Rapid diagnosis of resistance to imidazolinone herbicides in barnyardgrass (Echinochloa crus-galli) and control of resistant biotypes with alternative herbicides

The resistance of barnyardgrass (Echinochloa crus-galli) to imidazolinone herbicides is a worldwide problem in paddy fields. A rapid diagnosis is required for the selection of adequate prevention and control practices. The objectives of this study were to develop expedite bioassays to identify the resistance to imidazolinone herbicides in barnyardgrass and to evaluate the efficacy of alternative herbicides for the post-emergence control of resistant biotypes. Three experiments were conducted to develop methods for diagnosis of resistance to imazethapyr and imazapyr + imazapic in barnyardgrass at the seed, seedling and tiller stages, and to carry out a pot experiment to determine the efficacy of six herbicides applied at post-emergence in 13 biotypes of barnyardgrass resistant to imidazolinones. The seed soaking bioassay was not able to differentiate the resistant and susceptible biotypes. The resistance of barnyardgrass to imidazolinones was effectively discriminated in the seedlings and tiller bioassays seven days after incubation at the concentrations of 0.001 and 0.0001 mM, respectively, for both imazethapyr and imazapyr + imazapic. The biotypes identified as resistant to imidazolinones showed different patterns of susceptibility to penoxsulam, bispyribac-sodium and pyrazosulfuron-ethyl, and were all controlled with profoxydim and cyhalofop-butyl. The seedling and tiller bioassays are effective in the diagnosis of barnyardgrass resistance to imidazolinone herbicides, providing an on-season opportunity to identify the need to use alternative herbicides to be applied at post-emergence for the control of the resistant biotypes.

Limited occurrence of resistant radish (Raphanus sativus) to AHAS-inhibiting herbicides in Argentina

Radish has developed feral and weedy biotypes, which is a concern for agriculture around the world. In Argentina, it is one of the most widespread and troublesome crop weeds. In Brazil, this species has developed herbicide-resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides. The objective of this study was to record the presence of herbicide-resistant weedy radish plants in Argentina. In spring 2008, we found a small population of radish at the end of the flowering stage in an imidazolinone-tolerant canola field treated with imazethapyr. Screening and dose-response tests were conducted to two successive generations. They proved the biotype resistant status, and showed extensive survival (between 50 and 80% of control) to the application of a double dose of four AHAS‑inhibiting herbicides from two different chemical families (imidazolinones and sulfonylureas). Dose-response assays exhibited very high resistance for imazethapyr (LD50 = 2452.5 g a.i. ha-1, GR50 = 2926.9 g a.i. ha-1) and intermediate for metsulfuron (LD50 = 3.0 g a.i. ha-1, GR50 = 43.2 g a.i. ha-1). The acquisition of cross-resistance to different herbicide families would confer an adaptive and invasive advantage in agricultural environments to this biotype. Due to the herbicide rotation conducted in the field, the dispersion of this biotype was restricted. This is the first report of resistance in weedy radish in Argentina.

Germination and growth of Fimbristylis miliacea biotypes resistant and susceptible to acetolactate synthase-inhibiting herbicides

Weed biotypes resistant and susceptible to herbicides may have differences in their adaptive values. The aims of this study were to compare, under controlled and non-competitive condition, the growth analysis, germination features and seed weight of Fimbristylis miliacea (FIMMI) biotypes resistant and susceptible to acetolactate synthase (ALS) inhibiting herbicides. Experiments were conducted in a greenhouse and in a laboratory from October 2008 to February 2010. Two resistant biotypes (FIMMI 10 and FIMMI 12) and one susceptible biotype (FIMMI 13) were used for the studies. For the study on growth analysis, the treatments were arranged in a completely randomized experimental design with four replications and sampled at 21, 28, 35, 42, 49, 56, 69 days after emergence (DAE) and at flowering stage. For the studies on germination speed, germination and seed weight, the indexes for germination speed, percentage of germination at different temperatures and seed weight of the biotypes were determined. The results showed that the resistant biotype FIMMI 12 shows differences in all variables compared to the resistant biotype FIMMI 10 and compared to the susceptible biotype FIMMI 13, only for the evaluation at flowering. The susceptible biotype FIMMI 13 showed a higher germination speed index and higher germination rate when compared with the resistant biotypes. On the other hand, the resistant biotypes FIMMI 10 and FIMMI 12 showed higher seed weight.

Acetolactate synthase activity in Euphorbia heterophylla resistant to ALS- and protox- inhibiting herbicides

The objective of this study was to determine the activity of the enzyme acetolactate synthase in biotypes of wild poinsettia (Euphorbia heterophylla) with multiple resistance to ALS- and Protox- inhibitors in the presence and absence of imazapyr, imazethapyr and nicosulfuron. We conducted in vitro assay of ALS enzyme extracted from plants of Vitorino, Bom Sucesso do Sul and Medianeira biotypes (with multiple resistance) and a susceptible population in the absence and presence of imazapyr, imazethapyr and nicosulfuron. In the absence of herbicides, biotypes with multiple resistance showed higher affinity for the substrate of the enzyme compared with the susceptible population. The herbicides imazapyr, imazethapyr and nicosulfuron had little effect on the enzyme activity of ALS-resistant biotypes and, conversely, high inhibitory effect on ALS of the susceptible population. Resistance factors were very high, greater than 438, 963 and 474 for Vitorino, Bom Sucesso do Sul and Medianeira biotypes, respectively. The resistance to ALS inhibitors is due to the insensitivity of ALS to herbicides of both imidazolinone and sulfonylurea groups, characterizing a cross-resistance.

Dose-response curves of Lolium multiflorum biotypes resistant and susceptible to clethodim

Chemical control with herbicides, especially glyphosate, is the main method used to control ryegrass. However, the repeated use of glyphosate has selected resistant ryegrass biotypes. Thus, the ACCase inhibitor herbicides have become the main alternative to control glyphosate-resistant biotypes, being widely used by farmers in Rio Grande do Sul. Repeated use of ACCase inhibitors, in turn, have selected ryegrass biotypes resistant to this herbicide mechanism. Thus, the objective of this study was to evaluate the response of ryegrass biotypes to different clethodim rates by dose-response curves. Increasing doses (0, 12, 24, 48, 72, 96, 144 and 192 g a.i. ha-1) of the herbicide clethodim were applied at the 3-4 ryegrass leaf stage. The variables control at 14 and 28 days after treatment (DAT) and shoot dry weight were evaluated. The data were fitted by nonlinear regression log-logistic and C50 and GR50 were calculated based on the equation. The resistance factor was obtained by the ratio of C50 or GR50 of the resistant biotype by matching the susceptible biotype. Based on the equation parameters, the doses of GR50 64.7 and 234.5 g a.i. ha-1 clethodim and C50 11.2 and 172.1 g a.i. ha-1 clethodim were obtained, at 28 DAT for the susceptible and resistant biotypes, respectively. The ryegrass biotype denominated Cotril is resistant to clethodim, being controlled with a dose 15.3 times greater than that of the susceptible biotype, and a 50% reduction of this biotype occurs with a dose 3.62 times higher than that of the susceptible one.

Residual herbicides in weed management for glyphosate-resistant soybean in Brazil

In agricultural production systems where the glyphosate-resistant soybean crop (Glycine max) is grown and the practice of crop rotation with alternative herbicides is not adopted, the exclusive and continuous use of glyphosate has led to the occurrence of resistant weed populations that may limit or compromise the benefits of this technology. Thus, the efficacy of weed management programs, including the use of residual herbicides (sulfentrazone, flumioxazin, imazethapyr, diclosulan, chlorimuron and s-metolachlor) applied in preemergence and followed by in-crop postemergence applications of glyphosate (PRE-POST) were compared to glyphosate postemergence only programs - POST. The study was conducted across nine locations during the 2009/2010 and 2010/2011 growing seasons. PRE-POST programs were efficient in the control of Amaranthus viridis, Brachiaria plantaginea, Bidens pilosa, Commelina benghalensis, Eleusine indica, Euphorbia heterophylla and Raphanus raphanistrum, with the level of control being similar when comparing the program with two applications of glyphosate POST. Some PRE-POST programs were not efficient in controlling Cenchrus echinatus, Ipomoea hederifolia and Ipomoea triloba. Sulfentrazone and diclosulam PRE-POST programs improved the control of Ipomoea triloba compared to sequential applications of glyphosate alone. No significant differences in soybean yield were observed between any of the herbicide treatments or study locations. The use of residual herbicides in preemergence followed by glyphosate in-crop postemergence provides consistent weed control and reducing early season weed competition. Furthermore, these programs utilize at least two herbicide modes of action for herbicide use diversity, which will be needed to stay ahead of resistance build-up, regardless of when weeds may appear.

Determination of photosynthetic pigments in fleabane biotypes susceptible and resistant to the Herbicide Glyphosate

Chlorophylls and carotenoids are the main photosynthetic pigment in plants. In the weeds, the greatest amount of photosynthetic pigments can result in high competitiveness of the species. The aim of this study was to quantify the content of photosynthetic pigments in biotypes of fleabane (Conyza bonariensis) susceptible and resistant to glyphosate, by two different methods, as well as a correlation between chlorophyll content obtained by portable and classical methodology (extractable chlorophyll). An experiment was conducted in greenhouse and laboratory, 2 x 5 factorial scheme, where factor A was equivalent to biotypes of fleabane (resistant and susceptible to glyphosate) and factor B to developmental stages plants (rosette vegetative I, II and III and reproduction). At all stages of development, fleabane plants were evaluated with the portable determiner (chlorophyll content) and then the same leaves were subjected to classical methodology laboratory (extractable pigments). The resistant biotype of fleabane showed higher contents of chlorophyll a, b, and total carotenoids, inferring a greater competitive potential regarding the susceptible population to the herbicide. The portable determiner of chlorophyll showed high correlation with the classical method of determination of photosynthetic pigments, and can thus be used to accurately assess this, saving time and reagents.

Chemical control of different Digitaria insularis populations and management of a glyphosate-resistant population

This study aimed to control different populations of Digitaria insularisby glyphosate herbicide, isolated and mixed, besides the combination of methods (chemical and mechanical) to manage resistant adult plants. Three experiments were conducted, one in pots which were maintained under non-controlled conditions and two under field conditions. In the experiment in pots, twelve populations of D. insularis were sprayed with isolated glyphosate (1.44 and 2.16 kg a.e. ha-1) and mixed (1.44 and 2.16 kg a.e. ha-1) with quizalofop-p tefuryl (0.12 kg i.a. ha-1). The treatment of 1.44 kg a.e. ha-1 of glyphosate plus 0.12 kg a.i. ha-1 of quizalofop was sufficient for adequate control (>95%) of all populations. Population 11 (area of grain production in Itumbiara, GO) was considered sensitive to glyphosate. Others populations were moderately sensitive or tolerant to the herbicide. In the field, the plants of D. insularis of one of the experiments were mowed and, in the other, there were not. Eight treatments with herbicides [isolated glyphosate (1.44 and 2.16 kg a.e. ha-1) and mixed (1.44 and 2.16 kg a.e. ha-1) with quizalofop-p-tefuryl at 0.12 kg a.i. ha-1), clethodim at 0.108 kg a.i. ha-1) or nicosulfuron at 0.06 kg a.i. ha-1)] were assessed, in combination with or without sequential application of the standard treatment, sprayed 15 days after the first application. The combination of the mechanic control with the application of glyphosate (2.16 and 1.44 kg a.e. ha-1) plus quizalofop-p-tefuryl (0.12 kg a.i. ha-1) or clethodim (0.108 kg a.i. ha-1), associated to the sequential application, was the most effective strategy for the management of adult plants of resistant D. insularis.

DETECTION OF GLYPHOSATE-RESISTANT PALMER AMARANTH (Amaranthus palmeri) IN AGRICULTURAL AREAS OF MATO GROSSO, BRAZIL

ABSTRACT The recent introduction of Palmer amaranth (Amaranthus palmeri) in Brazilian agricultural areas may promote several changes on weed management, especially in no-till systems and in glyphosate-resistant crops, since glyphosate-resistant biotypes of A. palmerihave been frequently selected in other countries. Therefore, this research was developed in order to evaluate the glyphosate susceptibility of a Palmer amaranth biotype recently identified in the State of Mato Grosso, Brazil. For this purpose, glyphosate susceptibility of three Amaranthusbiotypes was compared: A.hybridus var. patulus, collected in the State of Rio Grande do Sul - Brazil; A.hybridus var. patulus, collected in the State of São Paulo - Brazil; and A.palmeri, collected in the State of Mato Grosso - Brazil. Dose-response curves were generated for all biotypes, considering eight rates of glyphosate and six replicates. All the experiments were repeated twice. Both A.hybridus biotypes were satisfactorily controlled by glyphosate, demanding rates equal to or lower than 541.15 g a.e. ha-1 for 80% control (LD80). The A.palmeri biotype was not controlled by glyphosate in any of the assessments and required rates greater than 4,500 g a.e. ha-1 to reach LD80, which are economically and environmentally unacceptable. Comparison of the Brazilian A.palmeri biotype to the A. hybridus biotypes, as well as, to the results available in scientific international literature, led to the conclusion that the Brazilian Palmer amaranth biotype is resistant to glyphosate.

ANTAGONISM IS THE PREDOMINANT EFFECT OF HERBICIDE MIXTURES USED FOR IMIDAZOLINONE-RESISTANT BARNYARDGRASS (Echinochloa crus-galli) CONTROL

ABSTRACTHerbicides mixtures are used in many situations without the adequate knowledge related with the effect on major target weeds. The objective of this study was to evaluate the effects of different herbicides mixtures used in irrigated rice in order to establish the adequate combinations for the prevention and management of herbicide resistance in barnyardgrass (Echinochloa crus-galli). Three experiments were performed at field conditions with all major post-emergent herbicides used in irrigated rice in Brazil. The first experiment was performed with barnyardgrass resistant to imidazolinone herbicides and herbicides applied at label rates. The second and third experiments were performed with barnyardgrass resistant and susceptible to imidazolinone herbicides applied at doses of 50 or 75% of the label rates. The occurrence of additive, synergistic and antagonistic effects was identified at 18, 18 and 64%, respectively, among the total of 50 different associations of herbicide and rates evaluated. In general, the mixture of ACCase inhibitors with ALS inhibitors, quinclorac, clomazone + propanil or thiobencarb resulted in antagonism. Sinergic mixtures were found in clomazone with propanil + thiobencarb, profoxydim with cyhalofop-butyl or clomazone, and quinclorac with imazapyr + imazapic, bispyribac-sodium or cyhalofop-butyl. The mixtures of quinclorac with profoxydim were antagonic. Rice grain yield varied according to the efficiency of weed control. Seveveral mixtures were effective for imidazolinone resistant barnyardgrass control.

MULTIPLE RESISTANCE OF Sagittaria montevidensis BIOTYPES TO ACETOLACTATE SYNTHASE AND PHOTOSYSTEM II INHIBITING HERBICIDES

ABSTRACT The objective of this research was to evaluate the occurance of multiple resistance of Sagittaria montevidensis (SAGMO) biotypes to acetolactate synthase (ALS) and photosystem II (PSII) inhibiting herbicides through dose-response experiments. The experiment was conducted in a greenhouse from October 2012 to March 2013, in Pelotas, RS. The experimental design was completely randomized, with four replications. Treatments were arranged in a triple factorial design: two biotypes of S. montevidensis(SAGMO 35 - susceptible to herbicides and SAGMO 32 - suspected to be multiple resistance to ALS and PSII inhibiting herbicides), four herbicides (penoxsulam, (imazethapyr+imazapic), bentazon and saflufenacil) and 8 rates of these herbicides (1/32x, 1/16x, 1/8x, 1/4x, 1/2x, 0x, 1x, 2x, 4x, 8x, 16x, 32x and 64x). SAGMO 32 biotype presented high levels of resistance to penoxsulam, (imazethapyr+imazapic) and bentazon. For a 50% reduction in dry matter of the resistant biotype rate of 138 and 2.46 times higher than the label required for the susceptible biotype of the herbicides (imazethapyr+imazapic) and bentazon, respectively, are required. Saflufenacil may be used successfully to controlSagittaria montevidensis resistant in irrigated rice.

Glyphosate Herbicide Use in Urochloa brizantha Management in Intercropping With Herbicide-Resistant Maize

The success of the intercropping among cultivated species depends on the adoption of practices that provide, in due course, greater competitive ability of a species over another. The objective of this study was to evaluate the use of glyphosate herbicide in the suppression of Brachiaria (signalgrass) intercropped with maize. The experiment was conducted in a randomized complete block design with four replications. The treatments were arranged in a 5 x 2 + 2 factorial arrangement, the first factor corresponding to the doses of glyphosate (48, 96, 144, 240, 480 g ha-1 of the acid equivalent (a.e)) and the second one to the vegetative stages of the signalgrass at the time of application (2 and 4 tillers). Two controls were added to the treatment list, comprising controls without herbicide application and hand removal of the signalgrass. The number of plants, tillers and dry matter of signalgrass was reduced with glyphosate. The increase of the glyphosate doses enhanced the injure to the forage plants, mainly when the compound was sprayed at the two-tiller vegetative stage. The dry matter of maize plants increased proportionally to the glyphosate dose. However, the height of the maize plants was not affected. The grain mass and productivity of maize grain increased with increasing dose of glyphosate. The maize yield was negatively influenced on the untreated control. Glyphosate at 96 and 144 g ha-1, when applied at 2 and 4 tiller stage, respectively, reduces the growth of signalgrass and does not affect the maize grain yield.

Choosing the target loci : heat shock factors HSF1 and HSF2 as regulators of cell stress and development

Heat shock factors (HSFs) are an evolutionarily well conserved family of transcription factors that coordinate stress-induced gene expression and direct versatile physiological processes in eukaryote organisms. The essentiality of HSFs for cellular homeostasis has been well demonstrated, mainly through HSF1-induced transcription of heat shock protein (HSP) genes. HSFs are important regulators of many fundamental processes such as gametogenesis, metabolic control and aging, and are involved in pathological conditions including cancer progression and neurodegenerative diseases. In each of the HSF-mediated processes, however, the detailed mechanisms of HSF family members and their complete set of target genes have remained unknown. Recently, rapid advances in chromatin studies have enabled genome-wide characterization of protein binding sites in a high resolution and in an unbiased manner. In this PhD thesis, these novel methods that base on chromatin immunoprecipitation (ChIP) are utilized and the genome-wide target loci for HSF1 and HSF2 are identified in cellular stress responses and in developmental processes. The thesis and its original publications characterize the individual and shared target genes of HSF1 and HSF2, describe HSF1 as a potent transactivator, and discover HSF2 as an epigenetic regulator that coordinates gene expression throughout the cell cycle progression. In male gametogenesis, novel physiological functions for HSF1 and HSF2 are revealed and HSFs are demonstrated to control the expression of X- and Y-chromosomal multicopy genes in a silenced chromatin environment. In stressed human cells, HSF1 and HSF2 are shown to coordinate the expression of a wide variety of genes including genes for chaperone machinery, ubiquitin, regulators of cell cycle progression and signaling. These results highlight the importance of cell type and cell cycle phase in transcriptional responses, reveal the myriad of processes that are adjusted in a stressed cell and describe novel mechanisms that maintain transcriptional memory in mitotic cell division.

Ionic liquid mediated biomass deconstruction: from analysis challenges to fermentable sugars

Ionic liquids, ILs, have recently been studied with accelerating interest to be used for a deconstruction/fractionation, dissolution or pretreatment processing method of lignocellulosic biomass. ILs are usually utilized combined with heat. Regarding lignocellulosic recalcitrance toward fractionation and IL utilization, most of the studies concern IL utilization in the biomass fermentation process prior to the enzymatic hydrolysis step. It has been demonstrated that IL-pretreatment gives more efficient hydrolysis of the biomass polysaccharides than enzymatic hydrolysis alone. Both cellulose (especially cellulose) and lignin are very resistant towards fractionation and even dissolution methods. As an example, it can be mentioned that softwood, hardwood and grass-type plant species have different types of lignin structures leading to the fact that softwood lignin (guaiacyl lignin dominates) is the most difficult to solubilize or chemically disrupt. In addition to the known conventional biomass processing methods, several ILs have also been found to efficiently dissolve either cellulose and/or wood samples – different ILs are suitable for different purposes. An IL treatment of wood usually results in non-fibrous pulp, where lignin is not efficiently separated and wood components are selectively precipitated, as cellulose is not soluble or degradable in ionic liquids under mild conditions. Nevertheless, new ILs capable of rather good fractionation performance have recently emerged. The capability of the IL to dissolve or deconstruct wood or cellulose depends on several factors, (e.g. sample origin, the particle size of the biomass, mechanical treatments as pulverization, initial biomassto-IL ratio, water content of the biomass, possible impurities of IL, reaction conditions, temperature etc). The aim of this study was to obtain (fermentable) saccharides and other valuable chemicals from wood by a combined heat and IL-treatment. Thermal treatments alone contribute to the degradation of polysaccharides (e.g. 150 °C alone is said to cause the degradation of polysaccharides), thus temperatures below that should be used, if the research interest lies on the IL effectiveness. On the other hand, the efficiency of the IL-treatment can also be enhanced to combine other treatment methods, (e.g. microwave heating). The samples of spruce, pine and birch sawdust were treated with either 1-Ethyl-3-methylimidazolium chloride, Emim Cl, or 1-Ethyl-3-methylimidazolium acetate, Emim Ac, (or with ionized water for comparison) at various temperatures (where focus was between 80 and 120 °C). The samples were withdrawn at fixed time intervals (the main interest treatment time area lied between 0 and 100 hours). Double experiments were executed. The selected mono- and disaccharides, as well as their known degradation products, 5-hydroxymethylfurfural, 5-HMF, and furfural were analyzed with capillary electrophoresis, CE, and high-performance liquid chromatography, HPLC. Initially, even GC and GC-MS were utilized. Galactose, glucose, mannose and xylose were the main monosaccharides that were present in the wood samples exposed to ILs at elevated temperatures; in addition, furfural and 5-HMF were detected; moreover, the quantitative amount of the two latter ones were naturally increasing in line with the heating time or the IL:wood ratio.

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were Km = 2.8 µM, kcat = 5.3 min-1, kcat/Km = 2 min-1 µM-1 and Km = 5.0 µM, kcat = 7.0 min-1, kcat/Km = 1.4 min-1 µM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), a-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by a-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations