Pathogenicity and mycotoxin profile of Fusarium spp. inducing wheat head blight in Algeria

doi:10.21203/rs.3.rs-3237786/v1

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Pathogenicity and mycotoxin profile of Fusarium spp. inducing wheat head blight in Algeria

https://doi.org/10.21203/rs.3.rs-3237786/v1

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Fusarium is one of the world's most harmful wheat pathogens, causing severe crop diseases like Fusarium head blight (FHB). They constitute a serious threat to the production of wheat by considerably reducing crop yield, quality, and safety due to grain contamination with mycotoxin. The focus of the current study emphasizes aspects related to the pathogenicity and toxigenicity of some FHB isolates recovered from symptomatic Algerian wheat samples. Three genotypes of durum wheat were subjected to two pathogenicity tests (in vitro and in vivo), and the results showed a significant difference in aggressiveness on different phenotypic parameters, indicating intrinsic genetic variation in the host-pathogen interaction. All of the Fusarium isolates tested for mycotoxinogenicity produced Deoxynivalenol, zearalenone, and T-2 toxin. With DON, the most common mycotoxin type, producing 7.128µ/kg in the ELISA test and 373196.19µg/kg by LC-MS/MS, exceeding the European limits (1750µg/kg), the FusBo59 isolate of F. culmorum stands out as the most toxic. Following DON (18.2%) and 3-ADON (9.1%), 15-ADON (63.6%) emerged as the most prevalent toxin in wheat grains, according to a toxin analysis. T-2 and zearalenone (ZEN) were present, but at concentrations below the permissible limits (100µg/kg). On the other hand, F. avenaceum FusBi7 stood out as being the most aggressive, and the Cirta variety was the most tolerant to attacks from the Fusarium isolates studied. Thus, our data demonstrate that disease induction and toxin generation evolve independently of each other.

Fusarium head blight

pathogenicity

toxigenicity

ELISA

LC-MS/MS

Wheat, as the main staple food, is a substantial source of energy for human nutrition, providing 20% of global calories and protein (FAO, 2018). Unfortunately, under favorable environmental conditions, wheat is often subject to many pathogens throughout the period of production, harvest, transport, and storage.

Fusarium spp. are the most harmful wheat pathogens worldwide, inducing severe crop diseases such as Fusarium head blight (FHB), Fusarium root rot (FRR), and Fusarium crown rot (FCR), all of which have negative consequences for food safety and security, agricultural production, and human and animal health. FHB can cause significant economic losses in wheat by causing grains to whiten early, wilt, discolor, and the demise of spikelets or complete heads (Petronaitis et al., 2021).

Worse, Fusarium spp. contaminate cereals with multiple mycotoxins such as trichothecenes, deoxynivenol (DON), nivalenol (NIV), T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), and zearalenone (ZEN), which are known to be a threat to global public health through dietary exposure and also have a great effect on pathogen fitness and food safety (Wang et al., 2020; Adnan et al., 2020). In this monocyclic disease, the level of aggressiveness is frequently measured by assessing disease severity. Higher levels of aggressiveness are associated with earlier symptom evolution and higher levels of mycelium and mycotoxins in plant tissue (Miedaner et al., 2004). In this context, the ability of Fusarium mycotoxins to be produced varies not only between species but also between strains of the same species. The level of mycotoxin accumulation in infected wheat grain, however, can vary due to climatic factors, cultivation system, method, and date of grain harvesting, as well as the degree of FHB-resistant wheat cultivars (Golinski et al., 2010; Bernhoft et al., 2012; Xu et al., 2014; Del Ponte et al., 2015). According to a Food and Agriculture Organization (FAO), approximately 25% of the world's food crops are contaminated by mycotoxins, with detectable mycotoxins expected to reach 60–80% in all food and feed crops globally by 2020 (Eskola et al., 2020). Currently up to 500 different forms of mycotoxins have been found (Haque et al., 2020).

The most important mycotoxins in terms of food safety and legislation include aflatoxins (AFs), ochratoxin A (OTA), DON, fumonisins (FUMS), ZEN, ergot alkaloids, T-2 toxin, HT-2 toxin, patulin, and citrinin (Eskola et al., 2020). Human exposure to mycotoxins is acquired directly by ingesting contaminated plant-origin products such as cereals, dried fruits, and nuts or indirectly by eating animal-origin products such as contaminated milk, eggs, and meat (Capriotti et al., 2012; Flores-Flores et al., 2015), leading to several dramatic health hazards, including cancer, immunosuppression, nephrotoxic, hepatotoxic, mutagenic, estrogenic, and gastrointestinal effects. FHB pathogen species can be classified based on the profile of toxic secondary metabolites produced, which can result in diverse chemotype profiles that can cause different forms of grain infection depending on the occurrence of each species in the crop.

According to several studies, chemotypes that produce DON, 3-acetyldeoxynivalenol (3-ADON), and 15-acetyldeoxynivalenol (15-ADON) are more virulent to plants than those that produce NIV (Gilbert et al., 2010; Puri and Zhong, 2010; Pasquali et al., 2016). Trichothecene compounds (TCT) (particularly DON), ZEN, and FUMS are the most typically produced by Fusarium spp. in wheat (Lemus-Minor et al., 2015; Sadhasivam et al., 2017). F. graminearum, for example, is the primary producer of type B trichothecenes (Type B TCT) (primarily DON and its acetylated forms 3-ADON and 15-ADON), whereas F. sporotrichioides and F. langsethiae are the primary producers of type A trichothecenes (T-2 and HT-2 toxins) (McCormick et al., 2011). Furthermore, the new A-trichothecene NX-2 is produced by a small percentage of F. graminearum isolates from the United States and Canada (Kelly et al., 2016; Kelly and Ward, 2018; Liang et al., 2014).

Given this diversity, crop protection against Fusarium spp. and associated mycotoxins is critical not only for ensuring the quality and safety of grains meant for consumption as seed, feed, and edibles, but also for refining management techniques to protect consumer health. There was a correlation between DON accumulation and the level of FHB symptoms in one trial, where DON-resistant cultivars also demonstrated Fusarium resistance (Lemmens et al., 2016), however in another, near to negative associations were detected (Ji et al., 2015). Furthermore, there was no link between the level of mycotoxin contamination of grain and the visual evaluation of etiological symptoms (Birzele et al., 2002).

As a fundamental immunoassay, the screening ELISA kit is the conventional analytical tool for mycotoxin determination due to its simplicity, specificity, and cost effectiveness. For the qualitative and quantitative analysis of mycotoxins, more precise and sensitive analytical approaches have been proposed. LC-MS/MS is chosen because of its high selectivity, sensitivity, and quick multi-mycotoxin analysis. Despite its prominence as a staple crop, research into the pathogenicity and mycotoxin levels of Fusarium spp. in durum wheat in Algeria is still limited. This study aimed to: 1) assess the pathogenicity profiles of Fusarium isolates in three durum wheat cultivars (Triticum durum L.) using in vitro and in vivo tests; 2) assess mycotoxin levels in cultures of toxigenic Fusarium spp. as well as in several durum wheat grain samples using ELISA kits and LC-MS/MS methods; and 3) determine the relationship between pathogenicity and mycotoxin production.

Plant material

Pathogenicity of the Fusarium strain was investigated on three major wheat (Triticum durum L.) cultivars grown in the departments of north-eastern Algeria, namely GTAdur, Cirta, and Waha. The seeds used in this experiment were generously given by Setif's National Center for Certification and Control of Seeds and Plants (CNCC). Previously, the germination capability was measured after 1 minute of superficial sterilization with 1% sodium hypochlorite (NaClO), followed by three successive rinses with sterile distilled water, and incubation on sterile filter paper moistened in Petri dishes at 25°C for 8 days. The germinated seeds were counted, and the three types' germination rates ranged between 90 and 100%.

For mycotoxin determination was performed on 11 samples of grains retrieved from the ears of six varieties of durum wheat displaying indications of Fusarium wilt, collected from several states in Algeria's north-east. The examination using an MFC-90D 16 micro-hammer mill (Culatti, Zurich, Switzerland), grain samples weighing 50 to 250g were ground to a particle size of 0.1mm. The ground flour is stored in plastic bags at room temperature until it is analyzed.

Fungal material

Eleven Fusarium isolates were isolated from the FHB-symptomatic grain samples of durum wheat and ears collected from various north-eastern provinces of Algeria. The set of isolates was taxonomically and molecularly identified in previous studies (Bencheikh et al., 2020; Belabed et al., 2022) and is codified as follows: F. avenaceum (FusBi7, FusBi21), F. acuminatum (FusBi15, FusBi23, FusBo33), F. culmorum (FusBo59), F. incarnatum-equiseti species complex (FusBi1, FusBi2, FusBi8), F. tricinctum species complex (FusBi6), and F. chlamydosporum species complex (FusBo26). The pathogenicity of Fusarium isolates was estimated through their potential for inducing symptoms (efficiency of infection, severity of disease) in addition to their ability to produce host necrosis-inducing mycotoxins (Pariaud et al., 2009).

Reagents and Chemicals

ELISA tests

For samples preparation, methanol and ethanol (MeOH and EtOH Carlo Erba Reagents) and water purified by the Milli-Q purification system (Millipore Corporation, Bedford, MA, USA) were used. Three commercial ELISA kits were provided by MyBiosource and used: (i) ZEN (Zearalenone) ELISA Kit (cat. No. MBS2548744, USA); (ii) Deoxynivalenol (DON) ELISA Kit (cat. No. MBS283277, USA); and (iii) T-2 toxin (T2) ELISA Kit (Cat. No. MBS920908, USA).

LC-MS/MS tests

The standards of aflatoxin-B2 (AFB2), aflatoxin-G2 (AFG2), HT-2 toxin (HT-2), T-2 toxin (T-2), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), and zearalenone (ZEN), all with purity > 98%, were purchased from Sigma (West Chester, PA, USA) and Fluka (West Chester, PA, USA). The Internal Standard (IS) Ochratoxin A-(phenyl-d5) (OTA-d5) with a purity of 95% was purchased from Fluka (West Chester, PA, USA). Acetonitrile (MeCN), Methanol (MeOH), and acetic and formic acids were all HPLC-grade from Merck (Darmstadt, Germany). Ammonium acetate (analytical grade) was also from Merck.

Analytical-grade solvents, dichloromethane and ethyl acetate, were purchased from Sigma-Aldrich. Ultrapure water, purified by a Milli-Q gradient system from Millipore (Milford, MA, USA), was used in the preparation of the mobile phase. Anhydrous magnesium sulfate (MgSO4) was purchased from Sigma-Aldrich and sodium chloride (NaCl) from VWR, both treated at 500°C for 5h before use. Octadecylsilica (C18, particle size 55–105mm) was purchased from Waters (Milford, MA, USA), and Z-sep⁺ was purchased from Supelco (Bellefonte, PA, USA). Standard stock solutions of 10mg.L^− 1 of each mycotoxin was prepared in MeOH, and from these two working solutions, 1000µg.L^− 1 each were prepared in MeOH. A stock solution of IS, d5-OTA, at 10mg.L^− 1 was prepared in dimethylsulfoxide (DMSO) and a work solution at 500µg.L^− 1 was prepared in MeOH. All standard solutions were kept at -18°C if not in use.

Instrument and analytical conditions

The high-performance liquid chromatography was performed using an HPLC system Waters Alliance 2695 (Waters, Milford) with a triple quadruple mass spectrometer, Quattro Micro (Waters, Manchester, UK). A Kinetex C18 2.6µm particle size analytical column (150 × 4.6mm) with pre-column from Phenomenex (Tecnocroma, Portugal), maintained at 30°C, was used for chromatographic separation, as previously described by Cunha et al. (2018). The mobile phase was water/methanol/acetic acid [94:5:1 (v/v) and 5mM ammonium acetate] (solvent A) and methanol/water/acetic acid [97:2:1 (v/v)] (solvent B). The elution was conducted in a gradient that started at 95% of phase A with a linear decrease to 35% in 7min. Then the mobile phase A decreased to 25% at 11min, decreased to 0% at 13min, and remained constant until 25min. Initial column conditions were reached at 25 minutes and remained for 2min until the next injection. The flow rate was 0.3mL.min^− 1 and the injection volume was 20µL. The optimized MS/MS parameters for each analysis are listed in Table 1. The MS/MS acquisition was operated in positive-ion mode with multiple reaction monitoring (MRM), and the collision gas was Argon 99.995% (Gasin, Portugal) with a pressure of 2.9 × 10^− 3 mbar in the collision cell. Capillary voltages of 3.0 kV were used in the positive ionization mode. Nitrogen was used as desolvation gas and cone gas, with flows of 350 and 60L.h^− 1, respectively. The desolvation temperature was set to 350°C and the source temperature to 150°C. Dwell times of 0. s/scan were selected. The data were collected using the software program MassLynx 4.1.

Table 1

Optimized parameters for mycotoxins analysis by LC-MS/MS.
Mycotoxin/ metabolite	Retention time (min)	Parent ion (m/z)	Product ions (m/z)	Cone energy (V)	Collision energy (V)
15-ADON	8.77	339.1 [M + H]⁺	137.1^*	22	13
15-ADON	8.77	339.1 [M + H]⁺	321.2	22	13
3-ADON	8.77	339.2 [M + H]⁺	203.2	21	13
3-ADON	8.77	339.2 [M + H]⁺	231.2^*	23	13
AFG₂	9.03	330.8 [M + H]⁺	245.3	35	30
AFG₂	9.03	330.8 [M + H]⁺	313.1^*	35	24
DON	9.60	297.0 [M + H]⁺	203.3^*	22	13
DON	9.60	297.0 [M + H]⁺	249.0	20	11
AFB₂	9.89	315.0 [M + H]⁺	259.2^*	40	33
AFB₂	9.89	315.0 [M + H]⁺	287.3	40	35
HT-2	16.31	442.1 [M + H]⁺	215.3	18	15
HT-2	16.31	442.1 [M + H]⁺	263.2^*	18	15
T-2	16.79	484.0 [M + H]⁺	214.9^*	21	18
			245.2	23	15
			305.2	23	15
ZEN	17.19	319.2 [M + H]⁺	187.0^*	20	18
ZEN	17.19	319.2 [M + H]⁺	283.3	20	16
OTA-d₅	17.50	409.0 [M + H]⁺	239.4^*	32	22
OTA-d₅	17.50	409.0 [M + H]⁺	257.1	32	22
^* - Quantification ion.

Pathogenicity of Fusarium isolates

The pathogenicity of the Fusarium isolates was evaluated in both in vitro and in vivo tests. The first one was performed to investigate the impact on coleoptile and root growth by seed inoculation, and the second one was used to determine aggressiveness on the crown by soil inoculation.

Pathogenicity towards wheat seedlings

A pathogenicity assay was performed using eight strains, which are: FusBi7, FusBi21, FusBi15, FusBi23, FusBi8, FusBi1, FusBi2, and FusBi6. Durum wheat seeds from each cultivar were surface sterilized for 8min in 2% NaClO, rinsed six times in sterile distilled water, and dried. A set of five healthy wheat seeds from three cultivars were each inoculated with a 5mm diameter fungal plug taken from a one-week-old culture and a blank PSA disc (as a control). For all combinations of Fusarium isolate and wheat variety, three replicates were set up. The inoculated seeds were placed on sterile double-layer filter paper soaked with Potato Dextrose Broth (PDB) in Petri dishes. To favor fungal growth, all Petri dishes were hermetically sealed with parafilm strips to maintain high relative humidity and then incubated at 25°C for 6 days. Thereafter, pathogenicity was attempted by determining the coleoptile and root system length as well as the seminal root number, germination rate, and severity of attack through the symptoms developed.

In vivo pathogenicity of Fusarium isolates

The same Fusarium isolates previously used in the in vitro pathogenicity test were studied in vivo according to the method described by Demirci and Dane (2003). Healthy wheat seedlings of three varieties were sown in plastic pots (12 x 10cm, diam. by depth), containing a combination of soil and peat in a ratio of 2:1 (v/v). Each of the pots was sown with four surface-disinfecting wheat seeds and maintained in the greenhouse at its natural temperature and photoperiod. The artificial infection was obtained through the direct contact of a mycelial explant 5mm in diameter from a 7-day-old Fusarium colony with each seed, followed by its cover with a thin layer of soil mixture 2cm in height. Control seeds were similarly inoculated with only an agar plug without fungus. The pots and later the plants were watered frequently, depending on the soil moisture and when needed. The experimental pattern adopted was a randomized complete block design with three replicates (pots) per variety, each with four seeds for each pathogen. Fifty days post inoculation (DPI), three seedlings were carefully removed from the soil of each pot and thoroughly washed to get rid of all adhering soil particles so as not to mask root symptoms and influence the weight of the root system. The fourth plant of each Fusarium strain x durum wheat variety combination was allowed to complete its development cycle to full maturity to serve as a source for the isolation of Fusarium strains from the ears produced.

The well-washed plants were placed on a sterile paper towel to remove excess water and thus the length of the root and vegetative systems (longest root, longest leaf) were measured as well as their fresh weight. Later, Koch's postulate was performed by comparing the morphological characteristics of Fusarium strains re-isolated from symptomatic plants (root and crown) with those of the original inoculated isolates.

Mycotoxin production capacity of Fusarium isolates

Determination by ELISA Kit

A set of eleven Fusarium isolates, including FusBi1, FusBi2, FusBi6, FusBi7, FusBi8, FusBi15, FusBi21, FusBi23, FusBo26, FusBo33, and FusBo59, were tested for their ability to produce zearalenone (ZEN), T-2 toxin (T2), and deoxynivalenol (DON). Each Fusarium strain was grown on PDA medium in the dark at 25°C for 15 days (Noorabadi et al., 2021). Then, ELISA kits were used for the analysis of DON (cat. No. MBS283277), ZEN (cat. No. MBS2548744), and T-2 toxin (cat. No. MBS920908). Mycotoxin extraction and determination for each immunoassay kit were performed according to the manufacturer’s instructions, and the intensity of the resulting yellow color was measured using a 96-well microplate absorbance reader set to 450/630nm (T2, DON) and to 450nm (ZEN). Calibration curves for the quantification of DON, ZEN, and T-2 toxin were performed with the OD values of the standard concentration established for each kit (Table 2). The concentration range (ppb) of T-2, DON, and ZEN can be obtained by comparing the average OD value of the sample with that of the standard solution, and sample concentrations in each toxin were calculated according to the manufacturer’s instructions using the Microsoft Excel program. The correlation coefficient (R²) of the calibration curve ranged between 0.990 and 1.000. Limits of detection (LODs) were 6ng/mL (ZEN), 150ng/mL (DON) and 30ng/mL (T-2).

Table 2

Standard concentration used in mycotoxin dosage analysis.
Mycotoxin	Concentration	C2	C3	C4	C5	C6
DON	ppb	3	9	27	81	243
ZEN	ppb	0,3	0,9	2,7	8,1	24,3
T-2	ppb	0,3	0,9	2,7	8,1

Determination by LC-MS/MS technique

LC-MS/MS analysis was also performed to determine the typology of the mycotoxins produced in this case: DON, 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), and zearalenone (ZEN). The test was performed in the Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, University of Porto, Portugal, and was applied to a set of six isolates, including FusBi1, FusBi6, FusBi7, FusBo26, FusBo33, and FusBo59. Each Fusarium strain was cultivated on PDA medium in the dark at 25°C for 21 days (Samson, 2010). Afterward, the lyophilization of the medium was performed for further extraction and mycotoxins quantification.

Extraction and mycotoxins determination

Mycotoxins were extracted according to Smedsgaard (1997), with some modifications. Briefly, 0.5g of dried culture was weighed into a 50mL centrifuge tube and 40µL of OTAd5 (IS) at 500µg.L-1 was added. After leaving the samples overnight for equilibration, 10mL of water was added and shaken for 30min. Thereafter, 10mL of methanol, dichloromethane, and ethyl acetate (1:2:3, vol/vol/vol) (HPLC-grade purity) containing 1% formic acid were homogenized for 1min and sonicated for 15min. The tubes were then centrifuged at 4000g for 15min to induce phase separation and mycotoxins partitioning. Then, the upper layer was transferred to an injection vial and evaporated to dryness under a stream of nitrogen (SBH CONC/1 sample concentrator from Stuart® (Staffordshire, OSA, USA). The final extract was reconstituted in 150µL of mobile phase B (methanol: water: acetic acid (97:2:1) with 5mM ammonium acetate) and transferred to a 200µl insert glass for LC-MS/MS analysis. Each sample was injected twice.

Mycotoxin analysis in wheat grains by LC-MS/MS

Mycotoxins extractions were performed by the QuEChERS method with some modifications (Cunha et al., 2018). Briefly, 5.0g of grounded sample was weighed into a 50 mL centrifuge tube and 200µL of OTAd5 (IS) at 500µg.L^− 1 was added. After leaving the samples overnight for equilibration, 5mL of water were added and shaken for 30min. Thereafter, 5mL of acetonitrile (HPLC purity) with 1% formic acid was added along with 2.0g of MgSO₄ anhydrous salt and 1.0g of NaCl, and tubes were mixed for 1h in an orbital shaker. The tubes were then centrifuged at 4000g for 15min to induce phase separation and mycotoxins partitioning. For the dSPE clean-up procedure, exactly 1.2mL of the organic phase was transferred to a 4mL vial containing 100mg C18 and 50mg Z-sep⁺, homogenized for 30s, and centrifuged for 4000g for 5min. Then, 0.80mL from the upper layer was transferred to an injection vial and evaporated to dryness under a stream of nitrogen (SBH CONC/1 sample concentrator from Stuart® (Staffordshire, OSA, USA). The final extract was reconstituted in 750µL of mobile phase B (methanol: water: acetic acid (97:2:1) with 5mM ammonium acetate) and transferred to a 2mL glass vial for LC-MS/MS analysis. Each sample was injected twice.

Statistical analysis

To determine the importance of pathogenicity on various types of wheat between isolated Fusarium species, a statistical analysis (ANOVA) was carried out using the SPSS V. 25 software package (SPSS, 2017) at a probability threshold of 5%. To reflect the measurement variability, the results are reported as the mean ± SE (standard error of the mean), and homogenous groups are identified using Tukey's HSD test. Additionally, Pearson correlation tests were used to look at the connections between the parameters that were evaluated for pathogenicity and the mycotoxin levels.

Pathogenicity of Fusarium isolates towards wheat seedlings

The susceptibility of the three durum wheat varieties most commonly sown in the study area, as well as the pathogenicity of isolated Fusarium strains, were determined by measuring various development parameters such as coleoptile and root system length, number of seminal roots, germination rate, and severity via symptom induction.

Figure 1.

According to the statistical analysis of the results, the 8 Fusarium isolates had a negative and significant (p < 0.01) impact on the length of the coleoptiles, the length of the root system, the number of seminal roots, and the germination rate (Table 3). However, the statistical analysis revealed no significant difference between the three durum wheat cultivars and the eight isolates in terms of the growth parameters investigated. In terms of coleoptile length, the FusBi7 and FusBi21 strains significantly lowered the CLs of the three varieties evaluated when compared to their respective controls. When infected with the FusBi7 strain, coleoptile lengths measured 1.57, 0.3, and 0.11cm for the Cirta, Waha, and GTAdur varieties, respectively. In contrast to these findings, the lengths of the FusBi1 and FusBi2 strains were extremely close to those of the controls. (Table 3)

In terms of the effect of Fusarium strains on root system length, the FusBi7 strain is the most aggressive, yielding lengths of 2.21, 1.09, and 0.62cm for the Cirta, Waha, and GTAdur varieties, respectively. This is much lower than the 8.89, 8.36, and 9.14cm achieved with their respective controls. Furthermore, the FusBi7 strain had the greatest impact on the number of seminal roots, once again demonstrating its aggressiveness and yielding an average of 0.53 for GTAdur, 1.87 for Waha, and 2.8 for Cirta. This is significantly lower than the control values of 5.50, 5.43, and 5.97cm for the same varieties (Table 3).

Regarding Fusarium isolates influence on the germination of durum wheat seeds, the FusBi7 strain caused a significant drop, reaching 20% with the GTAdur variety. In terms of average germination rates, the GTAdur appears to be the most susceptible to diseases, with a germination rate of 73.33%, followed by the Waha variety (87.5%), and lastly the Cirta variety (93.33%).

Finally, in terms of symptom induction, we note the emergence of root necrosis on both the coleoptile and the seminal roots (Fig. 1), which rarely resulted in the death of the infected plant. The estimated severity generated by the strains examined reveals that the FusBi7 strain is the most severe of the three studied varieties, with rates of 0.3, 0.53, and 0.67 for the Cirta, Waha, and GTAdur, respectively. This is supported by the short coleoptile lengths observed in the same types when infected with the FusBi21 strain, which resulted in a high severity rate as a result of root coleoptile necrosis symptoms (Table 3).

Pathogenicity of Fusarium isolates in vivo

The healthy wheat seedlings were injected with the mycelium of eight isolates to determine their pathogenicity, showed fifty days post-inoculation, same symptoms occurred with the initial symptoms reported. There were no symptoms in the control plants. The pathogenic fungi were re-isolated from damaged plants (root and crown), and their identity was confirmed morphologically when compared to the isolates used as inoculum, demonstrating that Koch's postulates were fulfilled. There was no re-isolation of a related fungus from the control plants. The findings of the vegetative system weight, root system length, and root system weight parameters show a highly significant difference (p < 0.001) in the pathogenicity of Fusarium strains and a significant difference (p < 0.05) in the vegetative system length.

In vivo pathogenicity data demonstrate that some strains had a detrimental impact on the development parameters tested, including root system length and weight and vegetative system length and weight, while others provided values remarkably comparable to those reported by control plants. In terms of the effect on the length of the vegetative system, all three cultivars were susceptible to a specific strain of Fusarium. Thus, the FusBi7 isolate produced the lowest VSL value of 24.89cm for the Cirta variety, FusBi15 produced 21.11cm for the GTAdur, and FusBi1 produced 15.83cm for the Waha variety, all of which are lower than the 34.33, 32.33, and 34.16 VSL values of control plants (Table 4). According to the findings, the effects on the length of the vegetative system were translated into the weight of the vegetative system. Thus, FusBi7 reduced the VSW on the Cirta variety to 1.19g, FusBi15 created 1.21g on the GTAdur variety, and FusBi1 produced 0.43g on the Waha variety. These VSW weights are much lower than the 1.92, 1.76, and 1.66g reported with the control plants for the same kinds (Table 4).

In terms of the impact on the root system, the FusBi15 strain caused the shortest length of the GTAdur variety's root system at 13.89cm, while the FusBi7 strain lowered this length to 17.33cm and 16.11cm for the Cirta and Waha varieties, respectively. When compared to the control plants, these strains had considerably (p < 0.01) lower RSL values (28.61, 27.77, and 27.88 cm for the same varieties, respectively) (Table 4). However, in absolute terms, the FusBi1 strain produced the lowest RSW of 0.28g on the Waha variety, while FusBi6 produced 0.44g on Cirta and FusBi15 produced 0.47g on the GTAdur type. When compared to the control, these strains had considerably (p < 0.01) lower RSW values compared to the control plants, 0.98, 0.97, and 1.18g for the same varieties, respectively (Table 4).

Based on the in vivo pathogenicity results, the genotypic responses to pathogenic isolates show varied behavior and a substantial difference at the 5% threshold. It appears that the Cirta variety's vegetative development parameters are least significantly influenced, followed by GTAdur and finally Waha. They are represented by a VSL of 31.29, 28.34, and 25.68cm, and a VSW of 1.57, 1.43, and 1.00g, respectively. However, no significant variation was seen at the 5% threshold in the parameters linked to the length and weight of the root system (Table 4).

Table 3

Mean of *in vitro* Pathogenicity on germination parameters
Fusarium Strains	Coleoptile Length (CL) (cm)			Root system length (RSL) (cm)			Seminal root Number (SRN)			Germination rate (GR) (%)			Severity
Fusarium Strains	Cirta	GTAdur	Waha	Cirta	GTAdur	Waha	Cirta	GTAdur	Waha	Cirta	GTAdur	Waha	Cirta	GTAdur	Waha
Control	6,540^c	6,350^c	6,117^bc	8,890^c	9,140^c	8,360^c	5,967^b	5,500^b	5,4333^bc	100,000^a	100,000^b	100,000^a	0,000^a	0,000^a	0,000^a
FusBi1	5,900^c	5,427^c	6,280^c	6,813^c	3,640^abc	6,413^bc	6,000^b	4,333^b	5,5333^bc	100,000^a	80,000^ab	93,333^a	0,400^a	0,133^a	0,333^a
FusBi15	3,480^ab	3,833^abc	3,467^b	9,267^c	7,5133^bc	7,587^c	5,600^b	4,933^b	5,0667^bc	100,000^a	86,667^ab	86,667^a	0,200^a	0,000^a	0,267^a
FusBi2	6,467^c	5,327^c	5,567^bc	8,807^c	6,860^abc	7,773^c	4,200^ab	4,067^ab	5,1333^bc	100,000^a	73,333^ab	86,667^a	0,267^a	0,133^a	0,133^a
FusBi21	2,127^a	1,000^ab	0,840^a	6,680^c	3,633^abc	3,713^ab	3,933^ab	3,40^ab	3,400^ab	100,000^a	93,333^b	86,667^a	0,467^a	0,533^a	0,533^a
FusBi23	3,773^ab	3,400^abc	4,533^bc	3,033^a	2,1400^ab	3,933^ab	5,467^b	2,933^ab	5,2667^bc	86,667^a	66,667^ab	93,333^a	0,133^a	0,733^a	0,133^a
FusBi6	4,993^bc	4,233^bc	5,980^bc	8,987^c	6,633^abc	7,740^c	2,800^a	4,467^b	5,800^c	100,000^a	86,667^ab	100,000^a	0,133^a	0,200^a	0,200^a
FusBi7	1,573^a	0,113^a	0,300^a	2,213^a	0,620^a	1,087^a	4,267^ab	0,533^a	1,867^a	80,000^a	20,000^a	60,000^a	0,300^a	0,667^a	0,533^a
FusBi8	3,700^ab	4,687^bc	5,667^bc	4,573^ab	4,693^abc	7,840^c	6,200^b	3,667^ab	5,2667^bc	80,000^a	80,000^ab	93,333^a	0,267^a	0,133^a	0,467^a
Mean	4,002	3,503	4,079	6,297	4,467	5,761	4,808	3,542	4,667	93,333	73,333	87,500	0,271	0,317	0,325

Different lowercase letters indicate significant differences analysed by ANOVA followed by Tukey’s HSD test (p < 0.05). Data are mean (n = 3).

Table 4

Mean of *in vivo* pathogenicity
Fusarium Strains	Vegetative System Length VSL (cm)			Vegetative System Weight VSW (g)			Root System Length RSL (cm)			Root System Weight RSW (g)
Fusarium Strains	Cirta	GTAdur	Waha	Cirta	GTAdur	Waha	Cirta	GTAdur	Waha	Cirta	GTAdur	Waha
Control	34,333^c	32,333^b	34,167^c	1,9167^ab	1,762^a	1,658^d	27,778^b	28,611^bc	27,889^a	0,977^bc	1,188^bc	0,980^b
FusBi1	33,000^bc	28,833^ab	15,833^a	1,669^ab	1,377^a	0,434^a	26,833^ab	27,333^bc	19,583^a	0,811^abc	0,861^abc	0,282^a
FusBi15	34,889^c	21,111^a	31,111^ab	2,087^b	1,257^a	1,451^cd	22,333^ab	13,889^a	21,556^a	1,158^cd	0,471^a	0,727^ab
FusBi2	35,167^c	30,333^ab	19,000^ab	1,872^ab	1,444^a	0,572^ab	25,500^ab	26,667^bc	14,833^a	1,454^d	1,106^bc	0,332^a
FusBi21	30,583^abc	28,167^ab	31,167^ab	1,248^a	1,210^a	1,256^bcd	24,667^ab	29,833^c	29,278^a	0,696^abc	1,129^bc	1,012^b
FusBi23	35,667^c	27,222^ab	30,889^ab	1,836^ab	1,619^a	1,445^cd	20,778^ab	19,556^ab	20,000^a	0,884^abc	0,749^ab	0,697^ab
FusBi6	26,333^ab	31,667^ab	27,500^ab	1,376^ab	1,341^a	1,2556^bcd	24,667^ab	29,167^bc	29,500^a	0,439^a	0,659^ab	0,642^ab
FusBi7	24,889^a	28,111^ab	24,111^ab	1,193^a	1,642^a	0,908^abcd	17,333^a	21,444^abc	16,111^a	0,498^ab	0,837^abc	0,418^ab
FusBi8	29,833^abc	31,333^ab	25,833^ab	1,341^ab	1,583^a	0,724^abc	28,500^b	29,167^bc	25,667^a	0,867^abc	1,426^c	0,639^ab
Mean	31,295	28,347	25,681	1,578	1,434	1,006	23,826	24,632	22,066	0,851	0,905	0,594

Different lowercase letters indicate significant differences analysed by ANOVA followed by Tukey’s HSD test (p < 0.05). Data are mean (n = 3).

Mycotoxin production capacity of Fusarium isolates

Fusarium isolates' mycotoxigenic potential was determined using two analytical techniques: Kit ELISA and LC-MS/MS. All examined strains were found to be capable of secreting the three categories of mycotoxins evaluated using serological methods. Because of the sensitivity of the technique, the number of toxins discovered when LC-MS/MS is valid is substantially higher. The results of the mycotoxin investigation utilizing ELISA tests revealed that the FusBo59 strain and the FusBi15 strain have very high ZEN production rates of 3.941 and 3.116µg/kg, respectively (Table 5). However, when it came to DON, the FusBo59 strain was the most toxigenic, with a high level equal to 7.128µg/kg in the ELISA test and 373196.19µg/kg by LC-MS/MS. The amounts of the third mycotoxin tested, T2, ranged from 0.281 to 0.349µg/kg and were not significantly different.

Table 5

Mycotoxins detection by ELISA and LC-MS/MS in the culture extracts.
Fusarium isolates	ELISA			LC-MS/MS
Fusarium isolates	ZEN (µg/kg)	DON (µg/kg)	T2 (µg/kg)	ZEN (µg/kg)	DON (µg/kg)
FusBi1	0,501	0,338	0,305	0	34,57
FusBi15	3,116	0,100	0,281	/	/
FusBi2	0,297	0,249	0,305	/	/
FusBi21	0,634	0,125	0,320	/	/
FusBi23	0,463	0,043	0,320	/	/
FusBi6	0,200	0,063	0,305	0	6,41
FusBi7	0,429	0,014	0,305	0	34,39
FusBi8	0,501	0,093	0,320	/	/
FusBo26	0,366	0,125	0,349	27.63	62,08
FusBo33	0,418	0,043	0,349	0	102,61
FusBo59	3,941	7,128	0,320	0	373196,19
“/” not analysed

The HPLC-MS/MS study revealed that all of the strains investigated produced DON in concentrations ranging from 6.41 to 373196.19µg/kg (Table 6). Only two strains stand out in terms of 15-ADON production, FusBo33 and FusBo59, with values of 8.62 and 2090.24µg/kg, respectively. Only one strain of FusBo26, on the other hand, was capable to producing ZEN (27.63µg/kg). In addition, for the production of 3-ADON toxin, a single strain, FusBi6, synthesized the mycotoxin; however, the amount was smaller than the LOQ. It should be noted that the interday precision was estimated using the relative standard deviation (RSD) of the two weeks apart data. The data show an overall percent RSD range of 1 to 14%, which is within acceptable variability limits.

Table 6

Levels (µg/kg) of mycotoxins detected by LC-MS/MS in the culture extracts.
Positive	DON		15-ADON		3-ADON		ZEN
Fusarium isolates	Average µg/kg	RSD %	Average µg/kg	RSD %	Average µg/kg	RSD %	Average µg/kg	RSD %
FusBi1	34.57	13
FusBi6	6.41	6			<LOQ
FusBi7	34.39	4
FusBo26	62.08	6					27.63	4
FusBo33	102.61	14	8.62	3
FusBo59	373196.19	3	2090.24	1
LOQ = 2.5 µg/kg

Production of mycotoxins in wheat samples

The LC-MS/MS analysis of the durum wheat samples from which the various mycotoxigenic Fusarium spp. were isolated reveals significant diversity in terms of mycotoxin type and quantity (Table 7). Thus, in the W59 durum wheat sample from which the FusBo50 and FusBo59 strains were isolated, levels of DON of 33.51 and 624.96µg/kg and 15-ADON of 2.45 and 18.14µg/kg were identified in the two strains, respectively. We also found ZEN at 2.22µg/kg and 3-ADON at roughly 61.18µg/kg, while the remaining strains were negative for this toxin.

Furthermore, 15-ADON was found to be the most common mycotoxin type in durum wheat grain samples, with six samples having values above the LOQs. AF-G2 toxin, on the other hand, was identified in only one sample, E55 (1.04µg/kg), and AF-B2 in two samples, E49 and E47, with values of 16.28 and 20.82µg/kg, respectively. Finally, T2 was found in only one sample, E40, but the value was less than the LOQ. It should be noted that the interday precision was estimated using the relative standard deviation (RSD) of the two weeks apart data. The results show an overall percent RSD of 1 to 15%, which is within the acceptable variable limits.

Table 7

Types and levels (µg/kg) of mycotoxins detected **by LC-MS/MS** in cereal samples
Positive Samples code	DON		15-ADON		3-ADON		AF-G2		AF-B2		T2		ZEN
Positive Samples code	Average µg/kg	RSD %	Average µg/kg	RSD %	Average µg/kg	RSD %	Average µg/kg	RSD %	Average µg/kg	RSD %	Average µg/kg	RSD %	Average µg/kg	RSD %
E39			8.49	12			2.30	0					<LOQ
E44			11.11	8
E49									16.28	7
E47									20.82	15
W26			<LOQ										1.82	2
W59	624.96	1	18.14	2	61.18	5							2.22	15
W50	33.51	1	2.45	8
E54			7.98	4
E55							1.04	5
E40											<LOQ
W33			1.72	2
LOQ = 1 µg/kg; RSD%: relative standard deviation

Correlation between pathogenicity and mycotoxins production

The correlation matrix analysis of plant and seed development parameters tested in vitro and in vivo indicated significant positive associations. Thus, the length of the coleoptile is proportional to the number of seminal roots, and the germination rate is related to the length of the root system and the number of seminal roots. The severity of Fusarium strains is adversely related to the length of the coleoptile and root system, as well as the number of seminal roots (Table 8).

Additionally, the correlation matrix between the productions of the various kinds of mycotoxins reveals that the production of the three categories of toxins is negatively connected; this finding is more prominent with ZEN and T2. The more ZEN produced, the less T2 is present in the examined sample. Furthermore, the two matrices of the in vitro and in vivo pathogenicity experiments show no link between disease induction and toxin generation, each factor evolving independently of the other (Tables 8, 9).

Table 8

Pearson correlation matrix of *In vitro* pathogenicity on growth parameters and mycotoxin types (ELISA test)
Parameters	Coleoptile Length	Root system length	Seminal root Number	Germination rate	Severity	ZEN	DON	T2
Coleoptile Length	1
Root system length	,669	1
Seminal root Number	0841^**	0,876^**	1
Germination rate	0,578	0,771^*	0,871^**	1
Severity	-0,816^*	-0,850^**	-0,843^**	-0,541	1
ZEN	-0,120	0,364	0,281	0,183	-0,328	1
DON	0,614	0,405	0,551	0,430	-0,286	-0,091	1
T2	-0,105	-0,489	-0,333	-0,054	0,531	-0,764^*	-0,116	1

**. The correlation is significant at the 0.01 level (two-sided).

*. The correlation is significant at the 0.05 level (two-sided).

Table 9

Pearson correlation matrix of *In vivo* pathogenicity on growth parameters and mycotoxin types (ELISA test)
Parameters	Vegetative System Length	Root system length	Vegetative System Weight	Root System Weight	ZEN	DON	T2
Vegetative System Length	1
Root system length	0,170	1
Vegetative System Weight	0,628	-0,559	1
Root System Weight	0,537	0,293	-0,001	1
ZEN	0,150	-0,406	0,571	0,070	1
DON	-0,370	0,205	-0,429	0,173	-0,091	1
T2	0,342	0,488	-0,321	0,340	-0,764^*	-0,116	1

*. The correlation is significant at the 0.05 level (two-sided).

As is well known, the pride of FHB outbreaks comes from an economic and public health standpoint. It's worthy to raise awareness and interest, especially given the scarcity of insights on the aggressiveness and mycotoxins patterns of Fusarium spp. occurring in Algerian wheat, in order to address knowledge gaps that may have a broad vision of the sanitary quality of the grains. Therefore, this study highlights the variability in aggressiveness among Fusarium isolates collected from the north-eastern regions of Algeria as well as the diversity of mycotoxins, which are implicated in plant-pathogen interactions during the infection process. Further, the correlation between pathogenicity parameters and chemotype patterns was investigated.

Our findings of seedling pathogenicity tests have revealed significant variation in aggressiveness between isolates and between species, with F. avenaceum FusBi7 has been found for being the most aggressive, and caused severe symptoms in the in vitro pathogenicity test (significant reduction in the germination rate, coleoptile lengths, root system lengths, and seminal roots number in addition to appearance of root necrosis both on the coleoptile and on the seminal roots which rarely led to the death of the infected seedlings) and even in the in vivo pathogenicity test (significant reduction in the vegetative system length, vegetative system weight ,root system length and appearance of crown browning symptoms). Not unlike our results, according to Moparthi et al. (2021), isolates of F. avenaceum were highly aggressive on the crown tissues of wheat seedlings. The isolate of F. avenaceum showing severe symptoms on crown tissues stood out as being severely aggressive on wheat head plants in South-western Ethiopia by Kebede et al. (2020). Recent investigations reported that F. avenaceum isolates were the most aggressive not just on wheat but also on diverse crops, such as chickpea (Armstrong-Cho et al., 2023) and barley (Inbaia et al., 2023). However, contrary to our findings, Özer et al. (2020) also observed that F. avenaceum was able to cause crown in wheat plants, but it was intermediate with a 2.72 disease severity score compared to F. culmorum (3.52), F. pseudograminearum (3.49), and F. graminearum isolates (3.23). What’s more, results reported by Fernandez and Chen (2005) reveal that F. avenaceum poses only a minor concern to wheat. Those disease severity discrepancies noted in this study and previous work could be attributed to several factors, including genetic diversity in aggressiveness within pathogen isolates tested, differences in the tolerance of the host cultivars used, growth conditions, inoculation techniques applied, and assessment methods that need to be taken into account as well.

In the greenhouse assay, the pathogen isolates from the heads can trigger the disease crowns of wheat, which may suggest that both diseases share a common etiological agent. The dynamics of Fusarium inoculum leading to underground illnesses may serve as a source of inoculum driving infections in wheat heads that can eventually result in Fusarium head blight in the next season (Fernandez and Chen, 2005).

The varietal resistance of the host cultivars was the most paramount variable affecting the severity of head and crown diseases in wheat. The examined durum wheat cultivars exhibited diverging responses to the initial infection, with Cv. Cirta being less susceptible to the pathogen infection than other genotypes tested. The findings of Bouanaka et al. (2021) are in close proximity to the present study, which used multiple methods to evaluate the susceptibility of durum wheat genotypes towards FCR and FHB in Algeria. The cited authors stated that Cirta and GTAdur cultivars presented the highest levels of tolerance for initial seed infection to the pathogen Fusarium culmorum strain FC11 with (GI% _Cirta = 25.51%, AUDPC1 _Cirta = 33.16%) and (GI% _GTAdur = 29.16%, AUDPC1 _GTAdur = 36.10%) compared to Waha cultivars with a susceptibility of 38.00% and 46.50% for GI and AUDPC1, respectively.

The data of the crown inoculation assay in the greenhouse show that Cirta and GTAdur genotypes were more tolerant to the disease than Waha genotypes, which is in close conformity with the conclusion of Bouanaka et al. (2021), who observed that Cirta and GTAdur cultivars were tolerant with an AUDPC1 of 33.16% and 36.10%, respectively, while Cv. Waha were moderately sensitive, with an AUDPC1 of 46.5%. The same finding was obtained by Bencheikh et al. (2018), where the Waha cultivar was noticed to be considerably more susceptible to the infection of Fusarium chlamydosporum, resulting in the following reduction: 71.15% of FWVS, 75.04% of LRS, and 82.58% of LVS.

We recorded that there are significant differences in Fusarium strains disease-causing ability as well as in the behavior of durum wheat genotypes. This suggests the existence of intrinsic diversity in the genetic origin of the host-pathogen interaction. Due to this diversity, the results recorded here not only offer valuable insights about the aggressiveness profile of Fusarium isolates responsible for head blight and crown rot in wheat but also offer important data about the resistance level of local durum wheat genotypes. This knowledge will be helpful for breeding programs intended to enhance cultivar resistance to decrease yield losses and mycotoxin accumulation in Algeria and worldwide.

The current occurrence of such pathogenic Fusarium species in durum wheat has raised further issues through their contribution to the multiple mycotoxin contamination of Algerian cereals, mitigating wheat quality and therefore threatening consumer health and agro-food systems. This situation should have raised public health awareness. Each pathogen species has its own specific mycotoxigenic profile. Hence, accurate mycotoxins risk assessment is closely associated with the application of various advanced detection tools. Overall, it’s obvious from the obtained results that the FusBo59 strain (F. culmorum) has a high toxigenic ability to produce DON compared to the rest of the strains, with a maximum level equal to 7.128ppb in the ELISA test and 373196.19ppb by LC-MS/MS exceeding the legal limits permitted by the European Commission at 1750µg/kg for durum wheat intended for human and animal consumption (European Commission, 2006). Similar analysis on F. culmorum strains collected from various cropping areas of North Algeria showed 75% of strains were able to produce a significant content of DON that reached 80000µg/kg, also exceeding EC limits. DON levels in F. culmorum detected in this work (62239µg/kg mean) were typically higher than the mean value for DON levels in wheat (12300µg/kg) (Miedaner et al., 2021). It’s worth noting the potential threat that could jeopardize human safety. Moreover, isolates of F. culmorum associated with FHB collected in Europe and Asia produce a new mycotoxin type A trichothecene, NX-2, simultaneously with DON, 3-ADON, or NIV (Schiwek et al., 2022).

The analysis of DON contents using the chromatographic method led to its detection in all the evaluated isolates, with significant contents ranging between 6.41 and 373196.19µg/kg. Subsequently, it should be presumed that these isolates have an inheritable ability to contaminate grain cereals with this toxin. Noteworthy, the harmfulness of DON nowadays resides not only in their detection with high contamination levels and prevalence in grain-based foods worldwide but also in the stability of these compounds during different food processing, which has attracted worldly attention through further research focusing on the application of multiple DON detoxification approaches (Feizollahi and Roopesh, 2022; Li et al., 2023). Another intriguing finding is that DON was the mycotoxin with the highest frequency, and no culture extracts containing DON derivatives (3-ADON and/or 15-ADON) alone were obtained, agreeing with the finding of Yan et al. (2020), which demonstrated that DON (95.51%) was more frequently encountered in infected wheat than its acetylated derivatives.

It is observed that the isolate FusBo26 (F. chlamydosporum species complex) produced a mix of DON and ZEN mycotoxins, thereby suggesting that grain-based foods are likely to be contaminated with multi-mycotoxins compounds that could have heavy impacts on human and animal health, owing to the possible antagonistic, additive, or synergic effects (Wang et al., 2018). In the Netherlands, the co-occurrence of DON, ZEN, enniatin B and B1, HT-2 toxin, and NIV in wheat samples has been recorded (Van der Fels-Klerx et al., 2021). A Brazilian survey on wheat flour reported the simultaneous occurrence of DON, ZEN, and T-2 with usually high concentrations and incidence (Dos Santos et al., 2021).

T-2 production was recovered in our study at trace levels with all the examined isolates, reaching a maximum 0,35ppb. Conversely, a significant T-2 and HT-2 toxins content was obtained in durum wheat grains from Southern Italy in 2014 (150µg/kg mean) (Haidukowski et al., 2022). However, T-2 toxins have been shown to contaminate other cereals from the UK at higher values in the range of 171 − 1426µg/kg mean HT2 + T2 amount in winter oat varieties (Edwards and Stancic, 2022) and 279.34µg/kg in corn (Zhang et al., 2021). Hence, the durum wheat in Algeria is less vulnerable to T-2 contamination. The very low amounts determined in the present research are not expected to have any adverse impacts on human health or food security (USDA, 2010).

Our monitoring study emphasized the natural co-occurrence of multi-mycotoxins in Algerian durum wheat, of which Fusarium mycotoxins were the most predominant, while Aspergillus mycotoxins were detected with low concentrations and frequency. Among the Fusariotoxins, DON and its acetylated derivatives (15-ADON and 3-ADON), type B trichothecenes, are extremely intoxicating through the impact of their metabolites on the gastrointestinal tract and hematopoietic progenitor cellular systems and contribute to intestinal epithelial cell necrosis (Polak-liwiska et al., 2021; Gab-Allah et al., 2023). In our survey, 15-ADON was more prevalent in wheat grains (63,6%) than DON (18,2%) and 3-ADON (9,1%). One of the explanations for this finding could be the significant occurrence of 15-ADON-producing species in Algerian durum wheat. However, as opposed to our results, a higher incidence of DON was observed in Chinese wheat (90.8%), followed by 3-ADON (69.2%) and 15-ADON (49.4%) (Zhao et al., 2021). Those discrepancies in incidence could be attributed to weather patterns and the agro-ecological regions where the samples were harvested (Liu et al., 2016). Besides, contrary to 15-ADON, DON occurred at a lower incidence but with high concentrations in wheat samples (329 g/kg mean), compared with those encountered in 100% of the samples of Brazilian wheat flour, with a range level from 53 to 2905µg/kg (Dos Santos et al., 2021). Otherwise, Yan et al. (2020) alluded to a greater prevalence of DON in wheat samples (100%), but at a relatively moderate level (165.87µg/kg mean). The main reason for the differences in DON accumulation in examined samples could be a consequence of fluctuations in the ratio between DON-producing and non-DON-producing species (Beyer et al., 2007), host cultivars, meteorological factors, particularly temperature and relative humidity, as well as soil type.

In the current study, 18.2% of zearalenone-contaminated wheat grains had contents below the EC limits (100 g/kg in raw cereals), with a mean content of 2µg/kg. According to an EFSA Panel on Contaminants in the Food Chain survey, ZEN occurred in maize at a rate of 33%, with an average level of 15µ/kg (EFSA, 2011), significantly lower than the EC standards. However, investigations in Belgium on thirty samples of maize, wheat, oats, and other cereal-derived foods showed an 80% incidence of ZEN with a mean of 106221ng/g (De Boevre et al., 2012).

A part of the regulated mycotoxins identified in that study, AF-G2 and AF-B2 aflatoxins, were observed twice with average levels of 1.5 and 18,5µ/kg, respectively, compared with those occurring at lower frequencies and contents of Iranian wheat samples (0.11–0.34µg/kg and 0.12µg/kg, respectively) (Kardani et al., 2023). Because of the high concentrations of AF-B2 in durum wheat analyzed, which are beyond the EC limit (4µg/kg in raw cereals and its products derived), it’s more prone to aflatoxins contamination. This further exacerbates food safety concerns. Interestingly, T-2 was detected in only one positive sample in our investigations with amounts lower than the LOQ, and this strongly confirms the findings of our assay on the ability of pathogen isolates to produce mycotoxins. It seems plausible that these results are due to many factors, like the lack of T-2-producing species, host cultivar type, and harsh local climatic conditions for T-2 toxin biosynthesis.

As was noticed, there is a non-significant correlation between the pathogenicity of Fusarium strains and the production of mycotoxins. Li et al. (2023) reported the absence of a link between toxin production and the pathogenicity level of Fusarium oxysporum f. sp. sesami isolates, as demonstrated by the non-significant correlation (P > 0.05). They suggest that the results contribute to a better understanding of the mechanisms of host-pathogen interaction and pathogen control.

The toxin data reported here revealed co-contamination of Algerian durum wheat with multiple Aspergillus and Fusarium mycotoxins owing to the occurrence of various toxin-producing species. This exerts a detrimental impact on consumer health, inducing acute and chronic diseases of even greater concern. No stringent regulation has been stipulated up to now. To the best of our knowledge, currently in Algeria, investigations on the risk of pathogenic species prevalence and their associated mycotoxins in harvested wheat seeds are scarce. For the first time, we indicate the potential mycotoxin profile of Fusarium isolates causing Fusarium wilt on wheat, in addition to the natural occurrence and co-occurrence of multi-mycotoxins in durum wheat produced in Algeria. Therefore, this underscores the urgent need to control and regulate mycotoxin levels in cereals as a major challenge that must be taken with more thoughtfulness to safeguard food security.

This study adds to our understanding of the pathogenicity profile of Fusarium strains, including the most aggressive, F. avenaceum FusBi7. Furthermore, differences in the behavior of durum wheat types toward pathogenic infections have emerged, highlighting the Cirta variety as the most tolerant to Fusarium strain attacks. It should also be highlighted that pathogen isolates recovered from symptomatic ears and kernels can cause wheat crown rot, implying that the two diseases are caused by the same culprit. Finally, the study found that the induction of Fusarium wilt occurs irrespective of mycotoxin synthesis.

Acknowledgements

Firstly, we would like to thank the Directorate General for Scientific and Technological Research and Development, Ministry of Higher Education and Scientific Research, Algeria for supporting this study and The authors would like also to thank the National Center for the Control and Certification of Seeds and Seedlings, Setif regional laboratory, Algeria, for providing the harvested samples and healthy seeds of durum wheat.

Author contribution

B.I. is the principal researcher of this study. R.N., C.S., and P.E. Conception of hypothesis for research and Planning methodology to reach the conclusion; B.I., B.A., A.H., and M.S.C. Material preparation, data collection and analysis ; B.I., and R.N. Manuscript writing, editing and critical reading.

Conflict of interest

The authors have no competing interests to declare that are relevant to the content of this article.

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Pathogenicity and mycotoxin profile of Fusarium spp. inducing wheat head blight in Algeria

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