一株新的多菌灵高效降解茵的筛选与降解特性分析

从长期施用多菌灵农药的土壤中,通过富集筛选,获得1株新的多菌灵高效降解菌株。通过生理生化实验和16SrDNA序列同源性分析鉴定该菌株,应用高效液相色谱法对纯培养条件下菌株的降解特性和粗酶提取液的降解性能进行了分析。结果表明,筛选所获得的菌株与Raoultella菌属的亲缘关系最近,将其命名为Raoultellasp．MBC,该菌株能在以多菌灵为唯-碳源的无机盐培养基中生长;25℃、pH7．0、200r·min。的最适生长条件下避光振荡培养72h,多菌灵的降解率达到100％;在最适培养条件下外加氮源和碳源在培养后期均可以提高多菌灵的降解率,外加氮源对多菌灵的降解效果优于外加碳源;该菌体的粗酶提取液具有降解多菌灵活性,且多菌灵降解酶为诱导酶。研究结果为多菌灵污染土壤的生物修复和酶修复提供了材料和理论依据。     

土壤中双氰胺降解及与降解菌的关系

采用灭菌土培、非灭菌土培、田间蔬菜种植、添加外源降解菌土培试验研究了土壤中双氰胺（DCD）降解及与降解菌的关系。结果显示,不论是单施DCD、尿素配施DCD,还是碳酸氢铵配施DCD的土壤,灭菌处理的土壤中DCD半衰期分别比不灭菌处理的长13.56、5.79、14.51d。降解菌生长期间,降解菌总量（x）与DCD降解呈显著正相关,拟合的线性方程为y=3.1841x-2.5452,r=0.9752。外源DCD降解真菌可在灭菌土壤中定殖并有效降解DCD,培养15d后,U＋DCD＋DCD降解菌处理土壤中DCD降解真菌的数量增加至36.40×105cfu,且DCD含量极显著降低。这些结果表明土壤中DCD降解与降解菌关系极为密切,添加外源真菌加速了土壤中DCD降解。     

木霉T12菌株对多菌灵的降解特性及生物修复

从耐药性木霉菌株的诱变选育过程中,得到一株能在含多菌灵2000mgL-1培养基上生长的变异菌株T12。该菌株在以多菌灵为唯一碳源的无机盐培养基中,于25℃、200rmin-1振荡培养5d,对多菌灵的降解率达到73.1%。在pH6.0、温度25℃、5%接种量和加入0.5%酵母粉为最适降解条件下,该菌株对多菌灵的降解率达到92.1%。对原土壤、自然风干土壤和高温烘干土壤中的多菌灵进行室内降解实验,在25℃～28℃,5%接种量,15%含水量的条件下处理10d,对多菌灵的降解率分别达到79.7%、76.5%和70.5%。研究结果为该菌株在土壤生物修复中的应用提供了科学依据。     

一株红霉素降解菌红圆酵母的选育及其降解特性

选取长期堆放红霉素药渣的土壤,经驯化富集后筛选到1株能高效降解红霉素的菌株,根据表型特征、生理生化特性及18S rDNA序列同源性分析,鉴定为红酵母属的粘性红圆酵母（Rhodotorula mucilaginosa）。该菌的18S rDNA序列片段长度为1 562 bp,在GenBank登录号为EU294522。该菌可以利用红霉素作为碳源生长,适用碳源与氮源分别为蔗糖和NH4Cl。该菌降解红霉素的最适温度为30 ℃,最适pH 5.0～5.5,最适接菌量6%,降解率与通气量成正相关。在最适反应条件下培养48 h, 该菌株对红霉素的降解率可达100%。     

多菌灵降解菌djl-6和啶虫脒降解菌D-2固体菌剂的研发

以蚯蚓粪肥、猪粪肥、鸡粪肥、秸秆肥、米糠、泥炭以及花生壳粉作为载体,以多菌灵降解菌Rhodococcus qingshengii djl-6和啶虫脒降解菌Pigmentiphaga sp. D-2为材料,开展了固体菌剂的研发。接种菌株djl-6及D-2后,各载体均能够促进供试菌株的生长。以蚯蚓粪肥作为载体时,菌株djl-6、D-2有效菌数释放率最高,分别为100.40%和82.03%。在保存120 d时,除花生壳粉外,其余各载体中的活菌数均高于10~7 cfu/g;以蚯蚓粪为载体的djl-6菌剂活菌数达到了7.00×10~8 cfu/g,以猪粪为载体的D-2菌剂活菌数达到了4.29×10~8 cfu/g。固体菌剂保存30 d后进行土壤农药残留降解试验,以蚯蚓粪为载体的djl-6菌剂对5 mg/kg的多菌灵降解率为94.30%,以蚯蚓粪为载体的D-2菌剂对10 mg/kg的啶虫脒降解率为81.87%。研究结果表明,以蚯蚓粪为载体的固体农残降解菌剂活菌数高、保存期长,具有较好的应用前景。     

玉米秸秆低温降解复合菌系降解能力及微生物组成研究

根据还田秸秆配施尿素的生产实际,对玉米秸秆低温高效降解复合菌系GF-20进行氮源培养基驯化,探明其菌种组成和功能多样性及其与菌源菌种结构差异,完善复合菌系筛选方法,促进其开发利用。本文以低温高效降解复合菌系GF-20为研究对象,在硫酸铵和尿素不同配比下连续继代培养10代,获得不同氮源菌系(硫酸铵处理N1,硫酸铵和尿素混合处理N2-N5,尿素处理N6),测定其玉米秸秆降解率,评价复合菌系秸秆降解效率;采用MiSeq高通量测序对菌源土壤样品及不同氮源下继代培养的复合菌系菌种组成和功能多样性进行研究。结果显示N2处理玉米秸秆降解率显著高于其他处理;菌源土壤的Alpha多样性指数显著高于继代培养后的复合菌系,不同处理间N2处理显著高于其他氮处理;菌源和复合菌间以及不同氮处理间菌种组成具有显著差异, N2处理菌种组成多样性较高,菌群结构更加丰富、均匀,且碳水化合物的代谢通路丰度较高。菌源经限制性继代筛选后得到了参与玉米秸秆降解过程的功能菌,能有效提高秸秆降解率,其在硫酸铵和尿素氮源为0.16%+0.04%的条件下,菌系的秸秆降解效率较高,这为复合菌的生产实际开发利用提供了理论依据。     

海藻酸钠固定化细菌对毒死蜱的降解特性

毒死蜱的生产和使用日趋广泛,由其造成的环境污染和危害不容忽视。微生物是影响有机磷农药在环境中降解的最主要因素,也被认为是降解有机磷农药最可靠而高效的途径。固定化技术是提高微生物降解农药效率的有效方法之一。本研究以海藻酸钠为载体,采用注射器滴定法将蜡状芽孢杆菌(Bacillus cer-eus)HY-1用海藻酸钠溶胶包埋,研究了反应时间、固定化菌接入量、pH和毒死蜱初始浓度对毒死蜱降解的影响以及固定化菌的重复使用效果。结果表明:海藻酸钠固定化菌能够高效降解基础培养基中的毒死蜱,制备固定化小球海藻酸钠溶胶的最适浓度为2.5%(w/v),小球的平均粒径为3 mm。在培养时间为60 h时,固定化菌对100 mg·L-1毒死蜱的降解率达到最大。固定化小球接入量为160 g·L-1时,对100 mg·L-1毒死蜱的降解率最高。固定化菌对毒死蜱的降解有着较宽泛的pH适应范围,碱性环境更有利于其对毒死蜱的有效降解。当毒死蜱初始浓度为80 mg·L-1和100 mg·L-1时,固定化菌对毒死蜱的降解率较高,达90%左右。固定化菌可重复利用降解毒死蜱,当利用4次后,固定化小球虽已发生崩解,但对100 mg·L-1毒死蜱的降解率仍高达47%。因此,海藻酸钠固定化蜡状芽孢杆菌对水体中毒死蜱的降解率较高,环境适应性较强,固定化菌可在毒死蜱污染的净化去毒方面发挥重要作用。     

普施特降解菌Bacillus sp.zx2和zx7生长及降解特性

芽孢杆菌zx2和zx7是普施特高效降解菌,研究其生长和降解特性旨在为普施特污染土壤的生物修复提供科学依据。采用瓶培养法,对芽孢杆菌zx2和zx7的生长特性及单菌和复合菌对普施特的降解特性进行了研究。结果表明,zx2和zx7均可在普施特初始浓度≤200mg·L-1的无机盐培养液中生长良好,zx2在温度25～35℃和pH4.0～7.0时生长良好,而zx7适宜在温度30～35℃和pH5.0～8.0时生长,可见在适应性上二者互补。在最佳条件（温度32℃、pH6.0和普施特初始浓度为200mg·L-1）下,zx2和zx7在无机盐培养液中对普施特降解动态均符合阻滞动力学,半衰期分别为3.8d和2.8d,培养6d时普施特降解率分别为85.81%和90.27%。在培养过程中,zx2的pH是降低的,而zx7的pH基本不变,可初步表明二者降解机理不同;zx2和zx7复合菌（1∶1）对普施特降解率比单菌低,为82.70%,这可能是因为zx2或zx7降解普施特的过程中利用了对方产生的降解产物。     

毒死蜱降解菌株Bacillus latersprorus DSP的降解特性及其功能定位

从土壤中分离到一株能有效降解毒死蜱的细菌DSP，该分离株鉴定为侧芽孢杆菌（Bacillus latersprorus）。在纯培养条件下测定了分离株DSP对毒死蜱的降解性能。在接种量为菌浓度OD415=0．2，pH7．0、25℃条件下，测得1、10mg L^-1毒死蜱的降解符合一级动力学特征，其降解半衰期分别为1．48d、5．00d，100mg L^-1毒死蜱对DSP菌有明显的抑制作用；分离株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℃。该菌株含有一个20kb左右的质粒，通过吖啶橙与升温法对质粒消除实验证实，随着质粒的丢失，菌株利用毒死蜱的能力也丧失，用热击法和CaCl2法将菌株质粒转人大肠杆菌JM109和质粒消除处理的DSP^-菌中，随着质粒的获得，这些转化子获得了降解毒死蜱的能力。研究结果表明，Bacillus latersprorus DSP降解毒死蜱的功能和质粒有关。     

多环芳烃高效降解菌的筛选

以多环芳烃(PAHs)菲、蒽、芘、■和苯并(a)芘为供试物,对土著混合菌和引进菌同时进行筛选实验。结果表明,引进菌和土著混合菌经过驯化后对菲、蒽、芘、■和苯并(a)芘均具有一定的降解能力。其中,在pH=6时,混合菌U03在48h内对5种PAHs的降解率均相对较高,分别为:72.38%;64.46%;65.77%;66.49%和64.77%,并且其的降解速率在各菌剂中同样最快,通过SPSS数理统计分析软件对数据进行处理后得出,混合菌U03可在较短时间内达到较好的降解目的。室内模拟试验证明混合菌U03具有较强的降解PAHs的能力,混合菌的协同作用有利于污染土壤中PAHs的降解。     

Direct action of the biocide carbendazim on phenolic metabolism in tobacco plants

In view of the essential role of phenolic compounds in the development of pathogen resistance in plants, and given the influence that fungicides exert over phenolic metabolism, the aim of the present study was to determine the effect of the application of different rates of fungicide on the metabolism of phenolic compounds in tobacco plants (Nicotiana tabacum L. cv. Tennessee 86). The fungicide applied was carbendazim, with a purity of 100%, at three different rates: 1.3 mM (carb(1)), 2.6 mM (this being the recommended concentration, carb(2)), and 5.2 mM (carb(3)). The control treatment was without carbendazim. The results in relation to control plants indicate that the application of carb(1) in tobacco plants not afflicted by damaging biotic and abiotic agents boosts phenolic accumulation. Therefore, in the case of carbendazim, the application of 50% less (carb(1), 1.3 mM) than the recommended dosage (carb(2), 2.6 mM) of this fungicide could be more effective, because the foliar accumulation of phenolics presented at carb(1) may imply an increased resistance of plants to pathogen infection. On the other hand, we found an inhibition of the phenolic oxidation by the application of carbendazim, principally at carb(3). These results suggest that the excessive application of carbendazim (5.2 mM) could be harmful for healthy plants, because, on inhibiting phenolic metabolism (biosynthesis and oxidation), such treatment would also sharply reduce the capacity of these plants to respond against pathogen attack.     

Dissipation and distribution behavior of azoxystrobin, carbendazim, and difenoconazole in pomegranate fruits

The dissipation behavior and degradation kinetics of azoxystrobin, carbendazim, and difenoconazole in pomegranate are reported. Twenty fruits/hectare (5 kg) were collected at random, ensuring sample-to-sample relative standard deviation (RSD) within 20-25%. Each fruit was cut into eight equal portions, and two diagonal pieces per fruit were drawn and combined to constitute the laboratory sample, resulting in RSDs <6% (n = 6). Crushed sample (15 g) was extracted with 10 mL of ethyl acetate (+ 10 g Na(2)SO(4)), cleaned by dispersive solid phase extraction on primary secondary amine (25 mg) and C(18) (25 mg), and measured by liquid chromatography tandem mass spectrometry. The limit of quantification was ≤0.0025 μg g(-1) for all the three fungicides, with calibration linearity in the concentration range of 0.001-0.025 μg mL(-1) (r(2) ≥ 0.999). The recoveries of each chemical were 75-110% at 0.0025, 0.005, and 0.010 μg g(-1) with intralaboratory Horwitz ratio <0.32 at 0.0025 μg g(-1). Variable matrix effects were recorded in different fruit parts viz rind, albedo, membrane, and arils, which could be correlated to their biochemical constituents as evidenced from accurate mass measurements on a Q-ToF LC-MS. The residues of carbendazim and difenoconazole were confined within the outer rind of pomegranate; however, azoxystrobin penetrated into the inner fruit parts. The dissipation of azoxystrobin, carbendazim, and difenoconazole followed first + first order kinetics at both standard and double doses, with preharvest intervals being 9, 60, and 26 days at standard dose. At double dose, the preharvest intervals extended to 20.5, 100, and 60 days, respectively.     

嘧霉胺替代多菌灵在柑橘贮藏过程中的降解及防效研究

多菌灵残留超标已成为影响我国农产品出口的一大制约因素,积极开展多菌灵安全用药研究,寻找多菌灵替代用药方案尤为必要.本研究针对柑橘罐头原料加工果分别开展了以多菌灵、嘧霉胺浸果后的农药残留动态和保鲜防效研究.研究结果显示,柑橘浸药处理后30d内的保鲜效果,嘧霉胺1 000倍液处理略好于多菌灵600倍液处理;嘧霉胺处理后快速...     

Monitoring residues of carbendazim (applied as benomyl) and thiabendazole in Wellspur apples

Residues of benomyl (determined as carbendazim, widely accepted as MBC) and of thiabendazole in Wellspur apples were monitored after the apples were treated postharvest by immersion in a bath with each of the fungicides separately and stored under refrigeration. Whole fruit and pulp analyses were conducted over the period beginning 1 day after treatment and lasting up to 200 days afterwards. Under the conditions described, both benomyl (expressed as parts per million of carbendazim) and thiabendazole were found in the fruits in the following average amounts: 0.44 +/- 0.03 and 0.11 +/- 0.03 ppm benomyl (as carbendazim) in whole fruit and in pulp, respectively; 0.98 +/- 0.10 and 0.39 +/- 0.10 ppm thiabendazole in whole fruit and in pulp, respectively.     

生物防治菌与多菌灵混用防治棉花黄萎病的效应研究

盆栽试验多菌灵与多种生物防治菌混用防治棉花黄萎病结果表明 ,大部分混用防治效果优于单用效果 ,而供试芽孢菌“B90 8”与多菌灵混用效果大于单用多菌灵但小于单用生物防治菌“B90 8”。     

Is the application of carbendazim harmful to healthy plants? Evidence of weak phytotoxicity in tobacco.

To understand the phytotoxic effects that certain bezimidazole fungicides exert on plant growth, the aim of the present study was to determine the effect of the fungicide carbendazim, on foliar biomass, pigment content, and nutrient levels in tobacco plants (Nicotiana tabacum L. cv. Tennessee 86). The fungicide applied was carbendazim with a purity of 100%, at three different rates: 1.3 mM (carb1), 2.6 mM (the recommended concentration, carb2), and 5.2 mM (carb3). The control treatment was without carbendazim. The application of dosages of this fungicide lower than recommended (1.3 mM) resulted, on the one hand, in greater dry weight and, on the other, higher carotenoid concentrations, as well as higher N and K concentrations with respect to control. On the contrary, the application of the carbendazim dosage higher than recommended (5.2 mM) caused a decrease in dry weight and in all of the foliar pigments, as well as all of the nutrients, with respect to the other dosages and control. These results appear to indicate that besides its direct antibiotic action against pathogens, the effects of this fungicide in plants could be dangerous, especially at higher dosages. Nevertheless, the negative effects of carbendazim can be avoided by reducing the amount applied in current agriculture.     

Toxicity of phenmedipham and carbendazim to Enchytraeus crypticus and Eisenia andrei (Oligochaeta) in Mediterranean soils

Purpose

The main objective of the present study was to evaluate the toxicity of two reference chemicals, Carbendazim and Phenmedipham, for the compostworm Eisenia andrei (effects of Carbendazim) and the potworm Enchytraeus crypticus (effects of Phenmedipham) in 12 Mediterranean soils with contrasting soil properties. The observed toxicity was also compared to that obtained for OECD standard soil, used as a control.

Materials and methods

The soils were selected to be representative for the Mediterranean region and to cover a broad range of soil properties. The evaluated endpoints were avoidance behavior and reproduction. Soils were also assembled in two groups according to their pedological properties.

Results and discussion

Toxicity benchmarks (AC50s) obtained for E. andrei avoidance behavior in carbendazim-contaminated soils were generally higher for sandy soils with low pH. The toxic effects on the reproduction of the compostworms were similar in the six tested soils, indicating a low influence of soil properties. The avoidance response of E. crypticus towards Phenmedipham was generally highly variable in all tested soils. Even though, a higher toxicity was observed for more acidic soils. The EC50s for reproduction of the latter species varied by a factor of 9 and Phenmedipham toxicity also tended to be increasing in soils with lower pH, except for the soils with extreme organic matter content (0.6 and 5.8%).

Conclusions

A soil effect on chemical toxicity was clearly confirmed, highlighting the influence that test soils can have in site-specific ecological risk assessment. Despite some relationships between soil properties and toxicity were outlined, a clear and statistically significant prediction of chemical toxicity could not be established. The range of soil properties was probably narrow to give clearer and more consistent insights on their influence. For the four groups of tests, the toxicity observed for OECD soil was either similar, lower, or generally higher if compared with Mediterranean soils. Moreover, it did represent neither the organic matter content found in Mediterranean soils nor their textural classes.     

多菌灵重复施药对其持久性及土壤微生物群落功能多样性的影响

选择浙江大学华家池校区大棚蔬菜试验田,研究了多菌灵(以active ingredient,a.i.计)0.94、1.88和4.70 kg hm-2三种剂量重复施药对其持久性及土壤微生物功能多样性的影响。结果表明,第1次施药后,多菌灵在土壤中的降解速率分别为0.11、0.25、0.74 mg kg-1d-1;第4次施药后,多菌灵在土壤中的降解速率分别达到0.18、0.39、1.00 mg kg-1d-1。随着施药次数的增加,多菌灵的降解速率逐渐增大。初次施药后,土壤微生物群落AWCD值明显低于对照,土壤微生物群落的Shannon、Simpson和McIntosh指数也均低于对照水平。第4次施药后,三个处理土壤微生物群落的AWCD值、Shannon、Simpson和McIntosh指数均恢复到对照水平。     

不同土壤湿度下多菌灵的连续施用对土壤氮矿化速率与脲酶动力学的影响

明确在不同土壤水分环境下农药多菌灵对土壤氮循环和生物学属性的影响,对于评价多菌灵对土壤氮循环与生态环境的影响有重要意义。研究了在不同土壤湿度下连续添加不同浓度的多菌灵对土壤氮矿化、几丁质酶活性、土壤脲酶动力学参数以及微生物生物量的影响。结果表明:多菌灵添加量对土壤铵态氮、硝态氮、几丁质酶活性和微生物生物量氮具有显著影响;土壤湿度和多菌灵添加量的交互作用对铵态氮、硝态氮、矿化速率、脲酶酶促反应初速度(Vmax/Km)值以及微生物生物量氮有显著影响。在60%土壤最大持水量(WHC)条件下,5 mg·kg-1多菌灵的连续添加显著增加了铵态氮含量;在不同土壤湿度条件下,25 mg·kg-1多菌灵的连续添加显著增加了硝态氮含量。在60%WHC条件下连续添加10 mg·kg-1多菌灵的处理矿化速率显著高于不添加多菌灵的处理,但在90%WHC条件下添加10和25 mg·kg-1多菌灵处理的矿化速率显著低于不添加多菌灵的处理。在不同土壤湿度条件下,几丁质酶活性随多菌灵施用量增加而降低。在60%WHC条件下连续添加25 mg·kg-1多菌灵处理的脲酶Vmax/Km值显著低于连续添加10 mg·kg-1多菌灵的处理。多菌灵添加量可以通过影响土壤矿质氮含量与生物学属性从而影响土壤脲酶动力学的参数。     

Microwave-assisted extraction for the simultaneous determination of thiamethoxam, imidacloprid, and carbendazim residues in fresh and cooked vegetable samples

Microwave-assisted extraction (MAE) was carried out for the simultaneous determination of the insecticides thiamethoxam [(EZ)-3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene(nitro)amine], imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine], and the fungicide carbendazim (methyl benzimidazol-2-ylcarbamate) in vegetable samples. Five crop samples consisting of cabbage, tomatoes, chilies, potatoes, and peppers were fortified with the three pesticides and subjected to MAE followed by cleanup to remove coextractives prior to analysis by high-performance liquid chromatography. Using the selected microwave exposure time and power setting, the recoveries of the three pesticides from the fortified vegetable samples ranged from 68.1 to 106%. The corresponding recoveries for samples processed simultaneously but without microwave exposure ranged from 37.2 to 61.4%. The recoveries by MAE were comparable to those obtained by the conventional blender extraction technique. The precision of the MAE method was demonstrated by relative standard deviations of <7% for the three pesticides. The cooked cabbage and tomato samples showed no breakdown of the parent compounds, and the recoveries of three pesticides were comparable to those obtained with the uncooked samples.