Effects of long-term chlorimuron-ethyl application on the diversity and antifungal activity of soil Pseudomonas spp. in a soybean field in Northeast China

Abstract

The herbicide chlorimuron-ethyl has been applied widely for weed control in farmland, especially in soybean fields in China over the past decade, but the chronic effects of this herbicide on soil microorganisms, particularly Pseudomonas spp., is not well understood. Taking a continuously cropped soybean field in the town of Fuyuan—a soybean production base of Heilongjiang Province in Northeast China—as a case study, soil samples were collected from plots having received 0-, 5-, and 10-year applications of chlorimuron-ethyl (30 g active component of chlorimuron-ethyl/ha/year) to study the abundance and diversity of Pseudomonas spp. Meanwhile, an in vitro assay was used to examine the antifungal activities of isolated Pseudomonas spp. against soil-borne pathogens (Fusarium graminearum, Fusarium oxysporum, and Rhizoctonia solani) causing soybean root rot disease. The production of siderophore, hydrogen cyanide (HCN), and lytic enzymes (cellulase, pectinase, and chitinase) by Pseudomonas spp. was also investigated. With 5- and 10- year chlorimuron-ethyl application, the numbers of soil Pseudomonas spp. decreased from 121 × 10² CFU/g dry soil in the control to 40 × 10² CFU/g dry soil and 13 × 10² CFU/g dry soil, and the Shannon index values decreased from 6.23 to 3.71 and 1.73, respectively. The numbers of antifungal Pseudomonas spp. also decreased, and the proportions of Pseudomonas spp. with antifungal activities against the different test pathogens altered. All the antifungal Pseudomonas spp. could produce siderophore and HCN but not lytic enzymes. The results suggest that long-term application of chlorimuron-ethyl in continuously cropped soybean field had negative effects on the abundance and diversity of soil Pseudomonas spp., including species with different antifungal activities against pathogens. Siderophore and HCN rather than lytic enzymes formed the antifungal metabolites of Pseudomonas spp., and the number of antifungal Pseudomonas that can produce siderophore and HCN decreased markedly under application of chlorimuron-ethyl, especially after 10-year application.

Introduction

Chlorimuron-ethyl is a member of the sulfonylurea family of herbicides, and is applied mainly in soybean fields (Zhang et al. 2011; Wang and Zhou 2006). Due to its broad-spectrum weed control at low application rate, good crop selectivity, and low toxicity to human beings and animals, chlorimuron-ethyl has been used widely (Ma et al. 2009; Zawoznik and Tomaro 2005). Only in Heilongjiang Province in Northeast China—an important soybean production base in this country— does consumption of chlorimuron-ethyl reach about 400 tons per year, covering more than 1.33 × 10⁶ ha soybean field (Zhao and He 2007). Nevertheless, the long persistence of chlorimuron-ethyl in soil has a definite inhibitory effect on the growth of soil bacteria (Teng and Tao 2008).

Some soil Pseudomonas spp. have antagonistic activity against soil-borne pathogenic microbes via the production of secondary metabolites such as hydrogen cyanide (HCN), siderophore, lytic enzymes (cellulase, pectinase, and chitinase), and antibiotics (phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin) (Ahmadzadeh et al. 2006; Raaijmakers et al. 2002; Haas and Défago 2005). However, the diversity of Pseudomonas spp. and their antifungal activity are often impacted by chemical pollutants (Evans et al. 2004; Bergsma-Vlami et al. 2005). Most research in this field has been conducted in short-term laboratory microcosm and field experiments (Wu et al. 2009). Due to the longer half-life of chlorimuron-ethyl in soil, and the resistance and resilience of soil microbes to short-term environmental stress, a long-term in situ investigation could be more appropriate to assess accurately the potential ecological risk of long-term chlorimuron-ethyl application on the diversity of soil Pseudomonas spp. and their antifungal activity.

In this study, by substituting time series with spatial series, 0–10 cm soil samples were collected from plots under 0-, 5-, and 10-year chlorimuron-ethyl application in a continuously cropped soybean field in the town of Fuyuan in Heilongjiang Province, Northeast China, with the abundance and diversity of Pseudomonas spp. as well as their antifungal activities and secondary metabolites production being investigated, with the aim of assessing the potential ecological risk of long-term chlorimuron-ethyl application on soil Pseudomonas spp. in soybean field.

Materials and methods

Site description and soil sampling

Soil (0–10 cm) samples were collected from three plots in a continuously cropped soybean field in the town of Fuyuan in Heilongjiang Province. The soil is classified as phaeozem (FAO Soil Taxonomy, ISSS/ISRIC/FAO 1998), and the three plots have uniform geomorphology, close geographical position, same climate conditions, same soil type, and same field management, with application of either no chlorimuron-ethyl (CK; 48°12′28″ N, 134°15′56″ E), 5-year application of 30 g active component of chlorimuron-ethyl/ha/year (T5; 48°12′34″ N, 134°15′83″ E), and 10-year application of 30 g active component of chlorimuron-ethyl/ha/year (T10; 48°12′25″ N, 134°15′54″ E), respectively. The chlorimuron-ethyl residues in plots T5 and T10 were 4.36 μg/kg and 8.46 μg/kg, respectively.

The samples were taken randomly throughout the plots after soybean harvest, passed through a 2 cm mesh sieve, mixed into one composite sample per plot, and stored at 4°C for further analysis.

Isolation of soil Pseudomonas spp.

Soil suspension was prepared by shaking 10 g soil in 90 mL sterile water for 30 min on a rotary shaker (180 rpm). Soil suspensions from the three plots were each diluted to 10⁻², and 100 μL of these suspensions was plated onto King’s B agar (King et al. 1954) augmented with ampicillin (40 μg/mL), chloromycetin (13 μg/mL), and actidione (100 μg/mL) (Liu et al. 2006). After incubating at 28°C for 48 h, colonies on the agar were counted, and a single colony was further cultured on the same medium to establish pure cultures.

Identification of Pseudomonas isolates

Pseudomonas isolates were checked by taxonomic identification of Gram staining and by flagellar staining. Gram-negative isolates with flagella were tested by chemical identification, and isolates that can ferment glucose and produce oxidase and catalase were identified by molecular methods (Kan et al. 2005).

Total genomic DNA was extracted from the pure cultures growing in King’s B broth at 28°C for 12 h. The cultures were centrifuged at 8,000 rpm for 5 min. After removing the supernatants, the pellets were re-suspended in 1 mL DNA extraction buffer (100 mmol/L Tris-HC1, 100 mmol/L EDTA, 1.5 mmol/L NaC1, pH 8.0), rotary-vibrated in a vortex instrument for 5 min, 0.1 mL 20 % SDS solution was added, and the suspensions incubated at 65°C for 30 min (mixed every 10 min) followed by centrifuging at 12,000 rpm for 10 min. The pellets were extracted twice with an equal volume of chloroform-isoamyl alcohol (24:1; v/v). The extract was precipitated with 0.1 volumes of NaAc (3 mol/L) and 0.6 volumes of ice-cold isopropanol, and washed by ice-cold 70 % ethanol. DNA extracts were then re-suspended in 50 μL deionized H₂O, and checked with UV light (254 nm) after agarose gel electrophoresis and Goldview (BioDev; Beijing, China) staining. The DNA was stored at −20°C for further research.

To confirm the isolates as Pseudomonas spp., the Pseudomonas sequence-specific primers PsF (5′-TTA GCT CCA CCT CGC GGC-3′) and PsR (5′-GGT CTG AGA GGA TGA TCA GT-3′) were used to amplify by PCR a fragment of about 1,000 bp (Widmer et al. 1998; Garbeva et al. 2004). Positive isolates were subjected to further characterization.

Analysis of genotypic diversity of Pseudomonas isolates by ARDRA analysis

Amplified ribosomal DNA restriction analysis (ARDRA) was performed to identify the genotypes of isolated Pseudomonas spp. 16S rRNA gene was amplified with the universal primer pair 27F and 1492R (Martin-Laurent et al. 2001). The PCR products were digested with restriction endonucleases HinfI and TaqI (TaKaRa, Tokyo, Japan) under the standard conditions suggested by the manufacturer (Wu et al. 2009). Restriction bands were separated by 8 % polyacrylamide gel electrophoresis (PAGE) in 1 × TAE buffer at 200 V for 1 h, and DNA banding patterns were visualized by staining with GenFinder (TaKaRa). Shannon diversity index (H) was used to estimate the diversity of the Pseudomonas isolates based on ARDRA patterns:

$$ H = - \sum\limits_{{i = 1}}^s {{p_i}\ln {p_i}} = - \sum\limits_{{i = 1}}^s {\left( {{N_i}/N} \right)} \ln \left( {{N_i}/N} \right) $$

where N _i is the total number of the ith ARDRA type isolates, and N is the total number of the isolates in the sample (Wu et al. 2009).

In vitro test of antifungal activity

The antagonisms of isolated Pseudomonas spp. against three pathogens (Fusarium graminearum, Fusarium oxysporum, and Rhizoctonia solani) causing soybean root rot disease were tested in vitro (Chen et al. 1999; Fu 2006). An agar plug (5 mm in diameter) taken from the perimeter of an actively growing pathogenic fungus colony was placed at the center of a potato dextrose agar (PDA) plate. After 48 h incubation, 100 μL cultures were put in an Oxford cup and then placed around the agar plug. A strain with known antifungal activity (P. fluorescens Q2-87) served as a positive control. The plates were incubated at 28°C for 4 days, and scored by measuring the distance between the edges of the bacterial colony and the fungal mycelium. Antifungal ability was divided into three levels according to the diameter of inhibition ozone (low: 0–1 cm, medium: 1–2 cm, and high: 2–3 cm).

Production of siderophore, HCN, and lytic enzymes by Pseudomonas isolates

Siderophore production was assessed by the universal chemical assay using chrome azurol S (CAS) agar medium (Schwyn and Neilands 1987). The formation of a yellow-to-orange halo on the blue CAS agar plates was recorded, indicating the presence of siderophore in the culture supernatants of the isolates.

HCN production was determined by the method of Verma et al. (2007). The Pseudomonas isolates were grown in KB broth at 28°C on a rotary shaker. Filter paper was cut into uniform strips of 10 cm long and 0.5 cm wide, saturated with alkaline picrate solution, and placed inside the test tube in a hanging position. After incubation at 28°C for 48 h, HCN production was identified if the filter paper was observed to change color.

Cellulase and pectinase productions were determined as described by Verma et al. (2007). M9 medium agar (Na₂HPO₄ 6.0 g, KH₂PO₄ 3 g, NaCl 0.5 g, NH₄Cl 1.0 g, MgSO₄ 0.5 g, glucose 2 g, CaCl₂ 0.015 g, distilled water 1 L) amended with 10 g cellulose and 1.2 g yeast extract per liter distilled water was used to test cellulase production (Miller 1974). The isolates were plated and incubated at 28°C for 3 days. Development of halos was considered positive. M9 medium agar amended with 10 g pectin and 1.2 g yeast extract per liter distilled water was used to test pectinase production. After incubated at 28°C for 2 days, the isolates were flooded with 2 mol/L HCl. Clear halos around the colonies were considered as positive for lytic enzyme production.

Colloidal chitin was employed to evaluate the chitinolytic capability of Pseudomonas isolates (Shi et al. 2008). A loopful of 48-h-old culture was inoculated on chitin agar medium (A: 2% colloidal chitin; B: 0.05% KH₂PO₄, 0.05% MgSO₄·7H₂O, 0.05% NaC1, 0.001 % FeSO₄·7H₂O, 4% agar). After 6 days incubation at 28°C, the development of clear halos around the colonies was recorded after flooding with 5% NaOH.

Results and discussion

Identification and individual number of Pseudomonas spp. isolates

A total of 174 isolates were collected from the three soil samples. All the isolates were identified as Pseudomonas spp. based on taxonomic, chemical and molecular identification. A dramatic decrease was observed in the number of Pseudomonas spp. from 121 × 10² CFU/g dry soil in CK soil to 40 × 10² CFU/g dry soil in T5 soil and 13 × 10² CFU/g dry soil in T10 soil, suggesting that chlorimuron-ethyl had a chronic effect on Pseudomonas spp., with the abundance of Pseudomonas spp. obviously decreasing under the repeated application of the herbicide over a longer period.

Diversity of Pseudomonas spp. isolates

For all the isolates, five ARDRA patterns existed, named A–E (Fig. 1). The total number of the ARDRA patterns was four in CK soil and two for each in T5 and T10 soils, and the total number of isolates, the number of isolates with antifungal activity, and the Shannon index values all decreased markedly in chlorimuron-ethyl applied soils, especially in T10 soil (Table 1), suggesting the impacts of longer term application of chlorimuron-ethyl on the diversity of Pseudomonas spp.

Fig. 1

Five representative ARDRA patterns for Pseudomonas isolates. Lanes: M Marker DL, 2000; A–E five amplified ribosomal DNA restriction analysis (ARDRA) patterns for Pseudomonas isolates

Table 1 Numbers and diversity of Pseudomonas isolates based on amplified ribosomal DNA restriction analysis (ARDRA) analysis

Antifungal activity of Pseudomonas spp. isolates

There were 30–80 Pseudomonas spp. isolates exhibiting antifungal activity to the test pathogens in CK soil, but only 14–38 and 6–10 isolates having this activity in T5 and T10 soils, respectively, suggesting the negative effects of chlorimuron-ethyl on antifungal Pseudomonas spp. All the isolates with antifungal activity belonged to ARDRA patterns A, B, C and D and the isolates with antifungal activity in the former three patterns disappeared in T5 and T10 soils (Fig. 2).

Fig. 2

Numbers of antifungal Pseudomonas isolates. F.g Fusarium graminearum, F.o Fusarium oxysporum, R.s Rhizoctonia solani; A–D ARDRA pattern. CK No chlorimuron-ethyl application, T5 5-year application of 30 g active component of chlorimuron-ethyl/ha/year, T10 10-year application of 30 g active component of chlorimuron-ethyl/ha/year

Figure 3 shows that the proportions of Pseudomonas spp. isolates with low, medium, and high antifungal activities against Fusarium graminearum, Fusarium oxysporum, and Rhizoctonia solani differed in the three soil samples. In CK and T5, no isolate with high antifungal activity against the pathogens was observed (except a few against R. solani in CK), isolates with low or medium antifungal activity against F. graminearum and F. oxysporum had less difference, but those with medium antifungal activity against R. solani decreased in T5. In T10, the isolates with medium antifungal activity against F. graminearum and F. oxysporum increased, and those with high antifungal activity against R. solani showed a marked increase, as compared with CK and T5 soils.

Fig. 3

Percentage of antifungal Pseudomonas isolates with different antifungal ability in each soil; 2–3 cm, 1–2 cm, and 0–1 cm represent high, medium, and low antifungal activity, respectively. Abbreviations as in Fig. 2

Some researchers have mentioned the possible side-effects of applying bensulfuron-methyl, metsulfuron methyl, chlorsulfuron, and thifensulfuron methyl—also members of the sulfonylurea herbicide family—on soil Pseudomonas (Chen et al. 2009; Tina and Carsten 1998). In this study, chlorimuron-ethyl, a member of the sulfonylurea group, also had negative effects on soil Pseudomonas spp. in soybean fields. The decrease in abundance and diversity of soil Pseudomonas spp. after long-term chlorimuron-ethyl application could induce an imbalance between soil microbial communities. Furthermore, the alteration of population structure of the Pseudomonas spp., especially the changes in the proportions of Pseudomonas spp. with different antifungal activities against test pathogens, reflect the resilience of soil Pseudomonas spp. to long-term chlorimuron-ethyl stress.

Production of siderophore, HCN, and lytic enzymes by Pseudomonas spp. isolates

All 128 isolates with antifungal activity in the three soil samples could produce siderophore, and the majority (119) could produce HCN. Regarding production of lytic enzymes cellulase, pectinase, and chitinase, all the isolates lacked this ability, with the exception of three isolates in CK soil, one in T5 soil, and two in T10 soil. Of the 46 isolates without antifungal activity in the three soils, only 1 isolate in T5 soil and 3 isolates in T10 soil had a weak reaction in the siderophore assay, only 1 in T5 soil could produce HCN, and none could produce lytic enzymes (Table 2). These results indicate that upon application of chlorimuron-ethyl, the number of antifungal Pseudomonas isolates that can produce siderophore and HCN decreased markedly, especially in soils with 10-year application of chlorimuron-ethyl.

Table 2 The number of Pseudomonas isolates producing siderophore, hydrogen cyanide (HCN) and lytic enzymes in different chlorimuron-ethyl applied soils

Siderophore, HCN, and lytic enzyme were considered as contributing to the antifungal metabolites of Pseudomonas. The production of antifungal metabolites by Pseudomonas is related closely to the species of Pseudomonas and environment factors such as soil pH. Berg et al. (2005) proposed that all antifungal P. putida isolates could produce siderophore but not chitinase or pectinase. Ahmadzadeh et al. (2006) asserted that there was no obvious link between the inhibition of fungal growth in vitro and the production of antifungal metabolites such as siderophore, HCN, and protease by P. fluorescens. Costa et al. (2006) claimed that the majority of antifungal Pseudomonas isolates were able to produce siderophore, and fungal antagonists had a common feature exhibiting protease activity. In this study, all the Pseudomonas spp. isolates with antifungal activity produced siderophore, and the majority produced HCN. Few of the isolates produced the lytic enzymes cellulase, pectinase and cellulose. It seems that siderophore and HCN rather than lytic enzymes were the main antifungal metabolites of these Pseudomonas spp. isolates.

Conclusions

Long-term application of chlorimuron-ethyl in continuously cropped soybean field decreased the abundance and diversity of soil Pseudomonas spp., and also decreased the antifungal Pseudomonas spp. and altered the proportions of Pseudomonas spp. with different antifungal activities against the causative pathogens of soybean root rot disease. Siderophore and HCN rather than lytic enzymes were the main antifungal metabolites of Pseudomonas spp., and the number of antifungal Pseudomonas able to produce siderophore and HCN decreased markedly under the application of chlorimuron-ethyl, especially after 10-year application.

This study provides a case for understanding the chronic effect of chlorimuron-ethyl on soybean soil Pseudomonas spp. It would be advisable to study the significance of the resilience of soil Pseudomonas spp. further under long term chlorimuron-ethyl stress from the viewpoint of soil microbial ecology to more accurately assess the potential ecological risk of long-term chlorimuron-ethyl application on soybean field.