摘要
近年来，我国大棚蔬菜生产得到快速发展。然而，大棚温暖潮湿的环境给病虫害大量繁殖提供了适宜的条件，导致农药超量、频繁的使用。毒死蜱作为高效、广谱的有机磷杀虫、杀螨剂，在我国大棚蔬菜栽培中广泛使用，蔬菜和土壤受到一定程度污染。为评价毒死蜱在大棚蔬菜和土壤中施用的安全性，开展了大棚和露地蔬菜和土壤中毒死蜱的消解特征、毒死蜱单独和与其它农药复合多次重复使用在土壤中的降解特征及其对土壤微生物多样性的影响、毒死蜱高效降解菌的分离和鉴定、毒死蜱的微生物降解特性及途径、微生物菌剂和酶制剂对蔬菜和土壤中毒死蜱降解的生物强化等方面的研究。主要研究结果如下：
毒死蜱在大棚和露地小白菜上的消解符合一级动力学特征，在土壤中的消解符合双室模型。大棚封闭的环境和季节更替能改变毒死蜱在小白菜和土壤中的消解行为，毒死蜱在大棚小白菜和土壤中的消解速率低于露地，在秋季的消解速率低于夏季。与露地相比，推荐剂量和双倍剂量毒死蜱在夏季大棚小白菜上的半衰期分别延长了26.0％和10.7％，在秋季大棚小白菜上的半衰期分别延长了6.6％和15.7％；在夏季土壤中的半衰期分别延长了44.4％和140.0％，在秋季大棚土壤中的半衰期延长了16.2％和63.1％。大棚小白菜收获期毒死蜱的残留量比露地高几乎50％。
低浓度(4 mg kg~(-1))、中浓度(8 mg kg~(-1))和高浓度(12 mg kg~(-1))毒死蜱在土壤中的降解符合一级动力学特征，其半衰期分别为14.32 d、16.70 d和18.00 d。随着毒死蜱浓度的提高，其在土壤中的半衰期明显延长。三个浓度处理初期(7 d)土壤微生物多样性均受到明显的抑制作用，作用大小与毒死蜱浓度呈正相关，但这种影响随着时间的推移逐渐缓解，到第21 d各处理土壤微生物多样性已恢复至对照水平，到第35 d，各处理土壤微生物多样性甚至超过对照水平。
毒死蜱、三唑酮和丁草胺单独和复合重复使用在土壤中的降解均符合一级动力学特征，随着施药次数的增加，除了毒死蜱+三唑酮处理中毒死蜱的半衰期略有延长外，其它各处理农药的半衰期均逐步缩短。各处理施药初期(3 d)土壤微生物多样性均受到明显的抑制作用，但这种影响随着时间的推移逐步缓解，到第21 d各处理土壤微生物多样性均已恢复至或超过对照水平。随着施药次数的增加，农药对土壤微生物多样性的抑制作用逐渐消失。
从污染土壤中分离筛选到一株能以毒死蜱为唯一碳源和能源生长的真菌DSP，经形态特征和18S rDNA序列分析鉴定为轮枝孢属(Verticillium sp．)。
在纯培养条件下真菌DSP对毒死蜱的降解与农药浓度、pH值和温度有关。Verticillium sp．DSP对毒死蜱的降解符合一级动力学特征，1、10和100 mg l~(-1)毒死蜱的降解半衰期分别为2.03 d、2.93 d和3.49 d，100 mg l~(-1)毒死蜱对真菌DSP有略微抑制作用。真菌DSP在pH 5.0、7.0和9.0时对1 mg l~(-1)毒死蜱的降解半衰期分别为2.03 d、1.93 d和2.11 d。真菌DSP在15、25和35℃时对1 mg l~(-1)毒死蜱的降解半衰期分别为3.31 d、2.03 d和1.88 d。真菌DSP在不同pH及温度下对毒死蜱的降解作用为pH7.0＞pH5.0＞pH9.0，35℃＞25℃＞15℃。通过毒死蜱微生物降解产物的GC-MS分析，TCP是最主要的降解产物，TCP在真菌DSP的作用下进一步烷基化生成TMP或脱卤素，鉴于真菌DSP可以利用毒死蜱为唯一底物生长，因此，毒死蜱降解产物可能进一步分解转化为二氧化碳，为一矿化过程。
DSP菌剂处理能有效促进土壤和蔬菜上毒死蜱的降解。灭菌土、以前未使用毒死蜱的土壤、以前使用毒死蜱的土壤中毒死蜱的降解均符合一级动力学特征，毒死蜱在DSP菌接种土壤中的半衰期比相应未接种土壤的半衰期分别缩短了81.2％、59.1％和24.4％。与未喷菌剂的对照相比，DSP菌剂处理后毒死蜱在大棚和露地小白菜上的半衰期分别缩短了10.9％和17.6％，在大棚和露地土壤中的半衰期分别缩短了12.0％和37.1％。
DSP粗酶制剂可以促进小白菜、空心菜、木耳菜、四季豆和辣椒上毒死蜱的降解。5种蔬菜上毒死蜱的降解均符合一级动力学特征，并且E(1：10)处理蔬菜上毒死蜱的降解速率要高于E(1：20)处理。与对照相比，DSP粗酶制剂E(1：20)处理小白菜、空心菜、木耳菜、四季豆和辣椒上毒死蜱的半衰期分别缩短了29.7％、34.4％、19.4％、19.4％和13.9％，E(1：10)处理蔬菜上相应的半衰期分别缩短了46.2％、56.8％、34.0％、45.7％和32.3％。结果表明，微生物酶制剂是控制蔬菜上农药残留的有效手段。
Greenhouse production of vegetables has been developed rapidly in China during the past decades. Compared to field conditions, greenhouse provides more favorable climate for fast reproduction of pests and diseases that result in extensive and frequent applications of pesticides. Chlorpyrifos is a broad spectrum organophosphorus insecticide and acaricide widely used for insect pest control on greenhouse vegetables. For the evaluation on the safety of chlorpyrifos application in the greenhouse, studies were conducted on dissipation of chlorpyrifos on vegetable and in soil in the greenhouse and open field, the influences of chlorpyrifos alone and in combination with other commonly used pesticides on soil microbial diversity. One fungal strain capble of degrading chlorpyrifos was isolated from soil. The fungal degradation of chlorpyrifos and its mechanism in pure cultures, and bioaugmentation of chlorpyrifos degradation on vegetable and in soil by the fungal strain preparation and its cell-free extracts were also investigated. The results were summarized as follows:
Dissipation of chlorpyrifos on pakchoi and in soil was fitted to the first-order and bi-exponential models, respectively. The hermetic environment of the greenhouse and changes of seasons alter dissipation behavior of chlorpyrifos on pakchoi and in soil. The dissipation rates of chlorpyrifos on pakchoi and in soil in the greenhouse were lower than those in the open field, and the rates in the autumn were lower than those in the summer. Compared to the open field, the half-lives of degradation (DT_(50)) for chlorpyrifos at the recommended and double dosages on greenhouse pakchoi were extended by 26.0% and 10.7% in the summer, and 6.6% and 15.7% in the autumn, the corresponding DT_(50) on greenhouse soil were extended 44.4% and 140.0% in the summer, and 16.2% and 63.1% in the autumn, respectively. Chlorpyrifos residues at pre-harvest time in the greenhouse were higher than those in the open field by almost 50%.
Degradation of chlorpyrifos at levels of 4.0, 8.0, and 12.0 mg kg~(-1) in soil were all fitted to the first-order function, its DT_(50) were measured to be 14.32 d, 16.70 d, and 18.00 d, respectively. The DT_(50) of chlorpyrifos in soil was extended with the concentration of chlorpyrifos. In all treatments, soil microbial diversity was inhibited significantly at 7 d after chlorpyrifos treatment, and the inhibitory effect was increased with the concentration of chlorpyrifos, but disappeared gradually with the time, and soil microbial diversity in all treatments was recovered to the level of the control at 21 d after treatment.
Degradation of chlorpyrifos, triadimefon, and butachlor alone and in combination after repeated treatments in soil were all fitted to the first-order function. The DT_(50) of pesticides in all treatments were shortened gradually with the increasing of application times, except that the DT_(50) of chlorpyrifos was slightly extended in the combined treatment of chlorpyrifos and triadimefon. In all treatments, soil microbial diversity was inhibited significantly at 3 d after pesticides application, but the inhibitory effect disappeared gradually with the time, and soil microbial diversity recovered to or exceeded the level of the control at 21 d. The inhibitory effect of pesticides on soil microbial diversity disappeared gradually with the increasing of application times.
A fungal strain DSP capable of utilizing chlorpyrifos as sole carbon and energy sources was isolated. Based on its morphological characteristics and 18S rDNA sequence analysis, the isolated strain was identified as Verticillium sp.
The ability of the fungal strain DSP to degrade chlorpyrifos in pure cultures depends on pesticide concentration, pH, and temperature. Degradation of chlorpyrifos by the fungal strain DSP was fitted to the first-order function. The DT50 of chlorpyrifos were measured to be 2.03 d, 2.93 d, and 3.49 d at concentrations of 1, 10, and 100 mg l~(-1), 2.03 d, 1.93 d, and 2.11 d at pH 5.0, 7.0, and 9.0, and 3.31 d, 2.03 d, and 1.88 d at 15, 25, and 35°C, respectively. Fungal degradation was inhibited slightly by chlorpyrifos at high concentration of 100 mg l~(-1). The degradation rates of chlorpyrifos by the fungal strain DSP were effected by pH and temperature following an order of pH 7.0>pH 5.0>pH 9.0, and of 35°C >25°C> 15°C, respectively. TCP was identified as a main degradation product of chlorpyrifos by GC-MS analysis, and it was likely that TCP was further transferred to be TMP or dechlorination. According to the capability of the fungal strain DSP to utilize chlorpyrifos as sole carbon and energy sources, it is reasonable to propose that chlorpyrifos is mineralized by the fungal strain DSP.
Degradation of chlorpyrifos in soil and on vegetable was significantly accelerated by the addition of the fungal strain DSP. Degradation of chlorpyrifos in sterilized soil, previously chlorpyrifos-untreated soil, and previously chlorpyrifos-treated soil were all fitted to the first-order function. Compared to the un-inoculated controls, the DT_(50) of chlorpyrifos in three inoculated soils were shorted by 81.2%, 59.1%, and 24.4%, respectively. In contrast to the controls, the DT_(50) of chlorpyrifos in the greenhouse and open field were shortened by 10.9% and 17.6% on pakchoi treated with the strain DSP preparation, and 12.0% and 37.1% in inoculated soils, respectively.
Degradation of chlorpyrifos on pakchoi, water spinach, Malabar spinach, haricot beans, and pepper was enhanced significantly by cell-free extracts. Degradation of chlorpyrifos on five vegetables were all fitted to the first-order function, and the degradation rates of chlorpyrifos on E (1:10) treated vegetables were higher than those on E (1:20) treated vegetables. Compared with the controls, the DT50 of chlorpyrifos were shorten by 29.7%, 34.4%, 19.4%, 19.4%, and 13.9%, and by 46.2%, 56.8%, 34.0%, 45.7%, and 32.3% on E (1:20) and E (1:10) treated pakchoi, water spinach, Malabar spinach, haricot beans, and pepper, respectively. The results indicate cell-free extracts is a promising method for the detoxification of pesticide residues on vegetables.
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