Penoxsulam‐resistant barnyardgrass (Echinochloa crus‐galli) in rice fields in China

Barnyardgrass (Echinochloa crus‐galli) proliferation seriously threatens rice production worldwide. Whole‐plant bioassays were conducted in order to test the sensitivity to penoxsulam of 52 barnyardgrass populations and the resistance of six penoxsulam‐resistant populations to 12 other herbicides that are commonly used in rice fields. Among the 48 populations that had escaped penoxsulam control in the rice fields, 8.3% showed a very high level of resistance, 58.3% showed a high level of resistance and 10.4% showed a moderate level of resistance. Multiple resistance was confirmed in all six penoxsulam‐resistant populations that were tested further. They exhibited at least a moderate level of resistance; that is, to 6–10 of the total of 13 herbicides that was tested. Most of the six penoxsulam‐resistant populations showed at least a moderate level of resistance to bispyribac‐sodium, quinclorac, metamifop, cyhalofop‐butyl and oxadiazon, three populations held at least a moderate level of resistance to oxyfluorfen and pretilachlor, two populations also held at least a moderate level of resistance to pyrazosulfuron‐ethyl, pyribenzoxim and fenoxaprop‐P‐ethyl, but the resistance indices of the six populations to pendimethalin were all low. This study has confirmed resistance to pretilachlor and oxadiazon in weeds for the first time.     

Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase

The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.     

Expression of human protoporphyrinogen oxidase in transgenic rice induces both a photodynamic response and oxyfluorfen resistance

A human protoporphyrinogen oxidase (Protox) coding sequence under the control of a ubiquitin promoter was introduced into rice to determine whether transgenic rice overexpressing the human Protox gene exhibits resistance against a peroxidizing herbicide. The transgenic rice lines (H3, H4, H5, H6, H9, and H10) transcribed the human Protox mRNA, whereas hybridizing RNA band was not detected in wild-type rice, indicating that the human Protox gene had been successfully transmitted into transgenic rice plants. The transgenic lines H9 and H10 showed growth retardation and light-dependent formation of necrotic lesions. Compared with wild-type rice plants, rice with a human Protox gene had increased Protox activity and content of the photosensitizer protoporphyrin IX, and reduced chlorophyll. The photosynthetic efficiency in lines H9 and H10, as indicated by F_v/F_m, was not different from that of wild type. The two transgenic lines had decreased levels of antheraxanthin, lutein, and β-carotene and similar levels of neoxanthin and violaxanthin as compared with wild-type plants. The staining activities of catalase, peroxidase, superoxide dismutase, and glutathione reductase were higher in transgenic lines than in wild type. Line H9 germinated in the presence of 20 μM oxyfluorfen, whereas 2 μM oxyfluorfen inhibited the germination of wild-type seeds. Thus, the transgenic rice plants exhibited enhanced resistance to oxyfluorfen.     

9种除草剂对花生白绢病菌的影响

在实验室条件下测定了氟乐灵、乙草胺、异丙甲草胺、异恶草酮、乳氟禾草灵、乙氧氟草醚、三氟羧草醚、恶草酮、二甲戊乐灵等9种除草剂对花生白绢病菌Sclerotium rolfsi Sacc.的影响.结果表明:9种除草剂对花生白绢病病菌的毒力有较大差异,三氟羧草醚和乙氧氟草醚的毒力较高,IC50分别为7.88mg·L-1和18.91mg·L-1;9种除草剂对菌丝干重均有抑制作用,且随剂量的升高而升高,乙氧氟草醚和三氟羧草醚抑制作用最明显,在100mg·L-1和50mg·L-1时抑制率均达90%以上;除乙草胺和异丙甲草胺部分剂量外,其他除草剂对菌核数量均有不同程度的抑制作用,三氟羧草醚作用最为明显,在供试剂量下抑制率均达96%以上;除乙草胺、氟乐灵在供试剂量下对菌核单重有抑制作用外,其他除草剂在多数剂量下对菌核单重均有刺激作用,三氟羧草醚在50mg·L-1时,是对照菌核单重的8.34倍;而各种除草剂在供试剂量下,对菌核萌发均没有影响.     

A tobacco plastidal transit sequence cannot override the dual targeting capacity of Myxococcus xanthus protoporphyrinogen oxidase in transgenic rice

The effect of a plastidal transit sequence in Myxococcus xanthus protoporphyrinogen oxidase (Protox) on gene targeting ability was investigated by generating transgenic rice that overexpressed M. xanthus Protox with the additional plastidal transit sequence (TTS line). In transgenic lines TTS3 and TTS4, the Protox antibody cross-reacted with the mature M. xanthus Protox protein of 50 kDa. In an in vitro import system using the M. xanthus Protox gene with the plastidal transit sequence, M. xanthus protein was detected in both chloroplasts and mitochondria, confirming that it was targeted into both organelles, as in transgenic rice line, M4, that overexpressed M. xanthus Protox lacking the plastidal transit sequence. A prominent increase in chloroplastic and mitochondrial Protox activity was observed in TTS3 and TTS4 relative to the wild type. However, the increase was lower than that in transgenic line M4. Seeds from all transgenic lines (TTS3, TTS4, and M4) were able to germinate when treated with up to 500 μM of the Protox-inhibiting herbicide, oxyfluorfen, whereas seeds from the wild type failed to germinate even when treated at levels as low as 1 μM. After foliar application of oxyfluorfen, TTS3 and TTS4 exhibited a reduced Protox activity, however, it was much greater than uninhibited Protox activity of wild type. The great increase in conductivity was followed by the great accumulation of photodynamic protoporphyrin IX only in oxyfluorfen-treated wild-type plants, not in oxyfluorfen-treated TTS lines. The presence of the plastidal transit sequence neither excludes the intrinsic ability of subcellular translocation of M. xanthus Protox nor changes herbicide resistance in TTS lines.     

Use of Myxococcus xanthus protoporphyrinogen oxidase as a selectable marker for transformation of rice

Protoporphyrinogen oxidase (PPO) is the target enzyme of peroxidizing herbicides. The overexpression of Myxococcus xanthus PPO (Mx PPO) confers a high level of herbicide resistance in rice. Among the peroxidizing herbicides, butafenacil has an efficiency ∼1000-fold that of oxadiazon, as judged by calli susceptibility tests upon herbicide treatment. Butafenacil (0.1 μM) was used to select transgenic rice plants expressing Mx PPO under the control of the constitutive maize ubiquitin promoter. The ectopic expression of the Mx PPO transgene was investigated in the T₀ generation by Northern blot and Western blot analysis. The T₀ transgenic plants expressing the Mx PPO gene were resistant to butafenacil based on in vitro leaf disk and in vivo foliar spray tests.     

我国水稻田杂草抗药性研究进展

水稻是我国最主要的粮食作物之一,杂草的危害严重影响了水稻的产量与品质。化学防除仍然是治理水稻田杂草最有效的途径。目前我国水稻田稗属杂草、千金子、马唐、雨久花、野慈姑、异型莎草、耳叶水苋、眼子菜、节节菜、萤蔺等多种杂草对二氯喹啉酸、五氟磺草胺、氰氟草酯、噁唑酰草胺、苄嘧磺隆、吡嘧磺隆、双草醚、噁草酮、乙氧氟草醚等多种常用除草剂产生了抗药性。面对日趋严重的水稻田抗药性杂草的危害,对抗药性杂草进行深入系统的研究以达到科学防治的目的显得尤为重要。本文总结了我国水稻田抗药性杂草的抗药性水平、靶标酶抗药性机理、代谢酶抗药性机理和其他抗药性机理,归纳了抗药性杂草的交互抗性、多抗性发生情况以及抗药性治理现状,分析了我们在杂草抗药性研究与治理方面面临的问题。     

Studies into the action of the diphenyl ether herbicides acifluorfen and oxyfluorfen. Part II: The interaction with photosynthetic electron transport reactions

The effects of acifluorfen and oxyfluorfen on photosynthetic electron transport reactions of pea chloroplasts were compared with those induced by paraquat and monuron. Monuron inhibited electron flow between photosystems I and II, and paraquat acted as an electron acceptor for photosystem I, promoting superoxide formation by illuminated chloroplasts. Neither acifluorfen nor oxyfluorfen at concentrations up to 50 μM affected non-cyclic electron flow or promoted superoxide formation. Both herbicides were shown to repress ferredoxin-dependent NADP⁺ reduction by illuminated chloroplasts. Further experiments showed that, in the presence of ferredoxin-NADP⁺ reductase and chloroplast membranes maintained in the dark, p-nitro diphenyl ether (DPE) herbicides promoted the rate of ferredoxin-dependent oxidation of NADPH, implying that these herbicides can accept electrons from reduced ferredoxin. The interaction between acifluorfen, ferredoxin and chloroplast membranes was examined further by following the effect of this herbicide on the peroxidation of illuminated thylakoids. Lipid peroxidation was promoted by acifluorfen, although this effect was abolished if thylakoids were washed prior to use. The effect of washing could be reversed by adding exogenous ferredoxin. These data demonstrate that interaction of DPE herbicides with photosynthetic electron transport in the vicinity of ferredoxin is necessary for light-dependent herbicide activation.     

QuEChERS-高效液相色谱-串联四极杆飞行时间质谱法同时测定大米中7种除草剂残留

建立了同时测定大米中西草净、异丙隆、苄嘧磺隆、莎稗磷、乙氧氟草醚、草酮和二甲戊灵7种除草剂残留的QuEChERS-高效液相色谱-串联四极杆飞行时间质谱 (HPLC-MS/MS Triple TOF) 方法。样品经V (乙酸乙酯) : V (水) = 80 : 20混合溶液提取,以N-丙基乙二胺 (PSA) 为吸附剂进行基质分散萃取净化,采用HPLC-MS/MS Triple TOF检测,外标法定量。结果表明:7种除草剂在一定的质量浓度范围内,其峰面积与对应的质量浓度间线性关系良好,相关系数均大于0.99;西草净、异丙隆、苄嘧磺隆和莎稗磷在0.001、0.01、0.05 mg/kg 3个添加水平下的平均回收率在75%~108%之间,相对标准偏差 (RSD) 在1.1%~7.0% (n = 5) 之间,定量限 (LOQ) 均为0.001 mg/kg;乙氧氟草醚、草酮和二甲戊灵在0.01、0.05和1 mg/kg 3个添加水平下的平均回收率在81%~108%之间,RSD在2.7%~9.0%之间,LOQ均为0.01 mg/kg。该方法样品前处理简单快速,方法灵敏度、准确度和精密度均符合农药残留检测要求,可用于大米中西草净、异丙隆、苄嘧磺隆、莎稗磷、乙氧氟草醚、草酮和二甲戊灵残留的同时检测。     

气相色谱-串联质谱法检测乙氧氟草醚、唑草酮和乙螨唑在4种香辛料中的残留

建立了气相色谱-三重四极杆串联质谱 (GC-MS/MS)检测留兰香、桂皮、薄荷和月桂叶中乙氧氟草醚、唑草酮、乙螨唑残留的分析方法。4种香辛料用超纯水饱和,乙腈提取,无水硫酸镁及氯化钠盐析,氨基/石墨化碳黑 (NH₂-Carb) 固相萃取柱净化,多反应监测模式,气相色谱-串联质谱测定。结果表明:乙氧氟草醚在0.002 5~2 mg/L范围内,唑草酮和乙螨唑在0.01~2 mg/L范围内,3种农药的进样质量浓度与对应的峰面积间呈良好的线性关系,r > 0.99;乙氧氟草醚在0.025、0.5和2 mg/kg 3个添加水平下,在4种香辛料中的平均回收率在86%~112%之间,相对标准偏差 (RSD)在2.4%~9.6%之间;唑草酮在0.2、0.5和2 mg/kg 3个添加水平下的平均回收率在87%~114%之间,RSD在2.4%~11%之间;乙螨唑在0.5、2和5 mg/kg 3个添加水平下的平均回收率在86%~116%之间,RSD在3.2%~11%之间。乙氧氟草醚、唑草酮和乙螨唑在4种香辛料中的定量限 (LOQ) 分别为0.025、0.2和0.5 mg/kg。     

转基因水稻对稻瘟病的抗性研究

 采用苗期初筛、复筛、抗谱测定和田间自然诱发试验等不同鉴定方法,对经分子检测证明已整合有碱性几丁质酶基因和β-1,3-葡聚精酶基因的22个转化系的转基因水稻植株进行稻瘟病抗性鉴定研究,筛选出对稻瘟病的抗性比原种对照七丝软占有明显提高的一系列转基因水稻品系,其中表现高抗的有来自F4-9转化株系的7个品系。高抗材料的R7代品系,经室内抗谱测定及田间病圃试验结果,仍然表现高抗稻瘟病。本研究通过转基因技术,成功地将优质感病品种改良成高抗品系,研究结果证明了利用基因工程手段培育抗病水稻新品种是一个非常有希望的育种途径。     

噁草酮耐光解作用与施药技术改进研究

噁草酮对阳光的分解有良好的耐性,用25％乳油2.5L/hm~2防除旱直播稻田杂草,施药后曝晒25d左右药效开始下降,曝晒35～40d的防效仍保持在80％以上,45d后防效较差。施药后等雨或等水灌溉出苗,防除效果好。土壤湿润有利于药效的发挥。     

Studies into the action of the diphenyl ether herbicides acifluorfen and oxyfluorfen. Part I: Activation by light and oxygen in leaf tissue

The effect of acifluorfen and oxyfluorfen on chlorophyll bleaching, lipid peroxidation and photosynthesis in pea leaf discs was studied. Both her- bicides induced light-dependent bleaching and lipid peroxidation, the level of damage being greater at higher light intensities. Photosynthetic carbon dioxide fixation was only partially inhibited in treated leaf discs incubated in darkness, thus indicating that these herbicides did not inhibit photo- synthesis as a primary mode of action. Leaf discs maintained in darkness showed no visible signs of injury, and light-dependent herbicide-induced damage was reduced by incubating discs under nitrogen, orpre-incubating them with the electron-transport inhibitor monuron. It is suggested that acifluorfen and oxyfluorfen are activated by a light-dependent process, which requires photosynthetic electron transport.     

噁草酮耐光解作用与施药技术改进研究

噁草酮对阳光的分解有良好的耐性,用25％乳油2.5L/hm~2防除旱直播稻田杂草,施药后曝晒25d左右药效开始下降,曝晒35～40d的防效仍保持在80％以上,45d后防效较差。施药后等雨或等水灌溉出苗,防除效果好。土壤湿润有利于药效的发挥。     

Potential involvement of alkoxyl and hydroxyl radicals in the peroxidative action of oxyfluorfen

The potential involvement of hydroxyl and alkoxyl radicals in the peroxidative action of the p-nitro diphenyl ether herbicides acifluorfen (5-[2-chloro-4-(trifluoromethyl)phenoxyl]-2-nitrobenzoic acid), acifluorfen-methyl (methyl ester of acifluorfen), nitrofen [2,4-dichloro-1-(4-nitrophenoxy)benzene], nitrofluorfen [2-chloro-1-(4-nitrophenoxy)-4-(trifluoromethyl)benzene], and oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene] was evaluated under laboratory conditions. Methional was added to illuminated thylakoids from peas (Pisum sativum L., cv Little Marvel) and its oxidation to ethylene was used as an indicator of hydroxyl and alkoxyl radical production. Oxyfluorfen stimulated the rate of methional oxidation by 138% at 10 μM and 175% at 1 mM. This oxyfluorfen-induced stimulation of the rate of methional oxidation was dependent on light, photosynthetic electron transport, and hydrogen peroxide since it was not observed under dark conditions or in the presence of DCMU and catalase. Addition of Fe-EDTA, a catalyst of the Fenton reaction, stimulated the oxyfluorfen-induced enhancement of methional oxidation sixfold, suggesting that hydroxyl radicals are synthesized through a Fenton reaction. Acifluorfen, nitrofen, and nitrofluorfen inhibited the rate of methional oxidation whereas acifluorfen-methyl had no effect on the rate of methional oxidation, even at high concentrations (1 mM). Nitrofluorfen at 1 mM was the only p-nitro diphenyl ether herbicide tested to inhibit photosynthetic electron transport of pea thylakoids. In experiments with pea leaf disks, acifluorfen at low concentrations stimulated the rate of methional oxidation, whereas acifluorfen-methyl, nitrofen, and nitrofluorfen had no effect. These data indicate that hydroxyl and alkoxyl radicals could be involved in the mechanism of cellular damage caused by oxyfluorfen but they are not important for the activity of the diphenyl ether herbicides acifluorfen, acifluorfen-methyl, nitrofen, and nitrofluorfen.     

Effect of four herbicides and two oils on leaf-cell membrane permeability*†

Leakage of electrolytes from leaf discs of treated Phaseolus Vulgaris L. plants was the main criterion used to study the effect of several chemicals on the permeability of leaf-cell membranes. Paraquat, diquat, dinoseb and oxyfluorfen (2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(Trifluoromethyl) benzene) increased leaf-cell membrane permeability after exposure for 12 h or less. An‘aromatic’oil caused a large increase in permeability at 2–5 min after treatment. Increases in electrolyte release were also correlated with release of soluble amino acids from the leaf discs but the former method was the more sensitive. Increase in cell membrane permeability was always associated with injury symptoms such as appearance of necrotic areas in leaves. Chlorpropham, linuron, sodium azide, glyphosate and 2,4-D at 10^?3M, as well as 1% X-77 surfactant and a non-phytotoxic isoparaffinic oil did not alter leaf-cell permeability at 12 h after treatment. Light was necessary for paraquat and oxyfluorfen to alter leaf cell permeability. Paraquat and oxyfluorfen caused a greater increase in leaf-cell permeability of a soybean mutant with yellow leaves as compared with the normal green leaves. With oxyfluorfen this difference in permeability was greater than with paraquat, and was associated with the appearance of severe necrotic injury symptoms in the yellow mutant; paraquat caused no injury symptoms.     

转苏云金杆菌毒蛋白基因水稻抗螟性的离体叶片测定法

提出了转苏云金杆菌ｃｒｙｌＡｂ基因水稻抗螟性的快速离体叶片测定法,并用该方法评价了转ｃｒｙ１／Ａｂ基因水稻对二化螟、三化螟和大螟的抗性程度。结果表明,３种螟虫蚁螟取食纯合株系ＰＲ１６及分离株系ＰＲ１８、ＰＲ５７或ＰＲ５８中抗性植株中片后,食叶量与对照和分离株系中的非抗性植株相比极显著下降,接虫后第４ｄ的幼虫死亡率高达１００％。该方法评价转ｃｒｙ１／Ａｂ基因水稻对３种螟虫的抗性结果与活化成株测定法评     

抗真菌转基因水稻纹枯病抗性的杂种优势分析

用抗纹枯病的12个转入l～4个外源抗真菌基因的转基因水稻作父本,分别与2～3个雄性不育系配组,采用田间人工接种纹枯病菌的方法,从3个抗性指标上研究了转基因杂交水稻纹枯病抗性的杂种优势.结果表明,杂交水稻的抗病性与转基因父本显著正相关,抗病性的广义遗传力为0.52～0.83.在29个杂交组合中,有5个组合具有显著负向超低亲(父本)优势,但群体平均超低亲优势大于0,为35.01%～50.79%;杂交稻群体的平均负向竞争优势明显,强度大,为-57.60%～-31.23%,有38%的组合具有显著负向竞争优势,说明转基因杂交水稻的抗纹枯病性普遍低于转基因水稻父本,但仍具有较好抗性,并可从中筛选出强优势组合.     

Development of insect‐resistant transgenic rice with Cry1C*‐free endosperm

BACKGROUND: Yellow stem borer (Tryporyza incertulas Walker), striped stem borer (Chilo suppressalis Walker) and leaf folder (Cnaphalocrocis medinalis Guenec) are three lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, novel insect‐resistant transgenic rice was developed in which Bt protein expression was nearly absent in the endosperm. The resistant gene, cry1C*, driven by the rice rbcS promoter (small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase), was introduced into Zhonghua 11 (Oryza sativa L. ssp. japonica) by Agrobacterium‐mediated transformation. RESULTS: A total of 83 independent transformants were obtained, 19 of which were characterised as single‐copy foreign gene insertion. After preliminary screening of the T₁ families of these 19 transformants in the field, six highly insect‐resistant homozygous lines were selected. These six homozygous transgenic lines were field tested for resistance to leaf folders and stem borers, and for their agronomic performance. The Cry1C* protein levels in leaves and endosperm were measured by ELISA. Subsequently, the elite transgenic line RJ5 was selected; this line not only possessed high resistance to leaf folders and stem borers, normal agronomic performance, but also Cry1C* expression was only 2.6 ng g^?1 in the endosperm. CONCLUSION: These results indicated that RJ5 has the potential for widespread utility in rice production. Copyright © 2009 Society of Chemical Industry     

Modifying Myxococcus xanthus protoporphyrinogen oxidase to plant codon usage and high level of oxyfluorfen resistance in transgenic rice

Protoporphyrinogen oxidase (Protox) of Myxococcus xanthus (Mx Protox) is a 49-kDa membrane protein that catalyzes conversion of protoporphyrinogen IX (Protogen IX) into protoporphyrin IX (Proto IX). Upon heterologous expression in transgenic rice plants, Mx Protox is dually targeted into plastids and mitochondria, increasing resistance against the herbicidal Protox inhibitor oxyfluorfen. Here, we describe the chemical synthesis of the Mx Protox gene by assembling several small synthetic DNA fragments derived by ligation-PCR. Codon usage in the resulting 1416-bp gene was modified to correspond to that of the Arabidopsis Protox gene, a change that resulted in a decrease in G+C content from 71 to 49%. The modified Mx Protox gene was used to generate transgenic rice plants via Agrobacterium-mediated transformation. Integration, expression, and inheritance of the transgenes were demonstrated by Southern, Northern, and Western blot analyses. In plants transformed with the modified, low G+C-content Mx Protox gene, levels of Protox expression and enzyme activity were low compared to the levels observed for plants transformed with the native Mx Protox gene. Nonetheless, like the native gene, the modified gene conferred a high level of resistance to the herbicide oxyfluorfen in a seedling growth test.