The research was supported by the National Research Council (CNR) of Italy. The authors thank GIRE members for collecting seed samples and are grateful to Alison Garside for revising the English.

1.种子取样和存储<ol><li>监视不合理的除草剂表现不佳耕地， 即不因不利的气候条件或低质量的除草剂处理。 </li><li>收集来自一个物种的种子样品的时间和分配一个唯一的代码。成熟的种子通常是前收割从幸存的除草剂处理（S）的植物收集。及时监测，观察，如果种子是由母厂棚成熟时。 </li><li>在每个样品填表说明分配的唯一代码在赛季中使用的物质，收集日期，GPS坐标，直辖市，农民的名字，字段的大小，为患水平，作物，除草剂（S）和外地的历史记录，名称。 </li><li>从至少30随机选择的植物是代表励磁为患收集种子。保证种子样品含有至少5000成熟种子。对于专异交杂草（ 例如，黑麦草属物种或苋属），降低植物的编号，以10-15，保持种子的总数大约5000 11。 </li><li>子样的领域，如果杂草补丁分散在大面积（超过一公顷），为不同的抗除草剂的生物型选择。 </li><li>商店的种子在标记有分配唯一码启封纸袋。 </li><li>让水分蒸发，但不暴露的种子在高温（ 即，避免在阳光下让他们在汽车）或极端温度波动，避免感应二次休眠。 </li><li>清洁（除去谷壳，脱船体种子等 ）和它们在环境温度下储存在干燥的房间。在进行第一电阻测试后，储存的种子的时间在暗室中长时间在4℃下，优选在真空密封的塑料袋。这样的种子保存其活力的显著更长的时间。 </li></ol>注:种子休眠提供了一个灵活，高效的机制，使杂草适应，坚持农业生态系统。打破休眠，允许种子萌发，不同的协议具有取决于杂草种类， 即，休眠16的类型中使用。 主要有三种方式来删除休眠: <ol><li>春化 注:要获得同时萌发和出苗，一期的种子春化从几天到一个星期需要从许多物种中删除生理休眠:例如，反枝苋 ， 藜 ， 黑麦草属，野燕麦，蓼，虉paradoxa的 17-19。需要长达15天周期较长的虞美人 ， 异型莎草和Ammania红花和长达30天的水毛花 <suP&gt; 20。 <ol><li>把一些去离子水的塑料菜。切滤纸两层浸泡在水中，去除多余的。放置风干种子上的文件内。转移的塑料菜冰箱，在4℃的时间所需的时间。 </li></ol></li><li>乱刺 注:某些杂草种类较多顽抗萌发比别人因机械休眠， 即种皮的特点，并需要使用用硫酸化学划痕的发芽21。 <ol><li>制备用浓硫酸（95-98％）的烧杯中。准备一个装满水的烧杯中。把种子中的非织造织物的信封。 </li><li>浸泡例如，稗属物种或假高粱种子20分钟或5分钟，分别在浓硫酸。 </li><li>就拿出来信封使用镊子的烧杯中，并把它放在烧杯中装满了水。打开信封，把种子小漏勺冲洗一下彻底的在流水下。 </li></ol>词:A.苡菜类需要一个不同的协议22，23 <ol><li>浸泡的种子在氯仿2分钟。冲洗种子用去离子水中，并用吸水纸干燥。扣篮的种子在80％硫酸进行5分钟。 </li><li>把种子在一个小漏勺冲洗一下彻底的在流水下。 </li></ol></li><li>收获后的种子成熟 注:其他杂草种子成熟后，不发芽，在所有的几个月，无论用来打破休眠的方法。 <ol><li>存储的种子的期间中的至少3-4个月在RT和低湿度，然后按照对打破休眠（ 例如，水稻变种山毛榉或P.虞美人 ）上述协议。 </li></ol></li></ol> 3，种子发芽<ol><li>地方种子在PLAS发芽含0.6％（M / V）琼脂硝酸钾0.1％（KNO 3）TIC菜补充说: <ol><li>使用去离子水制备的琼脂的溶液在0.6％+ 0.1％的KNO 3。溶解在微波炉中的琼脂上。 </li><li>倒入琼脂溶液倒入塑料菜。冷却该基材上，然后放于种子。 </li></ol>变体:土壤，也可以用来作为在塑料盘中的基板。这种做法是对稗属特别有效，S.假高粱和黑麦草属。 </li><li>将塑料菜在柜子发芽大约一个星期取决于每个杂草的最佳条件光照和温度条件。对于大多数冬季品种，温度范围为15/25℃，夜晚/白天和12小时光照与霓虹灯管提供15-30微摩尔米-2秒-1的光合光子通量密度（PPFD）。对于许多夏天物种，所述温度范围为15/30℃，夜/日。 变种:•青梅物种，如S.假高粱，需要进行热处理。在划痕后，因此，种子S.在发芽柜4小时的周期在45℃和20小时，在24℃，3天，然后三天正常条件: 假高粱经受以下条件。 </li></ol> 4.育苗移栽和生长<ol><li>移植十五到二十苗放入盛有标准的盆栽混合塑料托盘（325 x 265 x95毫米）（60％粉质壤土，15％的沙子，15％agriperlite和10％的泥炭 - 以体积计）。 注:不是直接播种移栽，允许获得在同一生长阶段的统一立场的植物，这是一个重要的先决条件，以优化除草剂处理的性能。 </li><li>识别每个托盘的条形码在内的所有信息的唯一识别:人口代码，除草剂被测试，复制数量和渐进盘号&lt;。/ LI&gt; <li>地方托盘中，以维持所述衬底处或附近田间持水量加热温室和水的植物。 注:根据杂草种类的生长温度变化。通常测试期间秋/冬/春完成，所以光用400瓦的金属卤化物灯，它提供了约150微摩尔米-2秒的PPFD -1和12小时光照24，19，夏季杂草与补充的C 4光合周期通常需要更高的光强度，因此测试完成在春末，夏季或补充光照强度约400微摩尔米-2秒-1用14小时光照。 </li><li>使用不同的协议进行一些杂草为害水稻， 例如，A。苡菜类，S松材线虫和C.异型莎草在22描述。 <ol><li>移植苗成聚苯乙烯托盘24的圆形细胞（55毫米直径64毫米深）FIL导致用60％粉质壤土，30％沙子，10％泥炭（体积）。 </li><li>深集塑料容器装满水和固定的向下拧不锈钢棒，以防止它们飘浮（ 图1）12厘米托盘。 </li><li>保持在容器中的水位在1-2厘米以下的土壤表面的水平，并添加1.5克硫酸铜到每个容器（其含有10-12升水），以避免藻类的增殖。 </li></ol></li></ol> 5.除草剂处理<ol><li>治疗用芽前除草剂: <ol><li>后在发芽柜约三天如第3描述的，移植的发芽种子成塑料托盘含有上述基板和覆盖一层土（约1厘米）。这是为了确保苗不会由于杀草效果，而不是过大的埋藏深度出现的一个关键步骤。 </li><li>就拿基板领域卡帕城市放置托盘，里面有几个洞的底部，碟子上装满了水。 </li><li>移栽后一天，把托盘与芽前除草剂25。 </li><li>保持在衬底处或附近田间持水量通过既从上面和下面通过从飞碟毛细需要加水。此过程有利于除草剂的永久性在适当的深度（ 即，这里的发芽种子是）有一个良好的治疗效果。 </li></ol></li><li>治疗用苗后除草剂: <ol><li>喷塑厂当他们达到2-3叶期（ 即成长阶段扩展BBCH规模增长26 12-13）。 </li><li>治疗后，从那天起，根据杂草种类的季节（ 比如，稗属水的要求和灌溉系统。它提供水3分钟，每天4次，每隔一定时间从上午9点到9 PM）。水是DIS张焕春使用自动喷灌系统。 词:草甘膦工厂在现阶段BBCH 14-21应用。 </li></ol></li><li>除草剂编写和分发。 注:所有除草剂（前和出苗后）是在两个剂量施加作为商业制剂与推荐的表面活性剂，推荐字段剂量​​（1x）和三倍（3×）。 <ol><li>如果需要的话，根据标签指示准备在本体表面活性剂溶液;最终浓度通常表示为最终体积的百分比（ 例如，0.3％）或作为卷到每单位面积（ 例如，1升公顷-1）进行分发。 </li><li>使用以表面活性剂溶液为溶剂的除草剂（溶质）溶液，以保持活性成分的权利浓度。首先准备最集中的除草剂溶液（3次）。计算溶解在表面活性剂溶液的商业产品的数量（或在去离子水，如果一个使用下列公式的表面活性剂是没有必要的）: 浓度中药 = [（剂量域x剂量最大 ）x垂直尾翼 ] / V 德尔 其中:剂量草本植物 =除草剂剂量（毫升），剂量字段 =除草剂字段剂量（毫升公顷-1），剂量最大 =最大剂量递送，V 鳍 =的溶液（L）的最终体积，V 德尔 =体积由输送替补喷雾器（L公顷-1）。 </li><li>稀释（2:1，体积/体积）的除草剂溶液3倍以制备的浓度最低的一（1×）。此过程减少了称重或吹打除草剂时犯错的机会。除草剂溶液浓度表示为体积要的每单位面积（L公顷-1）分布。 </li><li>开始与下部除草剂剂量（1×）处理的序列。以这种方式，没有必要用相同的除草剂两种治疗之间洗喷涂柜。 </li><li>分发除草剂搜索解决方案N使用一个精密台喷雾器递送300微升公顷-1（±1％），在215千帕压力下，为0.75m秒-1的速度，用装有三个平帆（扩展范围）液压喷嘴悬臂。 </li><li>洗喷涂柜两次时，除草剂使用漂白剂的1％变化（体积/体积）中，然后冲洗干净。 变体:草甘膦施加的200升公顷-1 27的喷雾体积。 注:特别注意有高度时，除草剂的生物，如磺脲类或嘧磺隆flazasulfuron，用于支付。在后一种情况下做一次洗涤用漂白溶液和另一用氨（2.5％体积/体积）中，然后仔细用清水冲洗。 </li></ol></li></ol> 6.收集和数据分析<ol><li>通过条形码阅读器，它自动地识别每个纸盒，记录植物存活的治疗以及视觉估计生物量（VEB）的数量。植物是屁股ESSED作为死了，如果他们没有表现出积极的增长无论颜色还是外观等的。 <ol><li>使后三或四周治疗（WAT）的评估取决于所测试的除草剂（ 例如，三WAT为ACC酶抑制剂和四个WAT为ALS抑制剂或草甘膦）。 </li><li>通过包括易感人群（检查S）在所有实验， 即收集在一个网站，是从来没有或很少与除草剂处理人口评估的一般治疗效果。 </li><li>明示植物存活处理的植物的数量的百分比，计算正好在除草剂处理前，并计算每平均值的标准误（SE）（指的是两次重复的值）。 </li><li>对VEB通过植物生物量的视觉比较所得之间处理和不处理的检查相同的人口25，28的一个得分，范围从10不受除草剂（与未治疗的检查相比）植物为0什么时候植物显然是死的，给予每个处理托盘。 </li></ol></li><li>归因于种群基于从处理用两个除草剂剂量获得的结果四类:S当植物小于5％存活的除草剂剂量1×，SR时幸存者从5％到20％的除草剂剂量1×中，R的情况相比更植物的20％存活的除草剂剂量1x和RR时幸存者在除草剂剂量1×20％以上，并在除草剂剂量3×17的10％以上。 </li></ol>

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J., Sattin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-site+resistance+to+ALS+inhibitors+in+the+polyploid+species+Echinochloa+crus-galli.">Target-site resistance to ALS inhibitors in the polyploid species Echinochloa crus-galli.</a> Pestic. Biochem. Phys. 105 (2), 93-101 (2013).</li><li>Sattin, M., Berto, D., Zanin, G., Tabacchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+ALS+inhibitors+in+rice+in+north-western+Italy.">Resistance to ALS inhibitors in rice in north-western Italy.</a> Proc. BCPC. Weeds. , 783-790 (1999).</li><li>Scarabel, L., Berto, D., Sattin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dormancy+breaking+and+germination+of+Alisma+plantago-aquatica.+and+Scirpus+mucronatus.">Dormancy breaking and germination of Alisma plantago-aquatica. and Scirpus mucronatus.</a> Aspects of Applied Biology. 69, 285-292 (2003).</li><li>Collavo, A., Strek, H., Beffa, R., Sattin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+an+ACCase-inhibitor-resistant+Lolium+rigidum.+population+based+on+the+use+of+ALS+inhibitors:+weed+population+evolution+observed+over+a+7+years+field-scale+investigation.">Management of an ACCase-inhibitor-resistant Lolium rigidum. population based on the use of ALS inhibitors: weed population evolution observed over a 7 years field-scale investigation.</a> Pest Manag. Sci. 69 (2), 200-208 (2013).</li><li>Scarabel, L., Panozzo, S., Savoia, W., Sattin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-site+ACCase-resistant+Johnsongrass+(Sorghum+halepense).+selected+in+summer+dicot+crops.">Target-site ACCase-resistant Johnsongrass (Sorghum halepense). selected in summer dicot crops.</a> Weed Technol. 28 (2), 307-315 (2014).</li><li>Hess, M., Barralis, H., Bleiholder, H., Buhur, L., Eggers, T., Hack, H., Strauss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+extended+BBCH+scale+-+general+for+the+description+of+the+growth+stages+of+mono-+and+dicotyledonous+weed+species.">Use of the extended BBCH scale - general for the description of the growth stages of mono- and dicotyledonous weed species.</a> Weed Res. 37 (6), 433-441 (1997).</li><li>Collavo, A., Sattin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+glyphosate-resistant+Lolium.+spp.+biotypes+found+in+a+European+annual+arable+cropping+system+also+affected+by+ACCase+and+ALS+resistance.">First glyphosate-resistant Lolium. spp. biotypes found in a European annual arable cropping system also affected by ACCase and ALS resistance.</a> Weed Res. 54 (4), 325-334 (2014).</li><li>Scarabel, L., Cenghialta, C., Manuello, D., Sattin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+and+management+of+imidazolinone-resistant+red+rice+(Oryza+sativa.+L.,+var.+sylvatica.)+in+Clearfield&#174;+Italian+paddy+rice.">Monitoring and management of imidazolinone-resistant red rice (Oryza sativa. L., var. sylvatica.) in Clearfield&#174; Italian paddy rice.</a> Agronomy. 2 (4), 371-383 (2012).</li><li>Zelaya, I. A., Anderson, J. A. H., Owen, M. D. K., Landes, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+spectrophotometric+and+HPLC+methods+for+shikimic+acid+determination+in+plants:+Models+in+glyphosate-resistant+and-susceptible+crops.">Evaluation of spectrophotometric and HPLC methods for shikimic acid determination in plants: Models in glyphosate-resistant and-susceptible crops.</a> J. Agric. Food Chem. 59 (6), 2202-2212 (2011).</li></ol>

The authors declare that they have no competing financial interests.

内协议的几个步骤是抗除草剂的人口的成功关键评估:1）种子应当收集成熟时从幸存的除草剂处理（S）的植物。在母株的种子成熟是至关重要的，以避免在种子发芽后的困难; 2）种子的存放建议避免霉菌，防止发芽的扩散; 3）苗应在合适的生长阶段来处理，所报告的除草剂包装的标签上。必须小心，使得所处理的植物已经大致达到相同的生长阶段; 4）将除草剂溶液应准备并准确处理，以便苗用的?...

<html>除草剂是最广泛使用的杂草控制措施，占全球植物保护市场1高达50％。他们是相对便宜的工具，避免劳动密集和费时的土壤耕作活动，并最终导致成本效益，安全和有利可图的粮食生产2。然而，巨大的物候和遗传变异存在于许多杂草，对除草剂的使用过度依赖在一起，经常导致抗除草剂杂草种群的选择。选择性除草剂的一个非常具体的目标代谢引进3-5急剧增加的阻力案件的数量多年来。到目前为止，世界各地的240种杂草（双子叶植物140和100单子叶植物）产生了抗性行动（SOA）4不同的除草剂站点。这是一个大问题杂草管理，更一般的可持续作物生产。 e_content"&gt;早检测电阻的基础上，可靠的试验，在温室中频繁地进行，是管理除草剂抗性杂草的关键步骤。根据该目标，精度，时间和可利用的资源所需的水平不同的方法已被开发，如以及考虑6-12的杂草种类。然而，当确认一个新的杂草生物型的电阻状态是必需的（ 即，一组个人共享几个生理特征，包括生存属于一种或多种除草剂的能力在通常将控制它们的剂量用于特定基），一个强大的整株植物生物测定需要在受控环境4,11进行。 一个生物型是刚刚一种除草剂抗性很少。因此，每个生物型的特征在于一定的阻力图案，SOA的即，数量和类型的除草剂是抗性，并且由给定的阻力级别每个除草剂13。早期的和可靠的决心交叉或多重耐药5的格局，14是现场管理性的重要。 值得一提的是，抗除草剂无关与自然容忍对某些除草剂， 例如，双子叶植物与ACC酶抑制除草剂，单子叶植物与2,4-D， 问荆对一些杂草展览草甘膦。 本文提出了一种可靠的方法来测试假定采样除草剂在控制通过除草剂​​（S）差已经报道领域耐生物型。相关的变体，以相对于所涉及的杂草种类的标准协议被提出。在仅使用一种除草剂剂量15或治疗种子在培养皿8根据任一整株植物生物测定法替代的技术/协议的优点都涉及到更高reliability和推断，因为在实验中列入两个除草剂剂量的阻力水平的可能性。然而，对于常规的电阻测试中，相同的方法可以在仅一种除草剂剂量施加，所以降低了成本。 以及允许确认电阻状态的，可以同时用于优化下面研究步骤和/或制定完善的抗性管理策略获得的信息。

<table><tbody><tr><td>Paper bags</td><td>Celcar SAS</td><td></td><td></td></tr><tr><td>Plastic dishes</td><td>ISI plast S.p.A.</td><td>SO600</td><td>Transparent plastic</td></tr><tr><td>Sulfuric acid 95-98%</td><td>Sigma-Aldrich</td><td>320501</td><td></td></tr><tr><td>Non-woven fabric</td><td>Carretta Tessitura</td><td>Art.TNT17</td><td>Weight 17 g m&lt;sup&gt;-2&lt;/sup&gt;</td></tr><tr><td>Chloroform &gt;99.5%</td><td>Sigma-Aldrich</td><td>C2432</td><td></td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A1296</td><td></td></tr><tr><td>Potassium nitrate &gt;99.0%</td><td>Sigma-Aldrich</td><td>P8394</td><td></td></tr><tr><td>Plastic containers</td><td>Giganplast</td><td>1875/M</td><td>600 x 400 x 110 mm</td></tr><tr><td>Plastic trays</td><td>Piber plast</td><td>G1210A</td><td>325 x 265 x 95 mm</td></tr><tr><td>Polystyrene trays</td><td>Plastisavio</td><td>S24</td><td>537 x 328 x 72 mm, 24 round cells (6x4)</td></tr><tr><td>Copper sulfate</td><td>Sigma-Aldrich</td><td>451657</td><td></td></tr><tr><td>Agriperlite</td><td>Blu Agroingross sas</td><td>AGRI100</td><td></td></tr><tr><td>Peat</td><td>Blu Agroingross sas</td><td>TORBA250</td><td></td></tr><tr><td>Germination cabinet</td><td>KW</td><td>W87R</td><td></td></tr><tr><td>Nozzles</td><td>Teejet</td><td>XR11002-VK, TP11001-VH</td><td>The second type of nozzles are used only for glyphosate</td></tr><tr><td>Barcode generator</td><td>Toshiba TEC</td><td>SX4</td><td></td></tr><tr><td>Labels with barcode</td><td>Felga</td><td>TT20200</td><td>Stick-in labels with rounded corners</td></tr><tr><td>Barcode reader</td><td>Cipherlab</td><td>8300-L</td><td>Portable data terminal</td></tr><tr><td>Bench sprayer</td><td></td><td></td><td>Built in house</td></tr><tr><td colspan="4">&lt;strong&gt;Herbicides included in the results:&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Commercial product&lt;/strong&gt;</td><td>&lt;strong&gt;Active ingredient&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Altorex</td><td>imazamox</td><td>BASF</td><td></td></tr><tr><td>Azimut</td><td>florasulam</td><td>Dow AgroSciences</td><td></td></tr><tr><td>Biopower</td><td></td><td>Bayer Crop Science</td><td>Surfact to be used with Hussar WG</td></tr><tr><td>Dash</td><td></td><td>BASF</td><td>Surfact to be used with Altorex</td></tr><tr><td>Granstar</td><td>tribenuron-methyl</td><td>Dupont</td><td></td></tr><tr><td>Gulliver</td><td>azimsulfuron</td><td>Dupont</td><td></td></tr><tr><td>Hussar WG</td><td>iodosulfuron</td><td>Bayer Crop Science</td><td></td></tr><tr><td>Nominee</td><td>bispyribac-Na</td><td>Bayer Crop Science</td><td></td></tr><tr><td>Roundup</td><td>glyphosate</td><td>Monsanto</td><td></td></tr><tr><td>Trend</td><td></td><td>Dupont</td><td>Surfact to be used with Granstar and Gulliver</td></tr><tr><td>Viper</td><td>penoxsulam</td><td>Dow AgroSciences</td><td></td></tr><tr><td>Weedone LV4</td><td>2,4-D</td><td>Isagro</td><td></td></tr></tbody></table>

protocols for robust herbicide resistance testing in different weed species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

为了评估推定抗性群体的电阻状态，这是基本的，以包括在测定中的易感检查以便核实除草剂效力。的筛选试验上进行P.结果虞美人种群，杂草为害麦田，报告于图2中，其中的上易感检查（09-36），并在可疑的抗性酮（10-91）4芽后除草剂的效力呈现。人口09-36完全被ALS抑制剂受控iodosulfuron而只有一个植物存活测试，florasulam和苯磺隆（ 图2）的另两个的ALS除草?...

Watch this Scientific Journal Video about Protocols for Robust Herbicide Resistance Testing in Different Weed Species at JoVE.com

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

协议的鲁棒抗除草剂试验在不同杂草

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

protocols-for-robust-herbicide-resistance-testing-different-weed

Universidad San Sebastian

Research

JoVE Journal

Environment

14.5K 次观看.  National Research Council (CNR), Italy. A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

视频: Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

科研

遗传学

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

测量除草剂代谢的双子叶杂草价格与切下的叶片分析

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

环境科学

Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes onto humans. For this reason, transferable pesticide residue experiments are required for registration and re-registration by the United States Environmental Protection Agency (USEPA). Although such experiments are required, limited specificity is required pertaining to experimental approach. Experimental approaches used to assess pesticide transfer to humans including hand wiping with cotton gloves, modified California roller (moving a roller of known mass over cotton cloth) and soccer ball roll (ball wrapped with sorbent strip) over three treated turfgrass species (creeping bentgrass, hybrid bermudagrass and tall fescue maintained at 0.4, 5 and 9 cm, respectively) are presented. The modified California roller is the most extensively utilized approach to date, and is best suited for use at low mowing heights due to its reproducibility and large sampling area. The soccer ball roll is a less aggressive transfer approach; however, it mimics a very common occurrence in the most popular international sport, and has many implications for nondietary pesticide exposure from hand-to-mouth contact. Additionally, this approach may be adjusted for other athletic activities with limited modification. Hand wiping is the best approach to transfer pesticides at higher mowing heights, as roller-based approaches can lay blades over; however, it is more subjective due to more variable sampling pressure. Utility of these methods across turfgrass species is presented, and additional considerations to conduct transferable pesticide residue research in turfgrass systems are discussed.

Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed.

协议在草坪系统量化转换农药残留

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes ...

Screening Cotton Genotypes for Reniform Nematode Resistance

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is described here in which the root system is visually examined for nematode infection. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.

Here, a protocol is presented for the rapid non-destructive screening of cotton genotypes for reniform nematode resistance. The protocol involves visually examining the roots of nematode-infected cotton seedlings to determine infection response. The vegetative shoot from each plant is then propagated to recover plants for seed production.

筛选抗内虫抗性棉基因型

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium ...

Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway, which is virtually universal in plants. The inhibition of the enzyme stops the production of three essential amino acids: phenylalanine, tyrosine, and tryptophan. EPSPS is also present in fungi and prokaryotes, such as archaea and bacteria; thus, the use of GBP may have an impact on the microbiome composition of soils, plants, herbivores, and secondary consumers. This article aims to present general guidelines to assess the effect of GBP on microbiomes from field experiments to bioinformatics analyses and provide a few testable hypotheses. Two field experiments are presented to test the GBP on non-target organisms. First, plant-associated microbes from 10 replicated control and GBP treatment plots simulating no-till cropping are sampled and analyzed. In the second experiment, samples from experimental plots fertilized by either poultry manure containing glyphosate residues or non-treated control manure were obtained. Bioinformatics analysis of EPSPS protein sequences is utilized to determine the potential sensitivity of microbes to glyphosate. The first step in estimating the effect of GBP on microbiomes is to determine their potential sensitivity to the target enzyme (EPSPS). Microbial sequences can be obtained either from public repositories or by means of PCR amplification. However, in the majority of field studies, microbiome composition has been determined based on universal DNA markers such as the 16S rRNA and the internal transcribed spacer (ITS). In these cases, sensitivity to glyphosate can only be estimated through a probabilistic analysis of EPSPS sequences using closely related species. The quantification of the potential sensitivity of organisms to glyphosate, based on the EPSPS enzyme, provides a robust approach for further experiments to study target and non-target resistant mechanisms.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. In this article, we introduce general guidelines to quantify the effect of GBP on microbiomes, from field experiments to bioinformatics analyses.

量化草甘膦产品对微生物组的潜在影响

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

生物测定对杀虫剂的抗药性监测

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

生物学

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

实施<em&gt;体外</em&gt;耐药性检测:最大化的潜力，揭开临床有关的耐药机制

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

医学

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

表征草食动物的耐药机制：Spittlebugs臂形 SPP。为例

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice (Oryza sativa ssp.)

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously applied herbicides has presented the need for additional management methods. Allelopathy is a physiological process that some plant species possess that provide the plant with an advantage over its neighbors. Allelopathic crop varieties would be equipped with the ability to suppress the growth of surrounding competitors, thus reducing potential yield loss due to weed interference. This paper focuses on the construction and operation of a stair-step assay used for the screening of the allelopathic potential of a donor species (Oryza sativa) against a receiver weed species (Echinochloa crus-galli) in a greenhouse setting. The structure described in this paper serves as a stand for the plant samples and incorporates a timed watering system for the accumulation and distribution of allelochemicals. Allelochemicals produced by the plant roots are allowed to flow downward through a series of four pots separately into a collection tank and recycled back to the top plant through electric pumps. This method of screening provides an avenue for the allelochemicals from the donor plant to reach receiver plants without any resource competition, thus allowing quantitative measurement of the allelopathic potential of the selected donor plant. The allelopathic potential is measurable through the height reduction of the receiver plants. Preliminary screening data for the effectiveness of this method demonstrated height reduction in the receiver species, barnyardgrass (E. crus-galli), and thus the presence of allelopathic residues from the donor plant, weedy rice (Oryza sativa).

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp.

Allelopathy has shown promise as a useful supplemental weed control strategy in cropping systems. To determine the allelopathic potential of a desired plant specimen, a stair-step screening method is provided.

可重复的阶梯步骤测定，以访问杂草水稻的过敏性潜力（Oryza sativa ssp.）

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously ...

协议的鲁棒抗除草剂试验在不同杂草

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植物病原体的遗传操作