BIODEGRADATION OF GLYPHOSATE CONTAINING HERBICIDE BY SOIL FUNGI

Ten fungal isolates were isolated from two herbicide-contaminated soil farms obtained from Amoyo and the University of Ilorin environment in Kwara State after enrichment with mineral salt medium (MSM) supplemented with glyphosate-containing herbicide. The growth of fungal isolates was efficiently stimulated by the organophosphorus herbicide. Fungi isolated were subjected to screening by varying the herbicide concentrations from 0.1 to 3%, which is prepared with MSM. This screening showed that all the fungal isolates had the ability to act in the biodegradation process. However, varying degradative potentials were observed, as some had heavy growth while others had only slight or no growth as the concentrations of the herbicide increased. The ten fungal isolates were characterized and identified as


INTRODUCTION
Organophosphorus pesticides are actually more widely used in the world; these pesticides affect the nervous systems of insects and humans, in addition to influencing the reproductive system [1,2].These chemical agents block prolonged inhibition of the activity of the enzyme cholinesterase, responsible for the nervous impulse in organisms [3].The excessive use of organophosphorus in agriculture has originated serious problems in the environment [4].Although these pesticides degrade quickly in water, there is always the possibility that residues and by-products will remain at relatively harmful levels in organisms [5].
Several biological techniques involving the biodegradation of organic compounds by microorganisms have been developed and are still being developed [6].The use of microorganisms, either naturally occurring or introduced, to degrade pollutants is called bioremediation [7].In addition, they are robust organisms and are generally more tolerant to high concentrations of polluting chemicals, such as some bacteria [8].However, the aim of this study is to exploit the ability of fungi to degrade herbicide residue from agricultural soils.

Sterilization of glassware and other materials
All glassware used was thoroughly washed with detergent, rinsed, and allowed to dry.The glassware was then wrapped with aluminum foil and sterilized in the hot air oven at 170°C for 60 min.The distilled water used for serial dilution was autoclaved at 121°C for 15 min.The work bench was swabbed with 70% alcohol before and after every experiment.

Sample site
Two agricultural sites contaminated with herbicide were selected: these were farms from Amoyo in Ifelodun Local Government Area of Kwara State and the Nursery Section of the Department of Forestry, University of Ilorin, Kwara State.At each location, four different samples were collected.

Soil sampling
Two soil samples were collected at four different points at each location and were labeled A1, A2, A3, A4, and B1, B2, B3, and B4, respectively.At each point, soil samples were collected randomly 5-10 cm beneath the surface of the soil using a sterile hand trowel and packed in sterile polythene bags properly labeled.The samples were immediately transferred to a microbiology laboratory for analysis.

Herbicide
Two brands of herbicides containing one active ingredient, glyphosate, were used in this study.The brands were Force Up and Golden Sate with the active ingredient of analytical standard glyphosate (44.1%), which was provided by an agrochemical shop in Ilorin metropolis, Kwara state.

Physicochemical analysis of soil samples
The physicochemical characteristics of the soil samples that were determined include temperature, pH, percentage moisture content, and organic matter content.Soil pH, percentage moisture content, and organic matter content were determined based on the method described by Pramer and Schmidt [9].

Preparation of stock mineral salt medium for fungi
Stocks were prepared inside plastic bottles with sterile distilled water using the following composition of mineral salts as described by Ashour et al. [10].The constituents of fungi are presented in Table 1.

Preparation of media
All these mineral salts were measured into different plastic bottles and dissolved with 100 mL of distilled water as stock.Calculated volumes of each mineral salt were taken from various stocks with sterile pipettes.In order to make a 150 mL solution, 100 mL of distilled water and 20 mL of tap water were added as sources of trace elements to make up the 150 mL, followed by 2.3 g of agar-agar.The mixture was heated, homogenized, and autoclaved at 121°C for 15 min.The volume of each stock solution used for 150 mL of MSA medium for fungi and bacteria is described earlier above.

Isolation of fungi from soil samples
The spread plate method was used to isolate fungi from soil samples [11].Fungi growths were examined after incubation at room temperature for 48-72 h, following the method described by Dubey and Maheshwari [12].

Screening of fungal isolates
Screening of fungal isolates and growth capability on the herbicide MSAmedium was done qualitatively following the method described by Ashour et al. [10].Fungal screening was carried out by monitoring the growth capability of glyphosate-containing media at different concentrations (0.1%, 1%, 2%, and 3%).

Characterization and identification of fungal isolates
The most efficient herbicides for degrading fungal genera were examined macroscopically and microscopically to determine their macroscopic and morphological characteristics, respectively.The isolates were identified according to the keys of Onions et al. [13].Selected fungal isolates were used for the biodegradation experiment.

Fungal biodegradation study
The composition of the mineral salt broth medium used for the fungal degradation study was the same composition of stock mineral salt medium for fungi, with the exemption of agar-agar.Four hundred milliliters of the medium were prepared in a 500 mL conical flask, and 6 ml of the test herbicide was added as a source of carbon and energy before the medium was sterilized in an autoclave at 121ºC for 15 min.The progress of the degradation study was assessed by measuring the change in pH and percentage of dry weight over the period of time.Changes in pH of the mineral salt broth were monitored, and the percentage of dry weight was determined quantitatively in the course of the biodegradation period based on the method described by Ekundayo and Osunla [14].

Isolation and characterization of fungal isolates
A total of seven fungal isolates grew on MSA supplemented with 3% (w/w) Force Up herbicide.Their growth was taken as indicative of their biodegradative ability.Fusarium oxysporum showed the weakest growth, while Aspergillus niger showed the heaviest in terms of the diameter of colony, on MSA.However, the best four fungi, A. niger, Aspergillus flavus, F. oxysporum, and Mucor spp., were used for the fungal biodegradation study.Details of the degree of growth of all fungal isolates are shown in Table 2.
Isolate F1: It appeared as whitish colonies that turned black due to the formation of black conidia, with colonies spreading rapidly.Under microscopic examination, the hyphae were septate and profusely branched.Conidia were bonded in chains at the hips of sterigmata.Conidial heads are globose.The conidiophores were long, smooth, and hyaline (colorless).It was identified as A. niger (Plate 1).Isolate F4: The colony has thick white mycelium and a white color at the bottom of the plate; microconidia are oval and produced on simple short phialides.Macroconidia are septate and chlamydospores are present.It was identified as F. oxysporum (Plate 4).

Screening of fungal isolates in MSA
A. niger, A. flavus, Mucor spp., and F. oxysporum grew heavily.Aspergillus tamari, Penicillium spinulosum, and Aspergillus terrus grew moderately.A. oryzae and Trichoderma koningi grew slightly on MSA plates at 2% concentration of Force Up herbicide.However, A. niger and A. flavus grew heavily on MSA plates, while Mucor spp., F. oxysporum, and A. terrus grew moderately at 3% on MSA plates.A. tamari and P. spinulosum grew slightly, while A. oryzae, T. koningii, and Rhizopus stolonifer showed no growth at 3% after a week.Details of the growth of all biodegrading fungal isolates on MSA plates are shown in Table 2.

Fungal biodegradation studies
Only four fungal isolates were selected and used for biodegradation, namely, A. niger, A. flavus, Mucor spp., and F. oxysporum, among all the fungi isolated from herbicide-contaminated soil were used for the biodegradative study of fungi.Parameters measured during the course of biodegradation were changes in pH and dried weight after 12 days.

Changes in the pH of mineral salt broth (MSB) during biodegradation
The initial pH of the MSB was 6.80; values of pH recorded from day 3 to day 12 include the following: For A. niger, the pH ranged from 6.60 of days; for A. flavus, the pH ranged from 6.70 to 6.90; this also increases directly with the number of days; for Mucor spp., the pH ranged from 4.40 to 5.30; there was a sharp decrease in the pH to 4.40 before later increasing gradually towards neutrality; and for F. oxysporum, the pH ranged from 4.50 to 4.80; this was similar with Mucor spp.Details of the changes in pH are shown in Fig. 1.

Percentage of dry weight
The dry weight of the fungal isolates for the two brands of herbicide used was 18% for A. niger, 9.90% for A. flavus, 8.90% for Mucor spp., and 8.60% for F. oxysporum.Details of the dry weight are shown in Fig. 2.

DISCUSSION
Microbial analysis of herbicide-contaminated soils yielded viable fungi for all fungal isolates.Fungal growth at concentrations ranging from 0.1 to 3% herbicide supplement medium.These fungal isolates are known to be indigenous soil flora.
Ten (10)  and T. koningii, were isolated.Aspergillus spp. was the only common genus isolated.Aspergilli groups are commonly found in soil and decaying organic materials by converting resistant organic chemicals such as pesticides and herbicides into simplified metabolites and eventually into soluble benefits molecules.Fungi such as Aspergillus spp.play an important role in carbon cycling [15].In this study, five species of Aspergillus were isolated.This observation was in agreement with Abdel-Hafez [16], who found the most frequent fungal genus, Aspergillus, in forty soil samples collected from desert soils in Saudi Arabia.Ashour et al. [10] also reported the isolation of 45 fungal isolates identical to the ten fungi isolated in this study.Similarly, these data were in agreement with Wardle and Parkinson [17], who studied the side effects of glyphosate on fungal species by applying a range of glyphosate concentrations (0, 2, 20, and 200 ppm herbicide).They found out that there were few fungal responses to glyphosate incubated in pure culture, where Mucor hiemalis, F. oxysporum, and Penicillium nigricans were largely unaffected by glyphosate at any concentration.Mortierella alpine, Trichoderma harzianum, and Arthrinium sphaerospermum were all significantly stimulated by 200 µg/g of glyphosate-containing herbicide.Cladosporium cladosporioides was significantly inhibited by higher glyphosate concentrations.
The increased use of herbicides in agricultural soils causes contamination of the soil with toxic chemicals.When herbicides are applied, the possibility exists that these chemicals may exert certain effects on nontarget organisms, including soil microorganisms [18,19].The microbial biomass plays an important role in the soil ecosystem, where it fulfills a critical role in nutrient cycling and decomposition [20].When an herbicide is added to a cultivated medium, the various microorganisms may have different types of responses.Some microorganisms become intoxicated and lyse.Other microorganisms are resistant and tolerant to a pollutant and can increase their numbers and biomass because of decreased competition.Specific microorganisms will actually grow on organic pollutants.
This degradation pathway of glyphosate produces the major metabolite aminomethylphosphonic acid and ultimately leads to the production of water, carbon dioxide, and phosphate [21].In the environment, Wardle and Parkinson [17,18] observed that the presence of glyphosate in the soil and the overall microbial activity of the soil, although the number of fungi and actinomycetes was not affected.In fact, studying the effect of glyphosate on the number of microorganisms in soil, microbial biomass, and soil respiration, Statton and Stewart [22] observed only a small increase in microbial biomass but no negative or positive effects with respect to the number of microorganisms or soil respiration.Haney et al. [23] and Busse et al. [24] evaluated the effect of glyphosate on the microbial community of soils and observed that microbial activity was stimulated in the presence of this herbicide.Glyphosate can stimulate the growth of mycorrhizal fungi in vitro [25].
The values of pH of MSM broth for the moulds over the period of biodegradation were slightly acidic, as values ranged from  [26], who found that after repeated application of glyphosate, microorganisms were better able to utilize it.The study by Partoazar et al. [28] concluded that glyphosate application may alter or increase soil microbial activity and population.Increased microbial activity may be beneficial or detrimental to plant growth, soil microbial ecology, and soil quality.Among the ten isolated species, four of the most tolerant fungi were used for biodegradation (A.niger, A. flavus, Mucor spp., and F. oxysporum), which were not or slightly influenced by herbicides up to 3%.

CONCLUSION
Findings made in this research revealed that most of the indigenous soil microflora has the potential to biodegrade glyphosate, whose product of biodegradation is neither harmful nor toxic.Biodegradation is a better and much more attractive method of mopping up herbicidecontaminated soil when compared to other conventional methods usually used; such as the use of incinerators and chemical methods, which are very costly and can degenerate into environmental problems.
In conclusion, A. niger, A. flavus, Mucor spp., and F. oxysporum can be employed as potentially effective fungal strains and environmentally safer alternative tools to protect the environment from the pollution of glyphosate-containing herbicides.

CONSENT FOR PUBLICATION
Not applicable Isolate F2: Bright to yellow, green colonies have the same color of green with a tinge of yellow on the reverse side of the plate.Conidiophores are coarsely rough; heads vary in size; loosely radiate; and phialides are borne directly on the vesicle.Conidia are globose.It was identified as A. flavus (Plate 2).Isolate F3: Colonies are grey and of loose texture.Sporangiophores are branched, and sporangia are globose and small.Presence of chlamydospores in the sporangiophores.It was identified as Mucus spp.(Plate 3).

Table 2 : Growth of fungal isolates in mineral salt agar medium supplemented with 2% and 3% (v/v) of force up and goldensate herbicides brands S. No Fungal isolates Degree of growth in 2% MSA
[26] to 5.30 for Mucor spp.and A. flavus, which ranged from 6.70 to 6.90, which is more acidic among the fungal isolates.A. niger and F. oxysporum steadily increase toward neutrality over the period of biodegradation.This result varies slightly for values reported by Lancaster et al.[26], who obtained pH values of 4.55 and 6.44 for A. niger and Rhizoctonia spp.during the biodegradation of herbicide-contaminated soils.
[27] a period of 12 days of biodegradation.Also, Ratcliff et al.[27]reported a transient increase in fungal propagules after glyphosate addition (50 mg/L).It is likely that the glyphosate provided nutrients for fungal growth, as evidenced by the significant increase in the microbial population.This conclusion is in agreement with Lancaster et al.