Soil microbial community response to surfactants and herbicides in two soils

Applied Soil Ecology 74 (2014) 12–20

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Soil microbial community response to surfactants and herbicides in two soils M.L. Banks a,∗ , A.C. Kennedy b,1 , R.J. Kremer c , F. Eivazi d a

University of Missouri, Department of Soil, Environmental and Atmospheric Sciences, 302 ABNR Building, Columbia, MO 65211, United States USDA-ARS, Land Management and Water Conservation Research Unit, Washington State University, 215 Johnson Hall, Pullman, WA 99164-6421, United States c USDA-ARS, Cropping Systems and Water Quality Unit, University of Missouri, 302 ABNR Building, Columbia, MO 65211, United States d Cooperative Research, Lincoln University of Missouri, Jefferson City, MO 65101, United States b

a r t i c l e

i n f o

Article history: Received 27 July 2012 Received in revised form 29 August 2013 Accepted 30 August 2013 Keywords: PLFA Adjuvant Surfactant Herbicides Texture Soil microbial community

a b s t r a c t The environmental impacts of herbicides on desirable plants and the soil biota are of public concern. The surfactants that are often used with herbicides are also under scrutiny as potentially harmful to soil biological systems. To address these concerns, we used two soils, a silt loam and a silty, clay loam from south central Missouri, to investigate the impacts of herbicides and surfactants on soil microbial communities using phospholipid fatty acid (PLFA) analysis. The surfactants used in this study were alkylphenol ethoxylate plus alcohol ethoxylate (Activator 90), polyethoxylate (Agri-Dex), and a blend of ammonium sulfate, drift reduction/deposition polymers and anti-foam agent (Thrust). The herbicides were glyphosate, atrazine and bentazon. Surfactants and herbicides were applied to soils at label rate, either alone or combined, to 4000 g soil per pot. The two soils differed in history, texture, some chemical characteristics and several microbial community characteristics. A few of the chemicals altered some of the components of the microbial community after only one application of the chemical at field-rate. The Cole County, MO silt loam showed larger changes in the microbial community with application of treatments. For the Boone County, MO silty clay loam, Activator 90, Agri-Dex and bentazon treatments increased microbial biomass determined by PLFA; Thrust decreased PLFA markers, bacteria to fungi ratio; and Agri-Dex at both rates decreased monounsaturated fatty acids. Changes in the microbial community due to herbicides or surfactants were minimal in this study of a single application of these chemicals, but could be indicators of potential long-term effects. Long-term studies are needed to determine the changes in the microbial community after several years of annual applications of herbicides and surfactants on a wide array of soil types and management practices. Published by Elsevier B.V.

1. Introduction Herbicides are routinely applied to more than 90% of most U.S. crops at rates varying from g to kg ha−1 (Gianessi and Reigner, 2007; Center for Food Safety, 2008; Singh and Ghoshal, 2010). In 2007, 84 and 35 million kg of glyphosate and atrazine applied in the U.S. (U.S.E.P.A., 2011), respectively, raise concerns regarding potential impacts on soil microbial activity and community structure. In addition, surfactants are often used with herbicides as additives to enhance foliar uptake of post-emergence herbicides (Liu, 2004). While some of these chemicals may not be applied

∗ Corresponding author at: USDA-ARS, Greenhouse Production Research Group, 2801 West Bancroft Street, Toledo, OH 43606, United States. Tel.: +1 419 530 1541. E-mail addresses: [email protected] (M.L. Banks), [email protected] (A.C. Kennedy). 1 Tel.: +1 509 335 1554; fax: +1 509 335 3842. 0929-1393/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.apsoil.2013.08.018

directly to soils, a substantial amount may contact soil during application or rainfall events (Haney et al., 2000). Some herbicides, such as glyphosate, move little in soil and may be easily adsorbed to clay and organic matter and slowly degrade in water and soil (Ahrens, 1994; Pessagno et al., 2008; Barja and dos Santos Afonso, 2005). Others persist for many years in soil or enter groundwater (Kolpin, 1996; Boyd, 2000). Herbicide degradation is affected by soil factors including nutrient composition and content, pH, temperature and moisture (Weber et al., 1993). Like glyphosate, bentazon and atrazine are degraded by biological processes (Li et al., 2008). Atrazine-degrading microorganisms may accumulate in soil receiving frequent atrazine applications and coexist with the indigenous soil microbial community while metabolizing the herbicide (Satsuma, 2009; Zablotowicz et al., 2002). The addition of nonionic surfactants to soil with herbicides reduced herbicide degradation compared with the herbicides applied alone (Li et al., 2008). Herbicides and surfactants differ in chemical composition and react differently when incorporated into the soil system due to

M.L. Banks et al. / Applied Soil Ecology 74 (2014) 12–20

differences in chemical properties and interactions with soil components and environmental factors (Smith and Hayden, 1982; Ray et al., 1995). Glyphosate and atrazine may reduce enzyme activity and populations of various organisms in soil (Toyota et al., 1999; Sannino and Gianfreda, 2001). Ratcliff et al. (2006) found that bacterial and fungal populations were altered with application of various herbicides. When pure herbicide (mesotrione as active ingredient) and formulated herbicide were added to soils, the microbial activity was affected by application of pure herbicide (mesotrione) and formulated herbicide only at rates 10 times and 100 times greater than the recommended rate (Crouzet et al., 2010). These chemicals can directly or indirectly affect microbial communities or sub-populations of the community and these changes can be expressed either as short-term or long-term effects (Bittman et al., 2005; Ratcliff et al., 2006; Dick et al., 2010). Soil management history can also affect microbial composition and responses (Girvan et al., 2003). Microbial community structure, often used as an indicator in monitoring soil quality, is affected by various environmental and growth factors, such as moisture, temperature, nutrient availability, and management practices (Petersen et al., 2002). Assessment of microbial community composition within the soil ecosystem is helpful in determining if management practices and environmental conditions are aggrading or degrading to soils. Phospholipid fatty acid (PLFA) profiles, characteristic of soil microbial communities, differ with management practices including tillage, cropping system, and addition of various chemicals (Acosta-Martínez et al., 2010; Dick et al., 2010; Ratcliff et al., 2006; Ibekwe and Kennedy, 1998). The objective of this study was to examine the short-term effect of surfactants and herbicides on soil microbial community composition using PLFA analysis. We hypothesized that surfactants and herbicides added to different soils may alter soil microbial community structure as indicated by PLFA profiles and these profiles could provide an indication of potential long-term effects on the soil biota.

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rates (alkylphenol ethoxylate plus alcohol ethoxylate [Activator 90] 2.3 L ha−1 ; polyethoxylate [Agri-Dex] 2.3 L ha−1 ; ammonium sulfate + drift reduction/deposition polymers + anti-foam agent blend [Thrust] 2.8 L ha−1 ). Surfactant–herbicide combinations used in commercial formulations were applied at field application rates and included Activator 90 + glyphosate, Agri-Dex + atrazine and bentazon + Thrust. Non-treated soils served as controls. 2.2. Herbicide and surfactant treatments To determine effects of surfactants, herbicides and surfactant–herbicide combinations on the soil microbial community, a greenhouse experiment was conducted twice. Two-gallon pots (20.3 cm dia. by 20.3 cm in height) were filled with 4000 g of air-dried soil, fertilized and limed in accordance with fertility recommendations for field corn (Zea mays L. cultivar ‘Indenta’) based on soil test analyses (Lory et al., 1998). Soils were brought to field capacity and watered daily to maintain field capacity. Surfactant and herbicide treatments were prepared at designated rates using deionized water and were applied directly to pot surface. Surfactants used in this study were Activator-90 (Actv), Agri-Dex (Agrd), and Thrust (Thrst). Herbicides used were glyphosate (Glyp), atrazine (Atrz), and bentazon (Bent). Application followed label rates for the surfactants and herbicides, and amounts per pot were for 4000 g soil per pot (Table 2). Six seeds of field corn were planted in each pot and later thinned to two plants per pot. Treatments were replicated three times and arranged in a randomized complete block design on greenhouse benches. The study was replicated twice with similar results each time and the second study is reported here. Temperature in the greenhouse varied from 18 to 27 ◦ C throughout the day. Supplemental lighting was used to increase the daylight period to 14 h light and 10 h dark. The corn grew equally well in each soil. Seven weeks after seeding, when small roots were found throughout the pot, the corn foliage was harvested by cutting at the soil surface and the roots were carefully removed from the soil. Samples were collected from root-free soil, stored in plastic bags at 4 ◦ C and processed for PLFA.

2. Materials and methods 2.3. Phospholipid fatty acid analysis 2.1. Soils and chemicals We conducted the study with soils from the A horizons of a Wrengart silt loam (20% clay; fine-silty, mixed, active, mesic Fragic Oxyaquic Hapludalfs) collected from the Lincoln University Carver Farm, Cole County, Jefferson City, MO (38◦ 31 36.1 N, 92◦ 8 22.9 W), and a Mexico silty clay loam (37.5% clay; fine, smectitic, mesic Vertic Epiaqualfs) collected from University of Missouri Bradford Farm, Boone County, Columbia, MO (38◦ 53 48 N, 92◦ 12 23.5 W). The soils were located 56 km (26 miles) from each other. The Cole County, MO silt loam prior to collection was under continuous tall fescue (Festuca arundinacea L.) with annual fertilizer applications of N-P-K (60-30-30) for no less than 5 years. The Boone County, MO silty clay loam was under permanent broomsedge grass (Andropogon virginicus L.) due to its low pH, and had not been fertilized recently. No known herbicide or surfactant applications had been made to the two sites prior to soil sampling. Bulk soils were air dried, sieved to pass a 2 mm screen and analyzed for chemical and physical characteristics (Buchholz et al., 1983; Table 1). The two soils differed in that Cole County, MO silt loam was slightly acidic with low CEC and high P and K, while the Boone County, MO silty clay loam was very acidic with a high CEC and low P and K. Herbicides were applied at a dose simulating recommended field application rates (atrazine, 2.24 kg a.i. ha−1 ; bentazon, 1.12 kg a.i. ha−1 ; glyphosate, 0.84 kg a.i. ha−1 ). Surfactants were applied at the recommended dose and at two times field application

Phospholipid fatty acid analysis (PLFA) of the soil samples followed the methods of Bligh and Dyer (1959), modified by Petersen and Klug (1994). Reagents used in the procedure were high pressure liquid chromatography (HPLC) grade supplied by Sigma (St. Louis, MO) unless otherwise stated. Two-gram soil samples were added to Teflon-lined screw cap culture tubes and extracted. The total lipid extract was fractionated into glyco-, neutral, and polar lipids (Ibekwe and Kennedy, 1998). The polar lipid fraction was transesterified with mild alkali to recover the PLFA as methyl esters in hexane. Solid phase extraction was used to separate the samples for phospholipid analysis with 100-mg silica columns (Varian, Palo Alto, CA) as described in Pritchett et al. (2011). A gas chromatograph (Agilent Technologies GC 6890, Palo Alto, CA) equipped with a fused silica column, flame ionizer detector, and integrator was used to analyze fatty acid methyl esters. Integration and analysis of samples were operated with ChemStation software (Agilent Technologies GC 6890, Palo Alto, CA). Microbial Identification Systems, Inc. (Newark, DE) software provided parameters that were used for peak identification and integration of areas. Peak chromatographic responses were converted to mole responses by using internal standards and recalculation of responses was done as needed. Various peaks were used as markers for bacteria as described in Pritchett et al. (2011). Briefly, peaks that correspond to carbon chain lengths of 12–20 carbons are generally associated with microorganisms and were handled

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M.L. Banks et al. / Applied Soil Ecology 74 (2014) 12–20

Table 1 Soil properties of two Missouri soils used in the 7-week greenhouse study of corn (Zea mays L. cultivar ‘Indenta’) grown with herbicide and surfactant treatments applied to the soil surface. County

Textural class

pHs (CaCl2 )

Org. C g kg−1

N g kg−1

Bray 1 P mg kg−1

Exc. K mg kg−1

CEC cmol kg−1

Exc. Ca mg kg−1

Exc. Mg mg kg−1

Cole County, MO Boone County, MO

Silt loam Silty clay loam

5.61 4.52

12.7 11.0

0.122 0.096

47.08 1.34

107 57

12.4 22.2

1214 1524

233 252

*Abbreviation: Exc., exchangeable; org., organic.

as follows. Peaks used as markers for bacteria were 12:0 3OH, iso14:0, 15:0, ante15:0, iso15:0, i15:0g, cyc15:1, iso16:0, 16:1␻7, cis16:1␻7, trans16:1␻7, a17:0, cyc17:0, iso17:0, 17:1␻6, i17:1␻7, 18:1␻7, cis18:1␻7, cis18:1␻9, cyc19:0, cyc19:0 C11-12, cyc19:0, cis19:1␻9 (Vestal and White, 1989; Bååth, 2003; Frostegård and Bååth, 1996; Zelles, 1999). Fungal markers were 16:1␻5, cis16:1␻5, 18:1␻9, 18:2␻6, cis18:2␻6, 18:2␻9, 18:3␻3, 18:3␻6, cis18:3␻6 (Zelles et al., 1995; Frostegård et al., 1993; Frostegård and Bååth, 1996; Sundh et al., 1997; Grigera et al., 2007). Biomass was calculated from mole response calculations using the relationship determined by Bailey et al. (2002). Mole percent values of biomarkers representing both bacterial (total aerobic, Gram-negative and Gram-positive markers) and fungal (total and mycorrhizal markers) components were summed individually and bacteria to fungi ratios were calculated for each sample. For Gram-positive bacteria, markers were iso14:0, iso15:0, ante15:0, iso15:0g, iso16:0, iso17:0, cis18:1␻9 (Zelles et al., 1995; Sundh et al., 1997). The markers for Gram-negative bacteria were 15:1␻6c, cis16:1␻7, cis16:1␻7t, cyc17:0, cis18:1␻7; cyc19:0, cis19:1␻9 (Zelles et al., 1995; Sundh et al., 1997). Mycorrhizal markers were 16:1␻5, cis16:1␻5, 18:2␻6, cis18:2␻6, 18:2␻9 (Balser et al., 2005; Belen Hinojosa et al., 2005; Madan et al., 2002; Olsson, 1999). Biomass was calculated using the relationship determined by Bailey et al. (2002). Bacterial:fungal ratios were calculated for each sample. Other indicators were calculated based on the ratios of the cyclopropyl fatty acids to monoenoic precursors and the total saturated to total monounsaturated fatty acids (Kieft et al., 1994; Bossio and Scow, 1998; Fierer et al., 2003). Ratios were calculated for the proportion of the cyclopropyl fatty acids to monoenoic precursors and the total saturated to total monounsaturated fatty acids (Kieft et al., 1994; Bossio and Scow, 1998; Fierer et al., 2003). Specific peaks used to calculate the cyclopropyl fatty acids to monoenoic precursor ratios were cyc17:0 to cis16:1␻7 and cyc19:0 to cis18:1␻7. The ratio of total saturated to total monounsaturated fatty acids used the ratio of the sum of 14:0, 15:0, 16:0, 17:0, 18:0, and 20:0 to sum of cis16:1 ␻ 11, cis16:1 ␻ 9, cis16:1 ␻ 7, cis16:1␻5, cis17:1␻9, cis17:1␻8, cis17:1␻7, and cis17:1␻5. Monounsaturated fatty acids from 14:0 to 19:0 were also evaluated (Bossio and Scow, 1998). 2.4. Statistical analyses The data for the different parameters was analyzed for soil types and treatments using SAS PROC GLM (SAS, 2008). ANOVA was used

to determine significant effects and, where F-values were significant (P = 0.05), mean separations were conducted using Tukey’s test. Fatty acid percentages spanned a wide range and were log transformed when needed. 3. Results 3.1. Microbial markers The two soils used in this study were a Cole County, MO silt loam and a Boone County, MO silty clay loam, which differed in texture as well as some chemical and physical characteristics (Table 1). The Cole County, MO silt loam had a higher pHs of 5.61 compared with the Boone County silty clay loam pHs of 4.52. The organic C and N values were similar between the two soils. The Cole County silt loam was higher in P and exchangeable K compared with the Boone County silty clay loam. Cation exchange capacity (CEC) values of the Boone County, MO silty clay loam were almost double that of the Cole County, MO silt loam; however, exchangeable Ca and Mg were similar for the two soils. The two soils also differed in their microbial community structure as determined by PLFA marker analyses (Tables 3 and 4). Soil microbial biomass was 119 ␮g C g−1 soil for the Cole County, MO silt loam, significantly higher than the Boone County, MO silty clay loam at 75 ␮g C g−1 soil. Bacteria to fungi ratios (BtoF) also differed with 7.6 for the Cole County, MO silt loam and 2.3 for the Boone County, MO silty clay loam. Bacteria markers (BAC), Gram-negative (GN), aerobic (AER) and anaerobic bacteria markers (ANA) were also greater in the Cole County, MO silt loam than the Boone County, MO silty clay loam. Variability within groups was greater for the Boone County, MO silty clay loam and may have influenced the statistical significance of treatment differences. 3.2. Herbicide and surfactant treatments-marker The microorganisms in the two soils responded differently to the herbicide and surfactant treatments (Tables 3 and 4). More microbial groups in the Cole County, MO silt loam (Tables 3a and 3b) were affected by treatments than those in the Boone County, MO silty clay loam (Tables 4a and 4b). In the Cole County, MO silt loam, Actv surfactant at the 2× rate increased microbial biomass (MB) to three times that of the control. Fungal (FUN) markers

Table 2 Chemicals used and treatment rates applied to pots in greenhouse experiment (L ha−1 ). Treatments

Activator-90 Glyphosate Activator-90 + glyphosate Agri-Dex Atrazine Agri-Dex + atrazine Thrust Bentazon Thrust + bentazon Control

Treatment rates Surfactant

Surfactant × 2

0.02 ml

0.04 ml

Herbicide

Surfactant + herbicide

0.0065 ml 0.02 ml + 0.0065 ml 0.02 ml

0.04 ml 0.0167 ml 0.02 ml + 0.0167 ml

0.01 g

0.02 g 0.0083 ml

0

0

0

0.01 g + 0.0083 ml 0

M.L. Banks et al. / Applied Soil Ecology 74 (2014) 12–20

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Table 3a The effect of herbicide and surfactant treatments on microbial biomarker groupings of phospholipid fatty acid analysis from a Cole County, MO silt loam used in 7-week study of corn (Zea mays L. cultivar ‘Indenta’) grown in a greenhouse. Microbial biomarker groupings were microbial biomass (MB), bacterial (BAC), fungal (FUN), bacteria to fungi ratio (BtoF), Gram-negative bacteria (GN), and Gram-positive bacteria (GP). Treatments

MB ␮g C g−1 soil

BAC nmole g−1 soil

FUN nmole g−1 soil

BtoF

GN nmole g−1 soil

GP nmole g−1 soil

Control

119.8b

1.10a

0.15bcde

7.6ab

0.83a

0.27def

Actv Actv2 Glyp ActvGlyp

108.5b 377.2a 144.3b 153.1b

0.93b 0.72d 0.90c 0.79bcd

0.16bcde 0.27a 0.23ab 0.18bcde

6.2abcd 3.4d 3.9cd 4.5cd

0.69b 0.51de 0.54cde 0.47e

0.24fg 0.22gh 0.36a 0.29abc

Agrd Agrd2 Atrz AgrdAtrz

151.5b 119.3b 164.0b 88.7b

0.94ab 0.93b 0.81bcd 0.71d

0.91abcd 0.21abc 0.18bcde 0.10e

5.0bcd 4.6bcd 4.6bcd 5.1abcd

0.66bc 0.63bcd 0.46e 0.57bcde

0.29cde 0.31bcd 0.34ab 0.15i

Thrst Thrst2 Bent ThrstBent

141.8b 109.4b 219.5ab 77.3b

0.79bcd 0.76 cd 0.81bdc 0.85bcd

0.14cde 0.16bcde 0.18abcd 0.12cde

5.8abcd 5.4abcd 4.4cd 6.8abc

0.58bcde 0.57bcde 0.57e 0.67cb

0.21gh 0.19ghi 0.34ab 0.18hi

Actv = Activator-90; Actv2 = Activator-90, 2× rate; Glyp = glyphosate; ActvGlyp = Activator-90 + glyphosate; Agrd = Agri-Dex; Agrd2 = Agri-Dex 2× rate; Atrz = atrazine; AgrdAtrz = Agri-Dex + atrazine; Thrst = Thrust; Thrst2 = Thrust 2× rate; Bent = bentazon; ThrstBent = Thrust + bentazon. Different letters following values within the same column indicate significant difference (P ≤ 0.05) based on Tukey’s test. Bold text indicates significant difference from the control.

Table 3b The effect of herbicide and surfactant treatments on microbial biomarker groupings of phospholipid fatty acid analysis from a Cole County, MO silt loam used in 7-week study of corn (Zea mays L. cultivar ‘Indenta’) grown in a greenhouse. Microbial biomarker groupings were aerobic bacteria markers (AER), anaerobic bacteria markers (ANA), ratio saturated to monounsaturated (StoM), and monounsaturated fatty acids (MONO). Treatments

AER nmole g−1 soil

ANA nmole g−1 soil

StoM

MONO nmole g−1 soil

Control

1.1a

0.83a

0.01b

2.55abcd

Actv Actv2 Glyp ActvGlyp

0.93b 0.72d 0.90bc 0.80bcd

0.69ab 0.52de 0.54cde 0.47e

0.01b 2.0ab 34.0ab 36.1a

2.86abc 1.86d 2.49abcd 2.34bcd

Agrd Agrd2 Atrz AgrdAtrz

0.94ab 0.92b 0.81bc 0.71d

0.66bc 0.62bcd 0.46e 0.57bcde

21.5ab 19.7ab 32.5ab 0.01b

2.62abcd 2.77abc 2.37bcd 3.02abc

Thrst Thrst2 Bent ThrstBent

0.79bcd 0.8cd 0.81bcd 0.85bcd

0.58bcde 0.57bcde 0.46e 0.67bc

25. 9ab 21.1ab 30.9ab 0.00b

2.80abc 3.10ab 2.12 cd 3.29a

Actv = Activator-90; Actv2 = Activator-90, 2× rate; Glyp = glyphosate; ActvGlyp = Activator-90 + glyphosate; Agrd = Agri-Dex; Agrd2 = Agri-Dex, 2× rate; Atrz = atrazine; AgrdAtrz = Agri-Dex + atrazine. Thrst = Thrust; Thrst2 = Thrust, 2× rate; Bent = bentazon; ThrstBent = Thrust + bentazon. Different letters following values within the same column indicate significant difference (P ≤ 0.05) based on Tukey’s test. Bold text indicates significant difference from the control.

Table 4a The effect of herbicide and surfactant treatments on microbial biomarker groupings of phospholipid fatty acid analysis from a Boone County, MO silty clay loam used in 7-week study of corn (Zea mays L. cultivar ‘Indenta’) grown in a greenhouse. Microbial biomarker groupings were microbial biomass (MB), bacterial (BAC), fungal (FUN), bacteria to fungi ratio (BtoF), Gram-negative bacteria (GN), Gram-positive bacteria (GP). Treatments Control

MB ␮g C g−1 soil

BAC nmole g−1 soil

FUN nmole g−1 soil

BtoF

GN nmole g−1 soil

GP nmole g−1 soil

75.0c

0.62

0.28a

2.27bcd

0.34ab

0.28ab

Actv Actv2 Glyp ActvGlyp

122.3bc 118.1bc 92.4c 111.5bc

0.68 0.73 0.64 0.55

0.24ab 0.23ab 0.20ab 0.25ab

2.57bcd 3.00abc 3.09abc 2.15cd

0.35ab 0.40a 0.33ab 0.28b

0.28ab 0.28ab 0.27ab 0.22b

Agrd Agrd2 Atrz AgrdAtrz

255.6ab 221.1abc 99.5bc 146.3bc

0.62 0.71 0.64 0.64

0.26ab 0.28a 0.24ab 0.21ab

2.33bcd 2.39bcd 2.44bcd 3.22ab

0.29b 0.29b 0.36ab 0.34ab

0.28ab 0.36a 0.24ab 0.30ab

Thrst Thrst2 Bent ThrstBent

104.8bc 125.0bc 305.4a 156.6abc

0.52 0.61 0.58 0.63

0.28a 0.22ab 0.30a 0.248

1.73d 2.58bcd 2.06cd 2.40bcd

0.27b 0.28b 0.28b 0.32ab

0.20b 0.28ab 0.25ab 0.26ab

Actv = Activator-90; Actv2 = Activator-90, 2× rate; Glyp = glyphosate; ActvGlyp = Activator-90 + glyphosate; Agrd = Agri-Dex; Agrd2 = Agri-Dex 2× rate; Atrz = atrazine; AgrdAtrz = Agri-Dex + atrazine; Thrst = Thrust; Thrst2 = Thrust 2× rate; Bent = bentazon; ThrstBent = Thrust + bentazon. Different letters following values within the same column indicate significant difference (P ≤ 0.05) based on Tukey’s test. Bold text indicates significant difference from the control.

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Table 4b The effect of herbicide and surfactant treatments on microbial biomarker groupings of phospholipid fatty acid analysis from a Boone County, MO silty clay loam used in 7-week study of corn (Zea mays L. cultivar ‘Indenta’) grown in a greenhouse. Microbial biomarker groupings were aerobic bacteria (AER), anaerobic bacteria (ANA), ratio saturated to monounsaturated (StoM), and monounsaturated (MONO). Treatments

AER nmole g−1 soil

ANA nmole g−1 soil

StoM

MONO nmole g−1 soil

Control

0.62

0.34abc

23.9ab

3.40ab

Actv Actv2 Glyp ActvGlyp

0.63 0.68 0.60 0.50

0.35abc 0.40a 0.33abc 0.28bc

39.5a 26.4ab 30.4ab 27.2ab

2.52abcd 2.70abc 3.38ab 3.27ab

Agrd Agrd2 Atrz AgrdAtrz

0.57 0.66 0.60 0.64

0.26bc 0.25c 0.36ab 0.069abc

17.1ab 24.2ab 29.8ab 47.4a

1.49d 1.82cd 3.44a 2.73abc

Thrst Thrst2 Bent ThrstBent

0.47 0.56 0.53 0.58

0.27bc 0.28bc 0.28bc 0.31abc

22.6ab 38.0a 33.8ab 41.3a

2.95abc 2.40abcd 2.24bcd 2.74abc

Actv = Activator-90; Actv2 = Activator-90, 2× rate; Glyp = glyphosate; ActvGlyp = Activator-90 + glyphosate; Agrd = Agri-Dex; Agrd2 = Agri-Dex 2× rate; Atrz = atrazine; AgrdAtrz = Agri-Dex + atrazine; Thrst = Thrust; Thrst2 = Thrust 2× rate; Bent = bentazon; ThrstBent = Thrust + bentazon. Different letters following values within the same column indicate significant difference (P ≤ 0.05) based on Tukey’s test. Bold text indicates significant difference from the control.

were increased by 1.5× for Actv2 and Glyp compared with the control. The markers for BAC, BtoF, GN, Gram positive (GP), AER, and ANA were reduced compared with the control for most treatments. All treatments except Agrd at the recommended rate led to decreases in AER markers in the Cole County, MO silt loam. Gram negative bacteria were reduced across all treatments relative to the control. The response of GP markers to treatments varied. Gram positive markers after application of Actv, Agrd, Agrd2 were equal to the control, while those markers declined with application of Actv2, AgrdAtrz, Thrst, Thrst2 and ThrstBent. Glyp, ActvGlyp, Atrz and Bent caused an increase in GP markers. ANA markers were lower for most treatments when compared with control. The StoM ratios increased with ActvGlyp when compared with the control, and other treatments applied were the same as the control for Cole County, MO silt loam. Decreases in biomarkers of the bacterial community occurred with nearly all treatments in the Cole County, MO silt loam. In the Cole County, MO silt loam, Glyp alone and combined with Actv decreased BAC, BtoF, GN, AER, ANA, and increased GP. Agrd at the recommended rate decreased GN and ANA, and at 2× rate showed decreased BAC, GN, AER, and ANA. Atrazine showed a decrease in BAC, GN, AER, ANA, and an increase in GP. Combinations of Agrd with Atrz and Thrst with Bent decreased BAC, GN, GP, AER and ANA. Thrust at the recommended and 2× rates decreased BAC, GN, GP, AER and ANA. Bent decreased BAC, BtoF, GN, AER, ANA and increased GP. Overall, all treatments except Agrd decreased bacterial markers relative to control. Fungi increased with Actv at the 2× rate in the Cole County, MO silt loam. Bacteria to fungi ratio values decreased relative to control for Actv2, Glyp, ActvGlyp, and Bent. Gram-negative bacteria biomarkers were reduced with all treatments compared with control. Gram-positive bacteria biomarkers were higher than the control for Glyp, ActvGlyp, Atrz and Bent and lower for Actv2, AgrdAtrz, Thrst and Thrst2. Aerobic and anaerobic bacteria biomarkers were significantly decreased by all chemical treatments except Agrd and Actv, respectively, applied individually at recommended rates. In the Boone County, MO silty clay loam, only Agrd and Bent applied at the recommended rate significantly increased MB to 256 and 305 ␮g C g−1 soil, respectively, compared with control MB of 75 ␮g C g−1 soil (Table 4a). Both rates of Agrd decreased monounsaturated fatty acids from 3.40 nmole g−1 soil of the control to an average of 1.66 nmole g−1 soil (Table 4b). No other microbial characteristics were affected by the application of the other treatments to the Boone County, MO silty clay loam.

3.3. Lipid chain lengths Several treatments resulted in higher levels of certain lipid chains that were significant factors in differentiating the microbial communities in this study. For the Cole County, MO silt loam those treatments were Actv2, Glyp, ActvGlyp, Agrd2, Atrz, Thrst and Bent (Table 5). For the Boone County, MO silty clay loam, Agrd, Agrd2, AgrdAtrz, Thrst2, Bent, and ThrstBent were found to cause changes (Table 6). Only Actv was not represented in the two soils, while Agrd2 and Bent were found to be significant in both soils. The lipid chains responsible for indicating differences for Cole County soil were 12:0 2OH, 14:0, 14:1 ␻ 8c, ante15:0, i15:0, 16:0, iso16:0, 16:1 ␻5c, 16:1 ␻7c, 19:0 cyclo 11-12 2OH; for Boone County, 14:0, 15:0, 15:0 3OH, ante15:0, iso15:0, 16:0 3OH, iso16:0, isog16:1, isol16:1, 16:2 ␻6c. In the Cole County, MO silt loam, Thrst increased only 12:0 2OH; and Activ2 increased only 19:0 cyclo11-12 2OH compared with the control. Bent increased 14:0 and six other lipid chains: ante15:0, 16:0, i16:0, 16:1 ␻ 5c, 16:1 ␻7c and 19:0 cyclo 11-12 2OH. Interestingly ActvGlyp, Atrz, and Glyp treatments resulted in higher concentrations of each of the same lipid chains, with the exception of 14:0. However, those three treatments increased concentration of iso15:0, where Bent did not. For the Boone County, MO silty clay loam, fewer lipid chains varied with treatment and fewer herbicides and surfactants affected the various lipid chains. Application of AgrdAtrz, Agrd, Agrd2 and Thrst2 increased ante15:0. AgrdAtrz had higher iso16:0 than the control. Agrd and Agrd2 increased 14:0 and iso15:0 with Agrd2 also increasing 15:0 and 16:2 ␻6c. Bent led to increases in 14:0, 15:0, and isol16:1. Thrst2 increased iso15:0 and ThrstBent increased 15:0 3OH, 16:0 3OH and isol16:1. 4. Discussion Based on PLFA analyses, some of the herbicide and surfactant treatments altered microbial community composition and variability and these changes were most likely influenced by soil management history, pH, texture, and chemical properties (Wardle, 2002). The soils in this study reacted differently to the herbicides and surfactants. The Cole County, MO silt loam (pH 5.6; CEC 12) responded with changes in microbial groups and individual lipid chains, while the Boone County, MO silty clay loam (pH 4.5; CEC 22) showed few significant changes in the groups or lipid changes. The Boone County, MO silty clay loam was less responsive to the additions of chemicals, but was also more variable in its overall profile.

M.L. Banks et al. / Applied Soil Ecology 74 (2014) 12–20

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Table 5 Phospholipid chains from Cole County, MO silt loam that were significantly higher than control after the soils were treated with the various herbicides or surfactants (P ≤ 0.10, 0.05, 0.01; *, **, ***). Chain length

Herbicide or surfactant

12:0 2OH 14:0 14:1 ␻ 8c ante15:0 iso15:0 16:0 iso16:0 16:1 ␻5c 16:1 ␻7c 19:0 cyclo 11-12 2OH

Actv2

Glyp

ActvGlyp

Agrd2

Atrz

Thrst

Bent

– – – – – – – – – ***

– – – *** *** *** *** *** *** ***

– – – *** *** *** *** *** *** ***

– – ** – – – – – – –

– – – *** *** *** *** *** *** ***

** – – – – – – – – –

– ** – *** – *** *** *** *** ***

Actv2 = Activator-90, 2× rate; Glyp = glyphosate; ActvGlyp = Activator-90 + glyphosate; Agrd2 = Agri-Dex 2× rate; Atrz = atrazine; Thrst = Thrust; Bent = bentazon. Not statistically different = –.

The pH of a soil can greatly affect microbial growth and function as in the case of bacteria that do not function as efficiently at acidic pH (Börjesson et al., 2012). The Boone County soil, with a pH of 4.5, had a pH that could limit microbial response to an amendment. Soil texture has been shown to be a major contributor to differences in microbial function and microbial community composition (Marschner et al., 2001; Girvan et al., 2003; Fang et al., 2005; Collins et al., 2011). In a comparison of soil quality indicators across farms with different characteristics, soil texture and recent management practices greatly influenced nitrogen mineralization and the formation of aggregates (Collins et al., 2011). Texture greatly influenced the type of microbial populations present. Carbon substrate utilization assays also detected differences in community composition between sandy loam and silty clays in which corn was grown (Fang et al., 2005). Another likely contributor to variations in the microbial community is the addition of plant-based carbon substrates from exudates released by roots as the plant develops. Root growth and exudate rate and quality may differ between soils and among treatments thereby altering the microbial community (Brimecombe et al., 2001). In the Cole County, MO silt loam soil, most of the bacterial biomarkers decreased in response to herbicide/surfactant treatments while fungal biomarkers showed little response except for an increase due to Actv2 treatment. Actv2, Glyp, ActvGlyp and Bent reduced BtoF in the Cole County, MO silt loam. Herbicides may alter the bacterial and fungal composition of soils in various ways (Ratcliff et al., 2006). For example, glyphosate applied to agricultural soils caused transient increases in functional diversity of bacteria in a short-term (21-day) incubation study (Zabaloy et al., 2012). While bacterial groups negatively reacted to treatments, the fungal groups were not influenced. The bacterial community of the Cole County, MO silt loam was possibly more

sensitive to such changes in the soil environment (Pennanen et al., 1996; Wilkinson et al., 2002). Cationic surfactants are more toxic to Gram-negative bacteria than to Gram-positive bacteria and the response to toxicity is dependent upon the extent of soil sorption of the surfactant (Sarkar et al., 2009; Nye et al., 1994). The monounsaturated markers and the ratio of StoM were not affected by treatments in the Cole County, MO silt loam except for a significant increase in StoM after exposure to ActvGlyp. This may be an indication of stress; however, we did not apply treatments at levels that would cause extreme stress of the soil microbial community and cannot conclude if the application of ActvGlyp created or contributed to a stressful soil environment. In the Boone County, MO silty clay loam, biomass increased after Agrd and Bent application relative to the control. In the Boone County, MO silty clay loam, application of Agrd at both rates decreased the monounsaturated fatty acid markers. Nonionic surfactants sorb tightly to clays (Podoll et al., 1987). Nonionic surfactants had a greater affinity for and sorbed more strongly to montmorillonite clay than kaolinite (Ray et al., 1995). This affinity may partly explain our results or the lack of changes in the microbial community in the Boone County, MO silty clay loam. Differences in composition of soil microbial communities imply differences in physicochemical properties of the soils directly or indirectly attributed to variability in soil properties (Fierer et al., 2003). The pH, CEC and texture differed between the two soils. The Cole County, MO silt loam was slightly acidic with low CEC and high P and K, while the Boone County, MO silty clay loam was very acidic with a high CEC and low P and K. Soil texture is a factor affecting the soil bacterial community, as demonstrated in previous studies (Fang et al., 2005; Girvan et al., 2003; Marschner et al., 2001). Treatment components might be adsorbed to soil mineral and organic

Table 6 Phospholipid chains from Boone County, MO silty clay loam that were significantly higher than control after the soils were treated with the various herbicides or surfactants (P ≤ 0.10, 0.05, 0.01; *, **, ***). Chain length

14:0 15:0 15:0 3OH ante15:0 iso15:0 16:0 3OH iso16:0 isog16:1 isol16:1 16:2 ␻6c

Herbicide or surfactant Agrd

Agrd2

AgrdAtrz

Thrst2

Bent

ThrstBent

*** – – *** *** – – – – –

*** *** – *** *** – – – – **

– – – *** – – * – – –

– – – *** *** – – – – –

*** *** – – – – – ** – –

– – ** – – ** – – ** –

Agrd = Agri-Dex; Agrd2 = Agri-Dex 2× rate; AgrdAtrz = Agri-Dex + atrazine; Thrst2 = Thrust 2× rate; Bent = bentazon; ThrstBent = Thrust + bentazon. Not statistically different = –.

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M.L. Banks et al. / Applied Soil Ecology 74 (2014) 12–20

particles. This process of sorption has been reported to limit microbial degradation due to decreased accessibility to microorganisms (Selim et al., 1999; Koskinen et al., 2001), contributing to variable microbial response between the soils and within each soil. Soil textural differences likely contributed to differences in aeration, moisture, temperature, degradation rate, substrate availability, and other physical conditions that may affect soil microbial community structure (Corre et al., 2002; Rogers and Tate, 2001; Chen et al., 2003; Beylich et al., 2010; Cable et al., 2008; Fierer et al., 2003). Overall, results imply that treatments interacted with the two soils differently to affect the microbial community. Glyphosate treatment did not affect the microbial community in the Boone County, MO silty clay loam, but did in the Cole County, MO silt loam. Glyphosate has limited mobility in the soil and can be readily adsorbed to particles of clay and organic matter (Pessagno et al., 2008; Barja and dos Santos Afonso, 2005). Haney et al. (2000) showed that glyphosate applications did not significantly affect microbial biomass. Glyphosate may either stimulate or inhibit soil microbial activity based on the concentration used and on soil properties (Carlisle and Trevors, 1986). Glyphosate may have been adsorbed to soil particles making it unavailable to microbial degradation (Pessagno et al., 2008). Other studies demonstrated that adjuvants or surfactants aid in the sorption of herbicides to soil by modifying the solubility and limiting bioavailability (Beigel et al., 1999; Krogh et al., 2003). Gram-positive bacteria increased with addition of herbicides in the Cole County, MO silt loam, but did not change in the Boone County, MO silty clay loam. In other studies, the GP component exhibited growth fluctuations during the degradation of soil-applied herbicides (Pipke et al., 1987; Strong et al., 2002; Schmalenberger and Tebbe, 2002; Seeger et al., 2010). Surfactants may enhance degradation of herbicides and other compounds leading to higher biomass (Li et al., 2008). In addition, microorganisms can modify the structure and activity of some of these chemicals to reduce effects on the environment (Ying, 2006). In this study, the addition of surfactants at both application rates did not significantly affect PLFA profiles for the two soils, but did change some lipid chains and microbial markers. Microbial stress indicators are used to detect shifts in microbial community due to unfavorable conditions such as temperature, pH, substrate and water availability, and toxicity caused by various substances including heavy metals. Cyclopropane is produced under limited carbon source (Bossio and Scow, 1998). Monoenoic acids are associated with high substrate availability (Guckert et al., 1986; Kieft et al., 1994). Ratio of cyclopropyl fatty acids to monoenoic fatty acids might be an indicator to monitor stress imposed on the microbial community. The ratio of cyclopropyl fatty acids to monoenoic fatty acids increased with the ActvGlyp combination treatment in the Cole County, MO silt loam. This biomarker decreased with both application rates of Agrd (Table 5). We did not see any changes in monounsaturated fatty acids except with the application of Agrd and Agrd2, which decreased MONO in the Boone County, MO silty clay loam. Heipieper et al. (1996) reported that a shift in monounsaturated fatty acids from a lower number to a higher number was evidence of stress. There were several lipid chains that were higher after treatment application including 15:0, iso15:0, 16:0, iso16:0, 16:1 ␻5c, 16:1 ␻7c. The 15:0, 16:0 and 16:1, 19:0 cyclo chains are biomarkers for bacterial and fungal groups and the increase in a string of 15:0 and 16:0 chain length lipids in soil may indicate that some processes are changed by the addition of these compounds. Changes occurred, but may not be large enough to detect as we did not see overall changes in profiles when analyzed together (data not shown). Variability between the two soils was greater than any variability seen among the treatments (data not shown). A change in the amount of one lipid chain by the addition of a given compound may not be

biologically significant; however, if several lipid chains are altered this may be of interest. Further, the number of lipid chains that increased due to the herbicide or surfactant application was small considering the number of peaks or different lipid chains found in soils. In addition, we do not know if any increase in a particular lipid chain is inherently good or bad. These changes may; however, be precursors to other effects found after continual annual application of these herbicides or surfactants. The one-time application of treatments showed some changes in the soil microbial community in this study, but those changes were not evident in both of the soils. This may suggest that soil properties, soil texture, pH and the chemical composition, and the number of applications together determine the effect of herbicides and surfactants on soil microbial community composition (El Fantroussi et al., 1999; Girvan et al., 2003; Seghers et al., 2003; Lupwayi et al., 2010). 5. Conclusion Microbial communities in untreated soils from south central Missouri differing in history, pH, texture, microbial and physicochemical characteristics responded differently to soil-applied herbicides and surfactants. Most of the herbicides and surfactants in this study did not significantly affect the microbial community. A few of the chemicals applied at field rates altered components of the microbial community after only one application. These changes varied between the soils with the Cole County, MO silt loam having larger differences in the microbial community components after treatment application. Few changes were seen in PLFA biomarkers in the Boone County, MO silty clay loam with pH of 4.5 except for the treatments Agrd and Bent that increased MB, and for Agrd at both rates that decreased monounsaturated fatty acid biomarkers. The PLFA profiles indicated that the microbial community responded to application of most of the surfactants and herbicides resulting in changes in the concentration of some biomarkers. The biological significance of these changes is not readily apparent; however, they might be used as indicators in tracking responses to applied agrichemicals in future research. The application of surfactants regardless of rates, herbicides and combinations caused variable changes among the various microbial biomarkers relative to nontreated controls. The changes observed were minimal and were the result of single applications of the compounds to the soil surface. In addition, the response by soil microbial populations to the herbicides and surfactants differed due to dissimilar soil properties and land management. Further long-term studies are needed to determine the full extent of changes in the microbial community after several annual applications of herbicides and surfactants. Disclaimer Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the Lincoln University, the University of Missouri, or the USDA-ARS. USDA is an equal opportunity provider and employer. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Acknowledgements The authors wish to thank J.C Hansen, J. Coon and T.L. Stubbs for excellent analytical assistance. Also special thanks to Dr. Nigel Hoilett and James Ortbals for their assistance in carrying out the greenhouse study and sample preparation.

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