Effects of Sole and Herbicides Mixtures on Soil Microbes in A Maize – Based Cropping System

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Published on International Journal of Agriculture & Agribusiness
Publication Date: June, 2020

Olaniran, O. A., Adelasoye, K. A. & Adesina, G. O.

Department of Crop and Environmental Protection, Ladoke Akintola University of Technology
P. M. B. 4000, Ogbomoso, Nigeria

Journal Full Text PDF: Effects of Sole and Herbicides Mixtures on Soil Microbes in A Maize – Based Cropping System (Studied in South West Nigeria).

The research investigated the effect of mixed and sole applications of pendimethalin and butachlor on soil microbial population and biodiversity in a maize plot. Experimental area was divided into 5 m x 5 m plots, replicated three times and laid out in randomized complete block design. The treatments were: pendimethalin (2.0 l/ha) and butachlor (4.0 l/ha) in the ratios 1, 1, 1:1, 1:3, 3:1 and control. Soil samples were collected with soil auger five times at two weekly intervals. Fungal and bacteria populations and species composition were determined using standard methods and results were subjected to ANOVA at ά0.05 and species diversity was expressed descriptively. Bacterial population under sole pendimethalin (130.00x104cfug-1) and butachlor (123.33×104 cfug-1) were significantly higher than the initial, control and other treatments at two weeks after spraying (WAS). At 4th WAS, bacterial population increased and were not significantly different from the sole butachlor and pendimethalin. The fungal population was generally stable up to 4 WAS and not significantly different but reduced significantly at 6 and 8 WAS compared to initial soil (186.7x104cfug-1).Microbial population from herbicide mixtures does not follow a particular trend. Bacterial species isolated varied according to the sampling periods from 12 at the initial sample to 22, 26, 39, and 35 at 2, 4, 6, and 8 WAS respectively. Seventeen bacteria species occurred more than 25% in all the samples. Some of the most frequent bacteria were Proteus vulgaris (44.48%), and Bacillus stearothermophilus (55.6%). The number of fungal species isolated at each sampling time follows the same trend as bacterial population. Twenty five fungal species were encountered on the plots more than 25% and these included Penicillium dangeardi (50%), Waksmania spp. (55.6%) and Stereosporangium spp. (44.48%). The herbicides enhanced multiplication of microbes, and species initially dormant in the soil were revived.

Keywords: Pendimenthalin, Butachlor, Proteus vulgari, Waksmania spp.

Farmers need to adopt herbicide use to enhance productivity, reduce the drudgery of weed control and reduce cost of production. The traditional and commonly used control method, hoe-weeding, employed by most farmers in Nigeria has become expensive and unreliable due to constant wage increase and unavailability of labour particularly at peak period of the growing season. The use of herbicides for weed control is considered to be a better alternative to hoe-weeding because it facilitates efficient weed control, reduces labour requirements and its attendant costs, with consequent higher profitability to the farmers.
Given that productivity remains low and food deficit remain high, the use of agrochemicals is understandably inevitable, if low productivity must be redressed and food deficit corrected. But the use of agrochemicals is fraught with a lot of environmental problems which include adverse effects on untargeted, ecofriendly organisms both in the soil and other environmental compartments. The microbial biomass plays an important role in the soil ecosystem where they fulfill a crucial role in nutrient cycling and decomposition (De-Lorenzo et al., 2001).
Zabaloy et al. (2010) reported a persistent 2, 4-D degrading population that is able to use the herbicide as C and energy source in an agricultural soil where herbicide applications had ceased 2 years before the study. The number of degraders increased immediately after treatment of soil microcosms with 2, 4- D and remained high until the end of the incubation, while culturable aerobic heterotrophic bacterial counts were not affected by the herbicide. The addition of succinate (S) as an alternative source of C to soil microcosms did not stimulate degrade population, which confirmed that 2, 4-D degradation in this soil was mainly a metabolic process performed by specific degraders.
Studies have been carried by a number of researchers that used a range of herbicide concentrations in different agricultural soils (Ka et al. (1995); Merini et al., 2007). One practical implication of the proliferation of soil microbes able to degrade some herbicides, such as foliar-applied chlorophenoxy acids, is that this phenomenon guarantees self-cleaning of herbicide-impacted agricultural soils, reducing the risk of contamination. The objective of the study was to investigate the effect of mixed and sole applications of pendimethalin and butachlor on soil microbial population and biodiversity in a maize-based cropping system.

The experiment was carried out at the Teaching and Research farm of the Faculty of Agricultural Sciences, Ladoke Akintola University of Technology, Ogbomoso (4o101 E and 8o101 N), Oyo State, Nigeria during the 2014 cropping season. Annual rainfall can be as high as 1286 mm/annum with average precipitation of 100 mm/annum extending from March to November, warm and moist conditions start from April to October and temperature ranges from 28oC to 33oC. Humidity is as high as 74% all year round except in January when dry wind blows from the north. Soil prevalent in the area is well drained and of a sandy loamy texture.
The land was manually prepared and then divided into 6 plots sizes of 5 m x 5 m, replicated three times. The experiment was laid out in a Randomized Complete Block Design (RCBD) with 5 treatments, each block separated by 3 m spacing.
Soil samples were taken at 0 – 15 cm depth using a soil auger (located three spots per plot), five times at two weekly intervals, starting from the first sample taken before spraying and after herbicide spraying. The Initial samples were mixed together and sub sampled. The initial sample was used along with the other samples for soil microbial population and species composition analyses.
Variable mixtures of two the herbicides, Pendimethalin (Force Top) and Butachlor constitute the treatments.
T1 – 100% of Pendimethalin (P) (2 l/ha)
T2 – 100% of Butachlor (B) (4 l/ha)
T3 – 25% of Butachlor (1 l/ha) + 75% of Pendimethalin (1.5 l/ha)
T4 – 50% of Butachlor (2 l/ha) + 50% of Pendimethalin (1.0 l/ha)
T5 – 75% of Butachlor (3 l/ha) + 25% of Pendimethalin (0.5 l/ha)
T6 – Control
Spraying was done with knapsack sprayer with spray volume of 200 l/ha for pendimethalin and butachlor, the control was not sprayed.

2.1 Method of isolation, enumeration and identification of microorganisms
The isolation of the associated microorganisms was carried out by standard microbiological techniques. Ten (10 g) grams of the soil samples were thoroughly mixed with 90 ml of sterile distilled water. The mixtures were serially diluted and 0.1 ml of 103 and 105 dilutions was used for bacterial and fungal isolation respectively. Pure cultures obtained were characterized and identified using Barnett and Hunter (1972) and Onions et al. (1995). The microbial density of the soil samples was done by carrying out the serial dilution fold and each sample was plated from the dilution fold on plate count agar in duplicates. They were then incubated at 35oC for 24 hours before the plates were counted. Another 20 g of the soil was collected using sterile sampling bottle. Analysis of the sample was performed in the laboratory. Standard inocula were prepared from the stock culture taking one looful of the isolate. The plates were incubated at room temperature for 24 hours. Isolates were identified on the basis of their characteristics by the identification schemes as prescribed by Raul et al. (2001).

Data on microbial population were analyzed using Analysis of Variance with means separated by Duncan Multiple Range Test (DMRT) at 5% probability level while bacterial and fungal species composition were displayed as percentage occurrence on bar charts.

Sole pendimethalin (130.00x104cfug-1) and butachlor (123.33x104cfug-1) were significantly higher than the initial, control and other treatments at two weeks after spraying (2WAS). By the fourth WAS, the population of the bacteria increased and were significantly different from the sole butachlor and pendimethalin but were significantly higher than the control and initial soil samples. The population generally crashed significantly in all the treatments including the control at sixth WAS but all indicated another rise at eight WAS (Table 1). Fungal population was highest but not significantly different from the other treatments and the initial sample from equal amount of butachlor and pendimethalin (793.3x104cfug-1) and 75% butachlor+25% pendimethalin (813.3x104cfug-1). The fungal population was generally stable up to 4WAS with no significant different but dropped seriously at 6 and 8 WAS with initial soil (186.7x104cfug-1) being significantly different (Table 2).
Initial bacterial abundance in the soil was shown in Figure 1. The species diversity was summarized for all the plots at each sampling period and depicted on bar charts to show percentage occurrence. Twenty-two bacterial species were encountered in the soil samples taken 2WAS with abundance of the few dominant ones such as Proteus vulgaris (44.48%), Bacillus subtilis (38.92%) and Bacillus stearothermophilus, Pseudomonas telluria and Salibacillus spp. having 33.36%. Four other species had more than 25% occurrence on the plots (Fig.2).
Four weeks after spraying (WAS), twenty six species were also isolated with six of them having over 25% occurrences. The highest occurred was Pseudomonas telluria (44.48%) (Fig.3). Thirty nine bacteria species were recorded with five of them occurring more than 25% at 6WAS. The highest occurrence was Bacillus stearothermophilus (Fig. 4). Thirty five bacterial species were identified. Six occurred more than 25% and the two most occurred species were Bacillus subtilis and Bacillus stearothermophilus (Fig. 5). Initial fungal abundance in the soil was shown in Figure 6 with 14 species recorded.
There were 24 fungal species encountered on the plots at 2WAS with eight of them more than 25% occurrence. Waksmania spp. had 50% abundance (Fig. 7). Thirty fungal species were identified from the plots at 4WAS. Only six of them recorded more than 25% occurrence with the mostly abundant as Penicillium dangeardi (50%) (Fig.8). The fungal species encountered 6WAS were 35 in number with 7 having over 25% abundance; the mostly occurred was Waksmania spp. (55.6%) (Fig. 9). At 8WAS, thirty nine fungal species were isolated from the soil samples with four occurring more than 25% and the most abundant was Stereosporangium spp. (44.48) (Fig. 10).

The increased bacterial and fungal populations and biodiversity recorded in this study will enhance the ecosystem functions. A healthy population of soil microorganisms can stabilize the ecological system in the soil (Chauhan et al, 2006) due to their ability to regenerate nutrients to support plant growth. Any change in their population and activity may affect nutrient cycling as well as availability of nutrients, which indirectly affect productivity and other soil functions (Wang et al., 2008).
Ni et al. (2016) reported that a bacterium strain Y3, capable of efficiently degrading pendimethalin, was isolated from activated sludge and identified as Bacillus subtilis according to its phenotypic features and 16S rRNA phylogenetic analysis. This strain could grow on pendimethalin as a sole carbon source and degrade 99.5% of 100 mg/L pendimethalin within 2.5 days in batch liquid culture, demonstrating a greater efficiency than any other reported strains. Three metabolic products, 6-aminopendimethalin, 5-amino-2-methyl-3-nitroso-4-(pentan-3-ylamino) benzoic acid, and 8-amino-2-ethyl-5-(hydroxymethyl)-1,2-dihydroquinoxaline-6-carboxylic acid, were identified by HPLC-MS/MS, and a new microbial degradation pathway was proposed.
Bacillus sp. strain hys-1 had been isolated from active sludge, could degrade >90% butachlor at a concentration of 100 mg/L within 7 days. The strain hys-1 could mineralize butachlor via the following pathway: butachlor was initially metabolized to 2-chloro-N-(2, 6-diethylphenyl)-N-methylacetamide by debutoxylation and then transformed to form 2-chloro-N-(2, 6-diethylphenyl)acetamide by N-demethylation. Subsequently, it was converted to 2, 6-diethylaniline and further mineralized into CO2 and H2O (Yang Gao et al., 2015). Microbial degradation is the major avenue of butachlor degradation from soils.
Sherif Abd-Alrahman and Mounir Salem-Bekhit (2013) reported that six bacterial strains were isolated from an agricultural soil and found to be actively utilized butachlor, as a sole source of carbon and energy. Based on their morphological and biochemical categorization, the six bacterial and fungal isolates were identified as Psedomonas alcaligens, Bacillus licheniformis, Bacillus megaterium, Trichoderma viride, Rhizobium huakuii and Bradyrhizobium japonicum. Results show that the Trichoderma viride and Psedomonas alcaligens quickly degraded butachlor and reached nearly 98 and 75% in a medium containing 50 mg/kg of butachlor after 15 and 21 days, respectively.
Sharef et al. (2013) reported that the remaining amounts of pendimethalin recovered from media inoculated with Pseudomonas aeruginosa for 3, 7, 15 and 30 days were; 75.5%, 69.25%, 29.75 % and 19.25% respectively, while the amounts recovered from media inoculated with Bacillus mycoides were; 48.75%, 46.25%, 39.25 % and 28.25% following the same order. On the other hand, the respective amounts recovered from media inoculated with Bacillus cereus were; 45 %, 32.5 %, 30.5 % and 19.75% at 3, 7, 15 and 30 days, respectively. Despite the significant drop in the starting material, no metabolites were detected in Bacillus cultures while only N-(1-ethylpropyl)-3-methyl-2, 6-diaminobenzine was detected in P. aeruginosa culture indicating the capability of these microorganisms of complete mineralization of pendimethalin. Pendimethalin biodegradation by the three types of bacteria followed a biphasic model with faster rate of disappearance in the first phase and slower rate in the second. The half-lives for the first phase ranged from 0.3 days to 0.58 days, while it ranged from 3.7 to 6.03 days in the second phase. Based on the half-lives, the efficiency of the three bacterial species to degrade pendimethalin can be ordered as follows; Bacillus mycoides was more efficient than Pseudomonas aeruginosa which was more efficient than Bacillus cereus.

The effects of the two herbicides on the soil microbes was positive as indicated by increased in number and abundance of both bacteria and fungi. The resuscitation of the dormant microbes could also allay the fear of herbicide persistent and contamination of the environment as well as putting the problem of follow-crop at bay. Herbicide contaminated soil can also be cleaned by culturing the organisms capable of utilizing the particular contaminants thereby enhancing environmental safety.

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