Abstract
Garlic (Allium sativum L.) being infected by mycotoxigenic fungi is one of the primary factors limiting its nutritional and medical value. Therefore, there is an urgent need to repress mycotoxigenic fungi utilizing safer treatments, possibly involving nanoparticles. Fusarium incarnatum was isolated from garlic (A. sativum L.) that showed fungal contamination and their identification was confirmed using amplified and sequenced internal transcribed spacer nuclear ribosomal DNA regions, which confirmed the isolation of F. incarnatum from all cultivars. Copper oxide nanoparticles (CuONPs) at different concentrations showed inhibitory activity against F. incarnatum growth and mycotoxins, particularly at 400 ppm. The production of F. incarnatum mycotoxins, i.e., beauvericins, fusarins, moniliformin, and enniatins, was inhibited to 62.8%, 45.4%, 58.1%, and 55.0%, respectively at 400 ppm of CuONPs compared to the control. Shrinkage of the F. incarnatum cell membrane and collapsing of the cell walls were recorded via transmission electron microscopy at 400 ppm, but negligible distortion appeared at 100 ppm of CuONPs. CuONPs at 100 ppm encouraged the activity of CMC-ase, zylanase, and amylase, while 200 and 400 ppm promoted less enzyme activity. The current findings suggest that CuONPs have a fungistatic effect on F. incarnatum and their mycotoxins.
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Copper Oxide Nanoparticles as Fungistat to Inhibit Mycotoxins and Hydrolytic Enzyme Production by Fusarium incarnatum Isolated from Garlic Biomass
Aisha M. H. Al-Rajhi,a Reham Yahya,b,c Mohamed M. Alawlaqi,d Mohamed A. Fareid,e,f Basma H. Amin,g and Tarek M. Abdelghani e,*
Garlic (Allium sativum L.) being infected by mycotoxigenic fungi is one of the primary factors limiting its nutritional and medical value. Therefore, there is an urgent need to repress mycotoxigenic fungi utilizing safer treatments, possibly involving nanoparticles. Fusarium incarnatum was isolated from garlic (A. sativum L.) that showed fungal contamination and their identification was confirmed using amplified and sequenced internal transcribed spacer nuclear ribosomal DNA regions, which confirmed the isolation of F. incarnatum from all cultivars. Copper oxide nanoparticles (CuONPs) at different concentrations showed inhibitory activity against F. incarnatum growth and mycotoxins, particularly at 400 ppm. The production of F. incarnatum mycotoxins, i.e., beauvericins, fusarins, moniliformin, and enniatins, was inhibited to 62.8%, 45.4%, 58.1%, and 55.0%, respectively at 400 ppm of CuONPs compared to the control. Shrinkage of the F. incarnatum cell membrane and collapsing of the cell walls were recorded via transmission electron microscopy at 400 ppm, but negligible distortion appeared at 100 ppm of CuONPs. CuONPs at 100 ppm encouraged the activity of CMC-ase, zylanase, and amylase, while 200 and 400 ppm promoted less enzyme activity. The current findings suggest that CuONPs have a fungistatic effect on F. incarnatum and their mycotoxins.
DOI: 10.15376/biores.17.2.3042-3056
Keywords: Fusarium incarnatum; Garlic; Hydrolytic enzymes; Mycotoxins; Nanoparticles
Contact information: a: Department of Biology, College of Science, Princess Nourah bint Abdulrahman University P.O. Box 84428, Riyadh 11671, Saudi Arabia; b: Medical Microbiology, College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, Riyadh 14611 Saudi Arabia; c: King Abduallah International Medical Research Center, Riyadh 21423 Saudi Arabia; d: Biology Department, Faculty of Science, Jazan University, Jazan 45142 Saudi Arabia; e: Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo 11725, Egypt.; f:Basic Science Department, Prep Year Deanship, University of Haʼil, Saudi Kingdom.; g: The Regional Centre for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt;
* Corresponding author: tabdelghany.201@azhar.edu.eg; tabdelghany@yahoo.com
GRAPHICAL ABSTRACT
INTRODUCTION
Numerous fungi usually attack cereal grains, seeds, vegetables, fruits, as well as their foodstuff products either in the field or storage places. The ability of these fungi to produce mycotoxins has been documented under particular conditions, which minimizes the marketable and dietary value of these products, as well as results in undesirable health impacts on consumers including humans and animals (Abdelghany 2006). Many scientific papers have reported that Fusarium is considered one of the greatest economically detrimental fungal genera for many reasons, i.e., its phytopathogenic activity, potent producer for mycotoxins, and illness impacts on humans and livestock (Abdelghany 2014; Villani et al. 2019; Agriopoulou et al. 2020; Nofal et al. 2021a). Multiple documents have reported that Fusarium species are responsible for the production of several mycotoxins, e.g., fumonisins, trichothecenes, zearalenone, enniatins, and fusaproliferin (Munkvold 2017; Jajić et al. 2019; Gautier et al. 2020). Recently, Choi et al. (2021) reported the presence of Fusarium spp. in millet samples with the highest frequency of mycotoxin being F. asiaticum, followed by F. acuminatum, F. graminearum, F. incarnatum, F. equiseti, and F. tricinctum besides other species of Fusarium. Occurrences of zearalenone, deoxynivalenol, fumnasine, and nivalenol in these millet samples were also recognized. The trichothecenes A and ZEN found in corn were F. incarnatum (Munkvold 2017). Yazid et al. (2021) showed that three isolates of F. incarnatum produced ZEN, while HT-2 and T-2 toxins were produced by one isolate.
Previously, different strains were isolated from Tunisian sorghum, and the presence of ZEA in sorghum samples varied according to the strain (Lahouar et al. 2017). Villani et al. (2019) mentioned that F. incarnatum–equiseti species complex (FIESC) covers 33 phylogenetically diverse species that have been isolated from diverse biological samples as well as agricultural soils. According to Villani et al. (2019), although miscellaneous mycotoxins were produced by FIESC, its potential to cause mycotoxin contamination in grains and other food products is unclear because the diversity of these species has only been recently documented.
Outside of the nutritional value, the therapeutic potential of garlic (Allium sativum L.) has been previously confirmed and is still the focus of studies, due to the presence of multiple bioactive molecules (Singh and Sahu 2020). Garlic is considered one of the oldest and most imported crops in all countries; therefore its worldwide production has been regularly increasing over the last two decades. Although numerous scientific reports mention the antifungal activity of garlic extract in vitro and in vivo, this activity is quite a paradox, since the garlic plant or its fruits can be colonized by numerous fungi, particularly Fusarium species comprising of F. equiseti, F. proliferatum, F. oxysporum, F. acuminatum, and F. solani (Stankovic et al. 2007; Ignjatov et al. 2018; Miriam et al. 2021; Paul et al. 2021). The deteriorative efficacy of Fusarium spp. as well as other fungi on plants are due to the secretion of cell wall-degrading enzymes (CWDEs), e.g., lignocellulases, lipases, and proteases, which are responsible for the damage of the cell wall of plants (Nofal et al. 2021b; Al-Rajhi et al. 2022). Furthermore, the produced fungal toxins suppress the immune system of the plant, which helps the fungi successfully attack the plant (Lakshmipriya et al. 2020). Mallebrera et al. (2018) mentioned that Fusarium toxins enniatins and beauvericins encourage DNA fragmentation in addition to apoptosis through disorder in the mitochondrial pathways.
The search for effective approaches to eliminate pathogenic fungi and their toxins has become a global demand, so scientists turned to applying nanotechnology as a promising and potentially safe method for controlling fungal proliferation. Nanoparticles (NPs) have been applied as fungicide to restrict growth and mycotoxin production (Abdelghany et al. 2018a; Ganash et al. 2018; Jesmin and Chanda 2020). However, the activity of NPs on the production of mycotoxins has not been researched enough yet. More recent attention has been focused on the application of copper nanoparticles (CuNPs) and copper oxide nanoparticles (CuONPs) in these fields (Khamis et al. 2017; Abdelghany et al. 2020). It has been found that CuONPs suppresses Fusarium culmorum, F. graminearum, F. oxysporum, F. solani, Penicillium chrysogenum, Aspergillus flavus, and Alternaria alternate, and Aspergillus niger growth (Ghasemian et al. 2012; Shende et al. 2015; Abdelghany et al. 2020). The effect of CuONPs exhibited genotoxicity in F. solani and Penicillium digitatum as a result of exposure to CuONPs (Khamis et al. 2017). The repression of mycotoxigenic fungi growth is an area of interest for numerous investigators; therefore, the current study aimed to evaluate the activity of CuONPs against the growth, ultrastructure, and mycotoxins, as well as hydrolytic enzyme production of F. incarnatum isolated from garlic (Allium sativum L.).
EXPERIMENTAL
Isolation and Identification of Mycotoxins Producing Fungus
Cloves of garlic (Allium sativum L.) that showed fungal infection (as shown in Fig. 1A) from a field in Menofia Governorate, Egypt were separated, peeled off, and the surface was disinfected using 5% NaOCl for 2 min. They were rinsed in sterile water using three successive baths of for 1 min each, then dried using sterile filter paper to remove the excess water. Small, infected tissue samples with symptoms of fungal appearance (1 cm in length) were plated onto potato dextrose agar medium (PDA) amended with streptomycin sulphate (3%) and incubated for 6 d at a temperature of 28 °C until fungal growth appeared. The hyphal tips of the appeared Fusarium-like colonies were purified by being transferred to PDA plates and were incubated at a temperature of 28 °C until the purified fungus appeared, which was kept for further study (Dhingra and Sinclair 1995). The purified fungus was morphologically identified by the presence of macroconidia, microconidia, chlamydospores, as well as by the observation of the growth rate and the color of the colony (Nelson et al. 1983; Leslie and Summerell 2006).
Molecular Identification of the Pathogen
The purified and morphologically identified fungus was subjected to molecular identification (Abdelghany et al. 2018b; Hami et al. 2021). For identification, fungal DNA was extracted using a Microprep Kit of quick-DNA fungi/bacteria (Zymo research; D6007) according to the procedure outlined by Sigma Scientific Services Company. Maxima Hot Start PCR Master Mix (Thermo; K1051) was utilized for the PCR. Amplification of the DNA was done using forward primer ITS1-F (5′- TCCGTAGGTGAACCTGCGG-3′) and reverse primer ITS4-R (5′- TCCTCCGCTTATTGATATGC-3′). Various thermal cycler conditions throughout the PCR protocol were used, starting with 1 cycle at a temperature of 94 °C for 6 min, which was required for the initial denaturation, followed by 35 cycles for 45 s at a temperature of 94 °C, which was required for another denaturation, then 35 cycles for 45 s at a temperature of 56 °C, which was required for annealing, and ending with 35 cycles for 60s at a temperature of 72 °C, which was required for extension. The fungus DNA was scanned via gel electrophoresis. Sequences homologous at NCBI (http://ncbi.nlm.nih. gov/BLAST) were analysis via BLAST. The neighbor joining manner was used for the reconstruction of the evolutionary tree.
Copper oxide Nanoparticles and Rizolex-T50
Copper oxide nanoparticles (CuONPs) were purchased from Nawah Scientific, Cairo, Egypt. Particles were less than 100 nm and came in powder form. The size was confirmed by scanning electron microscopy (FEI Company, Hillsboro, Oregon-USA) at EDRC, DRC, Cairo). Rizolex-T 50 (20% Tolclophos-methyl and 30% Thiram) was applied as a chemical fungicide, which was purchased from Sumitomo Chemical, Tokyo, Japan.
Assessment of Copper Oxide Nanoparticles (CuONPs) Activity on Growth and Ultra-Structures of Pathogen
The inhibitory action of CuONPs at different concentrations against F. incranatum growth was achieved using Petri dishes containing Czapek agar media. Rizolex-T50 was applied as a positive control for fungal growth. The plates were centrally point-inoculated with a 0.5 mm disc from 5-day-old colony cultures of the pathogen, and then incubated for 7 d at a temperature of 28 °C. At the end of the incubation period, the radius of the colony was measured to calculate the inhibition %, according to Eq. 1,
(1)
where CRC (mm) is the colony radius of the control and CRT (mm) is the colony radius of the treatment (Note: CuONPs nil medium was used as the control).
The tips of the growing fungus hypha at different concentration of CuONPs were cut and fixed using 5% gluteraldehyde for 1 d. Then, the samples were washed in phosphate buffer (pH 7.2) 3 times, followed by the elimination of the excess buffer. Next, the segments were rinsed for 2 h in osmium tetraoxide diluted up to 1%, followed by the removal of osmium tetraoxide. The prepared segments were dehydrated by a sequence of different levels of ethanol, ranging from 50% to 95%. The segments were dried with absolute ethanol, and then dipped in propylene oxide for 1 h. The segments of hyphae were placed in propylene oxide and Epon 812 resin at a 2 to 1 ratio, followed by being placed in pure resin for 12 h, before finally being placed in an oven for 48 h at a temperature of 60 °C . Blocks (50 nm) were segmented via ultra-microtome, then stained with uranyl acetate-lead citrate 500A and examined at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt via transmission electron microscopy (TEM) (C Joel Jem- 1200 EX II. Acc. Voltage 120 KV. MAG- medium) (Abdelghany et al. 2021).
Assessing Copper Oxide Nanoparticles (CuONPs) Activity on Mycotoxins Production
Sterilized potato dextrose broth medium supplemented with different concentrations of CuONPs (50 mL in a 250 mL conical flask) was inoculated with F. incranatum and then incubated for 10 d at a temperature of 28 °C. At end of the incubation period, the fungal mycelia were decanted, and the filtrate medium was extracted with 50 mL of chloroform and methanol (at a 2 to 1 ratio), followed by desiccation to concentrate the extracted solvent (El-Taher et al. 2012). Then the extract was subjected to LC1620A liquid chromatography for mycotoxin analysis. The inject volume was 20 uL, the mobile phase water to acetonitrile to methanol was 5 to 4 to 1, the flow was 1.540 mL/min, and the current detector UV-Detector was at a wavelength of 254 nm. The mycotoxins were identified by comparing the retention time of the analyte with labeled internal mycotoxin standards.
Assessment of the Copper Oxide Nanoparticles (CuONPs) Activity on Hydrolytic Enzyme Production
Three substrates, including 0.5% carboxymethyl cellulose, 0.5% xylan, and 2% starch (Sigma-Aldrich, St. Louis, MO), were used for stimulating the production of CMC-ase, xylanase, and amylase, respectively (El-Katatny 2010). The sterilized minimal synthetic medium (MSM) (NH4NO3 1.0 g/L, K2HPO4 0.9 g/L, KCl 0.2 g/L, MgSO4·7H2O 0.2 g/L, MnSO4 0.002 g/L, FeSO4·7H2O 0.002 g/L, ZnSO4 0.002 g/L, and agar 20 g/L) containing the appropriate substrate and different concentrations of CuONPs (at a pH of 5.5 adjusted by 50 mM acetate buffer) in a plate was inoculated with the isolated fungus in the center of plate and incubated for 5 d. At the end, the enzymes activities were visualized via staining the agar plates with 0.2% Congo red dye for 15 min, followed by washing with a 1 M NaCl solution for detecting the CMC-ase and xylanase (Herculano et al. 2011). The amylase was detected by flooding the plate with 1% iodine from 2% potassium iodide. The enzymatic index (EI) was determined by measuring the enzymatic halo (diameter of colony with the clear zone) according to Eq. 2,
(2)
Statistical Analysis
All the results were analyzed via SPSS software (version 14, IBM, Armonk, NY). Calculations were done for the mean ± SD (standard deviation) of three replicates.
RESULTS AND DISCUSSION
Fungal Identification of the Infected Cloves of Garlic Biomass
The collected samples of garlic were covered by a white mycelium (as shown in Fig.1A), with soft and spongy bulbs.
Fig. 1. Garlic samples infected with F. incarnatum (A); colony of F. incarnatum on a Petri dish containing PDA medium (B); and the phylogenetic tree of the F. incarnatum strain JL5-2 (C)
The isolated fungus was morphologically identified as F. incarnatum, where at the initial stage the aerial mycelia were white (Fig. 1B) and turned to off-white, pale pink, and grey in the later phases, in addition to the microscopic examination. Molecular characterization of the isolate was conducted to recognize and confirm that the identity isolate was closely related to F. incarnatum. The phylogenetic tree demonstrated the similarity of the isolate to F. incarnatum (Fig. 1C). A BLAST search in the NCBI database via ITS sequences (Fig. 2) showed 99% to 100% similarities with F. incarnatum. F.incarnatum has been isolated from various plant material in different countries as well as other species, as mentioned in numerous reports. For example, it was detected on rapeseed in Iran (Nemati et al. 2015). Other Fusarium spp. were isolated from garlic, e.g., F. proliferatum (Mondani et al. 2021). Recently, F. proliferatum, F. solani, F. acuminatum, F. oxysporum, F. redolens, and F. subglutinans have been detected in rot garlic (A. sativum) (Gálvez and Palmero 2021). Previously, Penicillium, Aspergillus, Botrytis, and Fusarium genera were detected in garlic bulbs in Egypt, but Fusarium spp. represented the most prevalent genera (Moharam et al. 2013).
Fig. 2. The ITS sequences of the closely related fungal strains retrieved from the NCBI GenBank database and the cluster analysis of F. incarnatum
Inhibitory Action of Copper Oxide Nanoparticles (CuONPs) Against Fungus Growth
The inhibitory action of CuONPs (size less than 100 nm shown in Fig. 3) in terms of F. incarnatum growth and sporogenesis was observed to be dependent on the concentration (as shown in Table 1). The obtained results indicated that the inhibition of growth reached up to 81.70% at 400 ppm when compared to the control zero %. According to the microscopic analysis and spore counting, sporulation (%) decreasing was recorded as the CuONPs concentration increased. Although the inhibitory action of the chemical fungicide (Rizolex-T50) and 400 ppm of CuONPs was approximately in a similar narrow spectrum (85.36 % and 81.70%, respectively), but the sporulation (%) was far apart from each other (10 and 29%, respectively).
Fig. 3. Scanning electron micrograph of CuONPs. Magnification 12000X
Table 1. Colony Diameter, Inhibition and Sporulation of F. incarnatum at Various Copper Oxide Nanoparticles (CuONPs) Concentrations
Recent results showed the substantial suppression of F. oxysporum spore propagation after exposure to CuONPs (Ashraf et al. 2021). The inhibitory activity of CuONPs and chemical fungicide on fungal growth was confirmed and evaluated also in broth medium, which indicated that growth reduced with increasing CuONPs concentrations (Fig. 4).
The fungicidal activity of CuONPs was reported against multiple fungi. For instance, Fusarium solani and Penicillium digitatum had full inhibition at 60 µg/mL and 20 µg/mL, respectively (Khamis et al. 2017). In another study, the controlling effect of CuONPs was recorded for Penicillium expansum, Alternaria alternata, and A. solani (Nemati et al. 2015).
Fig. 4. Growth of F. incarnatum at different concentrations of CuONPs (100, 200, and 400 ppm) in broth medium
A mechanistic study of NPs against various fungi has been investigated. For instance, injury of the lipid bilayer in fungal cell membranes, as well as the formation of unusual swells on the surface of the hyphae, which led to hyphae deformation, were observed as a result of exposure to ZnO nanoparticles (He et al. 2011).
In the current research, the TEM showed that CuONPs caused alteration in the cell wall, as well as disruption of the cell membrane of F. incarnatum. Similarly, but on another fungus, CuNP treatments stimulated the distortion of A. niger hyphae at 200 and 300 ppm (Abdelghany et al. 2020).
In addition, sharp changes were observed in cell organelles of Fusarium oxysporum and F. solani hyphae treated with CuNPs (Pariona et al. 2019). Severe shrinkage of the cell membrane and the collapsing of the cell wall at high concentrations of CuONPs (400 ppm) are shown in Fig. 5.
Different levels of damage were observed in hypha or conidiospores but were dependent on the concentration; negligible deformation appeared at 100 ppm and was obvious at 200 and 400 ppm of CuONPs. It can be predicted that the oxidative stress stimulated by high levels of CuONPs, in addition to its toxicity mechanism, cause the disturbances of the metabolic pathways of the enzymes. Morphological disruptions in the F. oxysporum mycelia were observed recently by Ashraf et al. (2021), as well as reactive oxygen species (ROS) generation on the surface of the cell wall surface, which led to complete destruction.
Fig. 5. The ultrastructures of F. incarnatum at different concentrations of CuONPs (100 ppm, 200 ppm, and 400 ppm) (Note: CW = Cell wall; S = septum; SCy = shrinkage cytoplasm; N = nucleus; V = vacuole; M = mitochondria; and CM = cell membrane)
Inhibition of Mycotoxin Production
NPs have been widely applied for inhibiting the growth of many microorganisms, but in terms of the inhibition of mycotoxins production is still in its early phases. Liquid chromatography detected the produced mycotoxins by F. incarnatum (as shown in Table 2 and Fig. 6) under the effects of CuONPs. The obtained findings indicated that at concentrations that did not fully inhibit growth, the CuONPs were able to limit and decrease the mycotoxin secretions of F. incarnatum (as shown in Table 2).
Fig. 6. The liquid chromatography chromatograms of the mycotoxins production of F. incarnatum at 100 ppm, 200 ppm, and 400 ppm of CuONPs
Table 2. Mycotoxin Production of F. incarnatum at Different Concentrations of Copper Oxide Nanoparticles (CuONPs)
The changes in the productivity of mycotoxins may be due to the interference of CuONPs with the metabolic pathways of secondary metabolites. Four mycotoxins, i.e., beauvericins, fusarins, moniliformin, and enniatins, were detected in the metabolized medium of F. incarnatum. Unfortunately, the complete inhibition of these mycotoxins did not occur until high concentrations of CuONPs were used. A recent idea focused on the application of engineered NPs at low levels to inhibit mycotoxins without killing the producer fungi, in order to avoid NPs toxicity in humans (Jesmin and Chanda 2020). A study conducted by Asghar et al. (2018) exhibited that the synthesis of aflatoxin B1 was reduced under the effect of CuNPs. The inhibition % of beauvericins, fusarins, moniliformin, and enniatins synthesis at 400 ppm compared to the synthesis under the control treatment was 62.8%, 45.4%, 58.1%, and 55.0%, respectively (as shown in Table 2). The occurrence of these compounds, even in low concentrations, still can be unsafe, particularly in more consumed plant products. Seefelder et al. (2002) reported the presence of mycotoxins in garlic bulbs infected by fungi in Germany. Reduction of mycotoxins production under the effect of CuONPs may explained on the base of fungal growth retardation or the adsorption of mycotoxin on the nanoparticles.
Hydrolytic Enzymes
The obtained findings showed that F. incarnatum isolated from garlic was able to decompose the substrates via the hydrolytic enzymes CMC-ase, xylanase, and amylase with EI values greater than 1 (Table 3) at all the tested concentrations of CuONPs. The EI of each enzyme varied, which suggested different amounts of enzyme production depending on the CuONP concentration. An unexpected result was observed, where 100 ppm of CuONPs stimulated the production of enzymes with EIs of 1.31, 1.36, and 1.23 for CMC-ase, xylanase, and amylase, respectively, when compared with the EIs of the control or at 400 ppm of CuONPs. This finding was consistent with recent documentation by Abdelghany et al. (2020), which indicated that CuNPs at low level were efficient in terms of encouraging laccase production by A. niger, while it suppressed enzyme production at higher concentrations. The copper metal may interfere as a complementary for the composition of some enzymes. As mentioned previously, the synthesis of β-glucosidase, cellobiohydrolase, and β-xylosidase were repressed while Mn peroxidase was unaffected by the presence of CuNPs (Shah et al. 2010).
Table 3. Enzymatic Index of the CMC-ase, Xylanase, and Amylase Activities at Different Copper Oxide Nanoparticles (CuONPs) Concentrations