Introduction

Agricultural practices include the application of different pesticides to maintain crop performance by controlling insects, diseases and weeds. In nature, pesticide residues are subjected to physical, chemical and biochemical degra-dation processes, although they persist in the environment due to their high stability and water solubility²². The inad-equate use of pesticides can affect microorganisms and their activity, causing alterations in the biological processes that impact on soil fertility and, consequently, on crop productivity⁸. Pesticides can reduce the pro-duction of microbial biomass; this change may cause alterations in the mineralization of organic matter, redox reactions, denitrification, nitrification, ammonification, and methanogenesis¹³. One of the most widely used pesticide is glyphosate, a potent, broad spectrum, non-selective, post-emergent herbicide that is capable of controlling 97% of the weeds. Glyphosate is generally used along with cypermethrin, an insecticide belonging to the family of pyrethroids and widely used in nontillage systems. Among the fungicides, carbendazym is a systemic fungicide used for controlling a broad range of fungi affecting crops.

In the soil environment there are different types of microorganisms, such as fungi that participate actively in all the processes of transformation of organic matter. Fungal endophytes are organisms that colonize plant tissues during a specific period of their life cycle but cause no symptoms of tissue damage to their hosts²⁹. Among this highly diverse group of endophytic fungi, dark septate endophytes (DSE) have received much attention in the recent years^27,31. The genera Alternaría sp. and Curvularia sp. are widely known as pathogenic fungi in several cereals of commercial value1,40; however, species of the genera Alternaría and Curvularia (teleomorph: Cochliobolus) have been found to colonize grasses, playing a role as DSE fungi and promoting plant growth^14,24. These DSE fungi are not thought to be pathogenic, as they are observed on healthy plants³.

Colonization patterns by DSE seem to be different accord-ing to the plant host and the cultivation system²¹. These fungi often inhabit oligotrophic soils that are associated with the roots of hundreds of plant species in all climate regions and major biome types³³ as well as promoters of plant growth under greenhouse conditions²⁷. Dark septate endophytes are able to hydrolyze major C, N and P polymers into usable subunits and therefore could provide N and P to the host plant⁵. Newsham²¹ performed a meta-analysis of independent studies to assess the inoculation of DSE in different crops, concluding that the contents of N and P in inoculated plant biomass were higher compared to non-inoculated plants. Conversely, another meta-analysis suggested negative to neutral effects of DSE inoculations18.

Wheat is one of the winter cereals of major importance in Latin America. Agricultural production is one of the fundamental economic activities of Argentina, which is among the main world producers of crops such as soybeans, wheat, sun-flower and corn. In Argentina, the production area destined for the wheat crop was estimated in 5.95 million hectares, producing 18 million tons of the cereal¹⁹. Most studies have evaluated mycorrhizal and DSE root colonization in different hosts and Argentine regions^17,28. Nevertheless, little information is available about the biology of DSE isolated from cereals^27,31. The widespread use of pesticides has raised con-cerns about their potential impact on microbial communities and in this sense there are no studies evaluating the effects of agrochemicals on DSE. Therefore, the aim of this study is to isolate DSE from wheat roots and to assess the tolerance of isolated strains to different chemicals commonly used in agricultural practices.

Materials and methods

Thirty wheat plants were randomly collected from two sites located in Arenales and Ferré in July 2011 (Buenos Aires Province, Argentina). The plants were at phenological stage Z5. Samples were transported to the laboratory and placed in a cold chamber at 4 °C until their use. The roots were washed under tap water and sterilized superficially by immersing them in 70% ethanol for 2 min followed by 1% sodium hypochlorite (NaOCl) for 3 min and thoroughly rinsed five times with sterile distilled water. Subsequently, twenty five root pieces (5mm) of each plant were placed into drops of Gel-Gro (ICN Biochemicals, USA) with strep-tomycin and tetracycline hydrochloride 1% (Sigma-Aldrich, USA) in sterile Petri Plates 30 Plates30. The root pieces were incubated in the dark at 25°C and were periodically observed under a stereoscopic microscope (Nikon H550S, Japan). The segments of roots in which fungal growth was observed were placed on plates containing Malt Extract Agar (MEA) and cultured on such medium at 25 °C, thereby obtaining several isolates. The endophytic nature of the thirty isolates was corroborated following the test of resynthesis (Koch’s postulates) under greenhouse conditions³¹. Five isolates were identified following classical^10,36 and molecular methodologies³¹. The strains were deposited at the Fungi Bank of the Facultad de Agronomía, Universidad de Buenos Aires²⁷. Two isolates were selected for this assay due to their extracellular enzymatic production³⁵, their phospho-rus solubilization³¹ and their tolerance to different sodium salts³².

Figure 1: Morphology of DSE’s colony grown in MEA plate. (A) Alternaria alternata. (B) Cochliobolus sp. 

A culture of each strain grown on MEA for 7 days at 25 °C was used as inoculum to evaluate their tolerance to pesti-cides. Subsequently, two successive picks were conducted on basal salt medium (BSM)15 with the purpose of causing them a nutritional stress (starvation). The BSM containing the herbicide (glyphosate), the fungicide (carbendazim) or the insecticide (cypermethrin) was used in an agronomic common dose (AD), 2ADand 10 AD. Doses perPetri dishwere 0.20 ppm, 0.40 ppm and 2 ppm for the herbicide; 18.12 ppm, 36.25 ppm and 181.25 ppm for the fungicide; and 2.02 ppm, 4.04 ppm and 20.2 ppm for the insecticide respectively. The control treatment was BSM medium plus sucrose (2%). The inoculum was standardized using 5 mm-diameter disks obtained from the zone with active growth of a colony with 7 days of growth for all strains and all tested media. Each agrochemical dose and its respective control were tested in quintuplicate. Fungal growth was assessed periodically from the third day by measuring the colony diameter at 12 days. Fungal growth rates were analyzed using the following formula26: Growth rate = (Fd -Id)/(Ft - ), where: Fd is the final diameter (cm), Id is the initial diameter (cm), Ft is the final time (days) and It is the initial time (days).

Moreover, the inhibition percentage was calculated for each fungi and each agrochemical at 6 and 12 days using the following formula: Inhibition (%) = (Cd -Td)/Cd x100, where: Cd is the mean colony diameter in the control treat-ment (cm) and Td is the mean colony diameter in the agrochemical treatment (cm).

Results

The physicochemical properties of the soil (Typic Argiu-doll) were: organic carbon 1.7%, available phosphorous 15.0mg/kg (Kurtz and Bray No. 1 method), pH 6.2, elec-trical conductivity 0.5d/Sm clay 27.5%, silt 50.5% and sand 22%. Figure 1 shows the morphology of the DSE colony of A. alternata and Cochliobolus sp.

The strain Cochliobolus sp. reached a diameter of 75 mm of the colony after 12 days of growth while A. alternata completed the culture dish (90 mm) after 9 days (data not shown). There was significant change in the kinetics of growth of the strains by adding the different kinds of agrochemicals to the medium. The results from the two-way analysis of variance showed that there were significant inter-actions between the DSE strains and the agrochemicals in growth rate (p < 0.0001). Therefore, this interaction was fur-ther analyzed. Table 1 shows that the growth of A. alternata was significantly faster than that of Cochliobolus sp. in the control treatment and was less affected than Cochliobolus sp. when the agrochemicals were added.

The presence of glyphosate reduced the growth rate of both DSE strains. A. alternata showed significant differences in AD and 2 AD regarding Cochliobolus sp.; however, when the dose was increased to 10 AD, no differences were found between both strains.

The fungicide carbendazim was the most tolerated agro-chemical by the DSE strains. The growth rate of A. alternata was reduced by the addition of carbendazim as its dose increased in the solid media. No differences were found in 2 AD between both fungi (Table 1). On the other hand, the insecticide (cypermethrin) was the most toxic agrochemical on growth rate. A. alternata reduced its growth rate as the dose of cypermethrin increased in the media, while a dose equal to or greater than 2 AD inhibited the growth of Cochliobolus sp. (Table 1).

The percentages of growth inhibition are shown in Table 2A-C. The herbicide (Table 2A) increased this parameter (p < 0.05) in all the agrochemicals and doses tested for both fungi and on both days evaluated (day 6 and day 12). The fungicide increased the growth inhibition percentage of Cochliobolus sp.; however, an induction of the growth of A. alternata was observed when the fungicide was added at AD and 2 AD evaluated at 6 days. In these doses, at the end of the growth (day 12) no significant differences were detected with respect to the control treatment. For both evaluation days, the addition of carbendazim at 10 AD generated a significant increase (p <0.05) in the inhibition percentage (Table 2B).

Table 1: Growth rate of Cochliobolus sp. and Alternaría alternata (DSE strains). Comparison between DSE strains A. alternata and Cochliobolus sp. and agrochemical type. 

Table 2: Inhibition of growth rate of Cochliobolus sp. and Alternaría sp. (DSE strains) growing at different concentrations of: (A) glyphosate, (B) carbendazim and (C) cypermethrin. 

Table 2C shows the percentages of growth inhibition generated by the insecticide. The DSE Cochliobolus sp. exhibited significant inhibition when the AD was placed in the culture medium (p <0.05); however, no changes were detected between 2 AD and 10 AD. On the contrary, A. alternata recorded increases in inhibition in all the doses studied (p <0.05).

The concentrations of the agrochemicals that caused a 50% reduction in fungal growth compared to the control (effective concentration: EC 50) varied depending on the agrochemical type (Fig. 2). In all the agrochemicals analyzed, the fungus A. alternata was the most tolerant, presenting higher EC 50. The insecticide was the agrochem-ical that caused the greatest effect on DSE growth, followed by the herbicide. The fungicide proved to be the least toxic agrochemical for these fungi (Fig. 2). Cochliobolus sp. did not differ significantly from A. alternata in the EC 50 against carbendazim.

Figure 2: Effective concentration (EC 50), the concentrations of the herbicide, fungicide and insecticide that caused a 50% reduction in fungal growth compared to the control. Means ± SD (n = 5). Different letters differ significantly (p<0.05). 

Discussion

This study evaluates the tolerance of two DSE fungi to different agrochemicals. The tested DSE fungi were isolates of A. alternata and Cochliobolus sp. It is known that these fungi are grass pathogens; however, several studies have shown that they can also be found in the roots as DSE as non-pathogenic fungi^3,37. Therefore, the fungal species classified as non-pathogenic cause disease symptoms in certain host plants but are neutral or beneficial in other hosts.

Moreover, although pesticides are designed to be as specific as possible, they have effects on non-target fungal organisms. In accordance with our results, glyphosate (using equivalent herbicide concentrations) had an in vitro toxic effect on culturable mycobiota (Aspergillus sec-tion Flavi) strains from agricultural soils⁶. Likewise, other authors¹² found a reduction of microbial biomass at a higher glyphosate concentration and a temporary inhibitory effect at recommended field doses. Furthermore, effects of glyphosate on soil microbial communities and its mineraliza-tion in soils after various applications of this herbicide have been found16. A recent study¹¹ also demonstrated behavioral differences between glyphosate and its major metabolite, AMPA, related to the physical properties of saturated hydraulic conductivity (Ks) and soil moisture. Other authors found that glyphosate affects arbuscular mycorrhizal fungi due to a reduction in spore viability, root colonization and arbuscule percentage⁹. However, DSEs are more tolerant to different types of stress than other soil fungi²³. This could be due to the fact that the chemical compounds act effectively on the mycelium, while spores and chlamydospores are more resistant to these compounds due to their low activity given their water content and the presence of a large amount of unsaturated fatty acids4.

There is much less research on the effect of carbendazim on fungi. However, this fungicide has detrimental effects to fungal and bacterial biocontrol agents such as Trichoderma harzianum, Trichoderma virens, Bacillus subtilis and Pseudomonas fluorescens²⁰. In this research, we found that DSE strains have the potential to tolerate high concentrations (181.25 ppm, 10 AD) of fungicide; these results are in accordance with those of others authors². One study showed that the type of fungicide applied to the soil must be seriously considered as the authors detected negative effects on Rhizophagus fasciculatus (arbuscular mycorrhizal fungi) with benomyl (systemic benzimidazole); however, they found no effects when captan (non-systemic fungicide)⁷ was applied. Nevertheless, our results showed high tolerance of DSE fungi against carbendazim, which is a derivative of benomyl. Bipolaris tetramera and A. alternata have potential to tolerate and degrade 100 ppm of carbendazim in vitro³⁸. In accordance with that article, we found negative inhibition values in agronomic doses, 2 AD and no differences compared with control. These results would indicate an Alternaria sp. promotion growth when grown with the addition of carbendazim.

In the case of cypermethrin, there are studies that demonstrate that this insecticide caused a negative effect on soil enzymatic activities and microbial diversity³⁴. Our DSE strains showed low cypermethrin tolerance, high inhi-bition percentage and low EC50. Nevertheless, a study revealed that when the pesticide is applied on pepper plants there is an increase in bacterial abundance and a change in the composition of the community of the Firmi-cutes phylum to Bacteroidetes and 7-Proteobacteria phyla39. Other authors showed the potential of Aspergillus niger, Aspergillus terreus, Monilochaetes and Fusarium strains in the biodegradation of 50-150 ppm of this pyrethroid²⁵.

This research has shown that agrochemicals commonly used in agriculture have detrimental effects on the growth of DSE fungi, even when these agrochemicals are used at recommended doses; however, these negative effects vary according to the agrochemical considered. Moreover, this paper makes evident that DSE response to this type of toxin depends on the studied species of DSE fungus. This research is a contribution to a better understanding of the behavior of DSE against xenobiotics, evaluating the tolerance and/or their use as a source of nutrients.

Conclusión

The DSE A. alternata tolerates all three agrochemicals at the tested doses while Cochliobolus sp. is glyphosate and carbendazim-tolerant in all three doses but shows no tolerance to cypermethrin when the agronomic dose is twofold. In general, there is a decrease in the growth of the strains when increasing the dose of the agrochemicals studied.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The present research was financially supported by the Agencia Nacional de Promoción Científica y Tecnológica, Project PICT 2016:1136 and the Universidad de Buenos Aires, UBA-CYT 068/2011, Facultad de Agronomía, Argentina. We thank Ing. Raul Silvio Lavado the critical review of this manuscript.