Studying the effects of herbicides on microbial community and urease activity in the rhizosphere soil of maize is helpful to clarify the mechanisms herbicides used to affect soil microbial environment. In this research, four common preemergence maize specific herbicides, nicosulfuron+atrazine (A1), alachlor+acetochlor+atrazine (A2), propisochlor+atrazine (A3), and acetochlor+atrazine (A4), were selected to use in a pot trial. A preemergence herbicide nonspecific for maize, dinitraniline (A0), was used as the positive control, whereas water instead of herbicide was considered as the negative control (CK). At the maturity stage, the microbial communities and urease activity in the 0-20 cm, 20-40 cm, and 40-60 cm rhizosphere soils of maize were analyzed. Results showed that A0 dramatically suppressed maize growth, with no grain got finally, while A1 displayed the weakest effect. The tested herbicides affected the microbial community in the 0-20 cm layer greater than in the 20-60 cm ones, with A1 displaying the greatest effect. In the 0-20 cm soil, A1 largely reduced the relative abundance of the top three dominant genera,
Maize (
Microorganisms play key roles in nutrient cycling and energy flow in soil and are important indicators for soil health, soil pollution, and ecological restoration [
Except for microorganisms, enzymes also are important evaluation indicators for soil quality as they are directly involved in the biochemical process and nutrient cycle [
The rhizosphere soil is closely in contact with roots and shows a larger influence on plant growth than the nonrhizosphere soil. After entering into soil, herbicides interacted with soil microorganisms and plant roots, hence showed different effects to soil environment when compared with the nonrhizosphere soil. Studying the effects of herbicides on the microorganisms and enzyme activity in rhizosphere soil is helpful to elucidate the mechanisms of herbicides affecting both plant growth and soil environment [
In this study, five preemergence herbicides: four maize-specific herbicides and one nonspecific herbicide for maize were sprayed on the potted soil surface immediately after sowing maize. At the maturity stage, the effects of herbicides on the total bacterial and fungal community structures in the 0-20, 20-40, and 40-60 cm rhizosphere soils were analyzed by high-throughput sequencing technology, and the soil urease activities in the same soil layers were determined. The weight and number of maize grains were also recorded. Results from this study may help enlighten the effects of preemergence herbicides on the microbial environment in rhizosphere soil at different layers after planting a season crop and provide guides for selecting suitable herbicides for maize.
The maize hybrid “Bingdan 16” used in this study was provided by the Institute of Crop Science, Shanxi Academy of Agricultural Sciences. The growth period of Bingdan 16 is 120 days. The five preemergence herbicides commonly sold in market that were selected to use in this experiment (Table
The tested herbicides information.
Number | Effective components and content | Actual dosage ( | Water dosage (ml pot-1) |
---|---|---|---|
A0 | 48% dinitraniline | 14.7 | 100 |
A1 | 2% nicosulfuron+20% atrazine | 14.7 | 100 |
A2 | 10% alachlor+14% acetochlor+18% atrazine | 22.1 | 100 |
A3 | 16% propisochlor+26% atrazine | 17.7 | 100 |
A4 | 26% acetochlor+26% atrazine | 14.7 | 100 |
The raw soil sample was taken from a 2 m layer from an uncultivated land in Taigu County, Shanxi Province, China. The soil was air-dried, sieved (1 mm), and evenly mixed before use. Nutrient status of the soil was as follows: 0.2 g kg-1 total nitrogen, 19.8 mg kg-1 available nitrogen, 2.9 mg kg-1 available phosphorus, 30.3 mg kg-1 available potassium, and 1.2 g kg-1 organic matter.
The experiment was conducted from June 3, 2014, to October 1, 2014, at the Loess Plateau Crop Research Institute, Shanxi Agricultural University, China. A special root tube device (25 cm diameter, 200 cm length) that consists of two semicylinders fixed with iron wire and steel plates was used. Soil bulk density in 0-200 cm soil layer was measured for local maize field by 20 cm interval. Based on the soil bulk density, soil mass was calculated and weighted for every 20 cm length in the tube. The soil prepared above was added to the root tube devices and compacted by every 20 cm layer. The top 0-20 cm soil was added after mixing with NPK nutrients (urea, 170 mg kg-1; superphosphate, 560 mg kg-1; potassium chloride, 170 mg kg-1). All root tube devices were placed vertically in the grooves in a way that the soil surface inside the tubes was leveled to the outer soil surface. Five maize seeds were sown in each tube on June 3, 2014. Herbicide solutions (100 ml) were then sprayed evenly based on the dosage as mentioned in Table
On October 1, 2014 (at the maturity stage), the aboveground maize plants were cut and all tubes were opened. The rhizosphere soils from 0-20, 20-40, and 40-60 cm layer from the soil surface were collected using a banister brush from the root surface after the untight soil was removed. All soil samples were collected into seal bags and divided into two parts: one was stored in -80°C before sending to the Sangon Biological Engineering Co. Ltd. Shanghai to analyze the bacterial and fungal community structures, while the other part was used to determine the urease activity.
The grain number and weight per plant were recorded, and the thousand grains weight was calculated at the maturity stage.
For each layer of soil in each pot, the collected soil samples at the maturity stage used for analyzing urease activity were divided into two even parts, and the total of six replicated soil samples were air-dried and sieved. The urease activity was analyzed by the indigo colorimetry method [
The Microsoft Excel 2016 software was used to analyze the data, and the Origin 9 was used to draw bar charts. The SPSS 18.0 software was used to analyze the differences between treatments or soil layers based on the least significant difference (LSD) method at the level of
Among the tested herbicides, nicosulfuron+atrazine (A1) did not affect either the early growth of maize seedlings or the grain structures during the maturity stage, whereas dinitraniline (A0) dramatically inhibited the growth of maize seedlings, and with no grain at the maturity stage (Table
Effects of herbicides on the grain structures of maize.
Treatment | Grain weight (g plant-1) | Grain number (plant-1) | Thousand grain weight (g) | |||
---|---|---|---|---|---|---|
Measured value | Measured value | Measured value | ||||
CK | — | — | — | |||
A0 | -100.0 | -100.0 | -100.0 | |||
A1 | -38.7 | -31.3 | -18.8 | |||
A2 | -12.5 | 36.2 | -38.5 | |||
A3 | -7.1 | 21.7 | -27.0 | |||
A4 | -35.4 | 6.4 | -39.9 |
Notes: data in the table are
Effects of herbicides on the growth of maize seedlings. Note: CK, the no herbicide negative control; A0, a preemergence nonspecific herbicide for maize, dinitraniline (the active control); A1-A4: four preemergence maize specific herbicides, nicosulfuron+atrazine, alachlor+acetochlor+atrazine, propisochlor+atrazine, and acetochlor+atrazine, respectively.
The total number of effective bacterial and fungal OTUs was 5075-6757 and 1103-1673, respectively, from different soils. Shannon index reflects the diversity degree of microorganisms, and ACE and Chao 1 indices reflect the richness of the microbial community. For bacteria, the five tested herbicides had little effect on the bacterial Shannon index in the 0-60 cm rhizosphere soil of maize. The effects of herbicides on ACE and Chao1 indices in the 0-20 cm rhizosphere soil were also weak, with only A1 changing the ACE index by more than 20% (20.9%). In the 20-40 cm rhizosphere soil, A0 and A3 reduced the ACE and Chao 1 indices, to 28.3%-40.4%, whereas increased these indices by 24.3%-55.2% in the 40-60 cm rhizosphere soil compared with the control. A5 also increased the ACE and Chao1 indices in the 40-60 cm rhizosphere soil by 46.7% and 36.8% than that of the control, respectively (Table
Effects of herbicides on the alpha diversity of bacteria in the rhizosphere soil of maize.
Soil layer (cm) | Treatment | Shannon | ACE | Chao 1 | |||
---|---|---|---|---|---|---|---|
Measured value | Measured value | Measured value | |||||
0-20 | CK | 7.6 | — | 26109.7 | — | 15204.3 | — |
A0 | 7.6 | 0.9 | 30042.1 | 15.1 | 16573.7 | 9.0 | |
A1 | 7.5 | -1.2 | 20645.4 | -20.9 | 13299.7 | -12.5 | |
A2 | 7.7 | 2.3 | 25091.6 | -3.9 | 15564.7 | 2.4 | |
A3 | 7.8 | 3.2 | 27539.3 | 5.5 | 16687.2 | 9.8 | |
A4 | 7.5 | -0.2 | 29388.9 | 12.6 | 16735.1 | 10.1 | |
20-40 | CK | 7.6 | — | 31795.4 | — | 17283.6 | — |
A0 | 7.5 | -1.6 | 21090.5 | -33.7 | 12400.6 | -28.3 | |
A1 | 7.4 | -3.1 | 24043.1 | -24.4 | 14098.2 | -18.4 | |
A2 | 7.4 | -2.3 | 30041.6 | -5.5 | 16472.0 | -4.7 | |
A3 | 7.4 | -2.4 | 18942.7 | -40.4 | 11330.8 | -34.4 | |
A4 | 7.6 | -0.3 | 33844.3 | 6.4 | 17851.1 | 3.3 | |
40-60 | CK | 7.5 | — | 19817.4 | — | 12125.5 | — |
A0 | 7.8 | 4.1 | 30746.0 | 55.2 | 18073.9 | 49.1 | |
A1 | 7.3 | -3.2 | 21884.8 | 10.4 | 13089.6 | 8.0 | |
A2 | 7.6 | 1.5 | 22149.7 | 11.8 | 13397.3 | 10.5 | |
A3 | 7.5 | 0.5 | 25644.3 | 29.4 | 15070.1 | 24.3 | |
A4 | 7.5 | -0.3 | 29077.7 | 46.7 | 16219.4 | 33.8 |
Note: CK, the no herbicide negative control; A0, a preemergence nonspecific herbicide for maize, dinitraniline (the active control); A1-A4: four preemergence maize-specific herbicides, nicosulfuron+atrazine, alachlor+acetochlor+atrazine, propisochlor+atrazine, and acetochlor+atrazine, respectively.
In contrast to bacteria, the Shannon index of fungi in the herbicides treated 0-20 cm soil was increased by 21.0%-39.9% than that of the control. In the 0-20 cm soils, the fungal ACE and Chao1 indices in the A3 and A4 treated soils were higher than other herbicides. For the 20-40 cm soil, A0 increased the fungal ACE and Chao1 indices by 32.8% and 29.8%, respectively, over that of the control, while the other four herbicides showed negligible effects. A0, A1, A3, and A4 increased the fungal ACE and Chao1 indices by 21.4%-62.1% in the 40-60 cm soil than the nonherbicide control treatment (Table
Effects of herbicides on the alpha diversity of fungi in the rhizosphere soil of maize.
Soil layer (cm) | Treatment | Shannon | ACE | Chao 1 | |||
---|---|---|---|---|---|---|---|
Measured value | Measured value | Measured value | |||||
0-20 | CK | 4.1 | — | 2952.8 | — | 2328.9 | — |
A0 | 5.6 | 38.1 | 3190.9 | 8.1 | 2572.6 | 10.5 | |
A1 | 5.6 | 36.7 | 2978.8 | 0.9 | 2589.3 | 11.2 | |
A2 | 4.9 | 21.0 | 3043.3 | 3.1 | 2303.8 | -1.1 | |
A3 | 5.7 | 39.9 | 3987.5 | 35.0 | 3026.1 | 29.9 | |
A4 | 5.4 | 31.8 | 3488.6 | 18.1 | 2825.7 | 21.3 | |
20-40 | CK | 4.8 | — | 3373.7 | — | 2575.8 | — |
A0 | 5.2 | 10.2 | 4479.9 | 32.8 | 3343.4 | 29.8 | |
A1 | 5.1 | 7.2 | 3495.0 | 3.6 | 2760.6 | 7.2 | |
A2 | 5.4 | 13.2 | 3637.7 | 7.8 | 2679.6 | 4.0 | |
A3 | 5.4 | 13.6 | 3261.7 | -3.3 | 2707.8 | 5.1 | |
A4 | 4.8 | 1.1 | 3019.6 | -10.5 | 2463.6 | -4.4 | |
40-60 | CK | 5.2 | — | 2584.1 | — | 2181.4 | — |
A0 | 5.3 | 1.9 | 3621.1 | 40.1 | 2647.5 | 21.4 | |
A1 | 5.3 | 2.9 | 4189.2 | 62.1 | 2806.1 | 28.6 | |
A2 | 4.5 | -12.5 | 2337.6 | -9.5 | 1843.0 | -15.5 | |
A3 | 5.4 | 3.7 | 3508.7 | 35.8 | 2670.5 | 22.4 | |
A4 | 5.3 | 1.7 | 3522.1 | 36.3 | 2875.7 | 31.8 |
Note: CK, the no herbicide negative control; A0, a preemergence nonspecific herbicide for maize, dinitraniline (the active control); A1-A4: four preemergence maize specific herbicides, nicosulfuron+atrazine, alachlor+acetochlor+atrazine, propisochlor+atrazine, and acetochlor+atrazine, respectively.
At the phylum level, herbicides affected the bacterial community composition in the 0-20 cm soil greater than in the 20-40 and 40-60 cm soils, with A1 showing the greatest effect. In the 0-20 cm soil, Bacteroidetes, Firmicutes, and Proteobacteria were the top three dominant phyla in the CK treatment. A1 decreased the relative abundance of Bacteroidetes and Firmicutes greatly, from 27.8% and 26.0% in the CK soil to 5.5% and 4.4%, whereas increased the relative abundance of Proteobacteria, from 22.3% in the CK soil to 37.7%. The relative abundance of Actinobacteria in the A1 treated 0-20 cm soil was 693.0% more than that in the control and made Actinobacteria become the second dominant phylum. The relative abundances of Gemmatimonadetes and Planctomycetes in the A1 treated 0-20 cm soil were also 423.5% and 225.0% more than that in the control. Compared with A1, A3 and A2 showed similar but weaker effects on the 0-20 cm soil bacterial community. A0 displayed the weakest effect on the 0-20 cm soil among the five tested herbicides. In the 20-40 cm and 40-60 cm soil, all herbicides showed weak effects on the bacterial community, with A1 and A0 displaying much greater effect than other herbicides, respectively (Figure
Relative abundance of the top ten bacteria in the phylum (a) and genus (b) level in the 0-20 cm, 20-40, and 40-60 cm rhizosphere soils. Note: CK, the no herbicide negative control; A0, a preemergence nonspecific herbicide for maize, dinitraniline (the active control); A1-A4: four preemergence maize specific herbicides, nicosulfuron+atrazine, alachlor+acetochlor+atrazine, propisochlor+atrazine, and acetochlor+atrazine, respectively.
At the genus level, similar to the phylum level, A1 showed the greatest effect on the bacterial composition in the 0-20 cm soil and followed by A3 and A2. A1 reduced the relative abundance of
At the phylum level, a total of seven phyla were classified. Different from those in the bacterial community, all the five tested herbicides showed similar and great effects on the fungi community in the 0-20 cm rhizosphere soil. Ascomycota was the top one dominant fungal phylum in the CK soil at 0-20 cm layer, accounting for 45.0% of the total fungal community. Herbicides reduced the relative abundance of this phylum to 5.2%-7.9%. The relative abundance of the unclassified fungal phylum (Others) was also increased greatly, from 9.6% in the no herbicide control soil to 44.0%-67.9% in the herbicide-treated soils. A1 also increased the relative abundance of Basidiomycota greatly, by 202.4% than that in the CK treatment. A1 and A2 also reduced the relative abundance of Glomeromycota, and Chytridiomycota, by 32.5%-43.7% than that in the no herbicide control. In the 20-40 cm soil, A0 and A2 increased the relative abundance of Glomeromycota but reduced the relative abundance of the unclassified phylum, with the variation being 31.9%-82.4% when compared with the control. The relative abundance of Basidiomycota in the A3-treated soil and Ascomycota in the A4-treated soil was 194.6% and 189.5%, respectively, more than that in the control treatment. In the 40-60 cm soil, A0 and A1 displayed similar effects on the fungal community, with increasing relative abundance of Glomeromycota, Chytridiomycota, and Basidiomycota, whereas reducing relative abundance of the unclassified phyla (Others), whereas A2 showed opposite effects (Figure
Relative abundance of the top ten fungi in the phylum (a) and genus (b) level in the 0-20, 20-40, and 40-60 cm rhizosphere soils. Note: CK, the no herbicide negative control; A0, a preemergence nonspecific herbicide for maize, dinitraniline (the active control); A1-A4: four preemergence maize specific herbicides, nicosulfuron+atrazine, alachlor+acetochlor+atrazine, propisochlor+atrazine, and acetochlor+atrazine, respectively.
At the genus level, similar to the phylum level, all herbicides displayed great and similar effects on the fungal community in the 0-20 cm soil, but different effects in the 20-60 cm soils.
A0 significantly decreased the urease activity in the 0-60 cm layer rhizosphere soil by 30.6%-38.6% than the control, whereas A1 did not affect the urease activity in the same layer soils. A2, A3, and A5 did not significantly affect the urease activity in the 0-20 and 20-40 cm rhizosphere soils, whereas A2 and A3 increased the urease activity in the 40-60 cm rhizosphere soil, by 27.1% and 19.8% than that in the CK soils. As the layer increased, the urease activity did not change significantly in the A0, A1, and CK treatments but significantly increased in the A2- and A3-treated soils. The urease activity in the A4-treated 40-60 cm soil was also 24.2% higher than that in the 0-20 cm soil (Table
Effects of herbicides on the urease activity in the rhizosphere soil of maize.
Treatment | Soil layer (cm) | |||||
---|---|---|---|---|---|---|
0-20 | 20-40 | 40-60 | ||||
Measured value (mg g-1) | Measured value (mg g-1) | Measured value (mg g-1) | ||||
CK | — | — | — | |||
A0 | -38.6 | -33.1 | -30.5 | |||
A1 | -18.8 | -15.6 | -6.3 | |||
A2 | 2.7 | 15.0 | 27.1 | |||
A3 | -8.0 | 10.2 | 19.8 | |||
A4 | -15.4 | -2.2 | 13.6 |
Note: CK, the no herbicide negative control; A0, a preemergence nonspecific herbicide for maize, dinitraniline (the active control); A1-A4: four preemergence maize specific herbicides, nicosulfuron+atrazine, alachlor+acetochlor+atrazine, propisochlor+atrazine, and acetochlor+atrazine, respectively. Data in the table are
Soil microorganisms are the main components of the agricultural microbial system, play key roles in nutrient cycling and energy flow, and are considered as indicators to reflect the effects of herbicides on the soil environment [
After entering into soils, herbicides affect the growth of soil microorganisms either by directly feed soil microorganisms as an energy source, or indirectly by influencing the chemical environment, and then regulating the growth of soil microorganisms [
Soil microorganisms may also reversely regulate the effects of herbicides on the soil environment by degrading the harmful active substances of herbicides. These herbicide-degrading microorganisms include
Soil enzymes take part in the biochemical process and nutrient cycling in soil, which influence soil microecology environment [
In general, the effects of herbicides on soil microbial environment varied based on herbicide species and soil depths. Among the five tested herbicides, only A0 contains dinitraniline, and the other four herbicides all contain atrazine. Among the four maize-specific herbicides, only A1 contains a sulfonylurea component (nicosulfuron), whereas the other three herbicides instead with chloroacetamide ones (alachlor, acetochlor, or propisochlor). When considering soil depth, the tested herbicides affected the microbial community stronger in the 0-20 cm layer than in the 20-60 ones, whereas A0 and A1 showed consistent effects, but A2-A4 first showed insignificant effect but, then, increased the activity of soil urease with the soil depth increased. The differences in chemical characteristic, degradation, and migration rates of active components in the five tested herbicides may partly explain these inconsonant results among different soil layers. Further experiments still need to be done to confirm this speculation.
The use of herbicide is still a common measure to control weed damage in maize field. Herbicides affect both soil microbial environment, and the activity of soil enzymes has been reported. However, the effects of preemergence herbicides on the microbial environment of maize rhizosphere soil after applying for a relatively long time are still unclear. In this research, we applied five preemergence herbicides on maize soil and investigated the effects of herbicides on microbial community and urease activity in the rhizosphere soil of maize in different layers at the maturity stage. We found that among the five tested herbicides, nicosulfuron+atrazine (A1) largely altered the bacterial community structure especially in the 0-20 cm layers and showed insignificant effect on the activity of urease in the 0-60 cm rhizosphere soils, with other three maize-specific herbicides (A2-A4) displayed mild effects. The nonspecific herbicide of maize, A0, changed the microbial community slightly but dramatically dropped the activity of urease in the 0-60 rhizosphere soils. Considering that A1 did not affect the grain yield of maize, but A0 totally suppressed the development of maize grain, we proposed that A1 was a suitable herbicide for maize, and both the bacterial community structure and the urease activity in the rhizosphere soil at 0-20 cm layers are suitable indicators for evaluating the effects of herbicides on maize plant growth and soil microbial environment. The differences in active components may contribute to explain the differences among herbicide species and soil depths.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare no conflict of interest.
This work was financially supported by the Key Project of Shanxi Key R&D Program of China (201703D211001-02), the Special Plan of Scientific Research for Shanxi Agriculture Valley of China (SXNGJSKYZX 201701), the Shanxi Collaborative Innovation Centre of Featured Crops High-quality and Efficiency Production in Loess Plateau ([2016]5), the “1331 Project” Crop Ecology and Dry Cultivation Physiology Key Laboratory of Shanxi Province (201705D111007, [2017]14), the “1331 Project” Organic Dry Farming and Cultivating Physiology Innovation Team Project of Shanxi Province ([2018]4), the Modern Agriculture Industry Technology System Construction (CARS-03-01-24), the Science and Technology Innovation Foundation of Shanxi Agricultural University (2019002), and the University Science and Technology Innovation Foundation of Shanxi Province (2020L0159).