Antifungal potential, chemical composition of Chlorella vulgaris and SEM analysis of morphological changes in Fusarium oxysporum.

In pursuit of an environmentally benign fungicide alternative, the current study explored the antifungal activity of Chlorella vulgaris extracts against six plant pathogenic fungi (in vitro). The well diffusion agar method was used to investigate the growth inhibition of Fusarium oxysporum, Fusarium sp., Fusarium solani, A. flavus, A. niger, and A. alternata using the three C. vulgaris extracts viz. methanol (CvME), acetone (CvAE), and diethyl ether (CvDE). Different concentrations of CvDE were also investigated against F. oxysporum. The morphological modifications in F. oxysporum treated with CvDE (5 mg/kg) were studied using SEM and the chemical composition of CvDE was also determined by GC-MS analysis. All extracts, with the exception of A. alternata, were found to be effective in inhibiting the growth of plant pathogenic fungi. The CvDE extract, followed by CvME and CvAE, was found to be efficient against tested fungi. The CvDE was most effective against F. oxysporum with a 73.3% growth inhibition. The effects of various CvDE concentrations on F. oxysporum were found to be dosage dependent. The SEM micrograph revealed that CvDE-treated F. oxysporum had substantially less conidia than the control. The CvDE treatment damaged the mycelial structure as well. Major chemical components detected in CvDE were Heptaldehyde (15.7%), Octadecenoic acid, methyl ester (12.6%), Hexadecanoic acid (12%), 3-Decyn-2-Ol (10.98%), (E)-3,7,11,15-tetramethylhexadec-2-ene (9.76%), heptadecane-1,2,3,4,5-pentol (8.7%), Docosane, 4-methyl (7.28%).

Introduction

Microalgae are widely distributed and can cultivate practically any environment. A few of them are well-known for producing bioactive chemicals that have various applications, including in drugs and medicine, bioremediation, and the food industry (Ranglová et al., 2021, Santhosh et al., 2016, Vaz et al., 2016). However, a large number of bioactive compounds remain in these microalgae that haven't been identified yet. Microalgae have been actively investigated in recent decades for new chemicals that could be used as medicinal agents, antimicrobial, antiprotozoal, and antiplasmodial agents (Balaji et al., 2017, Dinev et al., 2021, Shaima et al., 2022). Chlorella is a green alga that is widely utilized in pharmaceuticals and cosmetics, as well as a food supplement due to its high nutritional content. Protein, vitamins, lipids, polysaccharides, minerals, carotenoids and immune stimulator chemicals are the primary components of Chlorella (Vaz et al., 2016). Chlorella extracts have been shown to have antimicrobial effects. This genus has been linked to the antibiotic Chlorellin (Pratt et al., 1944).

Plant pathogenic fungi cause numerous diseases to plants, causing yield as well as economic losses (Fletcher et al., 2006). Species of Fusarium, Aspergillus, and Alternaria cause various plant diseases such as blight, fruit rot, root crown rot, and wilt. The genus Fusarium is among the most economically important genera of fungi in the world. Its species such as Fusarium oxysporum, F. solani, F. equisetti, F. chlamydosporum etc. cause root and stem rot, vascular wilt, and/or fruit rot in a number of economically crop species (Dean et al., 2012). The members of the genus Alternaria causes many pre and postharvest diseases and losses to plants, fruits and vegetables (Roy et al., 2019). Alternaria alternata infect more than 400 plant species with leaf spots, rots and blights. The genus Aspergillus cause plant and fruit rot with production of mycotoxins such as aflatoxin that contaminate several oil bearing seeds (Perrone et al., 2007, Wilson and Payne, 1994).

Chemical fungicides are the most effective strategy at the moment to treat fungal diseases, albeit they are not environmentally friendly. Consequently, natural chemicals that can substitute these hazardous fungicides are in high demand. Several types of algae have been reported to inhibit soil-borne fungus, including Anabaena spp., Scytonema spp., and Nostoc spp.. The mycelial growth of plant pathogenic fungus was entirely suppressed by extracts from certain algae (Galal et al., 2011, Kulik, 1995).

C. vulgaris extracts were tested against a variety of key plant pathogenic fungi in this investigation, and the fungus that was found to be most susceptible to the extract was examined under a scanning electron microscope for morphological changes, as well as the chemical constituents of C. vulgaris extract was examined using GC–MS.

Materials and methods

Cultivation of Chlorella vulgaris

C. vulgaris was cultivated for 4 weeks at 25 °C under constant light on BG 11 nutrition media. Filtration through Whatman no. 1 filter paper yielded the active cells. The harvested biomass was utilized to make crude extracts with various solvents (Perveen and Alwathnani, 2013).

Preparation of the extracts

Crude extracts of C. vulgaris were prepared using the solvents methanol, acetone, and diethyl ether. In the conical flask, five grams of C. vulgaris biomass and 100 mL of the aforementioned solvents were mixed separately and placed at 20 °C for three days on an incubator shaker. Whatman no. 1 filter paper was used to filter the mixture. The resultant filtrate was dried thoroughly in a hot air oven at 40 °C. The dried residue was dissolved in 10% dimethyl sulfoxide (DMSO) to make a stock solution (@ 5 mg/ml).

Plant pathogenic fungi

The plant pathogenic fungi; F. oxysporum (MT151384), Fusarium sp. (ITCC 8189.11), F. solani (MW265001), A. flavus (Identified at the Department of Botany and Microbiology), A. niger (Department of the Botany and Microbiology), and A. alternata (ITCC 7912.10) were procured from the Department of Botany and Microbiology. The fungal cultures were stored at 4 oC on Potato Dextrose Agar (PDA) slants. Prior to the antifungal assay the fungal cultures were grown on the PDA plates at 27 °C for five days.

Antifungal assay

By using the well diffusion method, the antifungal activity of C. vulgaris extracts such as methanol (CvME), acetone (CvAE), and diethyl (CvDE) against six fungi (F. oxysporum, Fusarium sp., F. solani, A. flavus, A. niger, and A. alternata) was evaluated (Balouiri et al., 2016). A well was made 10 mm inside the plate's edge, and a 100 μl aliquot of the C. vulgaris extract was transferred into it, a disc (5 mm) of actively growing fungi (5 days old culture) was placed and it kept on the opposite side of the well. In the control treatment, instead of extracts, the well was filled with 10% DMSO. Fungal growth was measured after 5 days of incubation at 25 °C. Fungal growth inhibition was determined with the help of following formulaWhere, I = percent growth inhibition, GC = growth of fungi in control plate, GT = growth of fungi in treated plate.

Effect of different concentration of CvDE on F. oxysporum

The influence of various concentrations of most potential extracts (CvDE) against the most susceptible fungus (F. oxysporum) was also evaluated in a similar way as mentioned in the antifungal assay. Seven different concentrations of the CvDE were prepared by double dilution in 10% DMSO (5 mg/ml–0.0785 mg/ml) and different concentrations of the extract were poured into wells.

Observation of morphological changes in F. oxysporum by CvDE

Scanning electron microscopy (SEM) was utilized to examine the morphological changes in the fungus (F. oxysporum) treated with CvDE (5 mg/ml). The samples were taken from the margin of the inhibition area adjacent to the well of CvDE or control. The samples for SEM observation were prepared according to the method described earlier (Alwathnani and Perveen, 2017). The samples were first fixed for two hours at room temperature with 3% glutaraldehyde. After washing twice with 0.1 M phosphate buffer, the samples were fixed in osmium tetroxide (1%) for 2 h at room temperature. Next step was dehydration by transferring samples through a graded series ethanol (10 to 100%) for 15 min each. The samples were critical point dried with CO2 in a drying apparatus (EMS 850, USA). With the help of Sputter Coater, samples were coated with gold. A scanning electron microscope (JEOL, JSM-5500LV) at an accelerating voltage of between 18 and 20 kV was used to observe the prepared samples.

GC–MS analysis of Chlorella vulgaris extract

In order to find the chemical components of C. vulgaris, the extract that yielded promising results was subjected to GC–MS analysis. The methodology by Perveen and Alwathnani (2013) was employed. Gas chromatography (Perkin Elmer) was utilized with a mass spectrometer. It was equipped with an RTx-DB5 column with a dimension of 30 × 0.32 nm for GCMS analysis. The oven temperature was set at 75 °C for 2 min, then increased to 175 °C at a rate of 50 °C/min for 7 min. Helium (3 mL/min) was used as the carrier gas. The NIST database was used for chromatographic analysis and interpretation of the resulting mass spectra.

Statistical analysis

For each treatment there were four replicates. The gathered data was statistically analyzed using ANOVA and Tukey (HSD) post hoc through XLSTAT (version. 2021.3.1.1162).

Results

The extracts of C. vulgaris prepared using solvents such as methanol (CvME), acetone (CvAE), and diethyl ether (CvDE) were tested against F. oxysporum, Fusarium sp., F. solani, A. flavus, A. niger, and A. alternata. Well diffusion assay was employed to assess the inhibition potential of the extracts. The outcomes are depicted in Fig. 1. It reveals that all of the extracts had different impacts on the fungus that were examined. CvDE was significantly effective in inhibiting the growth of all fungi tested except A. alternata. CvDE inhibited F. oxysporum, Fusarium sp., F. solani, A. flavus, and A. niger to a high degree with percent inhibition of 73.3%, 63.3%, 60.0%, 56.75%, and 50.0% respectively (Fig. 1). CvME, on the other hand, was proven to be effective against F. oxysporum (66.7%), Fusarium sp. (60.0%), A. flavus (53.3%), and A. niger (54.3%). Whereas, CvAE was discovered to be efficient in hindering the growth of Fusarium sp. (54.3%), F. oxysporum (41.0%), A. flavus (33.3%), and A. niger (32.3%). The positive control, carbendazim (2%) prevented the growth of all fungus completely.

Fig. 1

Percent growth inhibition of F. oxysporum, Fusarium sp., F. solani, A. flavus, A. niger, and A. alternata by the methanol (CvME), acetone (CvAE) and diethyl ether (CvDE) extracts of C. vulgaris.

Data is mean ± Sd for n = 4. Bars showing different letters are significantly different (P ≤ 0.05) according to Tukey’s HSD tests.

Among all extracts, CvDE was most effective and caused maximum growth inhibition of F. oxysporum (73.3%). Therefore, different concentrations of CvDE were evaluated against F. oxysporum. The findings revealed that the inhibition of fungal growth was concentration dependent; it ranged between 74% and 16.7%. The minimum reduction in the growth of fungus by CvDE, was 0.156 mg/ml. While a concentration of 0.078 mg/ml was failed to prevent the fungus from growing (Fig. 2).

Fig. 2

Percent growth inhibition of F. oxysporum by different concentration of diethyl ether extract of C. vulgaris (CvDE). Data is mean ± Sd for n = 4. Bars showing different letters are significantly different (P ≤ 0.05) according to Tukey’s HSD tests.

The morphological changes in F. oxysporum treated with CvDE were observed under SEM (Fig. 3). Variation in the number of conidia was the apparent difference observed in the F. oxysporum treated with CvDE (Fig. 3 b,c) and untreated control (Fig. 3a). In the control sample, the micro and macroconidia of F. oxysporum were observed in abundance while they were sparse in the treated sample. The difference in the morphology of the mycelium between F. oxysporum treated with CvDE and untreated control was also visible. The mycelium of the treated fungus was slender, distorted, and rough as compared to the control where mycelium was well formed and smooth (Fig. 3a).

Fig. 3

Morphological changes in F. oxysporum. The untreated control has a large number of conidia and smooth mycelium (a). The treated with (5 mg/ml) of CvDE has sparse conidia and deformed mycelium (b, c).

The chemical constituents of CvDE was identified by GCMS analysis, and the results are reported in Table 1. Fatty acids and esters were discovered to be abundant in the extract. A substantial amount of various alcohols were also found. Investigation of CvDE for chemical composition, identified 14 components. Major components were Heptaldehyde (15.7%), Octadecenoic acid, methyl ester (12.6%), Hexadecanoic acid (12%), 3-Decyn-2-Ol (10.9%), (E)-3,7,11,15-tetramethylhexadec-2-ene (9.7%), heptadecane-1,2,3,4,5-pentol (8.7%), Docosane, 4-methyl (7.2%).

Table 1

Chemical composition of C. vulgaris extract (CvDE) analyzed by GCMS.

S. No.	Compound	Formula	Area (%)
1	3-methylbutan-2-o	C5H12O	1.1
2	(S)-(+)-2-Hexanol	C6H14O	1.6
3	Heptaldehyde	C7H14O	15.7
4	N-Isopentylacetamide	C7H15NO	1.1
5	3-Decyn-2-Ol	C10H18O	10.98
6	2-isopropyl-5-methylhexan-1-ol	C10H22O	3.4
7	5,9-Dodecadien-2-one, 6,10-dimethyl-	C14H24O	4.7
8	Hexadecanoic acid	C16H32O2	12.0
9	Octadecenoic acid, methyl ester	C19H34O2	12.6
10	(E)-3,7,11,15-tetramethylhexadec-2-ene	C20H40O	9.76
11	Heptadecane-1,2,3,4,5-pentol	C17H36O5	8.7
12	Docosane, 4-methyl	C23H48	7.28
13	propyl (9Z,12Z)-octadeca-9,12-dienoate	C21H38O2	3.7
14	(3Z)-3-hexadec-15-ynylidene-4-hydroxy-5-methyloxolan-2-one	C21H34O3	6.2
Total			98.82

Discussion

Micro-algae produce a variety of metabolites to protect themselves from adverse conditions. These metabolites include polysaccharides, amino acids, enzymes, antibiotics, and toxic substances. Antimicrobial properties have been observed in several of these compounds. Present results show that the extracts of C. vulgaris have antifungal properties. The current findings corroborate previous observations made by a number of researchers. Extract of C. vulgaris reduced the growth of plant pathogenic fungi such as A. niger, A. alternata, and P. expansum (Vehapi et al., 2020). A study discovered that a methanol extract of C. vulgaris inhibited significantly the growth of human pathogenic fungi (Ghasemi et al., 2003). While, another study reported that among four microalgae evaluated for antimicrobial activity, C. vulgaris was superior in inhibiting the growth of bacteria and pathogenic fungi than other tested microalgae (Ahmed, 2016). The current study's findings prove unequivocally that the C. vulgaris extracts were more effective against species of Fusarium than against other species. Whereas, none of the extracts could inhibit the growth of A. alternata. The phylogenetic differences between the fungal species could explain this variation (Philip et al., 2009). The fact that antifungal activity varies depending on the extract reveals that extract bioactivity is solvent dependent (Mian et al., 2003, Salvador et al., 2007, Stramarkou et al., 2017). A growth inhibition of 53.8% in R. solani by 5% methanolic extract of C. vulgaris has been recorded (Al-Nazwani et al., 2021). Further, they have recorded that the fungal inhibition was concentration dependent. Present study noticed that the maximum zone of inhibition by C. vulgaris extracts was against F. oxysporum. A report has demonstrated that the lowest concentration (1 gL−1) of the extract of C. vulgaris could inhibit 40% growth of F. oxysporum (Ferreira et al., 2021). Earlier researchers have also reported a reduction in F. oxysporum growth due to an extract of C. vulgaris (Vehapi et al., 2020, Vehapi et al., 2018).

SEM analysis showed morphological changes in the F. oxysporum treated with CvDE. The bioactive compounds could be responsible for the morphological changes in the fungus. Recently, the morphological changes and a reduction in R. solani spore production due to C. vulgaris methanolic extract has been reported. Fatty acids with C10 or more have been shown to trigger protoplast lysis (Al-Nazwani et al., 2021). Damage to the cell wall, breakdown of the cellular membrane or its proteins, which could lead to leaking of cell contents and cytoplasm coagulation (Jyotirmayee et al., 2014). Present study show the presence of several such fatty acids. Various studies have reported the potential of algal and cyanobacteria extracts in inhibiting the plant pathogenic fungi as well as controlling plant diseases caused by Fusarium spp. (Al-Nazwani et al., 2021, Kim, 2006, Kim and Kim, 2008, Omar, 2012, Vehapi et al., 2020, Vehapi et al., 2018). However, no one has observed morphological changes in F. oxysporum as a result of C. vulgaris extracts.

Fatty acids, esters, and alcohols were detected in CvDE when analyzed by GCMS. Previous studies have recorded the presence of many of these compounds in the extracts of Chlorella sp. (Al-Wathnani et al., 2012, Alwathnani and Perveen, 2017, Pantami et al., 2020). A study identified the chemical content of methanol extract of C. vulgaris by GC–MS probe. It demonstrated the existence of n-hexadecanoic acid, eicosanoic acid, octadecenoic acid, oleic acid, and pentadecanone, among other fatty acids. Several fatty acids, methyl esters, and carotenoids were identified in C. vulgaris extracts after a detailed analysis (Al-Nazwani et al., 2021). Compounds from various chemical families have been associated with microalgae's antimicrobial activity. Chlorellin is a combination of fatty acids that is a known antibiotic extracted from chlorella (Pratt et al., 1944). The antimicrobial activity of bioactive chemicals found in the present extract, such as 3-Decyn-2-Ol, heptanal, hexadecanoic acid, and heptadecane-1,2,3,4,5-pentol, has previously been reported (Costa et al., 2019, Dinev et al., 2021, Shaima et al., 2022). In the present study, these chemicals may have aided in the suppression of fungal growth.

Conclusions

The results of this investigation clearly demonstrated that plant pathogenic fungi could be inhibited by the extracts of C. vulgaris. The damaging potential of the CvDE, as revealed by SEM analysis of CvDE treated F. oxysporum, and the presence of bioactive compounds in the extract of C. vulgaris, proves its potential as an effective component for the control of plant pathogenic fungi. Since the plant pathogenic F. oxysporum is difficult to control, the findings of this study will aid in the development of strategies for managing the pathogen in an environmentally friendly manner.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.