Selection of filamentous fungi that are resistant to the herbicides atrazine, glyphosate and pendimethalin

. The objective of the present study was to isolate fungi from agricultural soils and evaluate fungal growth in culture medium contaminated with atrazine, glyphosate and pendimethalin. Filamentous fungi were isolated from agricultural soils and cultured in a modified culture medium containing 0, 10, 20, 50, and 100 μ g mL -1 atrazine, glyphosate and pendimethalin for 14 days at 28°C. The fungi that presented optimal and satisfactory growth were plated in Sabouraud culture medium with 4% dextrose and containing the herbicides at concentrations of 0, 10, 20, 50, and 100 μ g mL -1 for seven days at 28°C. The mean mycelial growth values were submitted to analysis of variance and the Tukey test (p < 0.05%) for comparison and relative growth determination, and maximum inhibition rates were calculated. The isolated fungi Aspergillus fumigatus , Fusarium verticillioides and Penicillium citrinum were shown to be resistant to atrazine, glyphosate and pendimethalin. F. verticillioides showed higher mean mycelial growth in the culture media contaminated with atrazine and glyphosate than the other two fungi. In the culture medium contaminated with pendimethalin, F. verticillioides , and A. fumigatus presented the highest mean mycelial growth values.


Introduction
Herbicides are most commonly applied in vegetable production systems; approximately 2.5 million tons of pesticides are used annually in the world, and in Brazil, the annual consumption has exceeded 300 thousand tons (Moraes, 2019).The high consumption of pesticides is based on the agricultural improvements that these products present, such as a decrease in invasive plants and increased economic efficiency in the production process.
Despite the benefits, the final location of these products is the soil, and they may have negative impacts, such as intoxication to crops cultivated in succession, organic matter degradation, surface and groundwater eutrophication and loss of essential soil functions, including nutrient cycling and environmental buffering power.There may also be a reduction in microbiota and biodiversity (Moreno-Mateos, Meli, Vara-Rodríguez, & Aronson, 2015).
To restore soils that have become polluted and unproductive by herbicide addition, it is necessary to remove contaminants from the area.Among the technologies available for this purpose, we highlight bioremediation that consists of ecological technology to accelerate rates of natural degradation through plants, algae, bacteria and fungi (Alegbeleye, Opeolu, & Jackson, 2017;Khayati & Barati, 2017).
Fungi play an important role in bioremediation due to their metabolic ability to degrade different toxic and recalcitrant compounds.They show efficiency in degradation due to their ability to adapt their metabolism to different carbon sources.This metabolic flexibility occurs due to the production of enzymes, such as oxidative and hydrolytic enzymes, that degrade compounds, including polycyclic aromatic hydrocarbons, plastics (PET) and pesticides, such as atrazine, glyphosate and pendimethalin (Kanagaraj, Senthilvelan, & Panda, 2015;Deshmukh, Khardenavis, & Purohit, 2016).
Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) is a pre-emergent and selective systemic herbicide, an important representative of the triazine group.When applied for many years, atrazine and its residues accumulate in agricultural soils and can be mobilized through cycles between dry and wet periods and then leached.Therefore, considering the high amounts applied annually, together with research demonstrating its high persistence in soils, atrazine represents a potential threat to the environment (Peng et al., 2018;Zhao, Feng, Hu, Wang, & Lu, 2019).Some microorganisms degrade atrazine by N-dealkylation reactions, wherein alkyl groups attached to the ring nitrogen atom in amines, carbamates or amides are removed by oxidation and conversion to aldehydes, producing desethylatrazine (DEA) and desisopropylatrazine (DIA).These degradation products may further be dealkylated to be converted to desisopropyl desethylatrazine (Coelho & Bernardo, 2017).
Glyphosate (N-(phosphonomethyl) glycine) is a post-emergent desiccant and systemic herbicide.It has high adsorption capacity through the exchange of binders with iron and aluminium oxides and hydrogen bridges formed with humic substances present in the soil.Adsorption reduces the concentration of herbicides in the solubilized fraction of soil, reducing its potential action.Once adsorbed, glyphosate can remain as a bound residue in the environment until complete mineralization occurs (Sidoli, Baran, & Angulo-Jaramillo, 2016;Gill, Sethi, & Mohan, 2017).
Microbial activity is an important factor that determines the presence of glyphosate in the soil, and microorganisms use it as a source of energy, phosphorus and carbon, which are mainly responsible for its degradation (Zhan, Feng, Fan, & Chen, 2018).The pathway of glyphosate degradation by microorganisms involves the action of enzymes, such as oxidoreductases and transaminases, and glyoxylic acid, which cleave the glyphosate molecule at bonds other than those of carbon and phosphorus and may occur under aerobic and anaerobic conditions in the soil profile (Wang et al., 2016).
Pendimethalin (N-1-(ethyl propyl)-2,6-dinitro-3,4-methyl-toluidine) is a non-systemic herbicide belonging to the selective, pre-and post-emergent dinitroaniline chemical group.After application of this herbicide to soil, it may dissipate through evaporation, leaching and runoff.It has low adsorption capacity, which makes it more mobile in the environment; in groundwater, as in surface water, its presence has already been detected (Kpagh, Sha'Ato, Wuana, & Tor-Anyiin, 2016).
The degradation of pendimethalin in soil can occur under both aerobic and anaerobic conditions.The aerobic degradation route occurs by amino group dealkylation, followed by the reduction of the nitrile group.Under anaerobic conditions, there is a sequential reduction of nitro groups (Tobler, Hofstetter, & Schwarzenbach, 2007).
Therefore, fungi are an attractive alternative for herbicide biodegradation processes, which makes them convenient for application in bioremediation processes.The objective of the present study was to isolate fungi from agricultural soils and evaluate fungal growth in culture medium contaminated with atrazine, glyphosate and pendimethalin.

Material and methods
The experiments were conducted at the Laboratory of Environmental Biotechnology (LABITEC) of the Center of Exact, Natural and Technological Sciences belonging to the State University of the Tocantina Region of Maranhão (UEMASUL), Brazil.
In each area, 12 soil subsamples were collected with the help of a dutch piercer at a depth of 20 cm.Each subsample set was mixed to form a composite sample (Chan-Cupul, Heredia-Abarca, & Rodríguez-Vázquez, 2016).Filamentous fungi were isolated by serial dilution; first, 12.5 g of each composite soil sample was weighed and then added to an Erlenmeyer flask containing 125 mL sterilized mineral medium (NaCl 0.85%), forming a 1:10 suspension that was agitated for 30 minutes on a shaker table.Then, 1 mL of each suspension was diluted in 9 mL mineral medium to obtain a 1:1000 ratio (Oliveira, Lima, Ambrósio, Bezerra Neto, & Chaves, 2017).
Subsequently, in a laminar flow hood, aliquots of 1 mL of each final dilution were inoculated into sterile Petri dishes containing Sabouraud culture medium with 4% dextrose, and tetracycline and chloramphenicol were added to inhibit bacterial growth during incubation in an oven at 28°C for seven days.The isolation sequence was performed using the scratch marks technique.Ten fungi were obtained and were preserved in inclined Sabouraud culture medium with 4% dextrose at 4°C.
The qualitative selection of the fungi was based on macroscopic observations of fungal colony growth after 14 days of incubation: poor growth (5 mm), reasonable growth (10 mm), satisfactory growth (20 mm) and optimum growth (40 mm).At this stage, 10 fungi, three herbicides (atrazine, glyphosate, and pendimethalin), five concentrations, and triplicate analysis were used, resulting in 450 experimental units.
The fungi that presented optimal and satisfactory growth were considered resistant to the herbicides and sent to the URM Microbiology Department of the Biological Sciences Center of the Federal University of Pernambuco, Brazil, for identification.The resistant fungi were inoculated in sterile Petri dishes containing Sabouraud solid culture medium with 4% dextrose, contaminated with atrazine, glyphosate or pendimethalin at concentrations of 0, 10, 20, 50, and 100 μg mL -1 and incubated for seven days at 28°C.
The mean mycelial growth (MCM) of the colonies, which indicates the degree of adaptability of the fungi as a function of time, was evaluated after the first, third and seventh days.The relative growth rates (TCR) demonstrated the growth of each fungus after three days, considering only the herbicide as a carbon source, and the maximum inhibition rates (TIM) that were related to the growth inhibition of each fungus after 24 hours of exposure to herbicides were calculated using Equations 1 and 2 (Costa et al., 2015): x 100 (1) where: T r is the relative growth rate, T i is the maximum inhibition rate, T t is the minimum growth rate with atrazine, glyphosate or pendimethalin (μg mL -1 ), T z3 is the growth rate at a concentration of 0 μg mL -1 after the third day, and T z1 is the growth rate at a concentration of 0 μg mL -1 after the first day.
The fungal mycelial growth test was performed in a factorial scheme, with the factor levels including the herbicide resistant fungi (three microorganisms), herbicides (atrazine, glyphosate and pendimethalin), five herbicide concentrations (0, 10, 20, 50, and 100 μg mL -1 ) and five replications, resulting in 225 experimental units.The results were submitted to analysis of variance and the Tukey test (p < 0.05%) to compare the means using the statistical program Sisvar (Ferreira, 2019).

Filamentous fungi that are resistant to the herbicides atrazine, glyphosate, and pendimethalin
The fungi classified as optimal and satisfactory, after macroscopic observations of colony growth, were identified by the Micoteca URM of the Mycology Department of the Biological Sciences Center of the Federal University of Pernambuco and considered resistant to the tested herbicides (Table 1).Applications of fungal mycelial growth in culture media contaminated with atrazine, glyphosate and pendimethalin In culture medium contaminated with atrazine, F. verticillioides differed from A. fumigatus and P. citrinum, showing a higher MCM (Figure 1).The 0 μg mL -1 concentration was the most adequate for fungal growth, followed by the 10 and 20 μg mL -1 concentrations, the growth at which did not differ from each other (Figure 2).
Acta Scientiarum.Agronomy, v. 43, e51656, 2021  When observing the fungi at each atrazine concentration, there was no significant difference between the, at 0 μg mL -1 .A. fumigatus and F. verticillioides showed statistically significant MCM values at 0, 10 and 20 μg mL -1 .F. verticillioides showed a higher MCM at concentrations of 50 and 100 μg mL -1 than at the lower atrazine concentrations (Table 2).When observing the atrazine concentrations for each fungus, there were no significant differences at 0 μg mL -1 .At concentrations of 0, 10 and 20 μg mL -1 , F. verticillioides showed statistically equal MCM, but at 10 and 20 μg mL -1 , the fungi did not differ (Table 3).The fungus F. verticillioides presented higher MCM values in culture medium contaminated with glyphosate than in the other media (Figure 3).The highest MCM values were at glyphosate concentrations of 0 and 10 μg mL -1 (Figure 4).When evaluating the fungi at each glyphosate concentration, P. citrinum presented a higher MCM at 10 μg mL -1 than the other two fungi.At 20 μg mL -1 , P. citrinum and F. verticillioides presented higher MCM values µg mL -1 than A. fumigatus and were not statistically different.At 50 and 100 μg mL -1 , F. verticillioides was the only fungus to develop mycelia (Table 4).When observing glyphosate concentrations for each fungus, there were no significant differences at 0 and 10 μg mL -1 .At 20 μg mL -1 , F. verticillioides presented a higher MCM than at the other concentrations and was the only fungus able to grow at 50 and 100 μg mL -1 (Table 5).In culture medium contaminated with pendimethalin, F. verticillioides and A. fumigatus presented higher and statistically equal MCM values (Figure 5).The MCM values were highest at 0 μg mL -1 , followed by those at the 10 μg mL -1 concentration (Figure 6).When evaluating the fungi at each pendimethalin concentration level, A. fumigatus and F. verticillioides presented MCMs that were statistically equal at 0, 10 and 20 μg mL -1 .There was no mycelial growth for any fungi at 50 and 100 μg mL -1 (Table 6).

Relative growth
The results of the relative growth rates (TCR) show that the MCM tended to decrease with increasing concentrations of the herbicides tested in this study (Figure 7).The 20 μg mL -1 concentration was most favourable for fungal growth in culture medium contaminated with atrazine, except for F. verticillioides, which showed the highest growth at 10 μg mL -1 (Figure 7 A).The 10 μg mL -1 concentration was the most favourable for the microorganisms in culture media contaminated with glyphosate or pendimethalin, as seen in Figure 7  (B and C).

Maximum inhibition
The results described in Figure 8 demonstrate the maximum inhibition rates (TIMs) ; negative signs indicate growth stimulation.In the culture media contaminated with atrazine and glyphosate, the fungi presented TIMs at 50 μg mL -1 , and an increase in growth inhibition was observed at 50 and 100 μg mL -1 , according to Figure 8 A and 8 B. In culture medium contaminated with pendimethalin, the concentration with the highest TIM was 20 μg mL -1 , with total growth inhibition at concentrations 50 and 100 μg mL -1 (Figure 8 C).Bonfleur, Tornisielo, Regitano, & Lavorenti (2015) reported that atrazine toxicity causes stress to the soil microbiota.Marinho, Barbosa, Rodrigues, Aquino, & Pereira (2017) tested the effect of increasing atrazine concentrations in Aspergillus niger AN 400, and the fungus was able to grow at concentrations up to 30 mg L -1 .The authors concluded that this fungus is very resistant to atrazine.Chan-Cupul et al. ( 2016) concluded that the genera Aspergillus and Penicillium are capable of degrading atrazine, indicating that these fungi can be used in bioremediation studies of soils contaminated by this herbicide.Barberis, Carranza, Magnoli, Benito, and Magnoli (2018) indicated that Aspergillus oryzae was tolerant to high atrazine levels and may be considered a potential bioremediation agent.The fungus A. fumigatus was tolerant to all changes that occurred in this study.
Under natural conditions, the degradation of glyphosate in soil depends on microbial degradation.Therefore, it is necessary to identify degrading microorganisms and confirm their potential for the bioremediation of glyphosate-contaminated environments (Yu et al., 2015).Fu et al. (2017) concluded that the fungus Aspergillus oryzae degrades glyphosate through the AMPA and methylamine pathways.Carranza, Barberis, Chiacchiera, and Magnoli (2016) established in their studies that Aspergillus flavi and Aspergillus niger may develop in vitro in the presence of glyphosate, especially when it is used as the sole source of phosphorus or nitrogen.
The concentrations of 50 and 100 μg mL -1 glyphosate limited the mycelial growth of A. fumigatus; however, the fungus F. verticillioides showed excellent mycelial growth in the presence of glyphosate, using the herbicide as a carbon source.
A study by Rodríguez-Liébana, ElGouzi, and Peña (2017) stated that the dissipation of pendimethalin is related to the endogenous biota of the soil.Microbial metabolism is the most important factor in the degradation of pendimethalin in soil (Ni, Li, Qiu, Chen, & He, 2018).The fungi A. fumigatus and F. verticillioides presented satisfactory results in this study; however, the growth of these microorganisms was limited at concentrations of 50 and 100 μg mL -1 .

Conclusion
The isolated fungi A. fumigatus, F. verticillioides and P. citrinum were resistant to the herbicides atrazine, glyphosate and pendimethalin.
The fungus F. verticillioides showed a higher mean mycelial growth in culture medium contaminated with atrazine at 10 and 20 μg mL -1 and that contaminated with glyphosate at 10 μg mL -1 .In the culture medium contaminated with pendimethalin, the fungi F. verticillioides and A. fumigatus presented the highest mean mycelial growth values, and the growth was highest at 10 μg mL -1 .

Figure 1 .
Figure 1.Mean mycelial growth values (cm) in culture medium contaminated with atrazine.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Figure 2 .
Figure 2. Mean mycelial growth values (cm) at different atrazine concentrations.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Figure 3 .
Figure 3. Mean mycelial growth values (cm) in culture medium contaminated with glyphosate.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Figure 4 .
Figure 4. Mean mycelial growth values (cm) in glyphosate concentrations.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Figure 5 .
Figure 5. Mean mycelial growth values (cm) in culture medium contaminated with pendimethalin.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Figure 6 .
Figure 6.Mean mycelial growth values (cm) in pendimethalin concentrations.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Figure 8 .
Figure 8. TIM (%) of fungi in culture media contaminated with atrazine, glyphosate or pendimethalin at concentrations of 0, 10, 20, 50 and 100 μg mL -1 after 1 day of incubation.(A) TIM (%) of fungi in culture medium contaminated with atrazine.(B) TIM (%) of fungi in culture medium contaminated with glyphosate.(C) TIM (%) of fungi in culture medium contaminated with pendimethalin.

Table 1 .
Fungi identified and incorporated into the Micoteca URM Culture Collection of the Center for Biological Sciences of the Federal University of Pernambuco, Brazil.

Table 2 .
Fungal growth at each atrazine concentration.
Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Table 3 .
Mean mycelial growth as a function of atrazine concentration for each fungi.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Table 4 .
Evaluation of fungi at each glyphosate concentration.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Table 5 .
Evaluation of glyphosate concentrations for each fungus.
Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Table 6 .
Evaluation of fungi at each pendimethalin concentration.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.

Table 7 .
Evaluation of pendimethalin concentrations for each fungus.Mean values followed by the same letter do not differ according to the Tukey test at a 5% significance level.