Optimization and Characterization of Nicosulfuron-Degrading Enzyme from Bacillus subtilis Strain YB1

A strain of Bacillus subtilis strain YB 1, isolated and preserved in our lab., showed a high nicosulfuron-degrading activity. Optimization of culture conditions on production of nicosulfuron-degrading enzyme from Bacillus subtilis strain YB 1 was carried out through mono-factor experiments. The characterization of degrading enzyme（s） was studied in this paper. The results showed that B. subtilis YB1 can use nicosulfuron as sole carbon source under aerobic condition. The key enzyme（s） involved in the initial biodegradation of nicosulfuron was localized to extracellular proteins and showed to be induced expressed. Enzyme-specific activity was up to 89.34 U mg-1 at pH 8.0 and 30℃, incubation for 96 h, inoculum 4.5x108 CFU mL-1 in Luria-Bertani liquid medium with nicosulfuron of 40 mg L-1. The maximum degradation rate of extracellular crude enzymes on nicosulfuron was 66% at pH 9.0, 35℃ in the enzymatic reaction system with nicosulfuron of 5 mg L-1. This degrading enzyme（s） was sensitive to high temperature, but kept high activity under alkaline conditions.

Genomic and transcriptomic insights into cytochrome P450 monooxygenase genes involved in nicosulfuron tolerance in maize(Zea mays L.)

Postemergence application of nicosulfuron for weed control in maize fields can cause great damage to certain maize inbred lines and hybrids. Two maize genotypes, tolerant inbred(HBR) and sensitive inbred(HBS), were found to significantly differ in their phenotypic responses to nicosulfuron, with the EC50(50% effective concentration) values differed statistically(763.6 and 5.9 g a.i. ha–1, respectively). Pre-treatment with malathion, a known cytochrome P450 inhibitor, increased nicosulfuron injury in both HBR and HBS. Our results support the hypothesis that nicosulfuron selectivity in maize is associated with cytochrome P450 metabolism. Further analysis of the maize genome resulted in the identification of 314 full length cytochrome P450 monooxygenase(CYP) genes. These genes were classified into 2 types, 10 clans and 44 families. The CYP71 clan was represented by all A-type genes(168) belonging to 17 families. Nine clans possessed 27 families containing 146 non-A-type genes. The consensus sequences of the heme-binding regions of A-type and non-A-type CYP proteins are ‘PFGXGRRXCPG’ and ‘FXXGPRXCXG’, respectively. Illumina transcriptome sequence results showed that there were 53 differentially expressed CYP genes on the basis of high variation in expression between HBS and HBR, nicosulfuron-treated and untreated samples. These genes may contribute to nicosulfuron tolerance in maize. A hierarchical clustering analysis obtained four main clusters named C1 to C4 in which 4, 15, 21, and 13 CYP genes were found in each respective cluster. The expression patterns of some CYP genes were confirmed by RT-q PCR analysis. The research will improve our understanding of the function of maize cytochrome P450 in herbicide metabolism.

Efficacy and Phytotoxicity Trial of Different Dosages of 4% Nicosulfuron SC on Weeds in Corn Fields

[Objective]The paper was to study efficacy and phytotoxicity of different dosages of 4% nicosulfuron on weeds in corn fields.[Method]With summer corn variety Zhengdan 958commonly used in field production in Hengshui area of Hebei Province of China as the test material,different dosages（1 050,1 500 and 1 950mL/hm2）of corn postemergence herbicide 4% nicosulfuron SC were sprayed during five-leaf stage of corn,and the treatments with water spraying and manual weeding were set as controls.The effects of different dosages of 4% nicosulfuron SC on weed control efficacy and safety and yield of corn were analyzed in order to determine the optimal dosage.[Result]Spraying 4% nicosulfuron SC with dosage of 1 500mL/hm2 during five-leaf stage of corn not only had better control effect against various weeds,but also did not produce phytotoxicity on corn,and the yield of corn was higher.[Conclusion]The study provided scientific information for using 4% nicosulfuron to eradicate weeds in corn fields.

Control Effect of 80% Nicosulfuron·Atrazine Water-dispersible Granules on Weeds in Spring Maize Field

[Objective] This study aimed to investigate the control effect of different concentrations of 80% nicosulfuron·atrazine water-dispersible granules on weeds in spring maize field. [Method] Maize field was sprayed with 300, 375, 450, 750 g/hm280% nicosulfuron·atrazine water-dispersible granules respectively, with 40 g/L nicosulfuron suspending concentrate and 38% atrazine suspending concentrate as control agents. Artificial weeding and control(CK) plots were set. [Result] Fresh weight control efficiency of 375-750 g/hm280% nicosulfuron·atrazine water-dispersible granules was significantly higher than that of 40 g/L nicosulfuron suspending concentrate and 38% atrazine suspending concentrate; no obvious phytotoxicity symptoms were observed after application of 300-450 g/hm280% nicosulfuron·atrazine waterdispersible granules; 750 g/hm280% nicosulfuron·atrazine water-dispersible granules posed certain impact on the growth of maize seedlings. Compared with control plots, various doses of 80% nicosulfuron·atrazine water-dispersible granules significantly improved the yield of maize. [Conclusion] In the present study, 375-450 g/hm280% nicosulfuron·atrazine water-dispersible granules exhibited high control effect on weeds in maize field and were safe for the growth of maize seedlings.

31%硝磺·烟嘧·特丁津可分散油悬浮剂防除玉米田一年生杂草药效试验

为明确31%硝磺·烟嘧·特丁津可分散油悬浮剂对玉米田杂草的防治效果、最佳田间使用剂量及对玉米的安全性,本研究采用苗后茎叶喷雾处理,进行田间药效试验。结果表明,31%硝磺·烟嘧·特丁津可分散油悬浮剂在418.5~465.0 g·hm^(-2)的有效成分用量下,施药15 d后对本氏蓼、反枝苋、稗草的株防效最高可达95.27%、93.65%和97.06%;施药30 d后对杂草鲜重防效分别达96.91%、96.02%和97.98%,均显著优于对照药剂;药后7和15 d对玉米药害等级均为1.0,未见对玉米产生明显药害,增产率相比于人工除草为-6.03%~-1.93%。综合用药成本及效果考虑推荐有效成分使用量为418.5~465.0 g·hm^(-2)。31%硝磺·烟嘧·特丁津可分散油悬浮剂能够有效防除玉米田杂草并对玉米具有较好的安全性。

Biodegradation of nicosulfuron by a novel Alcaligenes faecalis strain ZWS11

A bacterial strain ZWS11 was isolated from sulfonylurea herbicide-contaminated farmland soil and identified as a potential nicosulfuron-degrading bacterium. Based on morphological and physicochemical characterization of the bacterium and phylogenetic analysis of the 16 S r RNA sequence, strain ZWS11 was identified as Alcaligenes faecalis. The effects of the initial concentration of nicosulfuron, inoculation volume, and medium p H on degradation of nicosulfuron were investigated. Strain ZWS11 could degrade 80.56% of the initial nicosulfuron supplemented at 500.0 mg/L under the conditions of p H 7.0, 180 r/min and30°C after incubation for 6 days. Strain ZWS11 was also capable of degrading rimsulfuron,tribenuron-methyl and thifensulfuron-methyl. Four metabolites from biodegradation of nicosulfuron were identified, which were 2-aminosulfonyl-N, N-dimethylnicotinamide（M1）, 4, 6-dihydroxypyrimidine（M2）, 2-amino-4, 6-dimethoxypyrimidine（M3） and2-（1-（4,6-dimethoxy-pyrimidin-2-yl）-ureido）-N,N-dimethyl-nicotinamide（M4）. Among the metabolites detected, M2 was reported for the first time. Possible biodegradation pathways of nicosulfuron by strain ZWS11 were proposed. The degradation proceeded mainly via cleavage of the sulfonylurea bridge, O-dealkylation, and contraction of the sulfonylurea bridge by elimination of a sulfur dioxide group. The results provide valuable information for degradation of nicosulfuron in contaminated environments.

33%烟嘧磺隆·特丁津可分散油悬浮剂高效液相色谱分析

采用高效液相色谱法,以乙腈和冰乙酸水溶液(pH=3.0)为流动相,使用SinoChromODS-BP色谱柱和紫外可变波长检测器,在230 nm波长下对33%烟嘧磺隆·特丁津可分散油悬浮剂进行分离和定量分析。结果表明,在该分析方法下,烟嘧磺隆和特丁津的线性相关系数分别为0.9991和0.9998,标准偏差分别为0.037和0.044,变异系数分别为0.73%和0.16%,平均回收率分别为99.91%和99.70%,说明该方法可适用于该产品的质量控制要求。

喷雾助剂对烟嘧磺隆药液特性及减施除草活性的调节作用

为了评价喷雾助剂对除草剂减量施用下的增效调节作用,采用仪器分析和田间试验的方法研究了7种助剂对烟嘧磺隆的药液特性和除草活性的调节作用。结果表明,7种助剂均能显著降低药液的表面张力、接触角,增加药液铺展直径。杰效利对药液表面张力和接触角的降低作用和药液铺展直径的增加作用好于其他助剂,添加杰效利0.05%~0.2%后烟嘧磺隆药液的表面张力降低了41.52%~47.69%,接触角降低了49.87%~66.23%,铺展直径增加了59.94%~150.13%;渗透者、倍创和迈丝可以显著延长液滴干燥时间,与未添加助剂相比干燥时间分别增加了3.84%~36.69%、17.20%~23.57%、3.02%~18.01%;田间试验结果表明,渗透者和迈丝对烟嘧磺隆的增效作用好于其他助剂,能够降低烟嘧磺隆使用量达25%~40%,可以作为烟嘧磺隆的有效助剂选择使用。

土曲霉DT2菌株固体菌剂修复烟嘧磺隆污染土壤研究

为解决玉米田烟嘧磺隆残留导致下茬高粱药害的问题,本研究通过单因素优化方法制备DT2固体菌剂,并采用室内盆栽和大田试验相结合的方法,在土壤中添加烟嘧磺隆并施用菌剂DT2,确定了DT2固体菌剂对烟嘧磺隆残留土壤的修复效果和对高粱药害的缓解作用。研究结果表明,烟嘧磺隆降解菌土曲霉DT2固体发酵最佳条件为p H值7、接种量10%、发酵时间168 h,室温储藏90 d有效活菌数仍可达3.3×10^(8)cfu/g。室内盆栽试验结果表明,土壤中烟嘧磺隆浓度分别为0.023和0.046 mg/kg时,分别添加菌剂6.25和12.5 g/kg处理30 d后,可将烟嘧磺隆对高粱株高的抑制率由26.95%和49.23%分别降低至1.79%和2.24%;当土壤中烟嘧磺隆浓度为0.094 mg/kg时,添加菌剂25 g/kg处理40 d后,可将烟嘧磺隆对高粱株高的抑制率由51.39%降低至4.89%。当土壤中烟嘧磺隆浓度为0.046 mg/kg时,添加菌剂12.5 g/kg,药后10 d,土壤烟嘧磺隆残留率下降56.42%,与对照差异极显著;药后30 d,土壤中烟嘧磺隆质量为0;DT2菌剂可以改善土壤环境,增强土壤脲酶、碱性磷酸酶、过氧化氢酶等土壤酶活性和土壤细菌、真菌等微生物多样性;施用菌剂后40 d,不仅能够缓解烟嘧磺隆对高粱叶绿素含量、净光合速率和根系活力等各项生理指标的抑制,而且对高粱株高、根长和鲜重等有一定的促生作用。大田试验结果表明,添加DT2菌剂能够有效减轻土壤中烟嘧磺隆残留对高粱的药害,提高出苗率和产量,降低病株率和株高抑制率;当土壤中烟嘧磺隆施药量为20、30和40 g a.i/hm^(2)时,分别添加DT2菌剂300、450和600 kg/hm^(2)能够将烟嘧磺隆残留对高粱的抑制降低至无明显药害水平。综上,土曲霉DT2菌剂对土壤中烟嘧磺隆残留有较好的修复作用,具有潜在的应用前景。

分散固相萃取超高效液相色谱串联质谱法同时检测大豆植株及土壤中烟嘧磺隆、莠去津及其代谢物残留

土壤中烟嘧磺隆和莠去津的高残留往往会导致后茬大豆药害问题。本研究建立了一种分散固相萃取超高效液相色谱串联质谱法同时检测大豆植株及土壤中烟嘧磺隆、莠去津及其代谢物残留方法。样品经含2%甲酸的乙腈提取,经分散固相萃取净化,以乙腈和0.2%甲酸水为流动相,采用Poroshell 120 EC-C18色谱柱梯度洗脱,基质匹配标准曲线外标法定量分析。结果表明:在0.01、0.10 mg/kg和1.00 mg/kg添加水平下,烟嘧磺隆、莠去津及其代谢物的平均回收率为70%~113%,相对标准偏差为0.3%~11.8%,目标化合物质量浓度与对应的峰面积之间在0.001~1 mg/kg范围内线性关系良好,决定系数R^(2)≥0.9841,方法的定量限为0.01 mg/kg。该方法简便、快捷、准确,适用于土壤和幼苗期至鼓粒期大豆植株中烟嘧磺隆、莠去津及其代谢物的检测。本研究为烟嘧磺隆和莠去津的科学使用及后茬作物的安全种植提供有效监测方法。

不同质地土壤中烟嘧磺隆降解特征与烟草致害阈值研究

为明确不同质地土壤中烟嘧磺隆残留对烟草致害阈值及安全间隔期,从而为烟草前茬除草剂合理使用提供理论依据,在毕节砂壤土与黏壤土烟田开展除草剂药害田间试验,研究烟嘧磺隆在2种质地土壤中的降解特征、烟草药害阈值与安全间隔期及对土壤理化性质的影响。结果表明,烟嘧磺隆在砂壤土中降解半衰期为9.5~14.7 d,在黏壤土中降解半衰期为9.0~17.3 d;在砂壤土中致害阈值和田间安全间隔期分别为1.50μg/kg和70.6 d,在黏壤土中分别为2.47μg/kg和52.4 d;在施药后80 d,随烟嘧磺隆施用剂量(0到1倍)增加,土壤有机质含量变化不大,碱解氮含量呈先增加后降低趋势,土壤有效磷和速效钾含量则呈降低趋势,砂壤土和黏壤土的理化性状变化趋势一致但变化幅度不同,这与不同质地土壤中烟嘧磺隆的降解及与烟草间的互作影响有关。研究结果可为不同质地烟田土壤中烟嘧磺隆除草剂的合理使用及其残留降解防控提供重要参考。

烟嘧磺隆在土壤中的吸附及与土壤性质的相关性研究

采用平衡振荡法研究了烟嘧磺隆在8种不同类型土壤中的吸附,结果表明,其吸附过程均符合经典的Freundlich模型,最大吸附常数为6.891,最小吸附常数为0.798。根据土壤有机吸附常数和吸附自由能的大小对该除草剂的移动性能进行了评价,认为其在8种土壤中均以物理吸附为主,且具有中等或较高的移动性能。通过对吸附常数Kf与土壤有机质含量、粘土含量和pH值的关系进行分析,发现土壤有机质含量、粘土含量和pH值在吸附过程中均属支配因素,Kf与土壤有机质含量、粘土含量呈正相关,而与土壤pH值呈负相关。

麦谷宁生测方法及其对玉米的安全性研究

选用种植面积较大且抗逆性较强的玉米品种农大 10 8为指示植物 ,研究了麦谷宁的玉米沙培试验方法。结果表明 ,浸种催芽 ,播种后先盖覆沙量的一半后加药 6 0 m L,再盖沙至 2 cm厚 ,于 2 5℃黑暗培养 3d,可获得满意的结果。回归分析表明 ,麦谷宁浓度的对数与玉米主根长抑制率相对应的机率值成很好的线性关系 ,r2 均大于 0 .90。就大面积种植的 16个玉米品种对麦谷宁的敏感性测定结果表明 ,掖单 12对麦谷宁较敏感 ,农大 3138对麦谷宁耐药力最强 ,各品种对麦谷宁的 IC50 值均大于 7.38μg· kg-1。

不同品种玉米对烟嘧磺隆的耐药性研究进展

烟嘧磺隆是广谱灭生性、内吸传导型除草剂,广泛应用于玉米苗期杂草的控制。由于不同品种玉米对烟嘧磺隆的耐药性不同,因此部分品种玉米因其发生严重药害的问题屡见报道。文章从烟嘧磺隆的作用机理、不同品种玉米对烟嘧磺隆的耐药性鉴定以及玉米抗烟嘧磺隆的遗传机制等方面进行了综述,阐述了目前对玉米抗烟嘧磺隆基因的定位尚不明确,烟嘧磺隆在玉米体内代谢机制的研究还比较困难等问题,认为未来可通过对玉米抗烟嘧磺隆内源基因的发掘以及实现基因的最终转化来提高不同品种玉米的耐药性。

玉米田主要杂草对烟嘧磺隆的抗性

为明确我国玉米田杂草对烟嘧磺隆的抗性水平及分布现状,于2010—2011年自山东、吉林、四川、河北4省采集连续多年施用烟嘧磺隆的玉米田杂草种子样本121个,在温室内采用盆栽法测定了其对烟嘧磺隆的抗性。结果显示:4个杂草样本对烟嘧磺隆产生了抗性,其中山东淄博张店区傅家镇高家村的牛筋草抗性种群的GR50为25.76g/hm2,是敏感种群(1.33g/hm2)的19.37倍,已产生明显的抗药性;四川彭山县谢家镇岳油村的稗草、河北大城县广安镇夏屯村的虎尾草和河北邯郸的狗尾草分别产生了6.14、5.43和5.65倍的低水平抗性;其余杂草样本均无明显抗性。同一杂草不同采集地点的敏感样本对烟嘧磺隆的敏感性存在差异。

可降解水体中烟嘧磺隆微生物的分离与筛选

为了筛选出可降解烟嘧磺隆的微生物,利用富集培养技术从水样中分离得到了5株能以烟嘧磺隆为唯一氮源、碳源和能源的微生物,分别编号为YF1、YB1、YB2、YB3和YB4,其中YF1为真菌,其余均为细菌。将所得菌株定量接种于含不同浓度烟嘧磺隆的培养基中,于30℃、150r/min摇床上振荡培养5d后,利用HPLC检测烟嘧磺隆含量,计算降解率。结果发现:这些菌株对低浓度烟嘧磺隆的降解率显著高于对高浓度烟嘧磺隆的降解率,当培养基中烟嘧磺隆的含量为2mg/kg时,YF1对该药剂的降解率最高,为80.31%,其次是YB1和YB2,降解率分别为78.18%和73.72%,而YB3和YB4对药剂的降解率只有36.82%和25.75%。对3株降解率较高的菌株进行了初步鉴定,发现YF1为黑曲霉Aspergillus niger,YB1和YB2均为芽孢杆菌Bacillus sp.。

土壤中烟嘧磺隆和莠去津残留对甘薯的药害评价

为了明确玉米田除草剂烟嘧磺隆和莠去津土壤残留对后茬作物甘薯生长的影响,于2017年分别在河北省保定满城区、文安县、霸州市和石家庄藁城区采集甘薯田土壤样品,采用QuEChERS法,结合超高效液相色谱-串联质谱法(UPLC-MS/MS)测定了土壤中烟嘧磺隆和莠去津的残留量;通过盆栽试验,测定了烟嘧磺隆、莠去津以及二者混用对甘薯的药害作用。结果表明:在甘薯药害表现严重的河北保定满城区、文安县、霸州市3地甘薯田土壤中烟嘧磺隆的残留量在11.80~87.97μg/(kg土)之间,莠去津残留量在2.71~8.40μg/(kg土)之间;而在石家庄藁城区无药害症状甘薯田土壤中只检测到莠去津,含量在3.43~4.42μg/(kg土)之间。盆栽试验结果表明,栽后20 d每千克甘薯田土壤中烟嘧磺隆含量大于1μg时,甘薯产生药害症状;烟嘧磺隆对甘薯根系IC_(50)值为3.74μg/(kg土),IC_(10)值为1.27μg/(kg土)。土壤中莠去津含量在2~40μg/(kg土)范围内,对甘薯茎叶和根系没有可见药害症状,但对根系生长具有轻微抑制作用。烟嘧磺隆与莠去津按质量比10:1、5:1、5:2和5:5混用对甘薯药害有一定的拮抗作用,共毒系数CTC_(50)值分别为66.25、59.16、61.30和79.47。烟嘧磺隆对甘薯药害症状主要表现为植株矮小,叶片小,整株或心叶黄化,根系发育受阻,侧根短粗。生产上造成甘薯药害的诱因是土壤中残留的烟嘧磺隆,对甘薯药害阈值为1μg/(kg土)。

农作物对磺酰脲类除草剂耐性的研究

不同作物品种对磺酰脲类除草剂的耐性有一定差异。春小麦不同品种对绿磺隆的耐性差异很小，但在不同温度条件下有一定差异。大豆品种问对立磺隆耐性不同，荆山朴、东农７７－８０４，哈３７－６４３７和７９０２的耐性最强，绿秣食豆、东农３９和龙６８０４耐性中等，合丰２５，礼炮２１６和东农３３耐性较差。玉米不同类型对玉农乐耐性不同，其耐性顺序为硬粒型＞一般型＞普甜型＞爆裂型。

不同缓解处理对玉米烟嘧磺隆药害的缓解效果研究

以郑单958玉米杂交种为供试作物,研究了除草剂烟嘧磺隆对夏玉米的药害情况以及不同缓解剂对药害的缓解作用。结果表明,120g/hm2烟嘧磺隆对玉米生长产生了严重影响,株高下降8.69%,产量下降11.18%。定量喷施氨基酸、萘乙酸、奈安、丙酰芸苔素内酯均能改善烟嘧磺隆对玉米株高的抑制,效果相对较好;萘乙酸、胺鲜酯可缓解烟嘧磺隆对玉米叶绿素的影响;药害出现前、后喷施各种缓解剂均能缓解烟嘧磺隆对玉米丙二醛含量的影响;药害后期喷施宝叶可缓解烟嘧磺隆对玉米可溶性糖含量的影响。测产表明,烟嘧磺隆药害出现后喷施奈安的处理玉米产量最高,较120g/hm2烟嘧磺隆对照产量提高24.47%。综合分析,1.2g/hm2奈安对玉米烟嘧磺隆药害的缓解效果最好。

烟嘧磺隆的微生物降解研究进展

烟嘧磺隆属磺酰脲类除草剂,因其对玉米安全,对一年生和多年生禾本科杂草及部分阔叶杂草、莎草科杂草高效而在玉米田广泛应用,但其在土壤中的残留则易对后茬敏感作物产生药害。微生物对烟嘧磺隆的降解有望成为修复污染土壤的有效措施。文章从烟嘧磺隆的使用及危害,可降解烟嘧磺隆的微生物种类及降解特性、降解途径、降解酶及其编码基因等方面进行了综述。目前有关烟嘧磺隆微生物降解的研究多集中于其降解菌的筛选、降解特性及降解途径等方面,对于其微生物降解过程中起关键作用的酶和基因方面的研究仍较少,因此未来的研究趋势将主要体现在降解复合菌系的培养、降解微生物的环境生态学、降解基因以及降解酶制剂等方面。