Non-Toxic Bioherbicides Obtained from Trichoderma koningiopsis Can Be Applied to the Control of Weeds in Agriculture Crops

Abstract

This study aims to evaluate the production of compounds (fungal extract) from Trichoderma koningiopsis with herbicidal activity, as well as to analyze the effects of its application and acute toxicity using the microcrustacean Daphnia magna. Plackett & Burman Experimental Design (PB) was selected, followed by a triplicate fermentation with the best conditions after a Central Composite Rotatable Design (DCCR 2³). The dependent variables (responses) evaluated were the enzymatic activities of amylases, cellulases, lipases and peroxidases, and the fungal biomass produced was also quantified. A statistical analysis was performed on the variables to verify their significance and evaluate the validity of the proposed model. Two different fungal extracts were used for evaluation of acute toxicity, and were also applied in the Euphorbia heterophylla plant to verify the effects of phytotoxicity. Thus, the results obtained for the acute toxicity tests were EC_50-48h of 58,637.99 mg/L and 13,583.17 mg/L for the first and second extract, respectively. The first one showed yellowing of the leaves of the plant. The results showed that the produced compounds are promising alternatives for reducing the use of chemical herbicides, since the microorganism showed potential for the biological control of weeds.

Introduction

The projected growth of the world population will require an increase in agricultural production, leading to an incessant search for new management technologies that increase productivity. At the same time, environmental degradation is being accelerated due to the indiscriminate and continuous use of herbicides, reducing harvest efficiency.^1
–3 In addition, the excessive use of pesticides leads to serious soil pollution and deteriorates the quality of the environment as they are dispersed through water, air, and soil.^3

–6 Traditional chemical control options are limited due to ecodegradation, health hazards, and the development of herbicide resistance in weeds.⁷ Herbicide-resistant weeds are the main problem in weed control, as a number of weed biotypes have become increasingly resistant to herbicides due to the continuous use of the same products for years.⁸ In the last 20 years, no chemicals with a different mode of action has been synthesized.⁹ Such compounds have the potential to be models for developing herbicides with new modes of action.^10,11

Because of the negative consequences of using chemical herbicides, it is important to consider the sustainable management of weeds integrated with new tools such as biological herbicides.^12,13 The use of herbicides produced by microorganisms offers other benefits such as reducing impacts on the environment, promoting sustainable agriculture, and reducing dependence on synthetic herbicides.¹³ Bioherbicides are obtained through living organisms, most of which are microbial (fungi and bacteria), minerals, or plant-based products.¹⁴ Phytopathogenic fungi produce toxins that may play a role in the development of plant diseases. Weeds are a significant problem in crop production, and their management is crucial for modern agriculture to avoid yield losses and to ensure food safety.¹⁵

Enzymes produced from the bioherbicide-formulation process, such as lipases or cellulases, can be a means of entry for these microorganisms or phytotoxins in the target plants, but may degrade the lipid membrane and the cell wall, so that process must also be studied.⁹

Viable alternatives to the unregulated use of synthetic herbicides—from both an environmental and an economic point of view—is necessary to ensure the sustainable management of weeds. The objective of this study was to optimize the production of composites via submerged fermentation using the fungus Trichoderma koningiopsis, for use as control of weeds in agricultural crops. In addition, we evaluated their phytotoxicity, through applications in the Euphorbia heterophylla plant, and we determined the acute toxicity of these bioproducts using the microcrustacean Daphnia magna.

Material and Methods

Microorganism

The microorganism T. koningiopsis was selected for use in this work because it presented promising results in previous studies carried out by the research group (data not shown), especially for its phytotoxic effects in the E. heterophylla plant.

To perform the analyses, a series of tests of the microorganism were carried out in petri dishes containing Potato Dextrose Agar (PDA) culture medium for fungus growth. Afterwards, they were incubated in a bacteriological incubator for a period of 7 d at a temperature of 28°C.

Production Optimization of Composites

For the production optimization of composites with herbicide potential, a sequence of experimental designs was proposed. The experiments were designed by the Plackett & Burman (PB) methodology with 12 trials and more than 3 central points. The experiments investigated pH (5.0–7.0), glucose (0.75–2.25 g/mL), yeast extract (0.75–1.50 g/mL), peptone (0.75–2.25 g/mL), ammonium sulfate ((NH4)₂ SO₄) (0.00–0.15 g/mL), magnesium sulfate (MgSO₄.7H₂O) (0.00–0.15 g/mL), ferrous sulfate (FeSO₄.7H₂O) (0.00–0.30 g/mL), and manganese sulfate (MnSO₄.H₂O) (0.00–0.30 g/mL). Then, fermentation with the conditions that presented the best results in PB was conducted in triplicate, with the fixed conditions of pH (6.00), glucose (1.50 g/mL), yeast extract (1.12 g/mL), peptone (1.50 g/mL), MgSO₄.7H₂O (0.07 g/mL), and MnSO₄.H₂O (0.15 g/mL). After this step, the Central Composite Rotational Design with three investigated factors (DCCR 2³) was carried out, which was composed of the factorial points, 6 axial and 3 repetitions of the central point, totaling 17 trials. The parameters evaluated in this last plan were glucose (0.99–3.51 g/mL), yeast extract (0.24–2.76 g/mL), and peptone (0.12–1.30 g/mL).

Submerged fermenation was used to obtain the enzymatic extract and the fungal biomass. The different proportions presented for each plan were added to Erlenmeyer flasks (300 mL), which were then supplemented with distilled water until a volume of 150 mL of culture medium was reached. The pH of the assays was adjusted according to the experimental design.

The culture media was autoclaved at a temperature of 121°C for 30 min. After reaching ambient temperature, they were placed in the laminar air flow cabinet, and a small amount of this medium was spilled into the petri dishes containing the already isolated T. koningiopsis. The spores were scraped with the aid of a platinum handle, and afterwards, added again to the Erlenmeyer flasks.

The fermentations were carried out at a temperature of 28°C in an orbital shaker (New Brunswick ™ Innova^® 42) under agitation of 120 rpm for 72 h. The medium obtained after the fermentation was filtered to obtain the fungal biomass and enzyme extract. The extract was stored in polyethylene packages and kept in a refrigerator at a temperature of approximately 5°C.

Quantification of Fungal Biomass

To quantity the fungal biomass of T. koningiopsis, the mass method of dry mycelium per mL of culture medium was used.

Enzymatic Activity Determination

The enzymatic activity was determined for amylases^16,17 and cellulases¹⁸ by measuring the release of reducing sugars using the dinitrosalicylic acid (DNS) method¹⁹ (Equation 1): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \left[ { \ } \right] ART = \frac { ABS } { { 0 , 2672 } } \tag { Equation \ 1 } \end{align*} \end{document}

The activity of lipases²⁰ was determined using Equation 2, and the activity of peroxidases²¹ was used to calculate the mean of the values of the triplicate multiplied by 1,000. \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} At = { \frac { \left( { \bar Aa - Vb } \right) *MNaOH } { t*Vc } } *1000 \tag { Equation \ 2 } \end{align*} \end{document}

where \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\bar Aa$$ \end{document} = mean values observed, Vb = value presented by control, MNaOH = molarity of sodium hydroxide, t = reaction time, and Vc = enzymatic volume used.

To determine the enzymatic activities, triplicates of each assay were performed, and the unit (U/mL), by definition, was the amount of enzyme capable of catalyzing the reaction with formation the 1 μmol/min of product under the reaction conditions.

Evaluation of Acute Toxicity

The evaluation of the toxicity of the composites was performed through the acute tests using the microcrustacean Daphnia magna, according to NBR 12,713.²² The tests had a duration of 48 h; young individuals (2–26 h of life) of the microcrustacean were placed in containers, and for 24–48 h, how many organisms were immobile was recorded. The tests were performed in duplicate, and in each container with capacity 50 mL, dilutions were added with a maximum volume of 25 mL. The tests were composed of a control and the dilutions 1:1, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128.²³

Preliminary Application of Biocomposts

The extract obtained after the fermentation process, in its crude and filtered form, was administered in the E. heterophylla plant with applications of up to three replicates. The filtered extract was sprayed with the aid of a garden sprayer, and the crude extract was applied with the aid of a brush applied directly to the aerial part of the plant. The analyses were performed visually, after 7 and 14 d of application. Three evaluators attributed percentage grades ranging from 0 to 100%, where 0% corresponds to no phytotoxicity and 100% corresponds to complete death of the plant, according to Brzilian Society of Weed Science methodology.²⁴

Analysis of Data

The data obtained from the fermentation assays were analyzed using the STATISTICA 8.0 Software through the analysis of variance (ANOVA). GraphPad prism Software was used to analyze acute toxicity.

Results and Discussion

Optimization of the Fermentation Process

Experimental design using Plackett & Burman (PB)

The results obtained in the first experiment showed that all the tests presented different responses to the evaluated parameters (data not shown). Thus, it can be affirmed that the quantity of each component present in the culture medium has a different influence on the activity of the enzymes analyzed, and also in the production of fungal biomass. This is because the enzymes are responsible for regulating the metabolism of microorganisms, and they can modify their metabolism in response to changes in the environment.^25
–27 Statistical treatment of the data verified that, for almost all the parameters, the salts MnSO₄.H₂O and MgSO₄.7H₂O presented negative and positive significance (p < 0.05), respectively, while the salt (NH₄) 2SO₄ had no significance (p > 0.05) for any of the parameters. However, this type of design is a fractional factorial, so not all possible combinations between variables are made.

Thus, a new fermentation in triplicate was proposed, where the salts (NH₄)₂SO₄ and FeSO₄.7H₂O have been removed and fixed values were established for the conditions of the variables that presented the highest results. Values were lower than the first planning, so a complete factorial design was chosen, since it provided all the possible combinations between the investigated factorial levels. However, the values obtained were lower than the first planning; in this way, a complete factorial design was chosen to provide all the possible combinations between the investigated factorial levels.

Central Composite Rotational Design (CCRD 2³)

The CCRD 2³ had pH, (NH₄)₂SO₄, and FeSO₄ 7H₂O values fixed at 5.0, 0.60 g/Ml, and 0.30 g/mL, respectively, and the MgSO₄ 7H₂O and MnSO₄ H₂O salts removed after evaluation of the preceding tests. In this way, we tried to evaluate the effects of carbon and nitrogen sources²⁸ on the production of fungal biomass and on the activity of the mentioned enzymes, looking for the possibility of applying the composites. The values for the enzymatic activities and the quantification of the fungal biomass resulting from the DCCR 2³ design are presented in Table 1.

Table 1.

CCRD 2³ Matrix Experimental (Real and Coded Values) Used to Optimize the Production of the Biocomposite; Results of the Enzyme Activity for Amylases, Cellulases, Lipases, and Peroxidases; and Quantification of Fungal Biomass Obtained

ASSAY	GLUCOSE	YEAST EXTRACT	PEPTONE	AMYLASES (U/mL)	CELLULASES (U/mL)	LIPASES (U/mL)	PEROXIDASES (U/mL)	BIOMASS (g/L)
1	(1.50) −1	(0.75) −1	(0.37) −1	3.45	6.51	1.89	36.33	1.44
2	(3.00) +1	(0.75) −1	(0.37) −1	17.25	10.62	3.22	41.00	1.85
3	(1.50) −1	(2.25) +1	(0.37) −1	0.36	0.16	0.61	116.33	7.97
4	(3.00) +1	(2.25) +1	(0.37) −1	0.82	0.38	8.22	56.00	7.43
5	(1.50) −1	(0.75) −1	(1.12) +1	9.06	5.15	3.62	46.33	2.18
6	(3.00) +1	(0.75) −1	(1.12) +1	14.43	8.53	0.00	60.00	2.37
7	(1.50) −1	(2.25) +1	(1.12) +1	0.65	0.21	0.00	125.67	9.71
8	(3.00) +1	(2.25) +1	(1.12) +1	0.71	0.36	0.00	90.67	9.81
9	(0.99) −1.68	(1.50) 0	(0.75) 0	5.94	3.08	0.00	54.00	3.43
10	(3.51) +1.68	(1.50) 0	(0.75) 0	12.64	7.59	0.00	73.33	6.80
11	(2.25) 0	(0.24) −1.68	(0.75) 0	12.11	6.76	0.05	44.00	1.57
12	(2.25) 0	(2.76) +1.68	(0.75) 0	0.79	0.34	0.41	127.00	9.25
13	(2.25) 0	(1.50) 0	(0.12) −1.68	11.57	7.49	1.89	71.33	2.38
14	(2.25) 0	(1.50) 0	(1.30) +1.68	0.68	0.34	0.00	169.00	6.17
15	(2.25) 0	(1.50) 0	(0.75) 0	9.76	6.37	0.00	78.00	4.60
16	(2.25) 0	(1.50) 0	(0.75) 0	0.53	0.35	0.00	132.33	5.58
17	(2.25) 0	(1.50) 0	(0.75) 0	8.38	4.55	0.00	72.67	3.62

Assay 8 was responsible for higher production of fungal biomass; this may be related to the composition of the medium since it was supplemented with higher amounts of glucose, yeast extract, and peptone, causing a high availability of carbon, amino acids, and nitrogen to the microorganism.²⁹ As for the enzymatic activities, the tests that presented the greatest responses were the amylase and cellulase Assay 2, composed of the highest concentration of glucose; Assay 4 for lipases, composed of the highest concentrations of glucose and yeast extract; and Assay 14 for peroxidases, composed of the highest concentration of peptone. As in PB design, none of the Assays excelled in all evaluated parameters. However, that enzymatic activities were not high in some Assays stands out; the large range of activities for the enzymes studied in all the biocomposites could inficate potential for various applications in the degradation of substrates.

Table 1 also shows that tests that presented greater amounts of fungal biomass and enzymatic activity for peroxidases also presented greater responses of enzymatic activity for amylases and cellulases. This may indicate that, as fungi do not have the capacity to degrade all the compounds of fermentation media, they produce extracellular enzymes to help in this task.³⁰

The data were analyzed statistically, and it was possible to identify which were the significant and not significant variables for the tests. It was also possible to check the validation of the proposed empirical models. Table 2 presents the validated models for the production of fungal biomass and cellulase activity.

Table 2.

Validated Models for the Production of Fungal Biomass and Enzymatic Activity of Cellulases After Statistical Analysis

	MODEL	R²	Fcalc/Ftab
Production of fungal biomass	4,57 + 0,42^v1 + 0,27^v1^v1 + 2,93^v2 + 0,37^v2^v2 + 0,86^v3-0,03^v3^v3-0,13^v1^v2 + 0,05^v1^v3 + 0,35^v2^*v3	0.93	2.71
Enzymatic activity for cellulases	3.92 + 0,98^v1 − 3.71^v2-0,43^v3 -0,89^v1^v2-0,10^v1^v3 + 0,44^v2^*v3	0.87	73.80

v1 = glucose; v2 = yeast extract; v3 = peptone.

The production of fungal biomass had its model validated with a confidence of 95%, and a coefficient of determination (R²) 93%, showing a good adjustment of data to the proposed model. The production of fungal biomass is thought to be closely related to the amount of yeast extract and peptone present in the fermentation medium, as two variables showed a positive (p < 0.05) significance. Therefore, it was verified that the best conditions for the production of biomass occurred in the maximum values of the three variables: yeast extract (2.76 g/mL), glucose (3.51 g/mL), and peptone (1.30 g/mL).

Thus, it is possible to observe the significant positive effect of nitrogen sources (yeast extract and peptone), emphasizing that high concentrations of these products cause increased biomass production. In a study³¹ on the production of mycelial biomass, it was found that peptone and maltose generate a significant positive effect, with the effect of peptone concentration being more important than of maltose.

The model of cellulase activity was validated with 90% confidence without using the axial points—generating a first order model. The enzymatic activity of cellulases was observed to be related to the amount of yeast extract present in the fermentative medium, and its significance (p < 0.1) is negative. Cellulase activity was highest in high concentrations of glucose (3.51 g/mL) and at the lowest concentrations of yeast extract (0.24 g/mL) and peptone (0.12 g/mL).

Cellulases are used more often than other enzymes to protect crops from plant pathogens by degrading cell walls.³² Some microorganisms, such as Trichoderma sp., ³³ produce a variety of enzymes, such as cellulases, responsible for the degradation of polysaccharides. These polysaccharides include cellulose, which plays a structural role and promotes mechanical resistance.³⁰ With the degradation of the main constituent of the plant's cell structure, entrance of the microorganism, or the secondary metabolites produced, is facilitated and increases the phytotoxicity of the composites.

In contrast, the mathematical models for the activity of amylases, lipases, and peroxidases were not validated when using a confidence of 95%. Thus, the Pareto Graph presented in Fig. 1 was generated to identify significant effects.

Fig. 1.

Pareto graphs generated to indicate the effects of the variables investigated on the activity of the enzymes (a) amylases, (b) lipases, and (c) peroxidases.

The activity of amylases showed a significant (p < 0.05) negative effect on the yeast extract. Amylases have the ability to catalyze hydrolysis of the starch into sugars,³⁴ since the starch³⁵ is present in the plant tissues as a source of energy storage. Thus the enzyme acts in the degradation of starch, demonstrating the benefit of the biocomposites. High activity values for this enzyme indicate the efficiency for the hydrolysis of starch in storage tissues and plant leaves, and these enzymes, produced by fungi, stand out in industrial applications and encourage further studies.³⁶

Lipase activity is linked to the amount of glucose, peptone, and yeast extract present in the medium, with peptone concentration being negative (p < 0.05). The relationship between glucose and peptone is likewise negative, but the relationship between glucose and yeast extract is positive.

The cell wall of plants contains organic substances such as lignin, lipids, and proteins, as well as lipid substances such as waxes that make the cell wall a hydrophobic barrier.³⁷ The fatty acid composition of the cell wall is responsible for protecting the plant against pathogens, and its degradation with enzymes is vital for the process of infection.³⁸ Lipases are responsible for lipid metabolism in living organisms, and in biocatalysis, these enzymes are of great interest because they have a high biotechnological potential.³⁹ Furthermore, lipase enzymes can degrade the plasma membrane, which controls the entry and exit of substances from the cell.²⁸

With the activity of peroxidases, it is possible to verify that the yeast extract presented a positive (p < 0.05) significance. In addition, the salts present in the medium also may have influenced the activity of this enzyme, because, when tested, the influence of solutions containing FeSO₄ and K₂SO₄, were induced by iron ions.⁴⁰ Peroxidases are appropriate for composites, since they are oxidative enzymes responsible for the oxidation of peroxides. Many authors report the enzymes degrades lignin present in the cell wall of plants, which are responsible for the structural support to the secondary wall and provide greater resistance against microbial aggression.⁴¹

Peroxidases in biocomposites can also be used for the deactivation of mycotoxins, such as deoxynivalenol (DON), and are responsible for the destruction its epoxy ring.¹⁶ In this case, the enzyme would prevent the action of mycotoxin, not induce toxicity through the biocomposite.⁴¹

In biomass production, peroxidases showed significant positive effect in the presence of yeast extract. If the production of the biocomposite in test 12 was encouraged, it is possible to increase the amount of yeast extract in the fermentation medium and direct its application to the control of plants that present peculiarities compatible with this enzyme. The activities of amylases and cellulases, demonstrated negative significance for the yeast extract. The biocomposite in test 2, which showed higher activity responses to these enzymes, could be stimulated for application in plants that are influenced by this composition. Thus, tests 2 and 12 presented better characteristics to be used as bioherbicide and were selected for acute toxicity evaluation and applied to the E. heterophylla plant to verify the effects of phytotoxicity.

Evaluation of acute toxicity

The results found in this evaluation expressed the effective concentration of the composites that caused immobility to 50% of the D. magna population exposed during the 48 h (EC_50-48h). Therefore, the result obtained for the EC_50-48h of the biocomposite of test 2 was 58,637.99 mg/L with a dilution factor of 32, and for test 12 was 13,583.17 mg/L with a dilution factor of 128. The lower EC_50-48h values indicate greater toxicity to the test organism.

In this sense, when comparing the values of EC_50-48 presented in this work with a diversity of commercial herbicides, a considerably lower acute toxicity of the composites is seen, demonstrating the environmental viability of the produced bioherbicides. It is also important to discuss the ecotoxicological effects of bioherbicides, since this information allows optimal application conditions, as well as the ability to develop and encourage the use of these bioproducts.³⁵

Preliminary application of composites

Phytotoxicity effects of up to 90% were observed in the leaf area of E. heterophylla that obtained applications with three replicates of the crude test 2 biocomposite. However, when filtered, phytotoxicity was not present. The biocomposite in test 12, when applied in its crude form, presented phytotoxicity of up to 60% using three replicates of applications. Unlike the other biocomposite, it presented a 50% phytotoxicity when filtered. This verifies that the crude composites presented greater phytotoxicity and that the microorganism is the agent of the damages; once filtered, the observed effects were not the same. In addition, it was observed that the greatest damages were caused when using the largest repetitions of applications; the species E. heterophylla is difficult to control, as it is resistant to some herbicides.⁴³ Many applications are required, as well as combinations of herbicides.

Conclusion

It was possible to optimize and develop the composition of a fermentative medium using the microorganism T. koningiopsis to be applied as a bioherbicide, as well as to verify the effects of phytotoxicity and analyze acute toxicity.

As enzymatic activities were registered for the different enzymes analyzed in the composites, they acted together in the test plant to degrade the substrates and allow the microorganism to act. This can be verified through the preliminary application, where the bioherbicide effects were confirmed, causing the yellowing of the leaves of the E. heterophylla plant. In addition, the acute toxicity presented by the two composites analyzed was considerably lower than that reported for commercial herbicides used in weed control, confirming their environmental functionality.

Footnotes

Acknowledgments

The authors thank National Council for Scientific and Technological Development (CNPq) and Foundation for Research Support of the State of Rio Grande do Sul (FAPERGS) for financial support of this work.

Author Disclosure Statement

No competing financial interests exist.

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