Ecological Indicators 103 (2019) 194–201
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Original Articles
Effects of successive metalaxyl application on soil microorganisms and the residue dynamics Fenghua Wanga,1, Tongtong Zhoua,1, Lusheng Zhua, Zhongkun Dua, Bing Lia
⁎,2
T
, Xiuguo Wangb, Jun Wanga, Jinhua Wanga,
a College of Resources and Environment, Shandong Agricultural University, Key Laboratory of Agricultural Environment in Universities of Shandong, National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, Taian 271018, PR China b Tobacco Research Institute of Chinese Academy of Agricultural Sciences (CAAS), Qingdao 266101, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Metalaxyl dissipation Soil microbe DGGE Bacterial community T-RFLP
Metalaxyl, a chiral fungicide, was widely used to control mold fungi and oomycetes in many plants. However, studies are insufficient on the effects of successive metalaxyl applications on soil microorganisms and the residue dynamics. In the present study, metalaxyl was applied in field at two different doses (the recommended field rate, and twice of the recommended field rate) for 2 and 3 times, respectively. The soil samples were collected on days 0, 1, 3, 7, 14, 28 and 45 after the second and third treatment to determine the residue dynamics and were collected on days 7, 14, 28 and 56 to assess the effects of successive metalaxyl application on soil cultivable microbial populations and microbial community structure. The results revealed the degradation rate slightly accelerated with the increasing applied times of metalaxyl. The second application of metalaxyl increased soil bacteria populations, inhibited soil fungi, and had a transient stimulated effect on actinomycetes. No distinct differences were observed in soil bacterial, actinomycic and fungal populations except for fungi population on day 14 after the third treatment. The analysis of terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) revealed soil microbial community structure could be affected by metalaxyl at all experimental doses. These obtained results could provide basic data for evaluating the ecological toxicity of metalaxyl.
1. Introduction
alaninate], a broad-spectrum fungicide, is widely used to protect a wide range of crops including horticultural crops, vegetables and fruits from damage by fungal diseases of damping-off, late blight, stem, downy mildew and fruit rots (Celis et al., 2015; Wang et al., 2014). Due to the favorable physicochemical properties such as its nonvolatility and the excellent stability under different conditions of pH, temperature and light, metalaxyl has been broadly used all over the world including the US, Europe, Australia, Asia, Egypt and India (Malhat, 2017). However, the good water solubility of metalaxyl might cause the permeation of metalaxyl into soil to result in potential toxicity by the rain-wash and irrigation (Wilson et al., 2001). Sukul et al. (2008) found that metalaxyl stimulated the growth of thiosulfate, nitrogen, oxidizing bacteria, phosphorus-solubilizing bacteria, total bacteria and actinomycetes and inhibited the fungi. The previous studies indicated metalaxyl caused a dramatic decrease in microbial population, firstly increasing and then decreasing in
Pesticides, the important part in agricultural production, which are used to maintain adequate quality of agricultural products, and are also used in human and animal hygiene, in the protection of feed, food, natural raw materials and products made of them (Chatterjee et al., 2013). However, for the widespread use of them, excess pesticides may release to environment through rainfall, irrigation and so on. Thus, the effect of pesticides on natural environment is still a major problem (Seiber and Kleinschmidt, 2011). Microorganisms as the important part of soil ecological environment, are playing an essential role in accelerating organic matter degradation, promoting plant growth and stimulating the nutrient element circulation. Microbes are sensitive to contaminants in soil. Thus, microbes can provide essential data on environmental changes (Zhang et al., 2006). Metalaxyl [methyl N-(2, 6-dimethylphenyl)-N-(methoxyacethyl)-DL-
⁎
Corresponding author at: College of Resources and Environment, Shandong Agricultural University, 61 Daizong Road, Taian 271018, PR China. E-mail addresses:
[email protected] (F. Wang),
[email protected] (L. Zhu),
[email protected] (J. Wang),
[email protected] (J. Wang). 1 Fenghua Wang and Tongtong Zhou contributed equally to this work. 2 ORCID: 0000-0001-6212-1965. https://doi.org/10.1016/j.ecolind.2019.04.018 Received 5 December 2018; Received in revised form 6 March 2019; Accepted 8 April 2019 1470-160X/ © 2019 Elsevier Ltd. All rights reserved.
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determine cultivable microbial population and at −20 °C to quantitatively analyze metalaxyl residues and to perform DGGE assay.
arylsulphatase, phosphatase, dehydrogenaseand β-glucosidase activities, and a slow reduction in the activity of urease (Sukul, 2006). Metalaxyl also inhibited enzymatic activities, populations of soil nitrogen-fixing and total bacteria (Monkiedje et al., 2002). Han et al. (2010) found that metalaxyl could inhibit the diversity of soybean rhizospheric azotobacter. Zheng et al. detected the degradation dynamics of metalaxyl in watermelons and soil by using high performance liquid chromatography through field test (Zheng et al., 2014). Those previous studies have focused on single metalaxyl application. Actually, repeated inputs of metalaxyl as frequently as every ten to fifteen days are essential for controlling the plant diseases during agricultural management in field. Consequences of repeated inputs of pesticides to soil may be not the same as those of single treatment (Fang et al., 2016; Zhang et al., 2016b), but studies on the differences of degradation rates and impacts on soil microorganisms with successive metalaxyl inputs to field are scarce (Celis et al., 2015). Thus, study about the effects of metalaxyl with repeated treatments on the degradation and soil microbial community is very important from an eco-toxicological and agricultural aspect. In general, the present investigationaimed to evaluate the variance of the metalaxyl degradation dynamic at different application frequencies and examined how repeated use of metalaxyl affected the soil microbial community structure and diversity by field experiments. The implications of the present study could provide basic data for future studies on the toxicity to soil organisms and have supervisory significance for rational use of pesticides and sustainable utilization of resources.
2.3. Quantitative analysis of metalaxyl The extraction of metalaxyl was conducted as previously published with minor modifications (Liu et al., 2012; Li et al., 2013). In a 50 mL centrifuge tube, 2.0 g of soil sample and 10 mL of acetonitrile were added. The tube was oscillated at 2000 rpm for 1 min, then 1.0 g NaCl, 0.5 g citric acid disodium salt, 1.0 g sodium citrate and 4.0 g MgSO4 were added and then shaken at 2000 rpm for 2 min followed by centrifuge at 4000 rpm for 10 min. Next, 150 mg of MgSO4 and 25 mg of Npropyl ethylenediamine bonded solid adsorbent were added into the supernatant liquid (1 mL), and vortexed at 2000 rpm for 2 min, followed by centrifuge at 6000 rpm for 2 min. Then, the supernatant (200 μL) was dissolved in 800 μL acetonitrile, and filtered using 0.22 µm filter, followed by HPLC-ESI-/MS/MS analysis. Metalaxyl was separated on a Thermo Hypersil GOLD C18 column (2.1 × 100 mm, 3.0 µm). The mobile phases were the mixture of acetonitrile (A) and 0.1% (v/v) formic acid in water (B) at 0.2 mL/min flow rate in gradient mode as follows: 40% A for 0.5 min, gradient to 80% A in 2.5 min and for 7.5 min, to 40% A in 12.1 min and for 2.9 min. A sample volume of 10 μL was injected and metalaxyl was monitored by positive ion mode and multiple reaction monitoring (MRM). The conditions for HPLCESI-/MS/MS analysis were according to previous research. Quantitative analysis was conducted with external matrix-matched standards. The linear standard curve from the standard solutions (0.01, 0.05, 0.1, 0.5, 1.0 μg/mL) of metalaxyl was y = 9.8731x + 0.981, R2 = 0.9934. The analysis showed a constant and high recovery of 90.6% (n = 5, 90.6 ± 5.1%) in the spike recovery test. The method was sensitive with the limit of detection (LOD) of 0.0001 mg/kg.
2. Materials and methods 2.1. Chemicals Metalaxyl at purity 98% was provided by Heben Pesticide & Chemicals Co., Ltd. (Zhejiang, China). The organic solvent-acetonitrile (Sigma–Aldrich) was of HPLC-grade. The other reagents were at least of analytical grade and were obtained from Shanghai Sangon Biological Engineering Technology and Service and Kaitong Chemical Reagent Co., Ltd. (Tianjin, China) and a Milli-Q system from Millipore (Bedford, MA) was used to prepare ultrapure water.
2.4. Microbial counting Soil microbial enumerations of bacteria, fungi and actinomycetes were measured by incubation of the plates with selective media (Zhang et al., 2014). Beef extract peptone agar medium was prepared for cultivation of bacteria, Potato Dextrose Agar (PDA, Difco) was used to culture the fungi and Gauze's medium No. 1 was used to culture the actinomycetes. Soil samples of 10.00 g from each treatment and sterile water of 90.00 mL were put into 250 mL Erlenmeyer flasks, shaken at 250 rpm for 20 min, with the supernatant at 10−1 of soil suspension. 10−4 of soil suspension was prepared for bacteria, 10−4 for actinomycetes and 10−2 for fungi. Soil suspension of 0.10 mL was incubated on the agar plates at 30 °C. After 36 h, 48 h and 5 d, the amount of bacteria, fungi and actinomycetes was calculated. The experiment was conducted in triplicates. The results were expressed as CFU/g dry soil.
2.2. Experimental design We conducted the field survey in the areas located in Shandong, Taian, China. Metalaxyl had not been previously applied in the soil used in the study. The soil is a sandy loam soil. It has 16.82% clay, 58.67% sand, 24.51% silt. The physicochemical characteristics of the soil were as follows: 11.85 g/kg of organic matter, 95.62 mg/kg of available nitrogen, 26.53 mg/kg of available phosphorus, pH 6.01, with 19.20% of maximal water holding capacity. A randomized complete block design was performed consisting of 18 plots (10 m × 15 m). Tobacco was cultivated in each plot. The water solution of metalaxyl was sprayed at the suggested field application rate (1 × FR) and at two folds of the field application rates (2 × FR). The maximum application dosage of metalaxyl was 600 g active ingredient/ha1 recommended for the application to tobacco in 25% wettable powders, two or three times of applications were included in the field, and the time gap of the application was ten days. Simultaneously, equal parts of water sprayed 2 or 3 times were used as controls (CK). The experiment was performed in three replicates. During the experimental period, normal field managements, protocols such as irrigation and weeding were performed. Soil sub-samples were taken at 0, 1, 3, 7, 14, 28, and 45 d after the second and third treatment to quantitatively analyze metalaxyl residues and at 7, 14, 28 and 56 d to assess soil cultivable microbial populations and bacterial community. Five soil cores randomly collected from each plot were mixed thoroughly. The soil was air-dried at room temperature, and was sieved by 2 mm mesh. Then the soil was stored at 4 °C to
2.5. The analysis of soil bacteria by DGGE and T-RPLP Effects of metalaxyl on soil microbial diversity and community structure were evaluated using DGGE and T-RFLP analysis. Method of extracting DNA: The MO BIO Power Soil DNA Isolation kit (USA) was used to extract the total DNA in soil according to the protocols. Agarose gel electrophoresis of 1.0% was used to assess the quality and yield of extracted DNA. Extracted DNA was stored at −20 °C until PCR analysis. PCR-DGGE: The bacteria 16S rDNA was amplified by the nested PCR assay (Zhang et al., 2014). Primer pair of 27F/1492R was used for amplifying 16S rDNA and 338F-GC/518R was used for V3 region of 16S rDNA (Muyzer et al., 1993). The reactive solution (50 μL) for PCR was composed of 0.2 μM each primer, 1 μL template DNA (10 ng), 1 × PCR buffer (free of Mg2+), 0.3 mM each deoxyribonucleoside triphosphate (dNTP), 5 U Taq polymerase and 5 μL MgCl2 (25 mmol/L). Amplification was done in a 96 well-plates ThermoCycler (iCycler, Bio-Rad, USA). Cycling parameters consisted of an initial denaturation for 5 min 195
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at 94 °C, 30 cycles of 94 °C for 1 min, for 30 s at 57 °C, for 1 min at 72 °C, and a final extension for 10 min at 72 °C. These PCR products were checked through agarose gel (1%) to evaluate the size and quality of amplicons. Denaturing gradient gel electrophoresis (DGGE) was conducted with a D-Code universal mutation detection system (Bio-Rad, USA). Polyacrylamide gel (acrylamide-bisacrylamide solution, 37.5:1, 8%) with a linear gradient varying from 30 to 60% denaturant was used for separating the amplified 16S r DNA gene fragments. Electrophoresis was run at 60 °C for 5 h at 150 V in 1 × Tris-Acetate-EDTA (TAE) running buffer. Prior to observing the gel under UV light and photographing with Gel Doc XR system (Bio-Rad Laboratories, USA), the DNA was stained with SYBR Green I for 30 min. T-RFLP: By using 27-F (5′ labeled with 6-FAM) and 1492R as primers (27F-FAM: AGAGTTTGATCCTGGCTCAG; 1492R: GGTTACCTTG TTACGACT, Heuer et al., 1997), the bacterial 16S rRNA gene amplification was performed by PCR reaction. The PCR system contained 5.0 μL of 10 × PCR Buffer, 1.5 μL of dNTP (10 mM), 1.5 μL of template DNA, 1 μL of each of the two primers, 0.7 μL of Taq polymerase, and nuclease-free water to a final volume of 50 μL. Then, a SanPrep® quick PCR purification kit (BBI Life Sciences Corporation, China) was used to purify the obtained PCR products to gain the 16s target fragment that was used for enzyme digestion. Next, the purified products were digested by Msp I enzymes (TaKaRa Bio Inc., Japan) at 37 °C for 8 h. The digested PCR products were analyzed using a DNA analyzer (ABI 3730, BBI Life Sciences Corporation, China). Peak Scanner Software (v1.0) was used to analyze the fragment sizes. To analyze the microbial communities, a web-based tool named MiCA (http://mica. ibest.uidaho.edu/pat.php; Shyu et al., 2007) was used.
Table 1 Half-lives for degradation under field conditions with two and three repeated metalaxyl treatments.
t 1 = 0.693/ k
(2)
2
Pseudo-first-order kinetic model
2 repeated applications
1 × FR 2 × FR 1 × FR 2 × FR
C C C C
= = = =
0.1354*exp(−0.049t ) 0.3285*exp(−0.038t ) 0.2315*exp(−0.069t ) 0.4107*exp(−0.057t )
Correlation coefficient
Halflife (days)
0.9925 0.9855 0.9748 0.9730
14.1 18.2 10.0 12.2
significant effect on the half-lives. To date, the metalaxyl degradation with single treatment has been well-studied, but the changes with dissipation rates after repeated metalaxyl applications in field have not been thoroughly investigated. However, in the actual production, tolerance of Phytophthora infestans, Pseudoperonospora cubensis and Phytophthora parasitica var. nicotianae to metalaxyl exists in many areas (Liu et al., 2011; Pavelková et al., 2014), hence metalaxyl should be sprayed for 2 or 3 times. The dissipation pattern of metalaxyl under repeated applications also fitted well to the first-order model, which was similar to that of single metalaxyl application (Hanumantharaju and Awasthi, 2004). And from the results listed in Table 1, the degradation rate was quickened slightly and the half-life was shortened slightly with the increase of the applied times of metalaxyl. The similar results that metalaxyl dissipation was accelerated under repeated applications and in soil where metalaxyl was applied previously also were found in previous researches (Papini and de Andrea, 2001; Vischetti et al., 2008). The above results might be due to a shift of bacterial composition towards those microbial strains capable of degrading the fungicide (Vischetti et al., 2008). In the research of Papini and de Andrea (2001), they found that higher biological activity caused by some physicochemical characteristics could lead the enhanced degradation of metalaxyl. And the obviously enhanced dissipation under re-application also occurred in other fungicides (Baxter and Cummings, 2008; Fang et al., 2012; Triky-Dotan et al., 2010).
The data analysis was performed using SPSS 19.0 software (IBM, USA) for the bi-factorial analysis of variance (ANOVA) of numbers of bacteria, fungi and actinomycetes depending on the field application rate, time of exposure to metalaxyl and their interaction and half-lives of metalaxyl according to the field application frequencies and application rates with a post-hoc test and the least significant difference (LSD) test at p < 0.05. At the same time, one-way ANOVA was performed for the changes of different application rates of metalaxyl in the field with LSD test at p < 0.05. Results are reported as the mean ± standard error (SE). The dissipation of metalaxyl fitted well with a pseudo-first-order kinetic equation, and the reaction rate constant (k) and half-life (t1/2) were calculated using Eqs. (1) and (2): (1)
Application dosages
3 repeated applications
2.6. Statistical analysis
c = c0*exp(−kt )
Application times
3.2. Effects of metalaxylon on soil cultivable microbial population with repeated applications The influences of successive field applications of metalaxyl on soil bacterial, fungi and actinomycetes numbers are shown in Fig. 1. Soil bacterial population did not change significantly in the first seven days, but with the prolonged sampling time, bacterial population was increased by the successive inputs of metalaxyl to soil, and microbial population in treatments with 2 × FR of metalaxyl were higher than those in 1 × FR of the fungicide (p < 0.05). On the contrary, soil actinomycic numbers were stimulated by 2 repeated applications of metalaxyl on the first 7 days, but they recovered to control levels after 14 days (p ˃ 0.05). For fungal populations, there were no significant differences between treatments with metalaxyl and controls in field on day 7, but obvious inhibition effect were observed on days 14, 28 and 56 with 2 × FR of metalaxyl application in field compared with the controls. Unlike the 2 repeated applications of metalaxyl, there were no distinct differences in soil bacterial, fungal and actinomycic populations, except for the inhibition of fungal population on day 14 for three repeated applications. In general, metalaxyl stimulated soil bacterial growth, inhibited fungi growth and had no obvious stimulatory effect on actinomycetes with 2 repeated application of metalaxyl, but had no significant stimulatory and inhibitory effect on soil bacterial, fungal and actinomycic populations with the 3rd successive application of metalaxyl in field. The effects of time (7 d, 14 d, 28 d and 56 d), dose (1 × FR, 2 × FR) and the interaction of them on the microbial populations are listed in Table S2. Both time and dose had an obvious impact on the populations
where c0 (mg/kg) means the initial concentrations, and c (mg/kg) denotes the concentrations of metalaxyl at time t (day). The principal component analysis (PCA) was conducted using the DGGE band position and intensity. 3. Results and discussion 3.1. Effects of successive applications on metalaxyl degradation in field The dissipation of metalaxyl with repeated applications fitted well to the first-order model (Table 1). The half-lives were 14.1 d and 10.0 d for recommended field application dosage and 18.2 d and 12.2 d for two folds of application dosage under the 2nd and 3rd treatment, respectively. Dissipation rate were 0.049 and 0.069 for recommended field application dosage and 0.038 and 0.057 mg/kg per day for two folds of rates for the 2nd and 3rd treatment, respectively. Two-way ANOVA of the half-lives values with dose (1 × FR, 2 × FR) and repetition of application (2-, 3-times) was performed. As is shown in Table S1, neither dose (1 × FR, 2 × FR) nor frequency of application (2-, 3-times) have 196
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0.25 0.20 0.15 0.10 0.05 0.00
a: bacteria CK a a a
b: fungi
1×FR c b
a
2×FR
a ab
b a
c
b
7 14 28 56 Days after treatments (days)
Fungal population (104CFU/g dry soil)
Bacterial population (107CFU/g dry soil)
A
8.00 6.00
CK a aa
1×FR
2×FR a
a
a a
b
bb
b
4.00
c
2.00 0.00 7 14 28 56 Days after treatments (days)
c: actinomycete Actinomycic population (106CFU/g dry soil)
CK
1.50 1.00
b a ab
2×FR a
a a a
aa
0.50 0.00
a: bacteria CK 1×FR
0.20 0.15
a a
a
a a
2×FR a a
a
a a a
a
0.10
56
0.05
0.00 7 14 28 Days after treatments (days)
56
Fungal population (104CFU/g dry soil)
7 14 28 Days after treatment (days)
B Bacterial population (107CFU/g dry soil)
1×FR aa a
5.00 4.00 3.00 2.00 1.00 0.00
b: fungi CK 1×FR a a a
2×FR
a
a
a
a
ab
a
a
a
b
7 14 28 Days after treatments(days)
56
Actinomycic population (106CFU/g dry soil)
c: actinomycete CK
0.20 0.15 0.10
aa
a
a
1×FR a
2×FR
a
a
a
a
a aa
0.05 0.00
7 14 28 Days after treatments (days)
56
Fig. 1. Responses of soil bacterial (a), fungi (b) and actinomycete (c) populations to the 2nd (A) and 3rd (B) repeated treatments of metalaxyl under field conditions. 1 × FR: the recommended field rate; 2 × FR: two folds of the field rate. Error bars indicate mean ± standard deviation (SD). Different lowercase letters characters represent significant differences compared with the control at p < 0.05.
actinomycetes can use metalaxyl and/or its metabolites as energy and carbon sources, and, on the other hand, reduced competition with soil fungi for essential nutrients, lack of antagonistic inhibition induced by soil fungi, and release of substrates from the dead fungi could promote the growth of soil bacteria and/or actinomycetes (Zhang et al., 2016a). The different doses and times of exposure to metalaxyl and the interaction of them could not significantly impact the populations of cultivable bacteria, fungi and actinomycetes after the 3rd application (Table S2). It might be possible that microorganisms sensitive to metalaxyl were killed, and the non-sensitive microorganisms developed resistance to the fungicide, as was shown in repeated chlorothalonil applications (Zhang et al., 2016b). Thus, repeated fungicide application had a transient stimulatory or inhibitory effect on soil microbial populations, and soil microorganisms adapted themselves to the environment with fungicides (Fang et al., 2012). It is worth noting that, the results were slightly inconsistent with those obtained from our previous work in laboratory (Wang et al., 2015), which revealed that besides the main influence factor-metalaxyl, sunlight, precipitation, surface runoff and temperature all affected the microbial population thus, further studies are needed to elucidate the mechanism.
of bacteria, but the interaction of them had no significant effect. The two factors respectively and the interaction of them all influenced the populations of fungi. Only dose significantly affected the populations of actinomycetes in the 2nd metalaxyl treatments. Nevertheless, none of the factors (time, dose and the interaction of them) could significantly impact the microbial populations in the 3rd metalaxyl treatments. Statistical analysis results indicated that the populations of bacteria and fungi in the 2nd treatments were significantly different in each metalaxyl concentration group (Table S3). However, no significant differences could be observed in the populations of actinomycetes at each sample time and each concentration group in the 3rd metalaxyl treatments. The results indicating decreased fungal population after the 2nd metalaxyl treatment revealed that metalaxyl exerted persistent toxic effect on fungi in field (Droby and Coffey, 1991; Sukul et al., 2008). The possible reason might be that metalaxyl is a kind of broad-spectrum fungicide and may inhibit soilborne not-target fungi. The results of increased soil bacterial population at the end of the experiment and actinomycetes population on the first 7 days with 2 repeated metalaxyl applications suggested that, on one hand, soil bacteria and 197
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Aa
Ab
Ba
Bb
Fig. 2. DGGE profiles of soil bacterial community with 2 (A) and 3 (B) repeated metalaxyl treatments under field conditions. a: DGGE patterns; b: lanes and bands in DGGE patterns. The numbers (1–33 and 1–49) in Ab and Bb denote the bands in each lane. Lanes 1–12 denoted CK-7d, 1 × FR-7d, 2 × FR-7d, CK-14d, 1 × FR-14d, 2 × FR-14d, CK-28d, 1 × FR-28d, 2 × FR-28d, CK-56d, 1 × FR-56d, 2 × FR-56d. 1 × FR: the recommended field rate; 2 × FR: two folds of the field rate.
significantly affect diversities of soil bacterial communities. Simpson’s index and evenness index did not show a downward trend during the whole experimental period. In general, repeated metalaxyl inputs to the soil did not affect the bacterial community diversities. Principal component analysis (PCA) of DGGE patterns is shown in Fig. 3. As shown in Fig. 3A, the first two axes displayed 65.9% of the variation among all the treatments with 2 repeated metalaxyl applications and controls. Samples 1–9 were located together, indicating the high similarity among the samples, and were far away from samples 10, 11 and 12, which suggested that soil bacterial community were altered on day 56 after the second treatment. The distances among soil samples in the third treatments were significantly different from those among the second treatment. All samples distributed evenly on PCA (Fig. 3B), showing the insignificant separation among samples. Bacterial community composition changed a lot after 2 and 3 repeated metalaxyl applications. Some bands disappeared indicating that the bacteria represented by the bands were inhibited by metalaxyl selective pressures (Macur et al., 2007). The observation was consistent with the previous study, which showed that metam sodium with three successive treatments inhibited some of the predominant bands compared with the controls (Triky-Dotan et al., 2010). Some bands emerged showing that specific bacterial floras appeared. It should be noted that, some other bands always existed during the whole experiment this is
3.3. Effects of metalaxyl repeated application on soil microbial diversity and community structure under field conditions As shown in Fig. 2, band numbers were lower in the 2nd than in the 3rd treatments. In the second metalaxyl treatments and DGGE banding patterns were different from each other (Fig. 2A). The band numbers in metalaxyl treated samples were lower than in non-treated ones on day 7, but it was reversed on days 14, 28 and 56. Similarly, there were differences in band profiles among the samples and controls in the 3rd metalaxyl applications (Fig. 2B). The different DGGE patterns indicated different effects of metalaxyl treatments depending on different application frequencies and exposure time on bacterial communities (Fig. 2A and B). Some of the disappearing bands such as bands 7, 14 in Fig. 2Ab, and the new emerging bands such as bands 6, 8, 10, 22 in Fig. 2Ab and bands 2, 3 in Fig. 2Bb, may be related to the inhibition and stimulation of successive inputs of metalaxyl. Still the other bands were present during the whole experiment regardless of the applied rates and sampling time, such as bands 9, 11, 18, 21 in Fig. 2Aa and bands 17, 35, 36, 43, 44, 48 in Fig. 2Bb, revealing that these bands were unaffected by applications of metalaxyl. The diversity indexes (Simpson’s index, Shannon–Wiener index and evenness index) are listed in Table 2. The results showed that two repeated metalaxyl treatments did not
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Table 2 Diversity index of bacterial community in field with re-application of metalaxyl after DGGE analysis. Sampling time
7d
14 d
28 d
56 d
Application rate
CK 1 × FR 2 × FR CK 1 × FR 2 × FR CK 1 × FR 2 × FR CK 1 × FR 2 × FR
2 repeated applications
3 repeated applications
Shannon–Wiener Index
Simpson’s Index
Evenness Index
Shannon–Wiener index
Simpson’s Index
Evenness Index
2.63a 2.28a 2.06a 2.31a 2.60a 2.42a 2.42a 2.40a 2.79a 2.43a 2.40a 2.75a
0.91a 0.86a 0.81a 0.84a 0.90a 0.86a 0.88a 0.90a 0.92a 0.89a 0.89a 0.92a
0.89a 0.82a 0.78a 0.80a 0.88a 0.82a 0.89a 0.94a 0.93a 0.90a 0.91a 0.92a
3.38b 3.45b 3.43b 3.46b 3.44b 3.37b 3.47b 3.42b 3.36b 3.21a 3.26a 2.75a
0.96a 0.97a 0.97a 0.97b 0.97b 0.96b 0.97b 0.97b 0.96b 0.96a 0.96a 0.93a
0.98b 0.99b 0.99b 0.97b 0.98b 0.98b 0.98a 0.98a 0.98a 0.98b 0.98b 0.97b
could have the ability to degrade metalaxyl (Bailey and Coffey, 1986). We conjectured the special bands might have great comparability on those strains; the soil bacterial strains induced by repeated metalaxyl application needed to be isolated in the further research. In the present study, plate count method and molecular biology technology were both used to assess the influence of successive treatments of metalaxyl on soil bacterial communities, but different results were observed. It is possible because plate count method could only account for cultivable microorganisms, while only 1–10% of the soil microorganisms could be cultured. So this method is not complete and sensitive enough to understand the changes of the total microbial community (Müller et al., 2002). In later research, the study of soil microorganisms should adopt both traditional and modern methods of molecular biology to evaluate the whole effects. The T-RFLP analysis was performed for deeper study of the effects of metalaxyl application on soil microbial community structure. Five ascendant T-RFs were detected and the relative abundances of them are shown in Fig. 4. These results indicated that relative abundances of 486 bp and 400 bp T-RFs were reduced while those of 92 bp, 138 bp and 148 bp T-RFs were enriched with 2 repeated metalaxyl treatments. As for 3 repeated metalaxyl treatments, the relative abundances of 486 bp, 148 bp and 400 bp T-RFs were reduced while that of 92 bp T-RFs was enriched. The relative abundances of 138 bp concentrated in the end of the treatment period. The diversity index (Shannon–Wiener index, Simpson’s index and evenness index) of bacterial community in field with re-application of metalaxyl are listed in Table 3. The results showed that the Shannon–Wiener index mildly increased with 2 and 3 repeated metalaxyl treatments. The Simpson’s index slightly decreased with 2 and 3 repeated metalaxyl treatments. There was no significant change in evenness index for 2 and 3 repeated metalaxyl treatments during the whole experimental period. In general, repeated metalaxyl inputs to the soil did not affect the bacterial community diversities. From these results, soil microbial community was impacted after matalaxyl application to the soil. After the corresponding strains speculation of the enzyme fragment (Du et al., 2018; Shyu et al., 2007), 92 bp may represent Bacteroide; 138 bp, Bacillus; 148 bp, Eubacterium; 400 bp, Rhizobium and 486 bp, Firmicutes. Soil microbial composition could make adaptive changes when survival environment changes. We used T-RFLP to illustrate the change of microbial community structure and diversity caused by metalaxyl application under field experiment. Obviously, the microbial community structure was changed under metalaxyl applications. After analysis, the 400 bp T-RF was mainly represent Rhizobium, which could fix nitrogen and then provide organic nitrogenous compounds to plants. The reduction of 400 bp T-RF might designate that metalaxyl would affect the nitrogen fixation capacity of soil. The 138 bp T-RFs mainly stand for Bacillus, which could produce
Fig. 3. PCA of bacterial community DGGE patterns with 2 (A) and 3 (B) repeated metalaxyl treatments under field conditions. The numbers represent sample numbers. 1 = CK-7d; 2 = 1 × FR-7d; 3 = 2 × FR-7d; 4 = CK-14d; 5 = 1 × FR-14d; 6 = 2 × FR-14d; 7 = CK-28d; 8 = 1 × FR-28d; 9 = 2 × FR28d; 10 = CK-56d; 11 = 1 × FR-56d; 12 = 2 × FR-56d. 1 × FR: the recommended field rate; 2 × FR: two folds of the field rate.
probably because some bacterial strains with resistance were unaffected by metalaxyl (Wang et al., 2012). Principal component analysis indicated the differences between samples on day 56 and the other samples, this may be due to the appearance of bands 10 and 20 induced by metalaxyl the bacterial strains represented by bands 10 and 20 may be tolerant to metalaxyl or even be able to degrade metalaxyl. We also suggest bands 9, 11, 18 and 21 in Fig. 2Aa might represent the bacteria capable of degrading metalaxyl. A previous study reported that Sphingomonas canadensis strain and FWC47Bacillus flexus strain NBRC 15715 199
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100%
has little observed effects on the soil microorganisms, which suggests its safety to the soil ecological environment. However, in order to avoid bacterial resistance, the long-term use of a single metalaxyl application should not be adopted.
A
90% 80%
486
70%
400
60%
148
50% 40%
138
30%
92
4. Conclusions In this research, a field experiment was carried out to elucidate effects of repeated fungicide (metalaxyl) applications on metalaxyl dissipation and soil microbial community structure and diversity. The primary conclusions were as follows:
20% 10% 0%
100%
(1) The metalaxyl degradation rate increased with the increase of the applied times of metalaxyl; (2) The second inputs of metalaxyl had a temporary stimulation effect on soil actinomycetes and persistent positive effect on bacteria and negative effect on fungi, respectively; (3) A third application of metalaxyl exerted a transient adverse effect on soil fungi, and had no significant stimulation effects on soil bacteria and actinomycetes during the incubation time, respectively; (4) Metalaxyl with repeated applications exerted a significant impact on soil bacterial community, including the inhibition of the sensitive bacteria and the induction of the bacteria which could degrade metalaxyl; (5) In a practical way, metalaxyl is safe for soil environments under reasonable use. But due to the potential for bacterial resistance, it is not recommended for single applications over a long period.
B
90% 80% 70%
486
60%
400
50%
148
40%
138
30%
92
20% 10% 0%
Fig. 4. Average relative abundances of bacterial 16S rDNA T-RFs digested using endonuclease Msp I in soil samples with 2 (A) and 3 (B) repeated metalaxyl treatments under field conditions. First number means the frequency of metalaxyl, and the second means time. The letters FR and TFR represent the recommended field rate and twice the recommended field rate). And the legend listed on the right is the relatively high proportion T-RFs.
Acknowledgements This work was supported by the National Key R&D Program of China [grant numbers 2016YFD0800202, 2017YFD200307]; the National Natural Science Foundation of China [grant numbers 41771282, 41701279]; and the Natural Science Foundation of Shandong Province, China [grant numbers ZR2017MD005, ZR2017BB075]; and Shandong Provincial Key Laboratory of Agricultural Microbiology Open Fund (SDKL2017015). And we would like to express our heartfelt gratitude to Dr. Sam Allen, who helps us to polish the paper.
resilient spores and mitigate the impact of exposure to the poisonous and harmful substance. The enrichment of its abundance might also be because of the anti-stress capability of soil. Further studies are needed to elucidate the mechanisms at work. Other than the structure changes, the diversity index of soil microbial community was calculated. And the results indicated that metalaxyl application caused no obvious changes in the diversity and evenness of the soil bacterial community. In a word, repeated metalaxyl inputs to the soil could cause changes in specific soil microbial bacterial relative abundances but did not affect the soil microbial diversity and community structures. From those researches, we should take the resistance of soil microorganisms into consideration in the practical production. Metalaxyl
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecolind.2019.04.018.
Table 3 Diversity index of bacterial community in field with re-application of metalaxyl after T-RFLP analysis. Sampling time
7d
14 d
28 d
56 d
Application rate
CK 1 × FR 2 × FR CK 1 × FR 2 × FR CK 1 × FR 2 × FR CK 1 × FR 2 × FR
2 repeated applications
3 repeated applications
Shannon–Wiener Index
Simpson’s Index
Evenness Index
Shannon–Wiener index
Simpson’s Index
Evenness Index
0.04a 0.06a 0.04a 0.04a 0.05a 0.04a 0.04a 0.14a 0.18a 0.04a 0.06a 0.03a
3.18a 2.99a 3.16a 3.06a 2.87a 3.07a 3.06a 2.33b 2.26b 3.17a 2.99a 3.18a
0.94a 0.89a 0.93a 0.94a 0.93a 0.93a 0.94a 0.84a 0.77a 0.95a 0.90a 0.96a
0.04a 0.03a 0.17a 0.04a 0.04a 0.06a 0.04a 0.11a 0.10a 0.04a 0.08a 0.10a
3.18a 3.27a 2.24a 3.16a 3.12a 2.81a 3.19a 2.70b 2.66b 3.20a 2.67a 2.59a
0.94a 0.96a 0.81a 0.92a 0.95a 0.92a 0.95a 0.83a 0.85a 0.93a 0.88a 0.85a
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