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. Semilla Muestreo y almacenamiento <ol><li> Monitorear campos cultivados para el rendimiento herbicida pobres injustificada, es decir, no se debe a las condiciones climáticas adversas o tratamientos herbicidas de baja calidad. </li><li> Se recoge una muestra de semillas de una especie a la vez y asignar un código único. Las semillas maduras suelen ser recogidos antes de la cosecha de cultivos de plantas que habían sobrevivido al tratamiento (s) herbicida. Monitor oportuna para observar si las semillas se desprenden de la planta madre cuando madura. </li><li> Rellenar un formulario para cada muestra que indica el código único asignado, el nombre de la especie, fecha de recolección, coordenadas GPS, municipio, el nombre del agricultor, el tamaño del campo, nivel de infestación, los cultivos, los herbicidas (s) que se utilizan durante la temporada y los registros históricos del campo . </li><li> Recoger las semillas de al menos 30 plantas seleccionadas al azar que son representativos de la infestación de campo. Asegúrese de que la muestra de semillas contiene al menos 5.000 semillas maduras. Por una especie de malezas obligados cruzamiento(Por ejemplo, Lolium spp. O Amaranthus spp.), Reducir el número de plantas a 10-15, manteniendo el número total de semillas alrededor de 5000 11. </li><li> Sub-muestrear el campo si los parches de malezas se encuentran dispersos en grandes áreas (más de una hectárea) como los diferentes biotipos resistentes a herbicidas son seleccionados. </li><li> Tienda semillas en bolsas de papel sin sellar marcadas con el código único asignado. </li><li> Deje que se evapore la humedad pero no exponga semillas de alta temperatura (es decir, evitar lo que les deja en un coche bajo el sol) o fluctuaciones extremas de temperatura para evitar la inducción de la latencia secundaria. </li><li> Limpio (quitar la paja, las semillas de-casco, etc.) y almacenar a temperatura ambiente en un lugar seco. Después de realizar las primeras pruebas de resistencia, almacenar las semillas por largos períodos de tiempo en un cuarto oscuro a 4 ° C, preferiblemente en bolsas de plástico selladas al vacío. En esta manera semillas preservar su viabilidad durante un tiempo significativamente más largo. </li></ol> NOTA: latencia de la semilla proporciona un mecanismo flexible y eficiente que permite a las malas hierbas para adaptarse y persistir en los agroecosistemas. Para romper la latencia y permitir la germinación de semillas, diferentes protocolos tienen que ser utilizados, dependiendo de las especies de malezas, es decir, el tipo de latencia 16. Hay tres formas principales para eliminar la latencia: <ol><li> Vernalización NOTA: Para obtener la germinación simultánea y emergencia de las plántulas, un período de vernalización semillas que van desde unos pocos días a una semana es necesario para eliminar la latencia fisiológica de muchas especies:. Por ejemplo, Amaranthus retroflexus, Chenopodium album, Lolium spp, Avena fatua, Polygonum persicaria , Phalaris paradoxa 17-19. Se requiere un período de hasta 15 días más para Papaver rhoeas, Cyperus difformis y coccinea Ammania y hasta 30 días para que Schoenoplectus mucronatus <sup&gt; 20. <ol><li> Ponga un poco de agua desionizada en platos de plástico. Corte dos capas de papel de filtro y remojar en el agua, eliminar cualquier exceso. Coloque las semillas secadas al aire en el papel. La transferencia de los platos de plástico de una nevera a 4 ° C durante el período de tiempo requerido. </li></ol></li><li> Escarificación NOTA: Algunas especies de malas hierbas son más recalcitrantes a la germinación que otros debido a la latencia mecánica, es decir, características de la capa de semillas, y requieren el uso de una escarificación química con ácido sulfúrico a germinar 21. <ol><li> Preparar un vaso de precipitados con ácido sulfúrico concentrado (95-98%). Preparar un vaso lleno de agua. Ponga las semillas en un sobre de tela no tejida. </li><li> Remojar por ejemplo, semillas halepense Echinochloa spp. O sorgo durante 20 min o 5 min, respectivamente, en ácido sulfúrico concentrado. </li><li> Tome el sobre del vaso de precipitados con un par de pinzas y lo puso en el vaso lleno de agua. Abre elsobre, poner las semillas en un pequeño colador y enjuague a fondo con agua corriente. </li></ol> Variante: A. plantago-aquatica necesita un protocolo diferente 22, 23 <ol><li> Remoje las semillas durante 2 minutos en cloroformo. Enjuague las semillas con agua desionizada y secar con papel absorbente. Dunk las semillas en ácido sulfúrico 80% durante 5 min. </li><li> Ponga las semillas en un pequeño colador y enjuague a fondo con agua corriente. </li></ol></li><li> Poscosecha maduración de la semilla NOTA: Las semillas de otras especies de malas hierbas no germinan en absoluto durante unos meses después de su vencimiento, independientemente del método utilizado para romper la latencia. <ol><li> Guarde las semillas por un período de al menos 3-4 meses a temperatura ambiente y la humedad baja y luego siga los protocolos anteriores para romper la latencia (por ejemplo, Oryza sativa var. Fagus o P. rhoeas). </li></ol></li></ol> Germinación 3. Semilla <ol><li> Coloque las semillas a germinar en plasplatos de tics que contienen 0,6% (m / v) de agar con nitrato de potasio al 0,1% (KNO 3) ha añadido: <ol><li> Preparar una solución de agar al 0,6% + 0,1% KNO 3 usando agua desionizada. Se disuelve el agar en un horno microondas. </li><li> Verter la solución de agar en platos de plástico. Enfriar el sustrato y luego poner en las semillas. </li></ol> Variante: del suelo también puede ser utilizado como un sustrato en platos de plástico. Esta práctica es particularmente eficiente para Echinochloa spp., S. halepense y Lolium spp. </li><li> Coloque platos de plástico en un armario de germinación por alrededor de una semana con condiciones de luz y temperatura en función de las condiciones óptimas para cada especie de maleza. Para la mayoría de las especies de invierno, el rango de temperatura es 15/25 ° C noche / día y 12 hr fotoperíodo con tubos de neón que proporcionan una densidad de flujo de fotones fotosintético (PPFD) de 15-30 mol m -2 seg -1. Para muchas especies de verano, el rango de temperatura es 15/30 ° C la noche / día. Variante: SOme especies, tales como S. halepense, necesitará un tratamiento térmico. Por lo tanto después de la escarificación, semillas de S. halepense se someten a las siguientes condiciones: ciclos de 4 horas a 45 ° C y 20 horas a 24 ° C durante tres días en el armario de la germinación, y luego tres días en condiciones normales. </li></ol> 4. El trasplante de plántulas y Crecimiento <ol><li> Trasplante de quince a veinte plántulas en bandejas de plástico (325 x 265 x 95 mm) llenos de una mezcla estándar de encapsulamiento (60% suelo franco limoso, 15% de arena, 15% y 10% Agriperlite turba - por volumen). NOTA: El trasplante, en lugar de la siembra directa, permite un soporte uniforme de las plantas en la misma etapa de crecimiento que se obtiene, que es una condición previa importante para optimizar el rendimiento del tratamiento herbicida. </li><li> Identifique cada bandeja con un código de barras que incluye toda la información para la identificación única: Código de la población, el herbicida se está probando, replicar número y número de bandeja progresiva &lt;./ Li&gt; <li> Coloque las bandejas en una sala climatizada plantas de invernadero y el agua según sea necesario para mantener el sustrato en o cerca de la capacidad de campo. NOTA: La temperatura de crecimiento varía dependiendo de las especies de malas hierbas. A menudo se realizan pruebas durante el otoño / invierno / primavera, así que la luz se complementa utilizando 400 W lámparas de halogenuros metálicos, que proporcionan una PPFD de aproximadamente 150 mol m -2 s -1 y un fotoperíodo de 12 h 24, 19. Especies de malezas de verano con C 4 ciclo fotosintético por lo general requieren una mayor intensidad de la luz y por lo tanto, las pruebas se realizan a finales de primavera-verano o la intensidad de la luz es complementado aproximadamente 400 mol m -2 s -1 con un fotoperiodo de 14 h. </li><li> Use un protocolo diferente para algunas especies de malezas que infestan arroz con cáscara, por ejemplo, A. plantago-aquatica, S. mucronatus y C. difformis como se describe en 22. <ol><li> Transplante las plántulas en bandejas de poliestireno con 24 células redondas (55 mm de diámetro, 64 mm de profundidad) filconducido con 60% de suelo franco limoso, 30% de arena y 10% de turba (en volumen). </li><li> Establecer las bandejas de 12 cm envases plásticos profundos llenos de agua y aseguradas mediante varillas roscadas de acero inoxidable para evitar que se flotante (Figura 1). </li><li> Mantener el nivel de agua en los recipientes a 1-2 cm por debajo del nivel de la superficie del suelo y añadir 1,5 g de sulfato de cobre a cada recipiente (que contiene 10-12 L de agua) para evitar la proliferación de algas. </li></ol></li></ol> 5. Tratamientos herbicidas <ol><li> Los tratamientos con herbicidas de pre-emergencia: <ol><li> Después de tres días en el gabinete de la germinación como se describe en la sección 3, transplantar las semillas que germinan en bandejas de plástico que contienen el sustrato descrito anteriormente y cubrir con una capa de tierra (aproximadamente 1 cm). Este es un paso crítico a fin de garantizar que las plántulas no surgirán debido al efecto herbicida en lugar de una profundidad de enterramiento excesiva. </li><li> Tome el sustrato de campo capaciudad mediante la colocación de las bandejas, que tienen unos agujeros en la parte inferior, en platillos llenos de agua. </li><li> Un día después del trasplante, el tratamiento de las bandejas con el herbicida de preemergencia 25. </li><li> Mantenga el sustrato o por la capacidad de campo cerca de la adición de agua, según sea necesario, tanto desde arriba y abajo por capilaridad desde el platillo. Este procedimiento favorece la permanencia del herbicida a la profundidad adecuada (es decir, donde las semillas en germinación son) para una buena eficacia del tratamiento. </li></ol></li><li> Los tratamientos con herbicidas de post-emergencia: <ol><li> Rociar las plantas cuando llegan a la etapa de 2-3 hojas (es decir, etapa de crecimiento 12-13 de la escala BBCH crecimiento extendido 26). </li><li> A partir del día después del tratamiento, ajuste el sistema de riego en función de las necesidades de agua de las especies de malezas y la temporada (por ejemplo, para Echinochloa spp. Que suministra agua durante 3 minutos 4 veces al día, a intervalos regulares, de 9 am a 9 pm). El agua es discontribuido mediante un sistema de riego por aspersión automática. Variante: glifosato se aplica en la planta de la etapa BBCH 14-21. </li></ol></li><li> Preparación de herbicidas y la distribución. NOTA: Todos los herbicidas (pre y post-emergencia) se aplican como formulaciones comerciales con surfactantes recomendados en dos dosis, la dosis recomendada de campo (1x) y tres veces mayor que (3x). <ol><li> Si es necesario, preparar la solución de tensioactivo a granel con arreglo a las instrucciones de la etiqueta; la concentración final se expresa generalmente como un porcentaje del volumen final (por ejemplo, 0,3%) o como volumen a ser distribuido por unidad de área (por ejemplo, 1 L ha -1). </li><li> Utilice la solución de tensioactivo como disolvente para la solución de herbicida (soluto) con el fin de mantener la concentración correcta del ingrediente activo. Preparar la solución herbicida más concentrada primero (3x). Calcular la cantidad de producto comercial que se disuelve en la solución de tensioactivo (o en agua desionizada si unatensioactivo no es necesario) usando la siguiente ecuación: Hierbas Dosis = [(Dosis campo x Dosis máxima) x V aleta] / V del Dónde: hierba = dosis Dosis herbicida (ml), dosis de campo = campo herbicida Dosis (ml ha -1), Dose max = dosis máxima entregada, V aleta = volumen final de la solución (L), V = volumen del emitido por el pulverizador de banco (L ha -1). </li><li> Diluir (2: 1, v / v) la solución 3x herbicida para preparar el menos concentrado (1x). Este procedimiento reduce la posibilidad de cometer errores al pesar o pipetear los herbicidas. Concentración de la solución herbicida se expresa como volumen a ser distribuido por unidad de área (L ha -1). </li><li> Iniciar la secuencia de tratamiento con la dosis de herbicida inferior (1x). De esta manera no hay necesidad de lavar el gabinete de pulverización entre dos tratamientos con la misma herbicida. </li><li> Distribuya el solutio herbicidan usando un pulverizador de banco precisión la entrega de 300 L ha -1 (± 1%), a una presión de 215 kPa, y una velocidad de 0,75 m s-1, con un brazo equipado con tres abanico plano (rango extendido) boquillas hidráulicas . </li><li> Lave el gabinete rociar dos veces cuando el herbicida se cambia el uso de cloro al 1% (v / v) y luego enjuague. Variante: glifosato se aplica con un volumen de pulverización de 200 L ha-1 27. NOTA: Especial atención tiene que ser pagado cuando, como el sulfometuron o flazasulfuron sulfonilureas, se utilizan herbicidas altamente biológicos. En este último caso hacer un lavado con una solución de lejía y otro con amoniaco (2,5% v / v), seguido de un enjuague cuidadoso con agua. </li></ol></li></ol> 6. Recopilación y Análisis de los Datos <ol><li> A través de un lector de código de barras, que identifica automáticamente cada bandeja, registre el número de plantas que sobrevivieron al tratamiento, así como la biomasa estimada Visual (VEB). Las plantas son culocomputan como estar muerto si no muestran crecimiento activo, independientemente del color o de otro aspecto. <ol><li> Hacer la evaluación de tres o cuatro semanas después del tratamiento (WAT) en función de los herbicidas probados (por ejemplo, tres WAT para los inhibidores ACCasa y cuatro WAT para los inhibidores de ALS o glifosato). </li><li> Evaluar la eficacia del tratamiento en general mediante la inclusión de una población susceptible (comprobar S) en todos los experimentos, es decir, una población recogida en un sitio que nunca o raramente tratados con herbicidas era. </li><li> Supervivencia de las plantas Express como porcentaje del número de plantas tratadas, contó justo antes del tratamiento con herbicida, y calcular el error estándar (SE) por valor medio (valor de las dos repeticiones significa). </li><li> El VEB se obtiene a través de una comparación visual de la biomasa vegetal entre tratado y verificación de la misma población de 25, 28 no tratados. Una puntuación, que van desde 10 para las plantas no afectadas por el herbicida (en comparación con la comprobación no tratada) a 0 Cuándolas plantas son claramente muerto, se le da a cada bandeja tratada. </li></ol></li><li> Poblaciones atribuyen a cuatro categorías en función de los resultados obtenidos de los tratamientos con dos dosis de herbicidas: S cuando menos del 5% de las plantas sobrevivieron los 1x dosis de herbicidas, SR cuando los sobrevivientes oscilaron entre 5% a 20% en 1x dosis de herbicidas, R cuando más de 20% de las plantas sobrevivió a los 1x dosis de herbicida y RR cuando los sobrevivientes son más de 20% en dosis de herbicida 1x y más de 10% en dosis de herbicida 3x 17. </li></ol>

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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.

Varios pasos dentro de los protocolos son fundamentales para una evaluación exitosa de resistencia a los herbicidas en una población: 1) las semillas deben recogerse en la madurez de las plantas que habían sobrevivido al tratamiento (s) herbicida. La maduración de las semillas de la planta madre es crucial para evitar dificultades en la germinación de las semillas más tarde; 2) Se recomienda el almacenamiento adecuado de semillas para evitar la proliferación de mohos que impidan la germinación; 3) las plántulas...

<html> Los herbicidas son la medida de control de malas hierbas más ampliamente utilizado, que representa hasta un 50% del mercado global de protección de las plantas 1. Son herramientas relativamente baratas, evitar prácticas de cultivo del suelo en mano de obra intensiva y requiere mucho tiempo, y en última instancia resultar en la producción rentable, segura y rentable de alimentos 2. Sin embargo, la gran variabilidad genética fenológico y presente en muchas especies de malezas, junto con un exceso de confianza en el uso de herbicidas, se traduce con frecuencia en la selección de poblaciones de malezas resistentes a los herbicidas. La introducción de herbicidas selectivos con un objetivo metabólica muy específico 3-5 se ha incrementado dramáticamente el número de casos de resistencia a lo largo de los años. Hasta la fecha, 240 especies de malezas (140 dicotiledóneas y monocotiledóneas 100) en todo el mundo han desarrollado resistencia a los diferentes sitios de herbicidas de acción (SOA) 4. Esta es una preocupación importante para el manejo de malezas y más en general, para la producción agrícola sostenible. e_content &quot;&gt; La detección temprana de la resistencia, con base en pruebas fiables, se realizan con frecuencia en un invernadero, es un paso clave para manejar malezas resistentes a los herbicidas. Los diferentes enfoques se han desarrollado de acuerdo con los objetivos, el nivel requerido de precisión, el tiempo y los recursos disponibles, como así como las especies de malas hierbas consideradas 6-12. Sin embargo, cuando se requiere la confirmación del estado de resistencia de un nuevo biotipo de malezas (es decir, un grupo de individuos que comparten varias características fisiológicas, incluyendo la capacidad de sobrevivir a uno o más herbicidas pertenecientes a una grupo particular utilizado en una dosis que normalmente controlar ellos), un robusto bioensayo de toda la planta se debe realizar en un ambiente controlado 4, 11. Un biotipo rara vez es resistente a un solo herbicida. Por tanto, cada biotipo se caracteriza por un patrón de resistencia determinado, es decir, número y tipo de SoA de los herbicidas es resistente a, y por una resistencia dadanivel para cada herbicida 13. La determinación precoz y fiable del patrón de cruz o resistencia múltiple 5, 14 es importante para la gestión de la resistencia de campo. Vale la pena mencionar que la resistencia a los herbicidas no tiene nada que ver con la tolerancia natural que algunos exhiben especies de malezas a algunos herbicidas, por ejemplo, las especies dicotiledóneas vs. herbicidas ACCasa inhibidoras, especies monocotiledóneas vs. 2,4-D, Equisetum arvense vs. glifosato. Este artículo presenta un enfoque sólido para probar biotipos resistentes a herbicidas putativo muestreados en los campos donde se había reportado un mal control por el herbicida (s). Se presentan variantes relevantes a los protocolos estándar en relación con las especies de malas hierbas que participan. Las ventajas sobre las técnicas / protocolos alternativos basados ​​en cualquiera de los bioensayos de plantas enteras utilizando sólo una dosis de herbicida 15, o el tratamiento de semillas en placas de Petri 8 están relacionados con la mayor reliability y la posibilidad de inferir el nivel de resistencia debido a la inclusión de dos dosis de herbicidas en los experimentos. Sin embargo, para las pruebas de resistencia de rutina, los mismos métodos pueden ser aplicados en una sola dosis de herbicida, por lo que la reducción de los costes. Además de permitir la confirmación de la condición de la resistencia, la información obtenida se puede utilizar tanto para la optimización de los siguientes pasos de la investigación y / o la elaboración de estrategias de manejo de resistencia.

<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.

Para evaluar el estado de resistencia de una población resistente putativo, es fundamental para incluir un cheque susceptible en el ensayo con el fin de verificar la eficacia herbicida. Los resultados de una prueba de detección llevados a cabo en P. poblaciones rhoeas, una mala hierba que infestan los campos de trigo, se presentan en la Figura 2, donde se presenta la eficacia de cuatro herbicidas de post-emergencia en un testigo susceptible (09-36) y, por un presunto resistente (10-9...

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.

Los protocolos para pruebas de resistencia a los herbicidas robusta en diferentes especies de malezas

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 de Cartagena UDEC

Research

JoVE Journal

Environment

14.5K vistas.  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.

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

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.

Medición de Precios de los herbicidas Metabolismo en Dicot malas hierbas con un ensayo extirpados Leaf

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

Investigación

Medio ambiente

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.

Protocolos para la cuantificación de residuos de plaguicidas transferibles en Sistemas de céspedes

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.

Selección de genotipos de algodón para la resistencia de los nematodos reniformes

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

Genética

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.

Cuantificación del impacto potencial de los productos a base de glifosato en los microbiomas

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.

Bioensayos para vigilar la resistencia insecticida

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

Biología

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.

Implementación de<em&gt; In Vitro</em&gt; Droga Resistencia Ensayos: Maximizar el Potencial de Descubrimiento clínicamente relevantes mecanismos de resistencia

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

Medicina

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.

Caracterización de los mecanismos de resistencia Herbívoro: salivazo en Brachiaria Spp. como ejemplo

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).

Una basada en PCR genotipificación método para distinguir entre las variedades de tipo silvestre y ornamental de Imperata cylindrica</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.

Ensayo repetible de escalera para acceder al potencial alelopático del arroz weedy (Oryza sativa ssp.)

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

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Método de alto rendimiento, robusto y altamente flexible en el tiempo para la esterilización superficial de semillas de Arabidopsis

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

Los protocolos para pruebas de resistencia a los herbicidas robusta en diferentes especies de malezas

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