from 1-5-2024 to 20-6-2024 from wheat fields distributed across the main five districts and sub-districts (Al-Hurr, Al-Jadwal Al-Gharbi, Sahrawiya, Al-Khairat and Ain Al-Tamr) of Karbala Province. Approximately 0.5 kg of grains of various common wheat cultivars was collected from each field. The samples were placed in tightly sealed plastic bags and properly labeled with relevant information. All samples were transported to the Postgraduate Laboratory, Department of Plant Protection, College of Agriculture, University of Kerbala for further processing.
           
    The wheat grain samples were initially washed under running tap water to remove surface debris. Surface sterilization was then performed using 2% sodium hypochlorite solution for 3 minutes. Subsequently, the grains were thoroughly rinsed with sterile distilled water to remove residual disinfectant, then immersed in 70% ethanol for 30 seconds (Lahuf et al., 2019b; Abdulmoohsin et al., 2019). Disinfected grains were plated on water agar (WA) at five grains per plate and incubated at 25±2°C for three days. To purify the emergent fungi, 5 mm marginal mycelial discs were subcultured onto potato dextrose agar (PDA) and incubated under identical conditions (Watanabe, 2010; Alamry et al., 2024).
     
    Pathogenicity test of fungi associated with wheat grains
     
    To evaluate pathogenic potential of the isolated fungi using the plate method (Christensen et al., 1988; Dakheel et al., 2024), 5 mm marginal agar discs from 7-day-old PDA cultures were centrally inoculated onto WA plates and incubated at 25±2°C for three days. Ten surface-sterilized wheat grains (cv. Aba 99) were placed around the periphery of each fungal colony, with three replicates per isolate. Uninoculated WA plates with sterile grains served as controls. All plates were incubated at 25±2°C until complete germination was observed in the controls. Subsequently, the percentage of seed germination was calculated according to Jaber and Lahuf (2020) using the following formula:

     
    The percentage of growth inhibition was also calculated according to Abbott’s formula (Abbott, 1925), using the following equation:


    The percentage of occurrence and frequency of fungal isolates were calculated using the following formulas (Sharma and Sumbali, 2021):.



     
    Morphological identification
     
    Pathogenic isolates were identified based on cultural characteristics (colony appearance, pigmentation, texture) and microscopic features of hyphae, conidiophores and conidia using light microscopy. Species were determined using established taxonomic keys (Simmons, 2007; Watanabe, 2010).
     
    Molecular identification
     
    Polymerase chain reaction (PCR) and internal transcribed spacer region of the rRNA (ITS)-based molecular analysis were employed to confirm the morphological identification, following the methodology described by Lahuf et al., (2019b) and Ali et al., (2024). For each isolate, a commercially available ready-to-use PCR master mix was utilized to facilitate reaction setup. Two µL of extracted genomic DNA and the universal primer set ITS1/ITS4 (White et al., 1990) were used in this PCR assay (Lahuf et al., 2022; Jasim et al., 2019). PCR amplicons underwent bidirectional sequencing at Macrogen Inc. (Seoul, South Korea). Raw chromatograms were evaluated and trimmed of low-quality bases using ApE software (Lahuf et al., 2020b). Edited sequences were then analyzed using BLAST against the NCBI GenBank database to confirm species-level identification through phylogenetic matching with global reference isolates (Lahuf, 2019b; Mohammed et al., 2025). Aligned sequences were used to construct phylogenetic trees comparing local isolates with global reference strains using MEGA 11 (Tamura et al., 2021).
     
    Assessment of genetic variability among fungal isolates associated with wheat grains in Karbala province
     
    Genetic variability among the prevalent fungal isolates from Karbala wheat grains was evaluated using ITS rRNA sequence analysis, a reliable marker for species-level phylogenetic discrimination (Lahuf et al., 2023). ITS sequences were aligned and analyzed for DNA polymorphism using ClustalX 2.1. Nucleotide conservation and variability were subsequently visualized using the Sequence Demarcation Tool. All obtained fungal ITS sequences were deposited in the NCBI GenBank database (Shehan et al., 2023).

    RESULTS AND DISCUSSION

    Isolation of fungi associated with black point of wheat grain
     
    Fungal isolation from randomly collected Karbala wheat grains revealed distinct pathological symptoms despite surface sterilisation (Fig 1). Infected grains exhibited dark brown to black discolouration, particularly at the embryo, alongside dark spots on the seed coat. Kernels were frequently shrivelled, malformed and brittle, with rough, chalky, or powdery surfaces. Advanced infections caused tissue softening and disintegration, which severely reduced germination rates and yielded abnormal seedlings with distorted radicles and shoots.

    Fig 1: Representative black point disease symptoms observed on infected wheat grains.


           
    Symptomatic grains and fungal isolates were widespread across all surveyed regions in Karbala Province. Infection was consistently observed in all 13 surveyed wheat cultivars (Adana, Baghdad, Jad, French, Aba 99, Jehan, Mexipak, Rasheed, Buhuth 22, Sham, Dijla 1, Dijla 2 and Wafiya), indicating broad pathogen adaptability and a lack of strong cultivar-specific resistance under local conditions. A total of 172 fungal isolates were recovered (Fig 2). The highest proportion originated from Ain Al-Tamr (56%), followed by Al-Khairat and Al-Jadwal Al-Gharbi (18% each), while Al-Hurr and the Desert (Sahrawiya) areas yielded the lowest (4%). The high prevalence in Ain Al-Tamr likely reflects its history of extensive, continuous wheat cultivation, which promotes inoculum accumulation in soil and crop residues, unlike the more recently cultivated Al-Hurr and Desert regions (Karbala Directorate of Agriculture, 2022, personal communication).

    Fig 2: Percentage distribution of fungal isolates associated with wheat grains across the surveyed regions of karbala province.


           
    This observation is consistent with findings reported by Bithell et al., (2015), who demonstrated that repeated cultivation of wheat in monocropping systems significantly elevates the levels of soil-borne fungal pathogens. Such practices promote the build-up of pathogens, such as Gaeumannomyces graminis, the cause of take-all disease, which can accumulate during the first year of wheat cultivation and subsequently infect the root system of seedlings when wheat is replanted in the same field.
     
    Morphological and molecular identification and pathogenicity testing of fungal isolates associated with wheat grains
     
    Morphological identification
     
    Of the 172 isolates recovered from symptomatic grains, 120 were morphologically identified as Alternaria spp. based on Simmons (2007). On PDA, these formed fast-growing, aerial colonies (averaging 4.9 cm radial growth) that transitioned from dark gray or olive-brown with white margins to deep brown over two weeks. Microscopically, they produced light brown conidiophores bearing oval to ellipsoidal conidia (24-28 × 9-12 µm) with 1-4 transverse septa. The remaining isolates belonged to Stemphylium (17) and Cladosporium (13).
     
    Pathogenicity test of the isolated fungi associated with wheat grains
     
    Pathogenicity assays confirmed all 120 Alternaria isolates were pathogenic, significantly reducing seed germination by 77.77% to 100% compared to the 100% germination in uninoculated controls (Fig 3). This varying virulence-including complete germination suppression by some isolates-substantiates their role in grain rot, black point and seedling damping-off.

    Fig 3: Pathogenicity test of selected fungal isolate belonging to Alternaria sp. on wheat grains.


      
    The findings of the present pathogenicity test are consistent with previous studies, these Alternaria isolates significantly impaired wheat seed germination (Soomro et al., 2020). This inhibition is largely attributed to the production of germination-suppressing phytotoxins, such as deoxyradicinin and maculosin (Terna et al., 2020). As well as, it is aligning with Greeshma (2023), Alternaria colonization compromises seed viability and seedling establishment by depleting essential energy reserves and inducing tissue damage via degradative enzymes and toxic metabolite.
     
    Molecular identification
     
    Successful rDNA-ITS amplification yielded 500-600 bp PCR products for all samples. BLAST sequence analysis confirmed morphological identifications, showing 98-100% similarity with GenBank reference strains, where these nucleotide sequences were subsequently deposited (Table 1). Molecular profiling identified eight Alternaria species comprising 74% of total isolates across all surveyed regions in Karbala Province. Alternaria alternata was the most prevalent (19.32% occurrence; 28% frequency), followed by A. chlamydosporigena (13.8%; 10%) and A. sorghi (7%; 10%), alongside five less frequent Alternaria species.

    Table 1: Molecularly identified fungal isolates deposited in the genbank database with their pathogenicity.


           
    Accurate identification of seed-borne fungi is critical, as these pathogens reduce germination, weaken seedlings and initiate field epidemics that threaten crop productivity. Consequently, molecular detection using the highly discriminatory rDNA-ITS region (comprising ITS1, 5.8S and ITS2) has become a reliable cornerstone for precise fungal identification (Al-Tememe et al., 2019). Offering greater specificity and sensitivity than traditional morphology, ITS sequencing substantially improves diagnostic resolution in fungal taxonomy (Nguyen et al., 2018), as demonstrated by its successful application in recent plant pathology studies (Jaber and Lahuf, 2020; Shehan et al., 2022). Notably, an extensive literature review indicates this is the first documented report of Alternaria chlamydosporigena, A. cumini and A. sorghi on wheat in Iraq. Detecting these previously unreported species expands the known Iraqi wheat mycobiota and underscores the efficacy of molecular tools in revealing hidden pathogen diversity.
     
    Assessment of genetic variability among the most prevalent Alternaria spp. isolates in Karbala province
     
    ITS sequence analysis (Fig 4) revealed >99% genetic homology among all Alternaria alternata isolates across the surveyed regions of Karbala (Ain Al-Tamr, Al-Khairat, Al-Hurr, Al-Jadwal Al-Gharbi and the Desert/Sahrawiya). Aligned sequences exhibited near-complete identity across the entire ITS region. Phylogenetically, all A. alternata isolates clustered into a single clade, distinctly separated from the Spongospora subterranea outgroup (Fig 5). This pronounced genetic uniformity suggests a stable population structure facing limited evolutionary pressure, likely stemming from a single regional introduction source.                

    Fig 4: Percentage similarity among rDNA-ITS nucleotide sequences of Alternaria alternata isolates recovered from wheat fields in Karbala province.



    Fig 5: Phylogenetic relationships among Alternaria alternata isolates recovered from wheat fields in Karbala province.

     

    Furthermore, the rDNA-ITS sequence analysis (Fig 6) revealed >99% genetic homology among all Alternaria chlamydosporigena isolates from the Ain Al-Tamr and Desert (Sahrawiya) regions. Aligned sequences exhibited near-complete identity across the entire ITS region. Phylogenetically, all A. chlamydosporigena isolates clustered into a single clade with minimal genetic divergence, distinctly separated from the Spongospora subterranea outgroup (Fig 7). This pronounced genetic cohesion strongly suggests a common origin for this local population.

    Fig 6: Percentage similarity among rDNA-ITS nucleotide sequences of Alternaria chlamydosporigena isolates recovered from wheat fields in Karbala province.



    Fig 7: Phylogenetic relationships among Alternaria chlamydosporigena isolates recovered from wheat fields in Karbala province.


           
    In contrast to the previously studied species, rDNA-ITS sequence analysis of Alternaria sorghi (Fig 8) revealed significant population divergence. While isolates from Ain Al-Tamr and Al-Khairat exhibited >99% genetic homology, the desert (Sahrawiya) isolate showed over 60% sequence divergence. Aligned sequences displayed multiple mismatches throughout even the typically conserved central regions. Phylogenetically, the Ain Al-Tamr and Al-Khairat isolates clustered into a single clade, whereas the highly divergent Sahrawiya isolate formed a separate branch, indicating long-term evolutionary separation (Fig 9).

    Fig 8: Percentage similarity among rDNA-ITS nucleotide sequences of Alternaria sorghi isolates recovered from wheat fields in Karbala province.



    Fig 9: Phylogenetic relationships among Alternaria sorghi isolates recovered from wheat fields in Karbala province.


           
    Mainly various members of Alternaria genus were found to cause different diseases on multiple hosts such as leaf spot (Lahuf et al., 2020b; Jangid et al., 2025; Singh et al., 2025) and leaf blight (Chavhan et al., 2015; Jain et al., 2025). However, limited studies were accomplished in regard of other Alternaria diseases. Therefore, the primary objective of this analysis was to assess genetic diversity among the most prevalent Alternaria species isolated from wheat fields across different regions of Karbala Province. The integration of morphological and molecular diagnostic approaches provides a highly reliable framework for identifying taxonomically complex and economically important fungal species, including many members of the genus Alternaria, which are known to cause substantial yield and quality losses (Lahuf et al., 2020a).
           
    Genetic relatedness analysis based on molecular markers is widely recognized as a standard and effective approach for characterizing and differentiating fungal species and strains (Chehri et al., 2015). In particular, the rDNA-ITS region has been extensively used in phylogenetic studies to clarify evolutionary relationships among Alternaria species and to provide insights into their genetic diversity and evolutionary history. For example, Pryor and Gilbertson (2000) conducted a comprehensive phylogenetic analysis of Alternaria species using nuclear rDNA sequences in combination with mitochondrial small subunit (mtSSU) markers. Their findings confirmed the utility of rDNA sequences in resolving species-level relationships within the genus. Similarly, Park et al., (2008) re-evaluated phylogenetic relationships among Alternaria species using ITS sequences alongside additional genetic markers, further emphasizing the importance of this region for species delimitation.
           
    More recently, Iturrieta-González and Gené (2023) highlighted that the rDNA-ITS region remains a cornerstone in fungal taxonomy, including within the genus Alternaria. Their study demonstrated that combining ITS data with additional genetic markers enhances the accuracy and robustness of species identification. Likewise, Woudenberg et al., (2015) noted that although ITS sequencing is effective for preliminary identification, supplementary genetic markers-or even whole-genome approaches-may be required to achieve a comprehensive understanding of interspecific relationships and to obtain a more detailed and precise genomic resolution within this complex genus.

    CONCLUSION

    This study provides the first comprehensive characterization of Alternaria spp. causing wheat black point disease in Karbala, Iraq. Morpho-molecular analyses identified eight species-predominantly A. alternata-and represent the first regional report of A. chlamydosporigena, A. cumini and A. sorghi on Iraqi wheat. Pathogenicity assays confirmed these isolates severely impair seed germination, threatening local agriculture. Furthermore, high genetic homology (>99%) among A. alternata and A. chlamydosporigena populations indicates a stable, single-source inoculum across districts. These findings provide a critical foundation for ongoing pathogen surveillance, targeted resistance breeding and sustainable management strategies to safeguard Iraqi wheat productivity.

    ACKNOWLEDGEMENT

    The current study was supported by the Iraqi Ministry of Higher Education and Scientific Research and University of Kerbala.
     
    Disclaimers
     
    The views expressed in this article are those of the authors and do not necessarily reflect those of their affiliated institutions. The authors assume no responsibility for any loss arising from the use of this information.

    CONFLICT OF INTEREST

    The authors declare no conflicts of interest regarding this publication. No funding or sponsorship influenced the study design, data collection, analysis, decision to publish, or manuscript preparation.

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