Physiological and Molecular Characterization of Active Fungi in Pesticides Contaminated Soils for Degradation of Glyphosate

Pesticide contamination is a substantial problem in controlling agricultural pests. Understanding the physiological and molecular characteristics of naturally occurring fungi in the pesticide contaminated environment is crucial to managing glyphosate contamination. The study was aimed at isolating and characterizing soil fungi for their physiological roles towards glyphosate degradation. Pure cultures of fungi were isolated from soil contaminated with glyphosate at farms in Lagos, Nigeria. The cultures were grown on minimal salt agar media amended with glyphosate. The best isolates exhibiting good tolerance to the glyphosate were characterized using molecular techniques. The BLAST search indicated that the fungi belong to four Aspergillus species (Aspergillus avus strain JN-YG-3-5, Aspergillus niger strain APBSDSF96, Aspergillus fumigatus strain FJAT-31052 and Aspergillus avus strain APBSWTPF130, Trichoderma gamsii and Penicillium simplicissimum. The biodegradation study of the glyphosate by the selected fungi species showed the presence of Aminomethylphosphonic Acid (AMPA) except for Aspergillus fumigatus strain FJAT-31052. This validates AMPA as a valid pathway for degradation of glyphosate by fungi. Annotation analysis of the partial gene sequence shows that the strains possess protein coding gene clusters for glyphosate utilization and other physiological activities. A comparative genome analysis revealed that the genomes of the fungi were highly similar with genomes of environmental samples especially to Clostridium perfringens The GhostKOALA output conrmed that CYP2W1 gene (Cytochrome P450, fungi type) was present in Aspergillus fumigatus strain FJAT-31052 which was absent in genome of other fungi. The physiological and molecular characteristics of Aspergillus fumigatus strain FJAT-31052 clearly show that this isolate is a useful organism for managing contamination by glyphosate pesticide. Consequently, the isolated microorganism strains can be used in other soils as microbial inoculants for bio-augmentation combining them for their probable ability to degrade pesticides along with their biotechnological applications like enzyme-based remediation.


Introduction
Glyphosate (N-(phosphonomethyl) glycine) pesticide is a widely used pesticides against broad spectrum of pests such as weeds, insects, fungi, nematodes and rodents in agriculture [1,2]. The intensive use of pesticides has led to an increased level and risk of contamination of the ecosystem and harmful effects on biodiversity, food security, water and other resources [3,4,5]. Glyphosate is a highly effective pesticide because it prevents biosynthesis of some notable proteins that are needed for plant growth and it also inhibits a speci c enzyme pathway known as the shikimic acid pathway. This pathway is important for plants and some microorganisms. Studies have shown that the inhibitors of the shikimate pathway enzymes are potential herbicidal because an inhibitor of a key enzyme of plant metabolism might be herbicidal without being toxic to animals. Also, this pathway operates only in plants and microorganisms [6].
The high solubility in water and strong binding capacity to soil organic matter by glyphosate is the reason for its fast and easy distribution in ecosystems compartments [7]. The half-life of glyphosate ranges from 8.3 to 141.9 days, and it has been reported to be up to 1 year in some extreme cases [8]. The differences in rates of glyphosate degradation might be due to the changing microbial activity and extent of soil-binding. The ability of microorganisms to degrade glyphosate assumes the occurrence of enzymes cleavages by utilization of glyphosate as sources of energy. According to Sviridov et al. [9] two pathways have been proposed for glyphosate degradation, which are the AMPA pathway and the C-P lyase pathway. In the AMPA pathway, glyphosate is cleaved into aminomethylphosphonic acid (AMPA) and glyoxylate by the presence of glyphosate oxidoreductase, whereas in the C-P lyase pathway, degradation of glyphosate is catalyzed by C-P lyase with the formation of sarcosine as an intermediate product, which in the end forms formaldehyde and glycine in a reaction catalyzed by sarcosine oxidase [9].
Microorganisms known to degrade glyphosate by way of glycine comprise of Arthrobacter sp. strain GLP-1 and Pseudomonas sp. (strain PG2982) [10]. Researches have also shown that the cleavage of the C-P bond of glyphosate to produce sarcosine and nally to glycine is mediated by sarcosine oxidase-dehydrogenase [11,12,13]. It has also been reported that some group of bacteria, represented by a Flavobacterium sp. (Strain GDI) as well as the earlier-reported mixed bacterial cultures from soil degrade glyphosate by cleaving its carboxymethyl carbon-nitrogen bond to produce AMPA [10]. Some of the AMPA generated in this way if not further metabolized is a great concern to the environment because of their potential toxicity. This has led to mounting concern of extensive contamination of the environment resulting to likely potential risks to non-target organism due to entry into the food chain.
Despite the imminent toxicity posed by environmental threats, some organisms can still withstand glyphosate. The ability of some organisms to survive and live in a polluted environment and remediate it depends on physiological, molecular, genetic and ecological traits possessed by such organisms. Fungi are extremely important for many physiological functions in environmental assessment and protection. Their physiological and molecular determination can be useful in ecotoxicity studies and ecosystem management. Based on this we selected a farm in Nigeria where glyphosate is used as herbicides, isolated the fungi in pure culture, grow them in the medium using glyphosate as energy and study their physiological and molecular roles towards glyphosate degradation.

Soil samples collection
The soil samples used for this study were obtained from two farms: Abeto Farm and Igbalu Farm in Ikorodu, Lagos, Nigeria. The selected farms have a history glyphosate organophosphorus herbicide for the last 5-6 years to control pests.
Glyphosate is effectually still used to control pests at the farms. Soil samples were collected from four different points in each farm's location at 100 metres apart as described by Asef [14]. The soils were collected using a spatula at a depth of 15 cm and transferred to sterile containers. The soils samples were transported to the laboratory and stored at 4 °C until further analysis. Soil samples were air dried 24 hours and sieved through a 10mm mesh prior to screening.

Quanti cation of glyphosate content and metabolites
Pesticides and their metabolites were analysed from the soil. The amount of glyphosate in the samples were quanti ed using Gas chromatography as described by Moye and Deyrup [15]. Also, aminomethylphosphonic acid (AMPA), a primary degradation product of glyphosate in the samples were assayed using the method of Ermakova et al. [16].

Soil incubations, isolation and colony characteristics
The isolation of glyphosate degraders from soil samples was done to screen for strains that could degrade glyphosate in liquid enrichment medium. Ten grams of soil sample was weighed on analytical balance. One gram of the sample was transferred into 90 ml of sterile Minimum Salt Medium (MSM) broth in a 250 ml of Erlenmeyer ask respectively and incubated at 30 o C in a gyratory shaker for seven days. After the incubation period, 1.0 ml of sample was withdrawn and was serially diluted using 9 ml amount of sterile distilled water up to 10 5 dilution. All the replicates of each soil type and treatment were treated separately for isolation purposes. Pesticide degraders were enriched in the dark on a shaker at 180 rpm for seven days. The colony characteristics were examined. A second enrichment was done thereafter as described below by transferring pre-grown culture from each of the replicates to the fresh media.

Enrichment of glyphosate degraders
The fungi isolated from the soil samples were afterward used for the enrichment of potential glyphosate degraders.
These media were further treated separately with glyphosate to enrich for glyphosate degraders. The enrichment of glyphosate degraders was carried out in liquid media by dissolving the components in 1000 ml of distilled water and adjusting the pH of the basal medium to 6.0 using 1 M NaOH Solution. 150 ml of the basal medium was dispensed into 250 ml Erlenmeyer ask and the pesticide substrate was introduced into each ask at 100 ppm after sterilization in an autoclave at 121 o C for 15 minutes. 1.0 ml aliquot of diluted broth culture of each isolate (10 4 cells/ml) were seeded into each ask and incubated in a gyratory shaker incubator at 150 rev/min at 30 o C for a period of 32 days. The growth and enrichment ability were monitored at four-day intervals. The utilization of the pesticide fractions by the fungal isolates was evaluated by monitoring the fungal growth measured by viable count on PDA, the Optical Density (OD) at 620nm wavelength with 770 UV/Visible Light PG Spectrophotometer and changes in ionic concentration pH was determined with pH meter (model P2II) [17].

Degradation of glyphosate
The capability of the isolates to degrade glyphosate was also determined. For this purpose, the medium was supplemented with glyphosate (100 ppm). The bacterial isolates were rst grown in the medium supplemented with glyphosate to determine activity of fungi in the degradation of glyphosate.

Biodegradation e ciency
The percentage loss of the pesticide was calculated as: While the e ciency of biodegradation was calculated using the formulae: LT -LC (Where LT is % loss of pesticides in treatments and LC is % loss of pesticides in control). The ampli ed fragments were puri ed by ethanol in order to remove the PCR reagents, before they were sequenced using Applied Biosystems Genetic Analyzer 3130xl sequencer and Big Dye terminator v3.1 cycle sequencing kit. Bio-Edit and MEGA 6 were used for all genetic analysis.
The classi cation/identi cation of organisms were performed by a local nucleotide BLAST search against the nonredundant version of the NCBI ref database [19]. Phylogenetic relationship analysis was performed on the sequences of isolated microorganisms using Molecular Evolutionary Genetics Analysis version 6 [19]. The sequences were prepared using FASTA format and aligned using ClustaW option of the program [19]. The phylogenetic dendrogram was constructed using the maximum likelihood with 1000 bootstrap.

Genome sequence annotation
The annotation of protein-coding genes was provided by FGENESB; further gene prediction and functional annotation were performed by GhostKOALA. The pathways of selective compounds were interpreted using KEGG pathway chart.

Statistical analysis
All data set were analyzed using GraphPad prism 7.0. Signi cant means were separated using multiple comparison of two-way analysis of variance. Correlation analysis was done to compare the relationship between pesticide degradation, colony count, optical density and pH. Data obtained from the enzyme studies were subjected to Dunnett's test and Tukey's multiple T-test and two-way analysis of variance (ANOVA) where it was applicable.

Characterization of the soil from the locations
In this study, the soils were initially characterized to have a baseline concentration of glyphosate in relation to fungal distribution. All the locations had evidence of glyphosate contamination though the contamination levels varied depending on location ( Fig. 1) and they were signi cant (p < 0.05). Location1 had the highest contamination comprising 319.1 mg/kg glyphosate and 194.2 mg/kg AMPA. Location 3 showed increased glyphosate level without transformation product (AMPA). Among the locations, the lower level of glyphosate was observed more rapid in location 4 with only traces of glyphosate present (6.98 mg/kg).
Correspondingly, the fungal density varied within the location (Fig. 2). There was high enumeration of fungal count in locations where AMPA concentration was high and with signi cant concentrations of glyphosate. However, other locations where AMPA concentration was low or absent had low fungal count. This implies active metabolic state of the fungi. As a result, this study evaluates the relationship between the fungal density, glyphosate and AMPA. The Pearson correlation shows that fungal load is signi cantly related to AMPA (r = 0.94965; p ≤ 0.05) in comparison to glyphosate.

Selection and isolation of glyphosate degraders
A total of 14 isolates were obtained from the farms ( Table 1). The fungal inoculum from soils enrichment were plated on MSM agar plates to check for their ability to grow in presence of glyphosate on solid media. It was observed that S1b, S1c, S2.3, S3.2, S3.3, S4.1 and S4.4 isolates did not grow from any of the enriched soils on MSM agar when glyphosate was added externally, during the set incubation time. They displayed poor (+) clear zone, therefore, no further analysis was conducted on them. The isolates that showed good (++) clear zone (S1a, S1d, S2.1, S3.1, S4.1 and S4.3) were further analyzed for their ability to degrade glyphosate ( Table 2). A total of six (6) potential degraders were obtained after successive sub-culturing from the soils.

Enrichment of glyphosate degraders
Six fungal isolates were stimulated to grow in the presence of glyphosate. The glyphosate mixed with MSM showed enhanced growth of some fungal isolates as shown by their optical density which ranged from 93.47% in P. simplicissimum SNB-VECD11G to 96.64% in T. gamsii P2-18 (Table 3). Furthermore, the growth of fungal isolates, A. fumigatus FJAT-31052 and A. avus EFB01 were promoted and they tolerated the glyphosate as they continued growing till the Day 32 where the experiment ended. Whereas, growth promotion of other fungal isolates was inhibited after the Day 28. More so, the fungal growth in the presence of glyphosate caused a decrease in the pH of the environment from 6.0 to 4.7 in all the isolates ( Table 4).
The changes were all signi cant (P < 0.05) with increasing incubation times but were not signi cant (P > 0.05) within the isolates. The change in pH was more obvious in A. avus EFB01 (22.06%) and lowest in A. avus JN-YG-3-5 (19.21%). Therefore, the fungal growth was inversely proportional with the pH.
The analysis of viable fungal count in all the samples at different days varied ( Figure 3). Three major phases of growth were identi ed in all the isolates. These includes lag phase, exponential phase and death phase. These phases were similar in all the fungi. The lag phase lasted for 8 days (day 0 to day 8), thereafter a slight but non increase in growth from day 8 to day 16. The exponential phase started from day 16 to day 24 and death phase started after day 24. The peak of the growth was on the 24 th day. On this day, the maximum fungal count was 1.09+E06 CFU by A. fumigatus FJAT-31052 and the lowest was 6.20+E05 CFU by T. gamsii P2-18. All other isolates growths were signi cant.

Degradation of glyphosate
The potential ability of the six fungal strains for glyphosate biodegradation were observed for 32 days (Fig. 4). The strain T. gamsii P2-18 sp. degraded 91.45% of glyphosate leaving 930.81 mg/kg of AMPA. In addition, it was observed that there was 92.07% glyphosate degradation when inoculated with A. niger APBSDSF96 leaving 113.53 mg/kg AMPA (Fig. 5). Interestingly, A. avus JN-YG-3-5 utilized 92.86% without accumulation of AMPA; this had the highest extent of degradation.
Overall, an analysis of the degradation e ciency of the fungi strains in glyphosate degradation showed that the isolates were e cient degraders with percentage degradation above 90% (Fig. 6). However, A. avus EFB01 had the poorest percentage degradation (27.17%) indicating poor metabolism of glyphosate. The degradation e ciency of A. avus JN-YG-3-5 was the most e cient fungi (85.6%) (Fig. 6).
3.6 Bacteria genome annotation Automated annotation identi ed several genes using a statistical signi cance threshold ( Table 6). The genome sequences of the fungi were compared to those of several organisms (Archae generic, C. pefringes, B. subtilis and P. putida) known to function in metabolic processes. Validation of the sequence annotation using the FGENESB database yielded the following result: Rhizobium huautlense comprises 5 potential protein coding genes, 1 operon and 4 transcription units. Pseudomonas aeruginosa strain MZ4A contains 11 protein genes, 1 operon and 7 transcriptional units. Pseudomonas aeruginosa strain 22ABUH7 had 5 protein genes, 1 operon and 3 transcriptional units. Bacillus subtilis strain VBN01 had 8 protein genes, 1 operon and 5 transcriptional units. Pseudomonas aeruginosa strain HS-38 sequence was made up of 6 potential protein coding genes, 1 operon and 5 transcriptional units. Pseudomonas aeruginosa strain MZ4A and Pseudomonas aeruginosa strain HS-38 had potential protein coding genes similar to

Discussion
Our ndings 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 eld 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 in uence bacteria growth. For instance, Yang et al. [23] found out that pH level of culture medium was one of the key factors in uencing 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 (µ max = 0.78 h − 1 ) than at other pH values including pH 5.0, 5.5, and 6.0 (µ max = 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 in uence 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, e ciency 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 ndings 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 signi cant growth and increase in microbial population.
Pseudomonas putida while others did not. A search of the identi ed proteins for speci c functions revealed that the genes are distributed in different functional categories majorly protein metabolism and respiration (Table 7). Numerous genes associated with pesticide degradation were identi ed.
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 signi cant 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. avus 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 identi ed 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 identi ed 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 avus 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 bene cial advantage to be utilized in a wide range of environmental applications. This could have also been an added advantage to Aspergillus avus 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 avus 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 identi ed 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 e ux of drugs from the cells of target organisms. Also, the Zinc nger protein gene and zinc nger chimera 1, were discovered along with many oxidoreductase genes. A search of the identi ed proteins for speci c functions revealed that the genes are distributed in different functional categories majorly protein metabolism and respiration. Numerous genes associated with pesticide degradation were identi ed. 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, identi cation of AMPA even to a signi cant 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 detoxi cation 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 rst 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.

Declarations
Availability of data and materials All generated or analysed data during this study are included in this published article and are also available from the corresponding author upon request.