Notes
Pretreatment with Stenotrophomonas maltophilia CGMCC 1.1788 increased the aphicidal activity of imidacloprid
2013 Volume 38 Issue 3 Pages 139-143
Details
2013 Volume 38 Issue 3 Pages 139-143
With the development of modern agriculture, environmental problems caused by insecticide abuse have drawn people’s attention. It has been noticed that wide use of imidacloprid (IMI) and its environmental residue cause a lot of problems, especially decreased honeybee population.1) One methods to reduce environmental residue is to decrease the dosage of insecticides by increasing their activities. It is becoming harder to increase the activity of insecticides through chemical methods because of the limited availability of novel active ingredients. Therefore, it is becoming popular to change and modify the structure of insecticides to obtain more active substances with biotechnology. Microbes, which can transform and degrade insecticides, are considered potentially powerful tools for improving the activity of insecticides. Our laboratory has shown that soil microbial activity to metabolize neonicotinoid insecticides played a key role in the persistence of insecticidal bioactivity after soil application.2) Microbes not only have the ability to transform insecticides to nontoxic substances in the soil, but they also can structurally modify insecticides to increase their bioactivities. It has been reported that triadimefon could be transformed by microbes or other biota in soil to triadimenol which has different stereoisomers and activity in fungi and rats.3) Sanchez-Sanchez et al. reported that evolved cytochrome P450 CYPBM3 “21B3” from Bacillus megaterium has the ability to transform the phosphorothioate group of organophosphorus pesticides, parathion and chlorpyrifos, to the oxon derivative, which is considered an activating transformation, but in the meanwhile, it also has the ability to detoxify organophosphorus pesticides.4) Our data also shows that Stenotrophomonas maltophilia can transform IMI to 5-hydroxy IMI, which can be further transformed to olefin IMI under chemical conditions.5) More recently, we found that when succinate was used as an utilizable substrate, S. maltophilia CGMCC 1.1788 cometabolized IMI directly to olefin IMI via 5-hydroxy IMI.6) The bioactivity of olefin IMI is higher than that of IMI, but the bioactivity of 5-hydroxy IMI is quite low.7) Microbes have been widely used as biological factories to synthesize value-added products.8,9) However, it is complicated and costly to separate and purify transformed products with regular methods because of the complex composition of transformed products and the low ratio of the active ingredient. Moreover, the application of microbes to synthesize highly active insecticides is limited by the higher cost and lower prices of common insecticides.
In this study, we utilized the transformative activity of microbes to pretreat insecticide and then we detected the activity of insecticide after its direct soil application without separation and purification.
IMI (>96% purity) was provided by the National Pesticide Research & Development South Center, Nanjing, China. The 5-hydroxy IMI and olefin IMI were synthesized as reported by Dai et al.5) Acetonitrile and acetic acid (High Performance Liquid Chromatography (HPLC) grade) were purchased from TEDIA Company, USA. Other analytic reagents were purchased from Bio Basic Inc.
2. Bacteria, medium, and cultivationS. maltophilia CGMCC 1.1788 was obtained from the China General Microbiological Culture Collection Center (Beijing, China). According to the methods of Liu et al.,6) S. maltophilia CGMCC 1.1788 was grown in 100-mL Erlenmeyer flasks containing 30 mL of Luria–Bertani (LB) broth (5 g/L yeast extract, 10 g/L peptone, 10 g/L NaCl, pH 7.2) on a rotary shaker at 220 rpm and 30°C. After 24 hr, 3 mL of the culture broth was transferred to a 1,000-mL Erlenmeyer flask containing 300 mL of LB broth and incubated for 12 hr under conditions as mentioned above.
3. Pretreatment of IMI by S. maltophilia CGMCC 1.1788The bacteria grown in the culture medium were harvested by centrifugation at 6,000×g for 5 min at 4°C and washed twice with 0.1 mol/L Na2HPO4/KH2PO4 buffer (pH 8.0). Washed bacteria were resuspended with an equal volume of the transformation solution containing 0.1 mol/L Na2HPO4/KH2PO4 buffer (pH 8.0), 20 g/L utilizable substrates (sucrose or succinate) and 0.5 g/L IMI. Transformation was done in a 50-mL conical centrifuge tube containing 2 mL of the transformation solution containing resting cells on a rotary shaker at 220 rpm and 30°C for the indicated time. After supplementing the solution with distilled water to compensate for water evaporation, the transformation solution was appropriately diluted and directly used to measure aphicidal activity. The concentration of IMI in the transformation solution was calculated at its initial concentration (500 mg/kg). To prepare samples for HPLC, the transformed solution was centrifuged at 10,000×g for 10 min and the supernatant was appropriately diluted for HPLC analysis after filtering through a 0.22 µM filter.
4. Analysis of the bioactivity of pretreated IMI by S. maltophilia CGMCC 1.1788The bioactivity assay was conducted at the National Pesticide Research & Development South Cent, Nanjing, China, as reported previously.2) Briefly, horsebean seeds were planted in sand containing no insecticide at 24°C in a greenhouse. When the seedlings grew to 5 cm in length, each well-grown seedling was transplanted into a fresh plastic cup containing 80 g of soil and watered with 20 mL of a water solution that contained the indicated concentration of pretreated IMI. Each cup contained one seedling. After cultivation for 24 hr, the well-grown seedlings were selected for bioactivity assay. Each seedling was inoculated with six aphid imagoes of Aphis craccivora Koch that were removed 24 hr later and the number of total nymph was counted. The dead and live nymphs were counted 48 hr later, and the mortality rate was calculated. Results were obtained form three independent experiments with three replicates. Tests for significance were done by one-way ANOVA followed by Duncan's multiple range test (significance set at a=0.05) in SPSS Statistics 17.0.
5. HPLC analysisHPLC analysis was conducted with an Agilent 1200 HPLC system equipped with an HC-C18 column (4.6×250 mm, 5-µm particle size) as described previously.6) Elution was carried out at a flow rate of 1 mL/min at 30°C with a mobile phase containing A: water with 0.01% acetic acid; B: acetonitrile with 0.01% acetic acid; A : B=75 : 25 (v : v). The signal was monitored at a wavelength of 269 nm using an Agilent G1314A UV detector. Standard curves for HPLC were prepared using IMI, 5-hydroxy IMI, or olefin IMI as standards from which the transforming peak areas to molar concentrations were derived.
We have previously shown that S. maltophilia CGMCC 1.1788 transformed IMI into more active metabolites.6) To evaluate the aphicidal activity of IMI after pretreatment with S. maltophilia CGMCC 1.1788, 500 mg/kg of IMI was mixed with the bacteria at logarithmic growth phase using succinate as the utilizable substrate for pretreatment, and the bioactivity was measured 8 days later. As shown in Table 1, when unpretreated IMI was applied at 0.10 mg/kg or 0.05 mg/kg, the mortality of aphid nymphs of A. craccivora on horsebean plants was 33.1% and 15.9%, respectively; while, when the IMI that was pretreated by bacteria in the existence of succinate was applied directly without further separation and purification at the same concentrations, the mortality of aphid nymph on horsebean plants treated with 0.10 mg/kg or 0.05 mg/kg of pretreated IMI was 71.5% and 36.0%, respectively. Therefore, the aphicidal activity of pretreated IMI increased by 116.0% and 126.4%, respectively, compared to untreated IMI. To exclude interference by bacterial cells, S. maltophilia CGMCC 1.1788 cells were applied alone, and the mortality of aphid nymphs on horsebean plants was <1%, suggesting that S. maltophilia CGMCC 1.1788 has no insecticidal activity on aphid nymphs. These data show that the activity of IMI is significantly improved after bacterial pretreatment.
| Mortality of Aphid nymph (%) | ||
|---|---|---|
| 0.10 mg/kg | 0.05 mg/kg | |
| IMI | 33.1±4.8c | 15.9±4.8c |
| IMI pretreated by bacteria plus succinate | 71.5±8.0d | 36.0±7.7d |
| IMI pretreated by bacteria plus sucrose | 24.1±5.0b | 8.3±3.2b |
| IMI pretreated by bacteria | 36.0±1.6c | 15.1±2.9c |
| Bacterial control | 0.8±1.1a | 0.9±1.3a |
| Blank control | 0.2±0.1a | 0.3±0.1a |
Data are expressed as the means±SE (n=9). Values marked with different letters are significantly different and ones with the same letter are not significantly different within one column (p<0.05).
Our laboratory has shown that S. maltophilia CGMCC 1.1788 cometabolically transformed IMI into different products by using different utilizable substrates.6) In this study, we tested the effects of different utilizable substrates in pretransformation solution on the insecticidal activity of IMI. As shown in Table 1, when IMI was pretreated without utilizable substrates, the aphicidal activities of pretreated IMI against aphid nymphs were not significantly different from unpretreated IMI at either 0.10 mg/kg or 0.05 mg/kg. However, when succinate was used as the utilizable substrate, the insecticidal activity of pretreated IMI against aphid nymphs at 0.10 mg/kg or 0.05 mg/kg increased by 101.4% and 120.5%, respectively, as compared with the control without utilizable substrates. On the contrary, when sucrose was used as the utilizable substrate, the aphicidal activity of pretreated IMI against aphid nymphs at 0.10 mg/kg or 0.05 mg/kg IMI decreased by 33.1% and 45.0%, respectively, as compared with the same control without utilizable substrates. It suggests that the aphicidal activity of pretreated IMI varies with the different utilizable substrates for transformation.
3. The effect of different pretreatment times on the aphicidal activity of IMIThe effect of different bacterial pretreatment times on the aphicidal activity of IMI was measured. As shown in Fig. 1, the aphicidal activity of pretreated IMI decreased for the first 2 days and then started to increase gradually. The mortality of aphid nymphs on horsebean plants treated with 0.10 mg/kg of pretreated IMI was 26.4% and 19.1% on days 1 and 2, respectively, which decreased by 20.2% and 42.3%, respectively, as compared with 33.1% on day 0; the mortality of aphid nymphs on horsebean plants treated with 0.05 mg/kg of pretreated IMI was 11.5% and 10.0% on days 1 and 2, respectively, which decreased by 27.7% and 37.1%, respectively, compared with 15.9% on day 0. However, the aphicidal activity of pretreated IMI was increased after 4 days of pretreatment. The mortality of aphid nymphs on horsebean plants treated with 0.10 mg/kg or 0.05 mg/kg of pretreated IMI was 40.5% and 20.1% on day 4, respectively, which increased by 22.3% and 26.4%, respectively, as compared with 33.1% and 15.9% on day 0, respectively. On day 8 after pretreatment of the bacterial cells, the mortality of aphid nymphs on horsebean plants treated with 0.10 mg/kg or 0.05 mg/kg of pretreated IMI was 71.5% and 36.0%, respectively, which increased by 116% and 126.4%, respectively, as compared with 33.1% and 15.9% on day 0, respectively. Therefore, it is reasonable to set the transformation time to 8 days.
To understand the potential mechanism of increase of the aphicidal activity of IMI after pretreatment by S. maltophilia CGMCC 1.1788, we analyzed the composition of the transformation solution with different utilizable substrates using HPLC on day 8. As shown in Table 2, the amount of IMI decreased by 29.4% when succinate was used as the utilizable substrate, of which 32.1% was transformed to olefin IMI and 5-hydroxy IMI and 67.9% was degraded to non-detectable substances. The ratios of IMI, olefin IMI, and 5-hydroxy IMI were 88.2%, 10.0%, and 1.8%, respectively. In contrast, the amount of IMI decreased by 31.9% when sucrose was used as the utilizable substrate, of which only 19.0% was degraded to non-detectable substances, while 81.0% was transformed to 5-hydroxy IMI as well as trace amounts of olefin IMI. The ratios of IMI, olefin IMI, and 5-hydroxy IMI were 72.5%, 0.5%, and 27.0%, respectively. As a control, the amount of IMI only decreased by 7.8%, and a small amount of 5-hydroxy IMI was produced in the existence of the bacterium cells without utilizable substrates. The ratios of IMI and 5-hydroxy IMI were 98.0% and 2.0%, respectively. In the control without bacterial cells, there was no significant change in the amount of IMI and no production of olefin IMI or 5-hydroxy IMI regardless of whether there were utilizable substrates. This suggests that the difference of aphicidal activity of IMI after bacterial pretreatment with different utilizable substrates is due to the different ratio of cometabolic products of S. maltophilia CGMCC 1.1788. When succinate is added as a utilizable substrate, the major cometabolic product is olefin IMI, which is more active than IMI, with the result that aphicidal activity of IMI increases after pretreatment. Meanwhile, some of the cometabolic products that are not able to be detected by the HPLC or LC/MS-MS analyses may also contribute to the activity increase of IMI after pretreatment. When sucrose is added, the major cometabolite is 5-hydroxy IMI which is less active than IMI,10) with the result that aphicidal activity of IMI decreases after pretreatment.
| Utilizable substrates | Pesticide (mmol/L) | |||
|---|---|---|---|---|
| IMI | Olefin IMI | 5-Hydroxy IMI | ||
| S. maltophilia CGMCC 1.1788 | Succinate | 1.246±0.040b | 0.142±0.009b | 0.025±0.003a |
| Sucrose | 1.202±0.065a | 0.009±0.004a | 0.448±0.043b | |
| Control | 1.629±0.009c | ND | 0.033±0.001a | |
| Control without bacterial cells | Succinate | 1.716±0.045d | ND | ND |
| Sucrose | 1.735±0.098d | ND | ND | |
| Control | 1.742±0.008d | ND | ND | |
The concentration of sucrose and succinate was 20 g/L. Data are expressed as the means±SD (n=9). Values marked with different letters are significantly different and ones with the same letter are not significantly different within one column (p<0.05). ND, not detected.
The dynamic change of IMI and its cometabolic products in the transformation solutions during pretreatment is shown in Fig. 1. According to Fig. 1, on day 1 and day 2, the major cometabolic product was the 5-hydroxy IMI without apparent accumulation of olefin IMI. On day 4, the part of 5-hydroxy IMI that was transformed to olefin IMI was accumulating with time. On day 8, the major cometabolite was olefin IMI rather than 5-hydroxy IMI. These results are consistent with the change of the activity of pretreated IMI at different times. Therefore, the decrease of activity of IMI in the first two days of pretreatment is due to the preferential accumulation of less-active 5-hydroxy IMI, while the activity of IMI increases after 4 days of pretreatment, which contributes to the accumulation of more active olefin IMI.
Our laboratory previously reported that the initial insecticidal activity of IMI was lower than that of other neonicotinoid insecticides (acetamiprid and thiacloprid) after soil application, but its activity gradually increased with time.2) One possible reason is that soil bacteria transform IMI to more active substances, such as olefin IMI, which is more active than IMI.7) In this paper, our results show that the activity of IMI could be increased more than twofold after pretreatment with S. maltophilia CGMCC 1.1788 using succinate as the utilizable substrate which leads to the production of more highly active metabolites. Therefore, our study suggests that pretreating insecticide with microbes that have the ability to transform insecticides to more active substances is an effective method of increasing activity. It can overcome the problem of lower initial activity of IMI and reduce the dosage and environmental residue of this insecticide.
Usually, transforming and modifying of insecticides with microbes has some problems, such as a low transformation ratio, complex composition of metabolites, and the high cost of separation and purification. Moreover, insecticides are bulk commodities with low prices. Therefore, applying microbial transformation during the production of insecticide derivatives was restricted by all of these factors. Our laboratory has previously separated a strain of S. maltophilia from soil that can transform IMI to olefin IMI under certain conditions.6) However, utilizing the bacteria to produce olefin IMI is different for the reason mentioned above. To improve the insecticidal activity of IMI, S. maltophilia CGMCC 1.1788 was applied together with IMI in soil, but it is difficult for S. maltophilia CGMCC 1.1788 to colonize in soil (data not shown). Possible reasons most of the bacteria selected in laboratories with transformative activity are difficult to use for in situ biotransformation in soil include the lack of homogeneity and complex environmental conditions of the soil, the space occupying effect of soil microbes, and colonization resistance.11,12) According to the method demonstrated in this study, the higher activity derivative is produced by pretreatment, while the untransformed IMI that remained in the transformation solution is still utilized for killing insects. Therefore, it is not necessary to separate and purify transformed products. Our method avoids the problem of bacteria’s inability to colonize in soil and reduces cost considerably as compared with the conventional method. This research provides a new solution for reducing the dosage of insecticides to decrease their environmental residue.
