除草剂降解真菌对氯嘧磺隆的降解作用

利用自行筛选和保存的3种高效降解真菌菌株,对氯嘧磺隆除草剂降解作用的影响进行了系统的研究,采用高效液相色谱法测定其降解率。真菌黑曲霉、黄曲霉、F8酿酒酵母均可以很好的降解氯嘧磺隆,其中黑曲霉的降解率最高,为96.52%;其次是黄曲霉为88.21%。混合菌株对氯嘧磺隆的降解效率均高于其单一菌株,真菌黑曲霉与黄曲霉混合菌株降解率最高,为98.74%;其次是真菌黑曲霉与F8混合菌株为98.22%。在培养基中随着氯嘧磺隆降解率的增加,反应液的pH逐渐降低。     

一株高效氯嘧磺隆降解菌的分离鉴定及其降解机理初探

消除农业生产中除草剂残留是现代农业绿色发展的关键,为发掘更多磺酰脲类除草剂高效降解微生物资源,系统阐述氯嘧磺隆的微生物降解途径。以氯嘧磺隆为唯一氮源,从某磺酰脲类除草剂生产厂废水处理活性污泥中分离出的高效氯嘧磺隆降解菌LAM2021。生理生化特性和16S rRNA基因序列比对分析结果表明,该菌株属小坂菌属(Kosakonia sp.)。单因素试验结果经RSM优化后,菌株LAM2021在无机盐培养基中对50 mg/L氯嘧磺隆的最佳降解条件为:接种量5%、培养温度30℃、pH 6.0,11 h后降解率可达94.6%。利用高效液相色谱(HPLC)对接菌后降解体系内代谢产生的酸类物质进行分离与鉴定,结果表明产物主要为柠檬酸。推测菌株LAM2021受高浓度氯嘧磺隆胁迫,利用葡萄糖产生柠檬酸,通过降低环境pH使氯嘧磺隆发生水解,从而解除其胁迫。从菌株全基因组信息中发现有参与氯嘧磺隆降解的酯酶编码基因。采用液相色谱-质谱联用(LC-MS)对菌株LAM2021与氯嘧磺隆混合培养后的代谢产物进行检测与鉴定,共有5种主要物质被检测到。根据氯嘧磺隆的化学结构式和中间代谢产物特征,推测菌株LAM2021降解氯嘧磺隆的代谢途径为:氯嘧磺隆中的脲桥先水解断裂成2-氨基-4-氯-6-甲基嘧啶和邻甲酸乙酯苯磺酰胺,随后酯键被脱酯断裂成邻甲酸乙酯苯磺酰胺,最后环化为N-醛基糖精。     

生物肥对氯嘧磺隆残留的降解及对水稻生长的影响

为筛选具有降解氯嘧磺隆残留和作物增产效果的优质生物肥, 采用田间小区模拟试验, 研究了不同浓度氯嘧磺隆施用下, 3种生物肥对氯嘧磺隆残留的降解及对水稻生长的影响。结果表明, 供试的农大生物菌剂、世绿环保生物肥和农大生物肥对施用量分别为3 μg·kg^-1和6 μg·kg^-1的氯嘧磺隆残留均有降解作用, 对施用量为6 μg·kg^-1的氯嘧磺隆残留的降解率分别为61.5%、58.3%和62.7%, 比自然降解率分别高17.8%、14.6%和19.0%。土壤中氯嘧磺隆施用量为0.03 μg·kg^-1时即对水稻株高、穗长、结实率和产量性状产生明显影响, 氯嘧磺隆施用量越大药害影响越显著。3种供试生物肥均可解除0.03 μg·kg^-1氯嘧磺隆的残留对水稻产量的影响, 但随氯嘧磺隆施用量的增加, 供试生物肥虽可减轻药害影响, 但不能完全解除残留药害。农大生物菌剂和农大生物肥解除氯嘧磺隆残留药害能力高于世绿环保生物肥。田间氯嘧磺隆的施用量小于0.03 μg·kg^-1时, 施用供试的3种生物肥均可保证水稻生产安全。     

苯磺隆降解菌的分离鉴定和特性研究

为解决苯磺隆残留问题,本试验利用富集驯化培养分离法,从连续多年使用苯磺隆的田间土壤中,分离筛出一株能以苯磺隆为唯一碳源、氮源生长的降解菌。通过形态学、生理生化测定及16S r DNA序列系统发育分析,鉴定该菌株为产碱杆菌。抗生素敏感试验和底物敏感试验表明:降解菌株BHL对试验浓度范围内的所有供试抗生素都表现为敏感,其中对硫酸阿米卡星最为敏感;菌株BHL可以很好的利用试验所用磺酰脲类除草剂和有机磷农药,同时还可以利用部分芳香族化合物。本研究为功能农药残留降解菌的研究提供了理论基础。     

氯嘧磺隆降解菌筛选及基于16S rDNA序列的系统学分析

氯嘧磺隆是长残留除草剂,污染土壤后影响土壤肥力和后茬作物生长。筛选氯嘧磺隆降解菌21株,纯培养条件下,7 d对初始浓度50 mg/L氯嘧磺隆的降解达到0.75%~80.77%。对筛选到的氯嘧磺隆降解菌进行了16S rDNA基因扩增、序列测定和系统学分析,结果显示,所选菌株在系统发育地位上分别属于肠杆菌、短杆菌、柠檬酸杆菌、志贺氏菌、寡养单胞菌、无色杆菌、假单胞菌等7个属。     

氯嘧磺隆对土壤微生物类群及土壤呼吸强度的影响

通过实验室培养试验研究了氯嘧磺隆对土壤微生物种群动态变化和土壤呼吸强度的影响。测定了0、5、10、20、100μg kg-1的氯嘧磺隆对土壤中三大类微生物种群动态变化的影响,结果表明,氯嘧磺隆促进土壤细菌和真菌的生长,抑制放线菌的生长,真菌是氯嘧磺隆胁迫下的优势菌群。密闭法测定了0.01、0.1、1、10、100μg g-1的氯嘧磺隆对土壤呼吸的影响,结果表明,0.01、0.1、1、10μg g-1的氯嘧磺隆轻微抑制土壤呼吸,但受抑制的土壤逐渐恢复,除10μg g-1外,其它处理可恢复到对照水平,高浓度100μg g-1的氯嘧磺隆促进土壤呼吸,不能恢复到对照水平,但根据危害系数法的分级方法计算,氯嘧磺隆属于无实际毒害的农药。     

氯嘧磺隆对土壤微生物数量,酶活性及呼吸强度的影响

氯嘧磺隆是一类高效广谱除草剂,研究其对土壤微生物的影响对其合理使用具有重要意义。通过室内瓶培养实验研究了不同浓度氯嘧磺隆对土壤呼吸强度、土壤微生物数量和土壤酶活性的影响。结果表明:氯嘧磺隆施入土壤2周后,可明显提高土壤呼吸强度,且浓度越大,提高效果越明显;氯嘧磺隆施入土壤1周后,不同浓度氯嘧磺隆处理均可提高土壤细菌和放线菌数量,45 d时表现出抑制作用,60 d可恢复到对照水平;氯嘧磺隆对土壤真菌数量表现为抑制作用,而后恢复到对照水平,45 d时田间施用量可抑制土壤真菌的增殖,但60 d可恢复到对照水平。土壤培养前1周,氯嘧磺对土壤过氧化氢酶、土壤转化酶、土壤脲酶活性均表现出不同程度的抑制作用,且浓度越大抑制作用越明显。之后三种酶表现出一定的激活作用,培养60 d可恢复到对照水平。以上试验结果可为合理施用氯嘧磺隆提供理论依据。     

N+注入选育漆酶高产菌株及其产酶优化研究

以糙皮侧耳Pleurotus ostreatus WY01为出发菌株,通过N+注入诱变处理担孢子、RBBR-PDA平板变色法初筛、ATBS法测定漆酶活性复筛,获得1株漆酶高产诱变菌株ADW-08。用高碳低氮无机盐制备的油菜秸固体培养基（SM）培养,其峰值酶活力比出发菌株高出2.80倍,达7.78 U/g,且产酶稳定。对ADW-08固体培养产酶条件的研究表明,以葡萄糖为碳源明显优于蔗糖、麦芽糖、麸皮和可溶性淀粉;酒石酸铵较有利于ADW-08漆酶的分泌;适宜初始pH为5.0或6.0;ABTS和藜芦醇对产酶均有明显的诱导作用,而吐温-80对产酶有一定的抑制作用;正交试验结果表明,葡萄糖、酒石酸铵和pH最佳参数分别为15.0g/L、0.2g/L和5.2,酶活峰值为8.33 U/g。     

除草剂配施安全剂对土壤酶活性与糜子根系生理代谢的影响

为探究安全剂与除草剂复配施用对除草剂药害的缓解以及对糜子田土壤酶活性、根系活性氧代谢和糜子生长发育的影响。于2019年5～10月在陕西省榆林市小杂粮试验示范站进行试验,选用糜子品种''榆糜2号''为试验材料,选取苗前除草剂：谷友（10%单嘧磺隆,2.4 kg/hm2）与3种安全剂：芸苔素（300 mL/hm2）、赤霉素（300 mL/hm2）、奈安（1.2 kg/hm2）复配使用,设置清水不除草对照与人工除草对照。测定杂草鲜重防效、药害指数、根际土壤酶活性、糜子根系抗氧化酶活性与丙二醛含量及糜子农艺性状与产量。结果表明：1）芸苔素、赤霉素、奈安与单嘧磺隆复配后药害指数显著下降,分别降低了20.27%、21.63%、20.94%;2）单嘧磺隆处理（H1）的土壤蔗糖酶、脲酶活性在施药后7～42 d时相较于处理CK1被显著抑制,活性抑制率随时间降低,添加安全剂后土壤蔗糖酶和脲酶活性显著提升,抑制率显著降低;3）与单施除草剂相比,安全剂复配施用的糜子根系SOD活性在施药后14～28 d时显著升高;CAT活性显著降低;MDA含量明显降低;4）单嘧磺隆导致糜子株高、穗长、主穗质量降低,安全剂复配施用减少了除草剂的不利影响,其中单嘧磺隆与赤霉素复配施用产量最高,达5 035 kg/hm2,较CK1增产1 114 kg/hm2,较单施单嘧磺隆处理的增产1 061 kg/hm2。安全剂与单嘧磺隆复配使用能延缓单嘧磺隆对糜子根际土壤酶活性的抑制,提高土壤蔗糖酶、脲酶活性,提高糜子根系SOD活性,减缓了药害胁迫造成的膜脂过氧化作用,减少细胞膜系统的损伤,延缓了根系衰老进程,提高了糜子的株高、茎粗、穗长、主穗质量及产量。其中赤霉素与单嘧磺隆复配使用在缓解除草剂药害,促进糜子增产方面效果较好。     

保护剂减轻氯嘧磺隆对玉米残留药害的作用机理

采用沙培方法,利用生理生化试验及半定量PCR方法,研究了保护剂HN对玉米ALS、GSTs活性及GSTs的mRNA表达量的影响。结果表明,保护剂对6个玉米品种的ALS、GSTs均具有诱导作用,但对不同品种的诱导增加百分率却不同。对氯嘧磺隆敏感的玉米东甜3号的GSTs诱导增加百分率达到59.18,对其ALS的诱导倍数为叶部1.29倍,根部2.30倍;对氯嘧磺隆耐性较高的玉米屯玉88GSTs诱导增加百分率为19.26,其ALS的诱导倍数为叶部1.07倍,根部2.07倍。氯嘧磺隆和保护剂均可以诱导玉米GSTsmRNA的相对表达量的增加,保护剂的诱导作用更强。     

Carbohydrate use and assimilation by litter and soil fungi assessed by carbon isotopes and BIOLOG assays

Soil fungi are integral to decomposition in forests and other habitats, yet identifying probable functional roles of different taxa is problematic. Here, we compared carbohydrate assimilation patterns derived from stable isotope analyses on cultures with patterns of metabolic activity measured on Biolog^® SF-P plates for 12 taxa of soil- and litter-inhabiting saprotrophic fungi isolated from Douglas-fir (Pseudotsuga menziesii) ecosystems. To determine the relative assimilation of carbon from malt extract versus sucrose by ¹³C stable isotope analyses, we cultured fungi with malt extract (consisting primarily of glucose and maltose) plus either C₃- or C₄-derived sucrose as carbon sources. Rhodotorula graminis and F. oxysporum assimilated the highest proportion of sucrose, a Mortierella isolate and an unidentified sterile isolate (FPC 341) assimilated the lowest proportion of sucrose, and remaining cultures assimilated similar and intermediate proportions of sucrose. On Biolog plates, low metabolic activity of Mortierella and FPC 341 on sucrose and R. graminis and F. oxysporum on maltose were qualitatively consistent with isotopic results. Assimilation of sucrose calculated isotopically was correlated with the ratio of sucrose: maltose activity calculated from Biolog assays (r²=0.45, P=0.0145, n=12). Metabolic activity on Biolog plates for six other common soil carbohydrates were also determined: glucose, fructose, galactose, cellobiose, lactose, and glycogen. Metabolic activity was greatest overall on maltose and glucose and lowest on fructose. Two of the isolates (Aspergillus flavus and F. oxysporum) had higher metabolic activity on the glucose-containing disaccharide cellobiose than on glucose, strongly suggesting preferential uptake of cellobiose compared to glucose and suggesting the potential ability to use cellulose. The high metabolic activity of these cultures on galactose, a primary constituent of hemicellulose, also suggested cellulolytic capabilities. Our results indicated that stable isotope studies and Biolog assays may provide complementary information to characterize metabolic potential of fungi in forest litter and soil.     

一种降解秸秆的丝状真菌的分离和定性方法

建立了一种新的简单的分离秸秆降解丝状真菌的方法,用此方法从东北寒地黑土中分离出真菌56株,其中丝状真菌46株。这种分离方法简述为：先用土壤或者堆肥浸渍秸秆至接近腐烂,再用酒精对接近腐烂秸秆进行表面灭菌,最后用土豆葡萄糖琼脂培养基培养并分离秸秆中微生物,这样分离的微生物大多为产丝真菌。用添加或者不添加硝酸铵、酵母粉和黑土浸液等辅助物的秸秆粉制成不同培养基,这些培养基用来比较其对分离的微生物的生长的影响。结果表明,同一微生物在不同培养基中生长区别很小,同一培养基不同微生物生长区别较大。选择了只含秸秆粉而不添加辅助物的培养基作为微生物是否降解秸秆的定性培养基,对分离的56株真菌以水稻、大豆和玉米3种秸秆进行降解定性鉴定,结果显示：该方法筛选的46株丝状真菌都能降解3种供试秸秆。     

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。     

阿特拉津降解菌ADH-2的分离、鉴定及其特性研究

从长期施用阿特拉津的玉米地中采集土样,通过富集培养的方法分离出一株能以阿特拉津为唯一碳、氮源生长的细菌ADH-2,结合生理生化特陛及16SrRNA基因的相似性分析将其初步鉴定为节杆菌属（Arthrobacter sp．）。该菌在10h内对100mg·L-1阿特拉津的降解率为99．9％。外加氮源能促进菌株的生长,但对阿特拉津的降解有轻微的抑制作用。外加蔗糖和葡萄糖能显著促进菌株的生长,但对阿特拉津的降解表现出显著的抑制。而淀粉既能促进菌株的生长又能促进阿特拉津的降解。对其降解基因的初步研究显示,该菌含有trzN、atzB和atzC3个阿特拉津降解相关基因。通过与本实验室另外两株阿特拉津降解菌比较,菌株ADH-2具有更好的应用潜力。     

Degradation of cyhalofop-butyl (CyB) by Pseudomonas azotoformans strain QDZ-1 and cloning of a novel gene encoding CyB-hydrolyzing esterase

Cyhalofop-butyl (CyB) is a widely used aryloxyphenoxy propanoate (AOPP) herbicide for control of grasses in rice fields. Five CyB-degrading strains were isolated from rice field soil and identified as Agromyces sp., Stenotrophomonas sp., Aquamicrobium sp., Microbacterium sp., and Pseudomonas azotoformans; the results revealed high biodiversity of CyB-degrading bacteria in rice soil. One strain, P. azotoformans QDZ-1, degraded 84.5% of 100 mg L(-1) CyB in 5 days of incubation in a flask and utilized CyB as carbon source for growth. Strain QDZ-1 could also degrade a wide range of other AOPP herbicides. An esterase gene, chbH, which hydrolyzes CyB to cyhalofop acid (CyA), was cloned from strain QDZ-1 and functionally expressed. A chbH-disrupted mutant dchbH was constructed by insertion mutation. Mutant dchbH could not degrade and utilize CyB, suggesting that chbH was the only esterase gene responsible for CyB degradation in strain QDZ-1. ChbH hydrolyzed all AOPP herbicides tested as well as permethrin. The catalytic efficiency of ChbH toward different AOPP herbicides followed the order quizalofop-P-ethyl ≈ fenoxaprop-P-ethyl > CyB ≈ fluazifop-P-butyl > diclofop-methyl ≈ haloxyfop-P-methyl; the results indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicides, whereas the substitutions in the aromatic ring had only a slight influence.     

A comparison of three atrazine-degrading bacteria for soil bioremediation

The ability of three atrazine-degrading bacteria, Pseudomonas sp. strain ADP, a Pseudaminobacter sp., and a Nocardioides sp., to degrade and mineralize this herbicide in a loam soil was evaluated in laboratory microcosms. These bacteria all hydrolytically dechlorinate atrazine, and degrade atrazine in pure culture with comparable specific activities. The Pseudaminobacter and Nocardioides can utilize atrazine as sole carbon and nitrogen source, whereas the Pseudomonas can utilize the compound only as a nitrogen source. The Pseudomonas and Pseudaminobacter mineralize the compound; the end product of atrazine metabolism by the Nocardioides is N-ethylammelide. At inoculum densities of 10⁵ cells/g soil, only the Pseudaminobacter and Nocardioides accelerated atrazine dissipation. The Pseudaminobacter mineralized atrazine rapidly and without a lag, whereas atrazine was mineralized in the Nocardioides-inoculated soil but only after a lag of several days. The Pseudaminobacter remained viable longer than did the Pseudomonas in soil. PCR analysis of recovered bacteria indicated that the genes atzA (atrazine chlorohydrolase) and atzB (hydroxyatrazine ethylaminohydrolase) were less stable in the Pseudaminobacter than the Pseudomonas. In summary, this study has revealed important differences in the ability of atrazine-hydrolyzing bacteria to degrade this compound in soil, and suggests that the ability to utilize atrazine as a carbon source is important to establish "enhanced degradation" by ecologically meaningful inoculum densities.     

二(2-乙基己基)邻苯二甲酸酯降解菌的分离和鉴定及其在污染土壤生物修复中的作用

Di-（2-ethylhexyl） phthalate（DEHP） is a high-molecular-weight phthalate ester（PAE） that has been widely used in the manufacture of polyvinylchloride and contributes to environmental pollution.The objectives of the present study were to isolate a DEHP degrader that can utilize DEHP as a carbon source and to investigate its capacity to biodegrade DEHP in both liquid culture and soil.A bacterial strain WJ4 was isolated from an intensively managed vegetable soil,which was contaminated with PAEs.The strain WJ4 was affiliated to the genus Rhodococcus and was able to remove DEHP from soil effectively.A period of only 7 d was required to degrade about 96.4%of DEHP(200 mg L^-1) in the liquid culture,and more than 55%of DEHP(1.0 g kg^-1) in the artificially contaminated soil was removed within 21 d.Furthermore,Rhodococcus sp.strain WJ4 had a strong ability to degrade DEHP without additional nutrients in liquid minimal medium culture and DEHP-contaminated soil and to degrade the homologue of DEHP in both liquid culture and soil.Strain WJ4 represents a novel tool for removing PAEs from contaminated soils and it may have great potential for application in the remediation of environmental pollution by PAEs.     

Productivity and some properties of immunoglobulin specific against Streptococcus mutans serotype c in chicken egg yolk (IgY)

Hens were immunized on thighs by using whole cells of Streptococcus mutans MT8148 serotype c strain as antigen through intramuscular (im) and subcutaneous (sc) routes to investigate the difference of immunization reactions and the changes in yolk antibody activities against antigen after initial immunization. Several properties of crude IgY were examined to evaluate the stability during food processing. Results showed that the specificity of IgY of im treated hens was nearly 10 times as high as those of sc treated antibody. IgY from the hens immunized with the serotype c strain showed significant cross-reactions against serotypes e and f, while minor reactions against serotypes a, b, d, and g were observed. In thermal stability tests, IgY activity in both yolk and crude IgY decreased with the increasing temperature, from 70 to 80 degrees C, but the thermal denaturation rates between those two samples were not significantly different. The addition of high levels sucrose, maltose, glycerol, or 2% glycine displayed effective protection against thermal denaturation of IgY. Lyophilized yolk-5% gum arabic powder showed better stability against proteases.