Our findings showed that the topsoil from the farms in various locations contain residues of herbicide chemical glyphosate and its metabolite, AMPA. This can be attributed to the over-reliance of this chemical in agricultural practices. However, the concentration of this glyphosate in the field was found to be relatively higher than published work on Environmental Health Criteria 159 under the sponsorship of United Nations Environment Programme, the International Labour Organisation, and the World Health Organization [7]. Therefore, this calls for serious remedial action to be taken as the accumulation of glyphosate is likely to pose serious danger to ecological receptors.
Notwithstanding the diversity of organisms present in the contaminated site, our interest was majorly on fungi as little is known on their role for biodegradation of glyphosate. There was high enumeration of fungal count in location where AMPA level was remarkably high as well as relatively high level of glyphosate compared to lower levels. This was also supported by the pearson correlation which shows high correlation with AMPA. A range of bacterial strains have been implicated to be abundant in glyphosate contaminated environment either because of their capability of using the compound as sole source of phosphorus, carbon or nitrogen [20]. As such they play a role in degradation. Therefore, investigation of the role of diversities of fungi in the degradation of glyphosate can be remarkably interesting.
In order to isolate potential fungi to degrade glyphosate we observed the enhanced growth of these microbes from the four soil locations. Only six isolates from all the location demonstrated enhanced growth in the presence of glyphosate. This shows their ability to use glyphosate as an energy source. However, the inability of the other isolates to survive could be the toxic effect of glyphosate on the organisms. Eman et al. [21] noted that application of pesticides has the possibility to exert some effects on non-target organisms, plus the soil microorganisms. Presence of pesticides makes some microorganisms to lyse while other microorganisms may be resistant and tolerant to a pollutant, hence, increase in their numbers and biomass due to decreased competition [21].
In studying the effect of glyphosate on the activities of the fungi in the enriched medium supplemented with glyphosate, the pure isolates induced changes in the medium such as changes in pH, optical density, and fungal counts. The decrease in the pH levels of the culture medium may be as a result of microbial metabolism and production of secondary metabolites. Analysis of supernatants by Montserrat et al. [22] also demonstrated a decrease in pH resulting from rapid production of lactic, acetic, pyruvic and citric acids. The implication is that such changes in pH can influence bacteria growth. For instance, Yang et al. [23] found out that pH level of culture medium was one of the key factors influencing the growth of four bacteriocinogenic strains. Furthermore, LeBlanc et al. [24] stated that the growth of Lactobacillus fermentum CRL 722 was noticeably slower at pH 4.5 (μmax = 0.78 h−1) than at other pH values including pH 5.0, 5.5, and 6.0 (μmax = 1.15 − 1.25 h−1). This is in agreement with results obtained in this study which shows that optimum growth of fungi under glyphosate was obtained at approximately 5.0. This might also influence bioremediation activity. Therefore, changes in metabolic state of the fungal can be the driving force of the pH suggesting active state of the fungal cells. Longer lag phase (16 days after inoculation) could suggest that fungal enrichment in glyphosate treated medium may be slower than bacteria. However, efficiency in transformation of glyphosate need to be evaluated. The short exponential phase could suggest active state of the fungi towards glyphosate remediation. Stratton and Stewart [25] observed a small rise in microbial biomass but no negative or positive effects in respect to the number of microorganisms. In addition, Haney et al. [26] and Busse et al. [27] assessed the effect of glyphosate on soil’s microbial community and their findings concluded that microbial activity was stimulated even in the presence of this herbicide. Therefore, it is prospective that the glyphosate provided nutrients for fungal growth, as shown by the significant growth and increase in microbial population.
Glyphosate is a nonselective, broad-spectrum, post-emergence herbicide that is widely used in agriculture; hence, degradation of the compound will be a positive obligation in agricultural practices. It was evident that the isolates of fungal strains degraded glyphosate. The growth ability of the fungal strains could ascertain a significant assimilation of glyphosate. One reason could be that the fungal strains exhibited optimal growth rates, in order to potentially adapt to the glyphosate concentration and to assimilate it. Also, it is possible that they possess enzymes capable of cleaving the C–P bond. It is remarkable to mention that this assimilation took place without enhancement by sucrose, nitrogen (N), and phosphorus source. For example, Eman et al. [21] reported that a concentration of 1% sucrose was important for the initial stimulation of fungal strains in glyphosate degradation. Studies reported that the development of enhanced degradation of xenobiotics or pollutants depends on multiple factors such as nutrient composition, chemical structure, soil properties including the presence of degrading microbes with appropriate metabolic functions [28, 29]. Our aim was to isolate the key player of glyphosate degrading fungi from soils which demonstrated rapid degradation capability. The intensity of glyphosate biodegradation with the indigenous microbial pure strain was highest in A. flavus JN-YG-3-5 which utilized 92.86% without accumulation of AMPA. Thus, this makes it so interesting for environmental application. Other strains had high capability but produced AMPA which might be detrimental to the environment.
Aspergillus sp have received tremendous interest for their suitability in bioremediation [30]. This could be the reason scientists and environmentalist are interested to develop various strategies for the use of Aspergillus sp. in bioremediation. This species will be useful in pesticide contaminated soil. Different species of fungi were identified using BLAST analysis. The high abundance of Aspergillus species in the samples may be due to their ability to tolerate and degrade pesticides. Similar studies have been conducted by Asef, [14] and have revealed the isolation, molecular characterization and pesticide degradation by Aspergillus species. Thus the reason Aspergillus sp have received tremendous interest for suitability to remediate wide range of xenobiotic compounds. The identified fungal strains observed in this study have high GC contents. This is likely to have made them tolerant to pesticide. One imperative property of the GC base pair is its higher thermal stability than the AT base pair. An increase in GC content correlates with a broader tolerance range of species [31].
The range of GC contents in the fungi suggests characteristics of microbe from soil. Aspergillus flavus JN-YG-3-5 can be a particularly important tools for use in biotechnology because it yielded high pure DNA quantity and has a GC content similar to well-known GC in soil for active physiological functions. The works of Smarda et al [31] and Njoku et al. [18] reported that GC-rich genes facilitate the response to environmental stress. In addition, it can also facilitate complex gene regulation. Thus, improved responses to environmental conditions might be enabled by GC-rich genes. The fungi having higher GC contents were better in glyphosate degradation thus giving a beneficial advantage to be utilized in a wide range of environmental applications. This could have also been an added advantage to Aspergillus flavus JN-YG-3-5 for complete mineralization of glyphosate pesticide.
Phylogenetic analysis explicitly showed that the polluted soil sheltered diverse fungi population belonging to three clusters of orthologous groups with Aspergillus flavus JN-YG-3-5 clustering together suggesting their similar ancestry. It can also be due to combination of selective factors, proximity and functional capacity [32]. The different groups that they belong to does not necessarily mean that they degraded the contaminant through different processes. It has been hypothesized that phylogenetically distant lineages might share mutual functions and functional features. The work agreed with the work of Ning and Beiko [32] who reported that functional similarities exist between operational taxonomic units (OTUs) that belong to different high-level taxonomic groups for fungi.
Automated annotation identified several proteins within the genome of fungal strains to include ABC transporters, these are members of a protein superfamily known to be involved in the efflux of drugs from the cells of target organisms. Also, the Zinc finger protein gene and zinc finger chimera 1, were discovered along with many oxidoreductase genes. A search of the identified proteins for specific functions revealed that the genes are distributed in different functional categories majorly protein metabolism and respiration. Numerous genes associated with pesticide degradation were identified.
Interestingly, The GhostKOALA output identified CYP2W1 gene (Cytochrome P450, fungi type) present in Aspergillus fumigatus strain FJAT-31052 which was absent in genome of other fungi. The cytochrome P450 enzymes are monooxygenases which catalyze many active reactions involved in the metabolism of wide variety of xenobiotics [33]. Previous studies have reported the activities of human CYPs involved in the metabolism of pesticides [34, 35]. CYP-pesticides interactions are by either the induction or inhibition of the metabolizing enzymes. In a study by Khaled et al. [33] HepaRG cells express a large panel of liver-specific genes including several CYP enzymes, which contrasts with HepG2 cell lines. Both immunoblotting and reverse transcription polymerase chain reaction (RT-PCR) techniques have been used to examine the pesticide-CYP induction [33, 36, 37].
Although fungi have received tremendous interest for their suitability in detoxifying a variety of contaminants, its ability to degrade glyphosate is a new area of research interest. Two different routes have been proposed to be utilize by soil microorganisms to metabolize glyphosate: The C-P lyase and AMPA pathways [9]. To demonstrate the valid pathway, identification of AMPA even to a significant amount shows that AMPA pathway is valid. This mechanism involves the oxidative cleavage of the C-N bond on the carboxyl side catalyzed by glyphosate oxidoreductase (GOX) which results in the formation of aminomethylphosphonic acid (AMPA) and glyoxylate. The mechanism for detoxification of glyphosate was suggested by activities of certain enzymes that catalyzes the reaction such as: oxidoreductases that cleave C–N with stoichiometric formation of glyoxylate and aminomethylphosphonic acid (AMP) [6]. Aminotransferase which catalyses the conversion of AMP to phosphonoformaldehyde. Phosphonatase which catalyze the cleavage of phosphonoacetaldehyde C–P bond to form acetalde-hyde. It was evident and validated from the annotated gene results that dehydrogenase/oxidoreductase related pathway is valid by the presence of dehydrogenase related protein (alcohol dehydrogenase) discovered in their genome.
Conclusion
In this study, novel glyphosate-degrading fungi strains were isolated from farm soils in Nigeria. All strains used for enhanced biodegradation grew in the presence of glyphosate and were able to degrade glyphosate. This is the first report to show fungal degradation of glyphosate from Nigerian soil. The use of these indigenous fungal strains promises to be effective in practical application of bioremediation of glyphosate since the microbes have already adapted to the localized habitat conditions. The essence of this is that isolated strains can also be added to other soils as microbial inoculants for their potential to degrade pesticides by improving soil quality for sustainable agriculture and environment. This study has provided strains with biodegrading genes, enzymes and pathways to be harnessed for a range of biotechnological and bioremediative applications. It provides novel insights into specialised organisms for active bioremediation. The physiological and molecular characteristics shows that Aspergillus species are useful organism for managing contamination by glyphosate pesticide.