Acta agriculturae Slovenica, 121/2, 1–7, Ljubljana 2025 doi:10.14720/aas.2025.121.2.20608 Original research article / izvirni znanstveni članek Pathogenicity assessment of Fusarium clavum associated with wheat head blight in Algeria Nedjet Rofida SEGMANE 1, 2, 3, Abdelkrim MEBARKIA1. 2, Alaeddine BOUTALBI 4, Abdelmalek LAOUAR 2, Kholoud M. ALANANBEH 5, Farah ALAMIR 5, Marwa BELALMI 1.2 Recived November 10, 2024; accepted March 29, 2025 Delo je prispelo 10. november 2024, sprejeto 29. marec 2025 1 Laboratory of Applied Microbiology, Ferhat Abbas University of Setif1, Algeria 2 Department of Agronomy, Faculty of Nature and Life Sciences, Ferhat Abbas University of Setif1, Algeria 3 Corresponding author (sagmane.rofida@univ-setif.dz) 4 Department of Biology and Plants Ecology, Faculty of Nature and Life Sciences, Ferhat Abbas University of Setif1, Algeria 5 Department of Plant Protection, School of Agriculture, The University of Jordan, Amman, Jordan Pathogenicity assessment of Fusarium clavum associated with wheat head blight in Algeria Abstract: Durum wheat (Triticum durum Desf) is one of the most important cereal crops in Algeria. During the agri- cultural season 2021-2022, typical symptoms of Fusarium Head Blight were observed in wheat fields in the Setif region. One of the causal agents was identified on the basis of morphological characters and DNA sequences as Fusarium clavum J.W. Xia, L. Lombard, Sand.-Den., X.G. Zhang & Crous, a member of the Fusarium incarnatum-equiseti species complex. It was isolated from symptomatic wheat glumes. In vitro and in vivo patho- genicity tests were conducted on three Algerian durum wheat varieties to assess the effect of this isolate on the seedling and the mass of durum wheat kernels. The results showed that Fu- sarium clavum caused a significant reduction in the coleoptile (38.9 %) and root length (42 %) and decreased kernels mass by 20.8 %. This study further confirms the presence of Fusarium clavum as an agent causing Fusarium Head Blight on wheat in Algeria. Key words: durum wheat, Fusarium clavum, pathogenic- ity, Algeria. Ocena patogenosti glive Fusarium clavum, kot povzročiteljice pšeničnega ožiga v Alžiriji Izvleček: Trda pšenica (Triticum durum Desf) je eno izmed najpomembnejših žit v Alžiriji. V pridelovalnih sezo- nah 2021-2022 so bili na pšeničnih poljih na območju Setifa opaženi značilni simptomi pšeničnega ožiga, ki ga povzroča gliva iz rodu Fusarium. Kot povzročitelj je bila na osnovi morfoloških znakov in DNK zaporedij prepoznana gliva Fu- sarium clavum J.W. Xia, L. Lombard, Sand.-Den., X.G. Zhang & Crous, predstavnica iz kompleksa vrst Fusarium incarna- tum-equiseti. Izolirana je bila iz simptomatičnih pčeničnih plev. In vitro in in vivo testi patogenosti so bili narejeni na treh alžirskih sortah trde pšenice za oceno učinka tega izolata na sejanke in maso pšeničnih zrn. Rezultati so pokazali, da je gli- va Fusarium clavum povzročila značilno zmanjšanje dolžine koleoptile (38,9 %) in dolžine korenin (42 %) ter zmanjšala maso zrn za 20,8 %. Raziskava potrjuje prisotnost glive Fu- sarium clavum kot fuzarijskega povzročitelja pšeničnega ožiga v Alžiriji. Ključne besede: trda pšenica, Fusarium clavum, patoge- nost, Alžirija Acta agriculturae Slovenica, 121/2 – 20252 N. R. SEGMANE et al. 1 INTRODUCTION Wheat (Triticum aestivum L.) is a key cereal crop and a major global source of food for people. Due to its strategic importance for human and animal nutrition, wheat occupies a privileged place in Algerian agriculture. For instance, it is used to make bread and other Algerian food as couscous, which is the country’s most popular dish (Kezih et al., 2014). Numerous phytopathogenic Fu- sarium species are found around the world, infecting a variety of crop plants, including cereals such as wheat, maize, oats, and barley (Boutigny et al., 2011). Fusarium drastically reduces grain quality and output. More sig- nificantly, several species of the Fusarium genus produce mycotoxins, which prevent the synthesis of proteins and as a result, exposure to these mycotoxins can lead to sev- eral health issues in humans and animals (Pestka, 2010). The wheat head, grain, and occasionally the peduncle are the only areas where Fusarium head blight (FHB) symp- toms can occur. Usually, when healthy heads are still green, the first discernible indication is the bleaching of a part of or all the spikelets. Spikelets above and below the initial spot of infection may also become bleached as the fungus spreads across the rachis. When examining infected spikelets, pink to orange masses of spores may be seen. It is during rainy, humid conditions that these spore masses are generated. Infected kernels, sometimes known as tombstones, have a shriveled, discolored ap- pearance and are low in mass (Freije & Wise, 2015). There is currently not enough information available in Algeria regarding the causative agent of Fusarium head blight of durum wheat (Laraba et al., 2017); besides, Fusarium cla- vum was discovered for the first time in Algeria in a study by Belabed et al. (2025), as a cause of this disease in du- rum wheat. This study was conducted to isolate and iden- tify Fusarium clavum species from infected wheat in the Setif region of Algeria and to evaluate their pathogenicity towards durum wheat varieties. Through morphological characterization, molecular identification, phylogenetic study, and pathogenicity tests conducted in vitro and in vivo, we aim to report Fusarium clavum as a head blight agent on durum wheat in Algeria. 2 MATERIALS AND METHODS 2.1 FUNGAL MATERIAL 2.1.1 Isolation of Fusarium clavum In this study, symptomatic infected wheat samples were collected randomly during the agricultural season 2021/2022 from several farms located in the Setif region, Algeria. Diseased samples (seeds, glumes, and crowns) were disinfected in 2 % sodium hypochlorite (NaClO) (com- mercial bleach, Bref, Henkel, Algeria) for 5 min and rinsed three successive times with sterile distilled water (Benhamou & Chet, 1996). They were then dried be- tween two sheets of sterile paper towels, plated on potato dextrose agar (PDA) medium in sterile Petri dishes, and incubated at 25 °C in the dark for seven days. Resultant colonies resembling Fusarium spp. were further sub-cul- tured on PDA for purification. 2.1.2 Macroscopic and microscopic characterization A mycelial disc subculture from strain F15B was placed on the PDA for evaluating macroscopic charac- teristics (Leslie & Summerell, 2006 and Xia et al., 2019). After a 21 - day incubation period in the dark at 25 °C, the morphological characterization was conducted based on the colony’s growth, appearance, and texture, and the pigmentation of the Petri dish’s face and reverse was ex- amined macroscopically (Leslie & Summerell, 2006 and Xia et al., 2019). Synthetic Nurtrient-poor Agar medium (SNA) (Nirenberg, 1976) was used for the diagnosis of micromorphological characteristics. The strain was ob- served under a light microscope to describe microscopic characters. The presence or absence of microconidia and macroconidia, their shapes and sizes, and the presence or absence of chlamydospores are diagnostic characteristics used for the identification of Fusarium species (Leslie & Summerell, 2006 and Xia et al., 2019). 2.1.3 Molecular identification and phylogenetic anal- ysis Molecular analysis was performed to validate iden- tification of strain F15B. The mycelium was harvested, and DNA was extracted using Nucleo Spin Plant II kit (Macherey-Nagel Germany). The internal transcribed spacers of ribosomal DNA (ITS) and the transcription elongation factor 1 alpha (TEF1) were amplified us- ing two primers ITS1/ITS4 (CTTGGTCATTTAGAG- GAAGTAA/ TCCTCCGCTTATTGATATGC) (White et al., 1990), and EF-728F/EF-2 (CATYGAGAAGTTC- GAGAAGG/ GGARGTACCAGT SATCATGTT) (O’Donnell et al., 1998; Carbone & Kohn, 1999). The PCR products were purified using the NucleoSpin® Gel and PCR Clean-up kit from Macherey-Nagel (Germany). Amplicons were sequenced with the Sanger technique Acta agriculturae Slovenica, 121/2 – 2025 3 Pathogenicity assessment of Fusarium clavum associated with wheat head blight in Algeria (Sanger et al., 1977). The sequences were edited using MEGA 11 software and compared with sequences in da- tabases by using Blastn (National Center for Biotechnol- ogy Information (http://www.ncbi.nlm.nih.gov). MEGA 11 was used to phylogenetically place our sample. The tree is based on TEF1 sequences and calculated by using neighbor-joining clustering and bootstrap analyses with 1,000 replications. 2.2 PATHOGENICITY TESTS 2.2.1 Pathogenicity test of F15B isolate towards wheat seedling A pathogenicity assay was performed following the protocol of Belabed et al. (2023) with some modifica- tions, using strain F15B to determine its pathogenic po- tential. Durum wheat seeds from each Algerian variety (Guemgoum Rkham, Djnah Khotaifa, and Oued Znati) were surface disinfected for 5 min in 2 % NaClO (com- mercial bleach, Bref, Henkel, Algeria), rinsed three times in sterile distilled water, and dried. Five healthy wheat seeds from the three varieties were each inoculated with a 5mm diameter fungal disc taken from a 21 day old PDA culture and a sterile PDA disc as a control. Three repli- cates were set up for all combinations of F15B isolate and wheat variety. The inoculated seeds were placed on ster- ile double-layer filter paper soaked with Potato Dextrose Broth (PDB) in Petri dishes. Petri dishes were sealed with parafilm and incubated in the dark at 25 °C for five days. The length of the root system, coleoptile, and germina- tion rate were measured to assess the pathogenicity. The reduction rate of coleoptile length (CLr %) and the re- duction rate of root length (RLr %) were calculated using the following equation: CLr (%) or RLr (%) = 2.2.2 Pathogenicity test of F15B isolate on wheat heads For the pathogenicity test on Algerian durum wheat heads (Guemgoum Rkham, Djnah Khotaifa, and Oued Znati varieties), the experimental spray inoculation pro- tocol of Mesterhazy (1995) was used. For each variety, three durum wheat seeds were sowed in plastic pots with four replicates distributed randomly. A spore suspension of 4 × 105 spores ml-1, obtained from F15B cultures was prepared using a Malassez hemocytometer. 3 spikes from each replicate were inoculated with spores during the full flowering stage, using glass sprayers, while the con- trol spikes were sprayed with sterile distilled water. The inoculated spikes were covered with a damp polyethyl- ene bag to retain humidity for 24 hours. For comparison purposes, the same experiment was conducted using a known Fusarium culmorum isolate (PV123206), which is recognized as pathogenic to wheat heads. This allowed assessing the potential impact of F15B isolate on wheat heads. After harvesting, the ears of each statistical unit were placed in a paper bag. Treated and untreated wheat spikes were harvested and threshed, and the kernels mass was measured. The mass of a thousand kernels (TKM) was then estimated. Subsequently, the reduction rate in the thousand-grain mass (TKMr %) of the Fusarium cul- morum and Fusarium clavum-inoculated spikes was cal- culated using the following equation: TKMr (%) = 2.3 STATISTICAL ANALYSIS Data was subjected to analyses of variance ANOVA using SPSS software (IBM SPSS Statistics version 26) at a 5 % level with a 95 % confidence interval. 3 RESULTS AND DISCUSSION 3.1 MACROSCOPIC AND MICROSCOPIC CHAR- ACTERIZATION Fungal colonies that resembled Fusarium spp. were obtained. Only a single Fusarium clavum-like isolate was obtained from durum wheat glumes and assigned as F15B. The morphological characters observed on SNA and PDA (not shown) agreed with the phylogenetic ana- lyses. 3.2 MOLECULAR IDENTIFICATION AND PHY- LOGENETIC ANALYSIS The obtained sequences of F15B isolate were depos- ited in GeneBank under accession numbers OR900216 (ITS) and PP035912 (TEF). The blastn search of ITS showed 98-99 % identity with several sequences of Fu- sarium clavum (e.g: OR582979, OR123379), while the Acta agriculturae Slovenica, 121/2 – 20254 N. R. SEGMANE et al. tested durum wheat varieties. On the contrary, the results showed that there was a significant difference (p < 0.05) between the infected plants and the non-infected control plants. This result shows that Fusarium clavum F15B iso- late was pathogenic and significantly affected coleoptile and root lengths (Tab.1). 3.3.2 Pathogenicity test of F15B isolate on wheat heads Twenty-six days after inoculation, typical Fusar- ium head blight symptoms appeared on the inoculated spikes, while the control spikes remained free of symp- toms. After harvest, inoculated grains exhibited deformi- ties compared to the negative controls. Re-isolation from the infected grains and glumes was conducted to satisfy Koch’s postulates. The results of TKM after means com- paring (Tab. 1) showed that Fusarium clavum reduced TKM from 55,9 g to 44,8 g in ‘Guemgoum Rkham’, and from 45,3 g to 26,5 g in ‘Djnah Khotaifa’, compared to the negative controls. On the other hand, no TKM reduc- tion was observed in ‘Oued Znati’. Concerning the rate of TKM reduction (TKMr %) by the isolate F15B, the re- sults were 21,1 % for ‘Guemgoum Rkham’ and -7,1 % for ‘Oued Znati’. However, it appears that ‘Djnah Khotaifa’ was the most susceptible to attacks of the isolate with a TKMr % of 41,3 %. The pathogenic isolate Fusarium culmorum (PV123206) exhibited a higher pathogenic potential, causing severe disease symptoms in the tested wheat varieties. This was reflected in the TKM, recorded as 29,9 g for var. Goumgoum Rkham, 16,5 g for var. Dje- nah Khotifa, and 29,1 g for var. Oued Zenati. Compared to the non-infected plants, this represents a TKMr % of 44,7 % for var. Goumgoum Rkham, 63 % for var. Djenah Khotifa, and 36,3 % for var. Oued Zenati. These reduc- tions were higher than those caused by Fusarium clavum isolate. Statistical analysis showed a significant difference (p = 0.01 < 0.05) between the inoculated and uninocu- lated control spikes. The reduction in TKM caused by the F15B isolate is directly associated with yield and quality loss, confirming its pathogenic impact on wheat heads. The most serious diseases affecting root, stem, and spike of wheat and at all stages of growth are caused by Fusarium species. In Algeria, some studies have been car- ried out on F. culmorum the main Fusarium Head Blight agent focusing on its occurrence, pathogenicity, and di- versity. Other pathogenic Fusarium species have not yet been studied well in Algeria (Touati-Hattab et al., 2016; Laraba et al., 2017; Abdallah-Nekache et al., 2019). Fusarium clavum is a species of the Fusarium in- carnatum-equiseti species complex (FIESC) which is a TEF sequence showed 99-100 % with Fusarium clavum isolates (e.g: GQ505672, MN170457). 3.3 PATHOGENICITY TESTS 3.3.1 Pathogenicity test of F15B isolate towards wheat seedling In vitro, the isolate F15B was responsible for 29,9 %, 60,4 %, and 26,3 % of coleoptile length reduction on Guemgoum Rkham, Djnah Khotaifa, and Oued Znati varieties. Furthermore, it induced 25,7 %, 64,3 %, and 35,9 % of root length reduction on Guemgoum Rkham, Djnah Khotaifa, and Oued Znati varieties (Tab. 1). After 5 days of incubation, it appears that ‘Djnah Khotaifa’ was more sensitive to F. clavum as coleoptile length and root length reduction were highest compared to vars. Guemgoum Rkham and Oued Znati. Also, it ap- pears that var. Djnah khotaifa was most susceptible to attacks of F15B isolate with a germination rate of 93 % compared to vars. Guemgoum Rkham (100 % germina- tion rate) and Oued Znati (95,3 %) (Tab. 1). There was no statistically significant difference be- tween coleoptile length reduction rate (p = 0.33 > 0.05), root length reduction rate (p = 0.31 > 0.05), and seed germination rate (p = 0.39 > 0.05) of the controls of the Figure 1: Phylogenetic tree generated from translation elonga- tion factor 1-alpha (TEF1) gene sequences of Fusarium spp. Bootstrap values (based on 1,000 replications) are indicated next to the branches. Based on the Neighbor-joining method, isolate F15B clusters with F. clavum isolates. The tree is rooted with Bisifusarium dimerum (Penz.) L. Lombard & Crous strain CBS 116527 Acta agriculturae Slovenica, 121/2 – 2025 5 Pathogenicity assessment of Fusarium clavum associated with wheat head blight in Algeria phylogenetically species-rich complex that includes over 30 recognized phylogenetic species (Xia et al., 2019). Fu- sarium clavum was reported for the first time in Algeria in the study of Belabed et al. (2025) as a causal agent of wheat head blight. The F15B isolate was morphologically identified as F. clavum. It was isolated from durum wheat glumes in Algeria’s Setif area in the North. The results of macroscopic and microscopic characteristics were similar to those described by Manganiello et al. (2021) and Belabed et al. (2025). The micro-morphological and plate-culturing characteristics of F15B isolate were also similar to those of Fusarium clavum (Wang et al., 2019; Xia et al., 2019). The phylogenetic tree, constru- cted using the Neighbor-Joining (NJ) method based on partial TEF1-α sequences, showed that the F15B isolate clustered with Fusarium clavum, indicating its genetic re- latedness to this species and confirming its classification within it. This study reports the presence of an F. clavum isolate in Algerian durum wheat fields. In contrast, Be- labed et al. (2025) identified multiple isolates of this spe- cies, further supporting its occurrence in the region. Fusarium clavum is a plant pathogen occurring worldwide as it can infect a wide range of plant hosts, such as Cucumis melo L. (Meshram et al., 2023), Solanum lycopersicum L. (Gilardi et al., 2021), Beta vulgaris L. (Khan et al., 2024), Rosa  spp (Manganiello et al., 2021) and Phoenix dactylifera L. (Rabaaoui et al., 2021). Few research was conducted on the pathogenicity of F. clavum on wheat. Fusarium clavum has been identified as a pathogenic species responsible for Fusarium head blight in wheat in Mexico (Leyva-Mir et al., 2022). Azil et al. (2021) investigated the Fusarium incarnatum-equiseti species complex associated with tuber dry rot and wilt of potatoes but did not report F. clavum. This highlights the need for further studies on the occurrence of F. clavum in different crops. The obtained results showed that strain F15B had a moderate aggressiveness toward wheat coleoptile length, root length, and seed germination, which concord with the finding of Belabed et al. (2023). They concluded that members of the F. incarnatum-equiseti species complex are moderate or weak pathogens. The F15B isolate had a moderate effect on wheat TKM (20.8 % TKM reduction), especially if compared with F. culmorum (Wm.G.Sm.) Sacc. one of the major FHB agents of wheat plants (48 % TKM reduction) (Abdallah-Nekache et al., 2019). The significantly greater virulence of F. culmorum aligns with its well-documented ability to induce severe disease symptoms in wheat, leading to substantial yield losses. This strong impact can be attributed to its aggressive in- fection mechanisms, including rapid colonization, toxin production, and disruption of host physiological proces- ses. Additionally, the reduction rate of TKM caused by F. culmorum and F. clavum strains may be linked to the susceptibility of the wheat genotypes used in the experi- ment, highlighting differences in host responses to fun- gal infection. This parameter plays a very important role and impacts the results. In this study, var. Djnah Khotaifa was the most sensible, comparing to vars. Guemgoum Rkham and Oued Znati. This is an important informa- tion benefiting breeding programs that aim at reducing susceptibilies of cultivars against Fusarium head blight agents. 4 CONCLUSIONS In Algeria, Fusarium clavum has been identified on wheat, and its pathogenic effects have been confirmed across three durum wheat varieties. Observed impacts included reduced seed germination, decreased coleop- tile and root lengths, and reduced thousand kernel mass. Our findings highlight the importance of expanding stu- Varieties Treatment Coleoptile length (cm) CLr (%) Germination (%) Root length (cm) RLr (%) TKM (g) TKMr (%) Guemgoum Rkham Djnah Khotaifa Oued Znati Control F15B Control F15B Control F15B 5,03 3,36 5,3 2 3,86 2,86 0 29,93 0 60,38 0 26,29 100 100 100 93 100 95,33 5,5 4,03 2,53 0,7 4,76 3,03 0 25,72 0 64,25 0 35,86 55,87 44,79 45,26 26,48 45,84 49,08 0 21,12 0 41,28 0 -7,06 Table 1: Observed effects of pathogenicity tests and thousand kernels mass reduction *Each number in the table represents the average of the replicates in each parameter (F15B: inoculated by F15B isolate) Acta agriculturae Slovenica, 121/2 – 20256 N. R. SEGMANE et al. dies focusing on determining the distribution, prevalen- ce, and toxigenic potential of Fusarium species associated with wheat diseases in Algeria. 5 REFERENCES Abdallah-Nekache, N., Laraba, I., Ducos, C., Barreau, C., Bouznad, Z., & Boureghda, H. (2019). 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