Introduction

Despite the significant importance of natural products such as fungal metabolites or toxins, which can pose risks to animals and humans, their full exploration has yet to be achieved. Mycotoxins are organic compounds that are typically non-volatiles and essentially produced by fungi in the form of secondary metabolites. For centuries, people have been aware of their toxic effects. However, due to the decreased use of antifungal medicines advised for use in food, the negative effects of toxins only recently have come to be given significant relevance, which inevitably threats our health (Weidenborner 2001).

Our study focuses on fungi known to produce toxins, namely Aspergillus, Trichoderma, Fusarium, Penicillium and Stachybotrys (Braise et al. 2009). These fungi and other fungus pathogens are known to harm plants severely, resulting in major losses. Furthermore, they seriously jeopardize food security, as emphasized by Xiao et al. (2014). In addition to these dangers, the evolution of drug resistance has emerged as a critical problem, demanding the creation of new and stronger compounds. Despite the efforts to discover new classes of antimicrobial agents, the control of pathogenic microorganisms remains a challenge due to the emergence of drug-resistant strains (Pinto et al. 2006). Microbial resistance to commonly used antibiotics and antifungals is the main component of the global health problem, given the large number of cases where conventional treatment of infections has failed. A review of the literature reveals that many plant extracts provide a variety of known and unknown compounds, such as essential oils, with antifungal activity (Pinto et al. 2006; Tabanca et al. 2007; Tullio et al. 2007; Dutta et al. 2007). It is trusty to mention that natural products have certain advantages over synthetic molecules, and are biodegradable, non-toxic, with high antifungal activity, and inexpensive and non-specific qualities. However, the development of resistance by pathogenic fungi against natural products poses a significant challenge. It would therefore be interesting to find a new family of synthetic compounds to search for molecules with interesting functionalities.

Morita-Baylis–Hillman (MBH) adducts have been shown to be an important class of bioactive products that can be easily synthesized and inexpensive (Lima–Junior CG, Vasconcellos MLAA 2012). Moreover, they exhibit potent biological activities such as antimicrobial (Sa et al. 2014; Kumar et al. 2013), antifungal (Das et al. 2007), antimalarial (Kundu et al. 1999; Narender et al. 2005), Leishmanicidal (S.C.O., Sousa et al. 2017) and anti-proliferative activity on human tumor cell lines (Kohn et al. 2006).

In order to search for new antimicrobial compounds, we have looked at the MBH adducts and their derivatives which have not shown any cytotoxic effects (Ketata et al. 2019) and antioxidant (Elleuch et al. 2018). Here, we plan to evaluate the antifungal activity of MBH adducts and their derivatives against Aspergillus, Trichoderma, Fusarium, Penicillium and Stachybotrys fungal strains.

To this end, four sets of cyclic MBH adducts (alcohols, acetates, phosphonates and hydrazono phosphonates) were newly synthesized. They were evaluated for their antifungal activity, using the “agar well diffusion” method.

Materials and methods

MBH adducts preparation

The MBH products 1, 2, 3 and 4 corresponding to alcohols, acetates, phosphonates and hydrazono-phosphonates, respectively, were prepared according to previous reports (Gatri and Gaied 2002; Kwong et al. 2007; Elleuch et al. 2016; Elleuch et al. 2017; Luo  et al. 2002 ; Moghadam et al. 2007) and tested at 10–3 dilution in DMSO.

Fungal cultures

Fungal strains were grown on potato dextrose agar (PDA) medium at 30 °C for 7 days until sporulation. Spores were collected by scraping the plates and filtering through sterile cotton. The spores were stored at 109 spores/mL in 20% Glycerol and at -80 °C. Liquid cultures were performed in Mandels’ medium. The composition of the slightly modified Mandels’ medium (Mandels et al. 1962) per liter was as follows: 2 g KH2PO4, 1.4 g (NH4)2SO4, 1 g yeast extract, 0.3 g CaCl2·2H2O, 0.3 g MgSO4·7H2O, 1 mL Tween 80 and 1 mL trace element solution composed of 1.6 g/L MnSO4, 2 g/L ZnSO4, 0.5 g/L CuSO4, 0.5 g/L CoSO4, was supplemented by adding 2% glucose. The medium was sterilized at 121 °C for 20 min. Before use, 100 μg/mL Ampicillin and 12.5 μg/mL Tetracycline were added to avoid bacterial contamination.

Biological screening

Biological Screening was assessed by the agar well diffusion method which is widely used to evaluate antimicrobial activities. This method is simple, inexpensive, easy to reproduce and easy to read andinterpret (Magaldi et al. 2004). Indeed, 100 µL of fungal inoculum of 109 spores/mL was spread on a solid PDA medium in square Petri dishes (120 mm × 120 mm). Subsequently, wells of 7 mm in diameter are made in these plates. Besides, 100 μL of each MBH product was loaded in wells dug in agar plates (7 mm diameter holes). DMSO was also added as a control as the MBH adducts were suspended in such a solvent, as well as Nystatin, the positive control. To serve as a control, DMSO was added as the solvent for the MBH adducts, and Nystatin was included as the positive control. The plates were incubated for 72 h at 30 °C, under aerobic conditions. After incubation, confluent fungal growth was observed. The diameter of the growth inhibition zone was measured in cm. The antifungal activities, in terms of the diameters of inhibition zones, are reported on the graphs. Tested fungi were Fusarium oxysporum (Skouri-Gargouri & Gargouri 2008) Trichoderma reesei (named RutC30) (Montenecourt & Eveleigh 1979), Stachybotrys microspora (N1) (Amouri & Gargouri 2006), Fusarium solani (S3) (Boudabbous et al. 2017), Penicillium occitanis (CT1) (Ayadi et al. 2011), Aspergillus niger (A1) (Hadj-Taieb et al. 1996), Fusarium aethiopicum, Alternaria alternataand Aspergillus flavus.

Fungicidal activities

The serial dilutions of MBH compounds were performed to produce volumes of 200 μL per Eppendorf tube. The final concentrations ranging from 10–1 to 10–4 M. 100 μL of the fungal suspension with a concentration of 108 spores/mL were added to each tube. Negative control wells contained the fungi in Mandels’ medium. After incubation at 30 °C for 24 h, an amount of 100 µL was spread on PDA media and incubated at 30 °C for 5 days. The fungicidal activity was determined by assessing the number of colonies-forming units.

Chemical analysis

Melting points were identified on a Kofler melting apparatus and were uncorrected. 1H and 13C-NMR spectra were performed on a Varian 500 MHz spectrometer. The chemical shifts (δ) and coupling constants (J) are expressed in ppm and Hertz, respectively. Microanalyses and mass spectrometry analyses were carried out on a Carlo Erba EA 1102 and on a 3200 QTRAP (Applied Biosystem SCIEX), respectively. All solvents and reagents were obtained from commercial sources and purified before use if necessary. Merck Kieselgel 60F254 plates were used for TLC, and Merck Silica gel 60 (0.063–0.100 mm) for column chromatography.

Statistical analysis

The results were expressed as the mean ± standard deviation (SD). The statistical significance of the differences was evaluated by Student’s t-test. P < 0.05 was considered to indicate a statistically significant difference.

Results and discussions

Cyclic acetates (2a-1) of MBH 2 were synthesized by the reaction of acetic anhydride on the corresponding alcohols (1a-1), in the presence of the triethylamine and DMAP as catalysts (Fig. 1, Table 1).

Fig. 1
figure 1

MBH alcohols and acetates synthesis

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Table 1 Chemical structure of cyclic MBH adducts
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Cyclic acetates of MBH (2a-1) were reacted with triethylphosphite in the presence of DMAP or imidazole under solvent-free conditions to derive the synthesis of allylic phosphonates (3a-1) (Fig. 1, Table 1). The synthesis of these MBH adducts is outlined in Fig. 1 and summarized in Table 1, providing a concise description of each step involved. It is described in detail in the previous works reported by Elleuch et al. (2016–2018) and Ketata et al. (2019).

The phosphonate 3a, in turn, reacts with hydrazine in refluxing methanol to form the hydrazono-phosphonates 4a-c (Fig. 2, Table 1) (Elleuch et al. 2016, 2018).

Fig. 2
figure 2

Hydrazonophosphonates 4a-c synthesis

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Study of structure–activity relationship

The fungistatic/fungicidal potential of all the synthetic MBH adducts and their derivatives were examined against the mentioned fungi. However, in the results section, only the compounds exhibiting significant antifungal activity are presented and discussed.

Antifungal activity of the alcohol family

The screening for antifungal activity of the alcohol family shows that only the six-membered adduct 1d, bearing the phenyl group (R = Ph), exhibits antifungal activity. It was against the following fungi: F. oxysporun, T. parceramosum, S. microspora and T. reesei with growth inhibition diameters, respectively: 1.5 ± 0.14, 4.05 ± 0.07, 2.6 ± 0.14, and 2.95 ± 0.07 cm (Table 2). Indeed, many synthetic or natural drugs contain this structural core of six-membered rings (de Carvalho et al. 2013; Khan et al. 2012). It should be noted that T. parceramosum is the most sensitive to alcohol 1d.

Table 2 Zone of inhibition diameter values of MHB antifungal products against Fusarium oxysporum, Trichoderma parceramosum, Stachybotrys microspora and Trichoderma reesei
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Antifungal activities of the acetate family

MBH 2 acetates were synthesized in a single step by there action between MBH alcohols and acetic anhydride in the presence of trimethylamine using DMAP as a catalyst. This synthetic approach is rapid (30 min) and inexpensive.

Most of the synthesized MBH2 acetates exhibit an antifungal activity that depends on theradical group R.

Antifungal activity against Trichoderma reesei RutC30 strain

The antifungal activity of the newly synthesized acetates having different radical groups was screened against the Trichoderma reesei (RutC30) strain. This adducts exhibited varying degrees of antifungal activities, depending on the radical group. Indeed, the MBH adduct 2a, a simple six-membered carbons acetate (R = H), presented an inhibition zone diameter of only d = 2.5 ± 0. This activity is lower than that of compound 2 g which is simple five-membered carbons acetate. It has a significant inhibitory effect (at p ≤ 0.05) (d = 4.85 ± 0.07) on the growth of T. reesei (Fig. 3). In addition, five-membered carbons acetate (R = Me) 2i (d = 3.9 ± 0.14) showed a significant antifungal effect compared to the effect of the six-membered carbons acetate (R = Me) 2b with d = 3.55 ± 0.07 (Table 2). Furthermore, by comparing the different diameters of the zone of growth inhibition (d) of the six-membered carbon acetates MBH with different radical groups, it is found that the antifungal effect depends on the radical group. Indeed, when R = Me, d(2b) = 3.55 ± 0.07 ≥ d(2a) = 2.5 ± 0 when R = H (Table 2).

Fig. 3
figure 3

Growth-inhibition zone diameters of the acetates family against Trichoderma reesei strain. The green color is used when the antifungal effect is related to the functional group and the red when it is related to the radical group. All tests were carried out in triplicate (mean ± SD; n = 3; *p < 0.05, **p < 0.005; p-values were calculated with Student’s test). All the MHB products were tested at (100 µL/well), while Nystatin was tested at (30 µL/well) from the same concentration (10–1 M)

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On the other hand, for MBH five-membered carbons acetates with different groups, the obtained results have revealed that when R = H (2 g), d = 4.85 ± 0.07, while the activity is significantly reduced when R = Me (d = 3.9 ± 0.14), as when R = Alkyl, Aryl (Table 2). Thus, the simple five-membered carbons acetate (R = H) 2 g has the highest inhibition potency against Trichoderma reesei (Fig. 3).

Antifungal activity against Trichoderma parceramosum strain

The present study on antifungal activity against T. parceramosum has shown that there is no significant difference in antifungal effect between the five membered-carbons acetates and their counterparts (six-membered carbons) as can be seen in Table 2. Indeed, the diameters of growth inhibition were d(2d(R = Ph)) = 3.35 ± 0.07 ≈ d(2 h(R = Ph)) = 3.7 ± 0.14, as well as for 2i(R = Me), d = 3.55 ± 0.07, and its counterpart 2b(R = Me), d = 3.95 ± 0.07 (Fig. 4; Table 2). Moreover, the simple five-membered acetate 2 g (R = H) has the highest antifungal potency (d = 4.35 ± 0.21), which is reduced in the case of R = Me or R = Ph.

Fig. 4
figure 4

Growth-inhibition zone diameters of the acetate family against Trichoderma parceramosum strain. The green color is used when the antifungal effect is related to the functional group and the red one when it is associated with the radical group. All tests were carried out in triplicate (mean ± SD; n = 3; *p < 0.05, **p < 0.005). p-values were calculated with Student’s test. All the MHB products were tested at (100 µL/well), while Nystatin was tested at (30 µL/well) from the same concentration (10–1 M)

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Antifungal activity against Stachybotrys microspora strain

Likewise, the five-membered carbon acetate has the highest antifungal activity against Stachybotrys microspora. Radical group replacement significantly reduces the antifungal activity against S. microspora. As shown in the Table 2, the acetate 2 g (R = H) has the highest antifungal activity (d = 6.1 ± 0.14). However, for R = alkyl, longer carbon chains have the least effective antifungal activity: d (2 g(R = H)) = 6.1 ± 0.14 ≥ d (2i(R = CH3)) = 4.1 ± 0.14 ≥ d (2j(R = CH2CH3)) = 3.95 ± 0.07 ≥ d (2 k(R = CH2CH2CH3)) = 3.55 ± 0.21 (Fig. 5; Table 2). In addition, the five-membered acetate 2 h (R = Ph), with a moderate steric hindrance, shows less antifungal activity (d (2 h) = 3.35 ± 0.21) than acetate 2 k (R = n-Pr) with d(2 k) = 3.55 ± 0.21. However, the antifungal activity of the six-membered ring acetate 2d with phenyl group (R = Ph), d(2d) = 3.60 ± 0.28 is greater than that of the acetate with methyl group 2b(R = Me), d(2b) = 2.5 ± 0.0.

Fig. 5
figure 5

Growth inhibition zone diameters of the compounds of the acetate family against Stachybotrys microspora strain. The green color is used when the antifungal effect is related to the functional group and the red one when it is linked to the radical group. All tests were carried out in triplicate (mean ± SD; n = 3; *p < 0.05, **p < 0.005). p-values were calculated with Student’s test. All the MHB products are tested at (100µL/well), while Nystatin was tested at (30 µL/well) from the same concentration (10–1 M)

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Antifungal effect of MBH against Fusarium oxysporum

Among the tested compounds, the MBH 2 g adduct displayed the most pronounced antifungal activity (d = 4.75 ± 0.21) against Fusarium oxysporum. Similarly, the radical substitution modifies the potential for antifungal activity: d (2 g(R = H)) = 4.75 ± 0.21 ≥ d(2i(R = CH3)) = 2.6 ± 0 ≥ d (2j(R = CH2CH3)) = 2.3 ± 0 ≥ d (2 k(R = CH2CH2CH3)) = 1.4 ± 0 (Fig. 6; Table 2).

Fig. 6
figure 6

Growth inhibition zone diameters of the compounds of the acetate family against Fusarium oxysporum strain. The green color is used when the antifungal effect is related to the functional group and the red one when it is linked to the radical group. All tests were carried out in triplicate (mean ± SD; n = 3; *p < 0.05, **p < 0.005). p-values were calculated with student’s t-test. All the MHB products were tested at (100 µL/well), while Nystatin was tested at (30 µL/well) from the same concentration (10–1 M)

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The MBH six-membered ring acetates 2b (R = Me, d = 1.4 ± 0) and 2d (R = Ph, d = 1.7 ± 0.14) show the weakest antifungal activity against F. oxysporum.

Antifungal activity against Fusarium solani, Penicillium occitanis and Aspergillus niger strains

The inhibitory effect of MBH five-membered carbon acetates on Fusariumsolani and Penicilliumoccitanis was observed, with the degree of inhibition varying depending on the specific radical involved. Acetate 2 g (R = H) exhibits the highest antifungal activity. In contrast, when R = Me, Alkyl or Ph, the respective compounds (2i, 2j and 2 h) share a rather moderate antifungal potential (Table 3).

Table 3 Values of the diameter of the zone of inhibition of MHB antifungal products against Fusarium solani, Penicillium occitanis and Aspergillus niger
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It should be noted that the five-membered MBH acetate 2 g (R = H) was the only compound capable of inhibiting the growth of Aspergillus niger (Table 3).

Compared to the antifungal activity of Nystatin as a potent commercial antifungal product, most of the tested MBH adducts exhibited moderate to strong inhibitory effects. Simple acetate 2 g (R = H) appears to have the highest antifungal activity against Fusarium solani and Aspergillus niger.

All the MHB products were tested at (100 µL/well) while Nystatin was tested at (30 µL/well) from the same concentration (10–1 M).

Compared to control nystatin, compounds 2i, 2j, 2 h, 2 k and 2a showed variable antifungal activity, while 2 g exhibited a broad spectrum of biological activity on all tested fungi.

Antifungal activity against Aspergillus flavus,Alternaria alternata and Fusarium aethiopicum strains

As shown in Table 4, the growth of Fusarium aethiopicum, Alternaria alternata and Aspergillus flavus, newly isolated strains in our laboratory, was strongly inhibited by the five-membered MBH acetate (R = H) (2 g). It is followed by a simple six-membered carbons acetate (R = H) and the antifungal activity weakens when the R group is longer (R = (CH2)2CH3) or even none against Alternaria alternata. Of the three fungi, Alternaria alternata and Aspergillus flavus strains exhibit the highest sensitivity to compound 2 g. When comparing the antifungal activity of 2 g to that of the standard fungicide Nystatin, 2 g is proven to exhibitgreat potential as a fungicide and may have a more promising future in this regard.

Table 4 Values of the diameter of the zone of inhibition of MHB antifungal products against Fusarium aethiopicum, Alternaria alternata and Aspergillus flavus strains
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It is worthy to recall that the MBH products were tested at (100 µL/well) and Nystatin was tested at (30 µL/well) from the same concentration (10–1 M). Despite this difference of the tested volume, the simple six-membered acetate 2a (R = H) has a good antifungal activity against F. aethiopicum, A. alternata and A. flavus strains. Acetate 2 k (R = n-Pr) demonstrated a weak antifungal activity. However, the antifungal activity of the five-membered ring acetate 2 g was found to be better than that of 2 k and 2a, except against F. aethiopicum which appears more sensitive to 2a. The results suggest that the introduction of an alkyl group would reduce the antifungal activity.

Regarding the strains under study, it should be noted that the majority of them are well known as phytopathogens (F. oxysporum, F. solani, A. alternata, A. niger and A. flavus) than others (T. reesei, S. microspora, T. parceramosum, P. occitanis, F. aethiopicum).

Fusarium oxysporum is a devastating fungal pathogen, already known to secrete degrading enzymes called “plant cell wall degrading enzymes” (CWDE) such as pectinases (Bravo-Ruiz et al. 2016; Bravo-Ruiz et al. 2017). Similarly, Fusarium solani is capable of causing disease in many agriculturally important crops (Arie 2019). Typically, the F. solani species complex (FSSC) causes stem and/or root decay of the infected host plant, and the degree of necrosis correlates with the severity of the disease (Coleman 2016). Aspergillus flavus is a plant pathogen that attacks economically important crops, such as corn and peanuts, spices, flax seeds, cereals, and sometimes dried fruits (e.g. figs) (Hedayati et al. 2007). A. flavus is often studied as a contaminant producing mycotoxins such as aflatoxins harmful to humans (Gravesen et al. 1994). Alternaria is also known worldwide as both a common plant pathogen and an air-borne allergen. More specifically, A. alternata is recognized as the aero-allergen type species. In the majority of cases, health problems among humans and animals have been associated with this species (Gravesen et al. 1994; de Luca, 2007). Finally, some of these phytopathogenic fungi, such as A. niger or T. reesei and Penicillium sp., can be used as biological control agents. They would protect the plant against the attack of other fungal strains and provide it with useful biomolecules (Bravo-Ruiz et al. 2017; Peng et al. 2021; Manzar et al. 2022). In this context, Aspergillus niger is known to solubilize potassium and phosphate, as well as to produce phytohormones (Mundim et al. 2022).

Assessment of fungicidal activity of 2 g compound

To further deepen our investigation, we determined the fungicidal activity of the synthesized compound 2 g, which stood out as having the best antifungal power against most of the tested fungi. It was tested against two fungi Aspergillus niger and Penicillium occitanis. Hence, not only does it offer good fungicidal power (70%) at a concentration of 0.1 mM, but also100% inhibition of fungal growth at 1 mM, as shown in Fig. 7.

Fig. 7
figure 7

Fungicidal activity of 2 g compound against Aspergillus niger and Penicillium occitanis. It was determined by observing the lowest concentration of the drug at which no visible growth exists, and it is expressed by the percentage of cell mortality

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The screening of antifungal activity demonstrated the ability of MBH adducts possess the ability to inhibit fungal growth. Indeed, among the tested adducts the alcohol 1d(R = Ph) and MBH acetates were the only antifungal products. Thus, the antifungal activity depends on the functional group, and the five-membered ring acetate (2 g) having R = H is the most active against all tested fungi.

The introduction of radical substituents at the ortho position of the n-membered carbon acetate increased or decreased antifungal activity depending on the core of the compound. Notably, compound 2 g is lipophilic (absence of OH group), suggesting that this property could facilitate its penetration into the fungal cell, and thus promoting its activity (Han al. 2019; (Elleuchet al. 2018). The acetate moiety would be responsible for the antifungal effect and the radical H would ensure the effectiveness of its antifungal power.

MBH Phosphonates and hydrazonophosphonates families

MBH phosphonates were synthesized by reacting MBH acetates with triethylphosphite in the presence of DMAP. The hydrazonophosphonates were synthesized by reacting MBH phosphonates with hydrazines. Although all of these adducts were tested, they failed to inhibit fungal growth.

Conclusion

MBH adducts are of great interest in different applications, as shown previously, thanks to their antioxidant activity (Sun et al. 2019). They can be used as additives to prevent lipid oxidation or as anti-melanogenesis(Ketata et al. 2019) as well as for their antifungal activities. We adopted the agar diffusion method to evaluate the antifungal activity of several MBH adducts (Magaldi et al. 2004). The present research work has shown that MBH adducts (alcohols (1), acetates (2)) are endowed with interesting antifungal activity against the most known fungal phytopathogens and/or toxin producers, namely the genera Aspergillus, Alternaria, Fusarium, Penicillium, Trichoderma. This allows us to conclude that these compounds can be potential candidates as effective antifungals in the fields of agriculture, agro-foods and pharmaceutical industry (Saremi et al. 2018; Han et al. 2019; Srivastava et al. 2019). The MHB products were then classified according to the effectiveness of their antifungal activity evaluated by the diameter of the fungal growth inhibition zone.

This study has also discovered that the 2 g five-membered carbon acetate has the most effective growth inhibitor activity against all tested fungi, compared to six-carbon acetate. Besides, it has demonstrated that the antifungal activity is often inversely proportional not only to the length of the radical but also to its complexity.