Abstract
In this study, Fusarium equiseti was isolated from the weed plant Tridax procumbens in an agricultural field and a crude extract produced with 75% ethanol for use as active ingredient material in natural herbicides. The herbicidal effect of F. equiseti extract was tested on water hyacinth (Eichornia crassipes), an invasive aquatic weed, by leaf disk assay at concentrations of 0.05%, 0.1%, and 0.2% w/v crude extract. Dose-dependent visual toxicity symptoms were evident after three days, namely chlorosis, yellow leaves surrounded by dark brown edges. Photosynthetic pigments (chlorophyll a, b, and carotenoids) and membrane integrity (as electrolyte leakage and malondialdehyde content) were evaluated following the leaf disk test. 3 days after treatment, photosynthetic pigment contents showed dose-dependent decreases, while both measures of membrane integrity showed dose-dependent increases with increasing extract concentration. In addition, a cytogenetic assay was conducted on Allium cepa L. root, in which mitotic index reduction and depigmentation were evident as early as 24 h after herbicide application. Finally, anatomical analysis of treated E. crassipes leaves revealed degradation or damage of the ground tissue. All told, our results support the F. equiseti crude-based natural herbicide cloud as a sustainable alternative in agriculture.
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Introduction
Water hyacinth (Eichornia crassipes (Mart.) Solms), a member of the Pontederiaceae1, is one among many harmful aquatic weeds in the world because of its detrimental effects on the environment and the economy2,3. It is well known that water hyacinth degrades the environment severely and is expensive to manage. Water hyacinth was first introduced globally with the intention of being given as presents, and it has unintentionally and intentionally found its way into the natural world. Africa, Asia, Europe, Central America, North America, and the Caribbean have all been the subject of invasion reports. According to the Invasive Species Specialist Group (ISSG), water hyacinth is ranked in the top 100 worldwide invasive species database2. The expansion of this weed and its ability to invade a wide range of freshwater habitats exerts harmful effects on fisheries and related commercial activities, access to clean water, irrigation, hydropower generation, tourism, and navigation along water courses3. Four biodiversity indicators have shown that water hyacinth also lowers the biodiversity of the invaded region. The invasion of water hyacinth has been found to decrease overall biodiversity in terms of evenness and richness of species2. Water hyacinth is among the fastest-growing plants known; it reproduces mainly by runners or stolons, which eventually give rise to daughter plants. Additionally, a single plant is capable of yielding thousands of seeds annually, and these seeds are viable for more than 28 years.
In general, water hyacinth may be managed by biological, mechanical, and chemical control methods. Biological control is a more compelling choice because it avoids release of harmful chemicals into the environment4. However, biological approaches are limited to small plants and take a long time to achieve control. Accordingly, chemical methods are frequently utilized. Synthetic herbicides are the most economical way to control weeds, but their extensive usage is harmful to farmers and consumers5. In natural ecosystems, chemical-based pesticides have become the main cause of contamination6. Additionally, some synthetic herbicides are no longer allowed; for example, many nations, including Austria, the European Union, and South Korea, have banned paraquat. In June 2020, the Ministry of Industry in Thailand likewise declared an outright ban of paraquat pesticides7. Thus, development of natural herbicides is required to manage water hyacinth and other agricultural weeds in a sustainable manner.
Allelopathy is a chemical interaction between plants, including microbes, that can be both advantageous and destructive. When used appropriately, this phenomenon can be very beneficial for controlling weeds, protecting crops, and restoring crops by releasing allelochemicals8,9,10,11. Manichart, et al.12 reported that allelopathic potential of secondary metabolites produced by fungi extract Alternaria brassicicola can control the weed Amaranthus tricolor. Microorganism-based natural products have received great attention for potential sustainable agricultural applications. Fungi offer an avenue for finding new natural herbicides with modes of action distinguishable from those already in general use, which is of increasing importance. Fungi are rich in secondary metabolites13, and fungal extracts have been demonstrated good sources of active ingredients with diverse benefits such as antimicrobial drugs14, antioxidative properties15, anticancer activities16, and antimalarial and antimycobacterial activities17. Fusarium equiseti (Corda) Sacc. is a widespread species found in subtropical, temperate, and tropical regions18. The secondary metabolites it produces include fusarochromanone, equisetin, zearalenone, and type A and B trichothecenes19. Therefore, F. equiseti may be a candidate source for natural herbicide production.
The present study aimed to characterize the phytotoxic effects of F. equiseti crude extract on the aquatic weed water hyacinth under laboratory conditions and included evaluation of some physiological properties in water hyacinth, namely electrical conductivity, lipid peroxidation, and photosynthetic pigment contents. Cytogenetic toxicity was evaluated on Allium cepa L., and an anatomical analysis was performed on E. crassipes assayed in vivo.
Material and methods
Fungal isolation
Samples of diseased tridax daisy (Tridax procumbens L.) were obtained from the Ladkrabang area and fungal strains were isolated by the tissue transplanting method. Briefly, the leaves were thoroughly washed in running tap water to remove dust and debris, and the samples were allowed to air dry. The cleaned samples were then surface sterilized with 70% ethanol, followed by 3% NaOCl for 4 min, another immersion in 70% ethanol, and finally washed twice with distilled water. All sterilized samples were cut under sterile conditions into small pieces (1 × 1 cm). Each piece of infected tissue was transferred to a Petri dish containing water agar medium, then incubated at 25 °C for seven days until fungal mycelia were observed. The emergent hyphal tips of the fungi were cut and placed on potato dextrose agar (PDA) medium (Merck, Germany) to obtain pure cultures. The purified fungal isolates were then transferred to PDA slants and maintained at 4 °C until further use.
Molecular identification and phylogenetic analyses
The pure fungal isolate was identified using a molecular technique. Fungal mycelia were grinded to a fine powder using a sterile mortar and pestle. Fungal genomic DNA was isolated using the CTAB method20. Genes encoding the RNA polymerase second largest subunit (rpb2), translation elongation factor 1-alpha (tef-1), and calmodulin (cam) were amplified by polymerase chain reaction (PCR) in a thermal cycler (BioRad, USA) using the primers in Table 1. Products were electrophoresed on a 1.5% agarose gel in 1X TBE buffer, colored with loading dye, and visualized under ultraviolet light. Correctly amplified samples were sent to Marcogen, Inc. (Kumchun-Ku, Seoul, Korea) for purification and sequencing. To identify cultured fungi, rpb2, tef-1, and cam sequences were combined and similarity searches performed using BLAST against the available sequences at NCBI (https://blast.ncbi.nlm.nih.gov). Table 2 lists accession numbers for the sequence from this study and published sequences used for phylogenetic analysis. Multiple sequence alignment was performed with MUSCLE21 and improved where necessary using BioEdit v. 7.2 with sequences from the GenBank database. Phylogenetic analysis utilized the maximum likelihood (ML) method with combination datasets of rpb2, tef-1, and cam, and was performed in MEGA 11 with 1,000 bootstrap replications. The outgroup consisted of Fusarium camptoceras CBS 193.65 and F. neosemitectum CBS 115,476 within the F. camptoceras species complex (FCAMSC).
Preparation of fungal extract
Fusarium equiseti was cultured through submerged fermentation. Briefly, fungal disks (5 mm) were transferred to fermentation flasks containing autoclaved medium broth under aseptic conditions. The fermentation broth contained 50.0 g/L potato extract and 20.0 g/L glucose. The final pH was adjusted to 5.0 ± 0.2, then cultures were incubated at room temperature for 30 days. Mycelia were collected from the medium by filtering through a sterilized muslin cloth and then incubated at 45 °C in a hot-air oven (Binder World FP400UL-208V, Binder, Germany) for 48 h to air-dry. The resulting mycelium powder was extracted with 75% ethanol (ratio 1.0 g of dried mycelia: 20.0 mL of solvents) for 24 h, then ultrasonic oscillated for 30 min at 50 °C. The obtained supernatants were filtered through one layer of Whatman No. 1 filter paper (Whatman Inc., Clifton, USA). The resulting filtrate solutions were evaporated using a rotary vacuum evaporator (BUCHI Rotavapor R255, BUCHI, Lausanne, Switzerland) under partial vacuum at 45 °C to obtain the crude extract. Finally, the crude extract was redissolved in 75% ethanol, adjusted to 2% (stock solution), and stored at 4 °C until further experiment.
Identification of F. equiseti extract constituents by gas chromatography/mass spectrometry (GC/MS)
Constituents of the F. equiseti extract were identified using GC/MS. A Scion 436 gas chromatograph was coupled to a triple quad (Bruker, USA) mass spectrometer. Operating parameters were as follows: helium flow rate of 1 ml/min; detection range of 30–500 amu; starting oven temperature of 50 °C (2 min); ramping to 250 °C (20 °C/min); and then holding for 18 min. The HP-5MS capillary column (30 m, film 0.25 µm, ID 0.25 mm) was filled with 1 mL sample in splitless mode. The temperature of the transfer line was 250 °C and that of the ion source 230 °C. Individual ingredients were identified by comparison of obtained mass spectra (molecular mass and fragmentation pattern) with the internal reference library (National Institute of Standards and Technology, NIST, 2014). Components were quantified in terms of the percentage peak area relative to total peak area.
Leaf disk test
Water hyacinth was collected from the Ladkrabang district of Bangkok, Thailand, in October 2023. The research on this plant species has comply with relevant institutional, national, and international guidelines and legislation.
The stock solution (2% F. equiseti crude extract) was diluted with 75% EtOH to produce concentrations of 0.05%, 0.1%, and 0.2% w/v. The optimal concentration was observed in the range 0.01 to 1% w/v by no experimental design (data not shown). Distilled water served as a control. Water hyacinth leaves were cut into 24 disks (diameter = 0.6 mm) and transferred to 9-cm Petri dishes containing the treatment solutions. All dishes were incubated in dark boxes at room temperature (25–27 ºC) for three days. Visual toxicity symptoms were observed at intervals every day.
Visual toxicity symptoms were evaluated after one day. As per Rao25, symptoms were scored on a scale of 0–10 where 0 = none (without toxicity symptoms), 1–3 = slight effect (slight injury or discoloration and some stand loss), 4–6 = moderate effect (moderate injury), 7–9 = severe effect (serious damage, stand loss and almost total destruction), 10 = complete (plant death).
Three days after treatment, membrane integrity and photosynthetic pigment contents were assessed. The experiment utilized a completely randomized design with four replicates.
Membrane integrity
Electrical conductivity
Electrical conductivity was evaluated in fresh leaves of the treated weed according to the method reported by Singh, et al.26 with modification. In short, fresh leaf disks were floated on 10 mL of water for an hour at room temperature, the conductivity of the medium was measured (EC1), boiling at 100 °C was performed for 20 min, and then the conductivity was measured again (EC2). Measurement was conducted using a Consort C3010 multi-parameter analyzer (Consort, Belgium). The experiment utilized a completely randomized design with four replicates. Relative electrolyte leakage (REL) was calculated by the formula:
Lipid peroxidation
Malondialdehyde (MDA) is a free radical generated as the final product in the lipid peroxidation process27. Its abundance is measured in terms of the concentration of thiobarbituric acid reactive substances (TBARs), which therefore is used as an index of lipid peroxidation28. Briefly, 0.1 g of treated leaves were ground in 4 mL of 0.1% (w/v) trichloroacetic acid (TCA), then centrifuged at 6000 × g for 20 min, from which the supernatant was collected. A reaction solution was then prepared, consisting of 1 mL of supernatant, 4 mL of thiobarbaturic acid (0.5% w/v in 20% w/v TCA), and 0.4 mL of 4% butylhydroxytoluene. The obtained mixture was boiled at 95 °C for 30 min, then centrifuged at 6000 × g at 4 °C for 10 min. The absorbance of the supernatant was recorded at 532 nm, subsequent to subtraction of non‐specific absorption at 600 nm. Finally, the TBAR concentration was calculated using an extinction coefficient of 155 mM/cm and expressed as nmol/g fresh weight29. The experiment utilized a completely randomized design with four replicates.
Photosynthetic pigments
Chlorophyll a, b, and carotenoid contents were evaluated. A fresh leaf disk from water hyacinth was ground in 80% acetone and kept in the dark for 3 h. Afterwards, pigment contents were measured by determining absorbance with a UV/Vis spectrophotometer at 663, 647, and 470 nm and calculating concentrations according to Lichtenthaler’s equation30. The experiment utilized a completely randomized design with four replicates.
Cytogenetic assay
Equal-sized bulbs of Allium cepa L. were used for cytogenetic experiments. The experiment utilized a completely randomized design with four replicates. Emerged onion roots were treated with the F. equiseti extract (0.1% active ingredient). The optimal concentration was observed in the range 0.05–0.5% w/v by no experimental design (data not shown). After 18 h, the root tips were washed and hydrolyzed in an enzyme mixture comprising 8.0% w/v cellulase and 6.0% w/v pectinase in a 0.01 M acetate buffer (pH 4.5) for 40 min at 37 °C. For slide preparation, the meristematic region was squashed onto a drop of 2% Giemsa solution (Merck Co., Ltd.). Photomicrographs of the slides were captured at 10X magnification using an EP50 digital microscope camera (Olympus CX23, Japan) and the EPview software. The following parameters were determined: mitotic index, mitotic phase index, and chromosome aberrations.
In vivo E. crassipes assay and anatomical analysis
The aquatic macrophytes were carefully placed in buckets under natural conditions (average temperature 28–30 °C, natural light, relative humidity 64–69%). F. equiseti crude extract was applied manually by wiping leaf surfaces (adaxial and abaxial) and petioles with a cotton ball dipped in extract preparation (here 2.0%, 4.0%, and 8.0% w/v) so as to thoroughly coat the foliage. The optimal concentration was observed in the range 0.1 to 16% w/v by no experimental design (data not shown). Water and a surfactant solution were used as control agents. The experiment utilized a completely randomized design with ten replicates. After treatment, plants were continuously monitored for visual toxicity symptoms. Treated leaf samples were sectioned into thin pieces by hand using a razor blade. All samples were stained with 0.1% safranin for 10 min. The slides were examined and photomicrographs captured at 4× and 10× magnification using an EP50 digital microscope camera (Olympus CX23, Japan) and the EPview software.
Statistical analysis
The data were expressed as mean value ± standard deviation (SD). Tukey’s multiple range test (p < 0.05) was used to identify significant difference between means using SAS version 9.00.
Results and discussion
Fungal identification
The genus Fusarium (Nectriaceae, Hypocreales) currently comprises over 400 species organized into 23 species complexes31. A multi-gene molecular phylogeny is therefore essential to accurately identify Fusarium species32,33. The cam, tef-1, β-tubulin (tub2), rpb1, and rpb2 genes have been reported effective for this purpose34,35. NCBI-BLASTn analysis revealed the combined sequence to have the highest pairwise similarity with Fusarium equiseti. Figure 1 presents the phylogram obtained from phylogenetic analysis, which was constructed concordantly and is supported by previous studies33,36,37. Our TP01 fungal isolates sorted into the Equiseti clade of the Fusarium incarnatum-equiseti species complex, specifically clustering with F. equiseti.
Phylogram derived from ML analysis of the combined rpb2, tef-1, and cam sequences of 25 taxa. Numbers above branches represent BS percentages; values ≥ 50% are shown. Scale bar indicates the expected number of nucleotide substitutions per site. The red arrow highlights the sequence obtained in this study.
Identification of F. equiseti extract constituents by gas chromatography/mass spectrometry (GC/MS)
Table 3 illustrates the complexity of F. equiseti extract. GC/MS analysis identified 43 components constituting 96.758% of the total extract. The main constituents were c (13.684% of total), hexitol (10.009%), pentadecanoic acid, ethyl ester (5.947%), 2,3-dimethylfumaric acid (5.613%), 2(3H)-furanone, dihydro-4-hydroxy- (5.470%) and 2-propenamide, N-(1-cyclohexylethyl)- (5.318%). Previously, Hestbjerg, et al.19 reported primary constituents of fusarochromanone, equisetin, zearalenone, and type A and B trichothecenes. However, different media are known to support production of different metabolites19. Moreover, extract composition can be influenced by diverse factors including species, climatic and environmental circumstances, and extraction technique7.
Reports indicate 2,3-dimethylmaleic anhydride to have insecticidal and antifungal properties38,39,40. Moreover, Rajashekar, et al.39 suggested that 2,3-dimethylmaleic anhydride-based natural products qualify as grain protectants because they are less hazardous to humans, and hence F. equiseti extract containing 2,3-dimethylmaleic anhydride may save human health and crop plants. In this work, the phytotoxic activity discovered in the current study may be connected to the abundance of these compounds and potential synergistic interactions.
Visual toxicity
Visual toxicity of F. equiseti crude extract on water hyacinth was evaluated for three days after application. The greatest toxicity score was obtained from the highest concentration (Fig. 2). This echoes the finding of Taban, et al.41 that visual toxicity score increases with the concentration of herbicidal agent. By day 3, leaf disks treated with 0.1% w/v and 0.2% w/v became yellow with dark brown edges (Fig. 3). Overall, disks treated at the highest concentration of 0.2% w/v exhibited necrosis, chlorosis, and leaf burning. Meanwhile, control disks changed only slightly, exhibiting some yellow color.
Mean visual toxicity scores of water hyacinth leaf disks treated with F. equiseti crude extract at different concentrations (% w/v). The same letters are not significantly different at p < 0.05.
Photo showing visual effect of F. equiseti crude extract on water hyacinth leaf disks at 3 days after treatment.
Electrical conductivity
Membrane damage was evaluated by measurement of electrical conductivity, which showed F. equiseti crude extract to induce electrolyte leakage (Fig. 4), significantly so at concentrations of 0.1% w/v and 0.2% w/v (p < 0.05). Necrosis was observed in treated leaves, and may be caused by electrolyte and cell component leakage leading to cell death42. Interestingly, the crude extract may increase membrane permeability, resulting in loss of membrane function. Dayan, et al.43 previously reported on the biological properties of sarmentine from Piper species as a contact herbicide, showing it to induce loss of plasma membrane integrity and consequently inhibit photosynthesis. Dayan and Watson44 also described membrane peroxidation caused by reactive oxygen species (ROS) to result in membrane failure. In addition, plant growth can be inhibited by increasing REL5. Therefore, F. equiseti crude extract may inhibit plant growth of water hyacinth.
Effect of F. equiseti crude extract on relative electrolyte leakage (%) from water hyacinth leaves. Means with the same letters are not significantly different at p < 0.05.
Lipid peroxidation
Lipid peroxidation is a biochemical marker of free-radical-mediated damage28, which can lead to tissue damage45. When membrane lipids are peroxidized, malondialdehyde (MDA) is generated as a secondary end product. MDA reacts with molecules of thiobarbituric acid, and hence the MDA or thiobarbituric acid reactive substances (TBARS) assay is commonly used to evaluate lipid peroxidation46. Accordingly, lipid peroxidation in water hyacinth leaf disks was measured as MDA content (Fig. 5), which increased significantly with extract concentration. Our results are in line with a study by Laosinwattana, et al.47 that showed natural herbicides from plant essential oils containing a variety of monoterpene active compounds to induce lipid peroxidation in Echinochloa cruss-galli (L.) Beauv. leaves, resulting in degradation of cell membranes. Lipid peroxidation in plants can damage integrity of membrane components and cause cell death27,42,48, particularly as loss of lipid bilayer integrity results in uncontrolled electrolyte leakage44. Water hyacinth demonstrated sensitivity to the cell damage by the extract, as evidenced by the significantly higher increase in MDA content in the leaf, which led to a bigger reduction in the plant's growth49.
Effect of F. equiseti crude extract on MDA content in water hyacinth leaves. Means with the same letters are not significantly different at p < 0.05.
Photosynthetic pigments
Chlorophyll a, b, and carotenoid pigments are fundamental to the photosynthetic conversion of light energy to chemical energy50. We observed treatment with F. equiseti crude extract to decrease pigment contents in a dose-dependent manner (Table 4). Notably, for carotenoids, which protect the photosystems against photodamage51, only the highest treatment concentration (0.2% w/v) had a significant effect. Overall, pigment decreases correlated with visual symptoms (Fig. 3); the chlorosis symptoms may be attributable to destruction of chlorophyll by the F. equiseti crude extract.
Photosynthetic pigments are localized in chloroplast membranes51; hence, it is also possible that the extract induced chloroplast membrane damage. Mahdavikia et al.52 described that altered membrane permeability could disturb physiological and biochemical functions. In particular, membrane leakage may result in increased lipid peroxidation, impeding cell growth and leading to cell death. Rai et al.53 further reported that lipid peroxidation leads to degradation of pigments.
Effect on mitosis index and chromosomal aberration
Table 5 summarizes the mitotic parameters observed following treatment of onion roots with F. equiseti extract. The mitotic index, which is equal to the number of cells in mitotic phases, can be used as an indicator of cell growth. The control sample displayed a mitotic index of 7.23% with normal distribution of divisional stages. Application of 0.1% F. equiseti extract resulted in a 72.06% reduction in the mitotic index (2.02%), with the fraction of cells in prophase increasing significantly (p < 0.05), making it the most prominent stage. The percentage of cells entering metaphase was not affected, but the fractions in anaphase and telophase were decreased. The reduction of mitotic activity may be related to the inhibition of DNA synthesis or nucleoprotein synthesis in the cell cycle. The accumulation of cells at prophase suggests the extract to disrupt cell division and reduce the number of cells entering the mitotic phase, which is supported by previous publications54,55.
Overall, 2.56% of dividing cells in treated roots were found to exhibit chromosomal aberration. Photomicrographs illustrating specific aberrations are presented in Fig. 6. A few occurrences of condensed nuclei (Fig. 6B) were observed in control and surfactant treatments. Meanwhile, roots treated with F. equiseti extract exhibited binucleate cells, spindle disturbance, C-mitosis, anaphase bridge, diagonal anaphase, and aberrant telophase (Table 6). Chromosomal aberration was predominantly seen in interphase (1.79%) and aberrant prophase (0.43%), consistent with the phase index values (Table 5). These aberrations may relate to the inhibition of DNA replication or nucleoprotein synthesis in the cell cycle56 and disrupt entrance into the next phase.
Representative images of the chromosomal aberrations induced in onion root tips by F. equiseti extract: (A) binucleate cell (asterisk); (B) normal and condensed nucleus (arrow); (C) spindle disturbance at late prophase; (D) C-mitosis; (E) anaphase bridge and diagonal anaphase; (F) aberrant telophase with precocious chromosomes (arrow).
Mechanistically, the chromosomal abnormalities may be explained by biochemical alterations in the chromosomes, spindle fibers, or cytoplasm of dividing cells. Binucleated cells (Fig. 6A) and condensed nuclei (Fig. 6B), which involve changes in nucleus structure, are related to the programmed cell death that occurs in response to abiotic stress57. Spindle fibers play crucial roles in chromosome movement and separation; as such, changes in fiber structure, such as spindle protein depolymerization, typically result in spindle disturbances (Fig. 6C), shifts in division poles, and the formation of multiple poles (Fig. 6E, diagonal anaphase)58. Alterations in cytoplasmic viscosity have also been linked to spindle assembly, leading to the formation of C-metaphase (Fig. 6D)59. In addition, the presence of C-mitosis could be the result of the complete inactivation of mitotic spindles. Additionally, aneuploidy and polyploidy may be induced at the final stages of cell division54. Another factor that can cause difficulty in chromosomal separation are improperly folded “sticky” chromosomes, where cross-linking of chromosomal proteins causes difficulty of separation60,61 and leads to formation of chromosome bridges (Fig. 6E). The translocation of the dicentric chromosomes may also cause metaphase, anaphase, and telophase bridges, potentially causing unequal exchange and structural mutations within the chromosomes62. Finally, presence of precocious chromosomes (Fig. 6F) at telophase is linked to laggard chromosomes63 that fail to combine into daughter nuclei, an abnormality that ultimately causes formation of micronucleated cells. Abnormal chromosomal structures could cause cell division to cease. Plant growth depends on these division processes; therefore, any interference with cell division activity eventually results in reduced plant growth.
Effect on E. crassipes leaf macro- and microstructure
Figure 7 shows E. crassipes plants treated with F. equiseti extract, which exhibited increasing symptoms corresponding to the extract concentration. Injuries to foliar tissues, such as necrosis and depigmentation, were visible as early as 24 h after herbicide application. Phytotoxic symptoms observed included leaf burning and folding, wilting, chlorosis, and necrosis. At the lowest concentration (2.0% w/v), plants displayed minor signs of injury and discoloration. Although the visual toxicity was overall mild, degraded tissue and bruising areas were evident, illustrated by the twisted leaf blade (Fig. 7B; red arrow); viewing of the adaxial surface under a stereomicroscope revealed bruising and eroded epicuticular epidermal tissue (Fig. 7F). These effects are in agreement with prior reports that herbicidal fungal extracts result in foliar lesions and occasional yellowing64,65. Additionally, degradation of epidermal cells and reduction of epidermal thickness have been reported in studies on chemical herbicides66,67. This phenomenon could potentially enhance penetration of the extract, leading to degradation or damage of ground tissue. Such loss of supportive tissue results in a non-uniform arrangement of vascular tissue, as depicted in Fig. 8C, with disorganized vascular bundles and larger aerenchyma chambers (Fig. 8C and D). These observations align with the findings of Pereira, et al.68, who demonstrated similar effects following application of sethoxydim to brachiaria grass.
Phytotoxicity symptoms observed in E. crassipes plants exposed to F. equiseti extract at 24 h after treatment: (A–D), visual symptoms; (E–H), stereomicroscopic views of adaxial surface in green leaf area (1X). Red arrow indicates a twisted leaf blade.
Phytotoxic effect of F. equiseti extract on E. crassipes leaf anatomy 24 h after treatment.
In conjunction with the mitosis herbicidal activity described above, our observations of toxicity symptoms in treated plants support that the F. equiseti extract, with its complex mixture of phytotoxic compounds, can have multiple modes of action. Similarly, Manichart, et al.54 studied the effect of Diaporthe sp. ethyl acetate extract applied to Amaranthus tricolor in concentrations of 0.187–0.750 mg/mL, and through via germination and initial growth assays found it to have anti-mitotic and enzyme inhibitory activities.
Conclusion
The present study characterized a natural herbicide based on F. equiseti crude extract for controlling the aquatic weed water hyacinth. The results showed the extract to induce toxicity symptoms, loss of photosynthetic pigments, and failure of membrane integrity in E. crassipes leaves. In addition, the extract affected mitosis index and chromosomal aberration in Allium cepa L. As a result, F. equiseti crude extract demonstrated its multiple modes of action on the treated plant. Developing natural herbicides and providing farmers with a safer alternative could benefit greatly from the crude extract. Thus, this crude extract is a candidate for the production of natural herbicides for sustainable agriculture. Further improvement of the F. equiseti crude extract is necessary to enhance its herbicidal potential and stability in formulation. However more research is needed to determine whether using the extract to manage weeds under various hydrogeological and geochemical conditions in the field is efficient, effective, does not harm non-target species, and is practically feasible economically.
Data availability
DNA sequence data of Fusarium equiseti TP001 from this research have been deposited in the NCBI repository (https://www.ncbi.nlm.nih.gov/) with the following accession numbers: rpb2 (PP266450), tef-1 (PP266449), and cam (PP314014).
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Acknowledgements
This research was supported from “National Science, Research and Innovation Fund (NSRF)” (Grant number RE-KRIS/FF66/05) from King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand.
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Laosinwattana, C., Manichart, N., Thongbang, M. et al. The effect of natural herbicide from Fusarium equiseti crude extract on the aquatic weed water hyacinth (Eichornia crassipes (Mart.) Solms). Sci Rep 14, 19542 (2024). https://doi.org/10.1038/s41598-024-70694-y
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DOI: https://doi.org/10.1038/s41598-024-70694-y
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