During the last century, farming and agricultural activities released many persistent and toxic chemical pesticides into the environment including insecticides, fungicides, nematicides, rodenticides and herbicides21. They comprise a variety of molecules possessing different properties that confer some degree of environmental persistence and mobility, as well as different toxic, carcinogenic, mutagenic and teratogenic potentials10,17,21. Such substances may also affect the endocrine systems of non-targeted organisms, including humans38.

The use of herbicides in Mexico increased strongly in the last decade, where according to recent studies more than 45% of pesticides marketed were herbicides, trazine being the most frequently used7. The use of atrazine is not regulated and is widely used in Mexican agriculture, where the application rates range from 0.1 to 4kg/ha/year. At these application rates, the annual consumption of atrazine in Mexico has been estimated to be around 1078 tons/year with an annual consumption increase of 10%18,19.

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) is a selective herbicide that belongs to the s-triazine family, which contains an aromatic hexameric and symmetrical ring, constituted by three carbon and three nitrogen atoms in alternate positions18. Atrazine is used to control broadleaf and grass weeds in corn, sorghum, sugar cane, coffee crops and conifer reforestation. Atrazine kills susceptible plants by binding to the quinone protein in photosystem II and inhibits photosynthetic electron transport36.

Despite its apparent biodegradability, atrazine has led to the contamination of terrestrial ecosystems and can be found and measured in ground and surface waters in many countries7,30. The removal of atrazine from the environment is an ecological responsibility, and finding a safe and economical method is both a major concern for land management agencies and a challenge to science13. Bioremediation with microbial organisms is one approach. In the last two decades, the use of ligninolytic macrofungi and their enzymes in the mycoremediation of environmental contaminants has become a promising solution40. This fungal ability to bioremediate is generally attributed to the production of extra-cellular ligninolytic enzymes such as laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP), which are substrate non-specific enzymes and are able to degrade a wide range of recalcitrant compounds including several herbicides that are structurally related to lignin3–5,9,39.

The first step to establish future studies of atrazine degradation through mycoremediation in contaminated sites is finding tolerant strains to atrazine28. The selection through dose-response bioassay is considered a way to identify these strains, which could be used in subsequent studies on soil mycoremediation8,29. With this in mind, we proposed the hypothesis that tolerant strains will not show mycelial growth inhibition and reduction in their enzyme activities upon exposure to the herbicide, therefore they will obtain a high mean effective concentration (EC50). To corroborate this hypothesis, the goals of this work were to assess mycelial growth rates and ligninolytic enzyme activities under atrazine stress in eight native ligninolytic macrofungi isolated from Veracruz, Mexico.

Mycelial growth was inhibited by the atrazine concentrations at different rates in the studied strains; however, none reached 100% inhibition under all tested doses. The lowest dose of atrazine (468mg/l) did not cause inhibition in D. elegans and Pleurotus sp. strain 1. The concentration of 937mg/l of atrazine did not inhibit the growth of Pleurotus sp. strain 1. These results suggest that moderate concentrations of atrazine are not toxic to these strains. In a previous study Nwachukwu and Osuji28 showed 8, 11 and 14% inhibition in the mycelial growth of Lentinus subnudus in soil extract agar supplemented with 5, 10 and 20mg/l of atrazine, respectively. These inhibition percentages were low, which could be due to the low atrazine concentrations used in Nwachukwu and Osuji´s study. In another study, Ribas et al.32 assessed the susceptibility of Agaricus subrufescens (basidiomycete) to atrazine and found high inhibition rates of mycelial growth in PDA supplemented with 10 (18.8%), 20 (31.3%), 30 (43.8%), 40 (50%) and 50 (48%) mg/l of atrazine. The concentrations used by Ribas et al.32 and Nwachukwu and Osuji28 were lower compared with the concentrations used in the present study (from 468 to 3750mg/l), which corresponded to field application rates.

The study of growth rates of ligninolytic macrofungi is important in order to understand their capacity for colonization and their potential to settle on a particular substrate. However, several pollutants have a negative effect on the growth rate of ligninolytic macrofungi27. In previous works, Casale and Hart5 reported a 69% reduction in the rate of growth for ascomycete Sclerotinia sclerotiorum when exposed to 500mg/l atrazine in the culture medium. Furthermore, the daily growth rate of filamentous microfungi was negatively affected by exposure to atrazine; some susceptible genera are Trichoderma, Penicillium and Aspergillus7.

On the other hand, the daily growth rate of Pleurotus sp. strain 1 and C. elegans was stimulated by low concentrations of atrazine (468mg/l). This physiological response has been previously reported in Microsporum fulvum (hyphomycete)1, when cultivated in Sabouraud agar supplemented with atrazine concentrations ranging from 5 to 50mg/l. However, the increase in the daily growth rate by atrazine in basidiomycetes has not been previously reported. In an earlier study, Maciel et al.23 reported an increase in the mycelial growth rate of Trametes sp. in malt extract agar supplemented with 0.1mM (24.1mg/l) of picloram (an herbicide). In this case, the increase in mycelial growth rate in fungi by herbicides could be related to the auxin-like action16,23.

The parameter used to determine fungi sensitivity or tolerance to atrazine was the median (EC50) effective concentration42. Based on this concept, Pleurotus sp. strain 1 was the most tolerant fungus to atrazine, presenting the highest EC50. There is little information about the EC50 of atrazine in ligninolytic macrofungi; therefore it is difficult to compare our results to other references. Only Nwachukwu and Osuji28 calculated the EC50 of atrazine in L. subnudus (EC50 at 15°C=20mg/l and EC50 at 25°C=11.9mg/l), which was smaller than that achieved in this work in all tested strains (from 706.1 to 2281.0mg/l). The available information only mentions that atrazine exposure in fungal organisms results in the growth inhibition of some ligninolytic macro- and filamentous micro-fungi6.

In addition, xenobiotics affect the synthesis and activity of fungal enzymes11,25. Laccase is one of the main enzymes produced by ligninolytic macrofungi, which plays an important role in fungal morphogenesis, lignin degradation and fungal plant-pathogen/host interaction3. Under the culture conditions used in this study all fungal strains produced laccase as the main ligninolytic enzyme. However, there was an increase in laccase activity for all tested strains due to the effect of the atrazine concentrations. In earlier works, Mougin et al.25 added several xenobiotics (among them atrazine), in a Trametes versicolor culture. Atrazine increased the laccase activity of T. versicolor by 369% (0.61U/ml) and 443% (0.87U/ml) when compared to the control (0.13Ug/g). Bastos and Magan5 found high laccase activity (19.9U/g soil) in T. versicolor growing in soil contaminated with atrazine at 0.9 and 2μg/g, in comparison to soil without atrazine (9.0U/g soil).

With regard to MnP activity, only C. elegans and P. sanguineus strain 1 showed MnP activity under the culture conditions. Atrazine concentrations in PDA increased MnP activity in C. elegans; however, in P. sanguineus strain 1 cultures resulted in a reduction. However, a particular physiological response was observed for Pleurotus sp. strain 2, which did not show MnP activity in the control treatment (without atrazine), but produced MnP activity in the presence of atrazine. The increase in laccase and MnP activities could be linked to oxidative stress on ligninolytic fungi generated by atrazine. This response is considered a defense mechanism for ligninolytic fungi26,40–42. Nevertheless, this response is not a general rule. Recently, Maciel et al.20 found a reduction in laccase activity of Ganoderma lucidum due to the effect of picloram. The latter is unusual because, in general, the addition of xenobiotics to the culture of ligninolytic fungi enhances laccase activity5,23,25. However, we must not overlook that individual species could respond dissimilarly to different pesticides.

Under natural conditions, fungi are usually not limited in growth by low atrazine concentrations in the environment30. However, in locations with an extremely high atrazine content an obvious effect on fungal growth has been observed. It should be noted that the impact of atrazine on fungal physiology is not only limited to inhibition or alteration of mycelial growth. In particular, the activities of extracellular enzymes, such as laccase and MnP, were enhanced with atrazine concentrations. For this reason, more research in the field of fungal ecology for atrazine-polluted habitats could prove helpful.

Based on the results, we conclude that Pleurotus sp. strain 1 was the most tolerant fungus to atrazine, as it did not show mycelial growth inhibition due to atrazine at either 468 or 937mg/l concentrations, thus achieving the highest EC50; however, it did not show high laccase activity and did not enhance to a great extent its enzyme activity by atrazine stress. Nevertheless, there were other fungal isolates such as D. elegans, P. sanguineus strain 2 and T. maxima, which obtained a moderate EC50 and high enzyme activity, principally laccase; therefore, we reject the proposed hypothesis. On the other hand, the most susceptible fungus was Pleurotus djamor. Atrazine stress increased laccase activity for all the strains. The increase in MnP activity caused by the effect of atrazine occurred to a lesser extent than increases in laccase activity. This study demonstrated, for the first time, the ability of C. elegans to produce laccase and MnP, including its ability to increase enzyme activity by atrazine stress. Overall, we propose the use of native macrofungi such as D. elegans, P. sanguineus strain 2 and T. maxima for future studies in atrazine mycodegradation.

Protection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this study.

Confidentiality of data. The authors declare that no patient data appear in this article.

Right to privacy and informed consent. The authors declare that no patient data appear in this article.

The publication of this study was supported by research grant CB 169124 from the National Council of Science and Technology (CONACyT) of Mexico.

The authors declare that they have no conflicts of interest.