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.

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

Cytokines produced by susceptible and resistant mice in the course of Paracoccidioides brasiliensis infection

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-a and TGF-ß) and type-1 (IL-2 and IFN-g) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected ip with P. brasiliensis produced early and sustained levels of IFN-g and IL-2; type-2 cytokines (IL-4 and IL-5) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-g concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-ß and low levels of TNF-a. In contrast, macrophages from A/Sn animals released high levels of TNF-a associated with a small production of TGF-ß. The in vivo depletion of IFN-g not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.A animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-a and IFN-g followed by a sustained secretion of IL-2 and IFN-g plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-a and IFN-g associated with production of IL-5, IL-10 and TGF-ß characterizes the progressive disease of susceptible animals.

Activity and functional interaction of alternative oxidase and uncoupling protein in mitochondria from tomato fruit

Cyanide-resistant alternative oxidase (AOX) is not limited to plant mitochondria and is widespread among several types of protists. The uncoupling protein (UCP) is much more widespread than previously believed, not only in tissues of higher animals but also in plants and in an amoeboid protozoan. The redox energy-dissipating pathway (AOX) and the proton electrochemical gradient energy-dissipating pathway (UCP) lead to the same final effect, i.e., a decrease in ATP synthesis and an increase in heat production. Studies with green tomato fruit mitochondria show that both proteins are present simultaneously in the membrane. This raises the question of a specific physiological role for each energy-dissipating system and of a possible functional connection between them (shared regulation). Linoleic acid, an abundant free fatty acid in plants which activates UCP, strongly inhibits cyanide-resistant respiration mediated by AOX. Moreover, studies of the evolution of AOX and UCP protein expression and of their activities during post-harvest ripening of tomato fruit show that AOX and plant UCP work sequentially: AOX activity decreases in early post-growing stages and UCP activity is decreased in late ripening stages. Electron partitioning between the alternative oxidase and the cytochrome pathway as well as H+ gradient partitioning between ATP synthase and UCP can be evaluated by the ADP/O method. This method facilitates description of the kinetics of energy-dissipating pathways and of ATP synthase when state 3 respiration is decreased by limitation of oxidizable substrate.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80ºC for 20 min). N-Acylation increased its ordered structure by 4% and decreased its ß-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the mycobacteria growth indicator tube (MGIT) system

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has increased the need for rapid drug susceptibility tests, which are needed for adequate patient treatment. The objective of the present study was to evaluate the mycobacteria growth indicator tube (MGIT) system to detect multidrug-resistant M. tuberculosis strains. The MGIT system was compared with two standard methods (proportion and resistance ratio methods). One hundred clinical M. tuberculosis isolates [25 susceptible to isoniazid (INH) and rifampicin (RIF), 20 resistant to INH, 30 resistant to INH-RIF, and 25 resistant to INH-RIF and other drugs] obtained in the State of São Paulo were tested for INH and RIF susceptibility. Full agreement among the tests was found for all sensitive and all INH-resistant strains. For RIF-resistant strains results among the tests agreed for 53 (96.4%) of 55 isolates. Results were obtained within 6 days (range, 5 to 8 days), 28 days and 12 days when using MGIT, the proportion method and the resistance ratio methods, respectively. The MGIT system presented an overall agreement of 96% when compared with two standard methods. These data show that the MGIT system is rapid, sensitive and efficient for the early detection of multidrug-resistant M. tuberculosis.

Heat storage rate and acute fatigue in rats

Thermal environmental stress can anticipate acute fatigue during exercise at a fixed intensity (%VO2max). Controversy exists about whether this anticipation is caused by the absolute internal temperature (Tint, ºC), by the heat storage rate (HSR, cal/min) or by both mechanisms. The aim of the present study was to study acute fatigue (total exercise time, TET) during thermal stress by determining Tint and HSR from abdominal temperature. Thermal environmental stress was controlled in an environmental chamber and determined as wet bulb globe temperature (ºC), with three environmental temperatures being studied: cold (18ºC), thermoneutral (23.1ºC) or hot (29.4ºC). Six untrained male Wistar rats weighing 260-360 g were used. The animals were submitted to exercise at the same time of day in the three environments and at two treadmill velocities (21 and 24 m/min) until exhaustion. After implantation of a temperature sensor and treadmill adaptation, the animals were submitted to a Latin square experimental design using a 2 x 3 factorial scheme (velocity and environment), with the level of significance set at P<0.05. The results showed that the higher the velocity and the ambient temperature, the lower was the TET, with these two factors being independent. This result indicated that fatigue was independently affected by both the increase in exercise intensity and the thermal environmental stress. Fatigue developed at different Tint and HSR showed the best inverse relationship with TET. We conclude that HSR was the main anticipating factor of fatigue.

Hormone replacement therapy increases levels of antibodies against heat shock protein 65 and certain species of oxidized low density lipoprotein

Hormone replacement therapy (HRT) reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp) and oxidized low density lipoprotein (LDL) have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 ± 0.03 vs 0.558 ± 0.11) and against LDL with a low degree of oxidative modification (0.100 ± 0.01 vs 0.217 ± 0.02) (P<0.05 and P<0.001, respectively, ANOVA). The hormone-mediated immune response may trigger an inflammatory response within the vessel wall and potentially increase plaque burden. Whether or not this immune response is temporary or sustained and deleterious requires further investigation.