Chitosan/tripolyphosphate nanoparticles loaded with paraquat herbicide: An environmentally safer alternative for weed control

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chitosan nanoparticles loaded the herbicide paraquat: The influence of the aquatic humic substances on the colloidal stability and toxicity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aplicação de subdoses dos herbicidas glyphosate, 2,4-d e paraquat em algodoeiro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of the detection and quantification limits in electroanalysis using two popular methods: application in the case study of paraquat determination

In this work, the reduction reaction of paraquat herbicide was used to obtain analytical signals using electrochemical techniques of differential pulse voltammetry, square wave voltammetry and multiple square wave voltammetry. Analytes were prepared with laboratory purified water and natural water samples (from Mogi-Guacu River, SP). The electrochemical techniques were applied to 1.0 mol L-1 Na2SO4 solutions, at pH 5.5, and containing different concentrations of paraquat, in the range of 1 to 10 mu mol L-1, using a gold ultramicroelectrode. 5 replicate experiments were conducted and in each the mean value for peak currents obtained -0.70 V vs. Ag/AgCl yielded excellent linear relationships with pesticide concentrations. The slope values for the calibration plots (method sensitivity) were 4.06 x 10(-3), 1.07 x 10(-2) and 2.95 x 10(-2) A mol(-1) L for purified water by differential pulse voltammetry, square wave voltammetry and multiple square wave voltammetry, respectively. For river water samples, the slope values were 2.60 x 10(-3), 1.06 x 10(-2) and 3.35 x 10(-2) A mol(-1) L, respectively, showing a small interference from the natural matrix components in paraquat determinations. The detection limits for paraquat determinations were calculated by two distinct methodologies, i.e., as proposed by IUPAC and a statistical method. The values obtained with multiple square waves voltammetry were 0.002 and 0.12 mu mol L-1, respectively, for pure water electrolytes. The detection limit from IUPAC recommendations, when inserted in the calibration curve equation, an analytical signal (oxidation current) is smaller than the one experimentally observed for the blank solution under the same experimental conditions. This is inconsistent with the definition of detection limit, thus the IUPAC methodology requires further discussion. The same conclusion can be drawn by the analyses of detection limits obtained with the other techniques studied.

A mechanism of paraquat toxicity involving nitric oxide synthase

Paraquat (PQ) is a well described pneumotoxicant that produces toxicity by redox cycling with cellular diaphorases, thereby elevating intracellular levels of superoxide (O2⨪). NO synthase (NOS) has been shown to participate in PQ-induced lung injury. Current theory holds that NO reacts with O2⨪ generated by PQ to produce the toxin peroxynitrite. We asked whether NOS might alternatively function as a PQ diaphorase and reexamined the question of whether NO/O2⨪ reactions were toxic or protective. Here, we show that: (i) neuronal NOS has PQ diaphorase activity that inversely correlates with NO formation; (ii) PQ-induced endothelial cell toxicity is attenuated by inhibitors of NOS that prevent NADPH oxidation, but is not attenuated by those that do not; (iii) PQ inhibits endothelium-derived, but not NO-induced, relaxations of aortic rings; and (iv) PQ-induced cytotoxicity is potentiated in cytokine-activated macrophages in a manner that correlates with its ability to block NO formation. These data indicate that NOS is a PQ diaphorase and that toxicity of such redox-active compounds involves a loss of NO-related activity.

Paraquat-loaded alginate/chitosan nanoparticles: Preparation, characterization and soil sorption studies

Agrochemicals are amongst the contaminants most widely encountered in surface and subterranean hydrological systems. They comprise a variety of molecules, with properties that confer differing degrees of persistence and mobility in the environment, as well as different toxic, carcinogenic, mutagenic and teratogenic potentials, which can affect non-target organisms including man. In this work, alginate/chitosan nanoparticles were prepared as a carrier system for the herbicide paraquat. The preparation and physicochemical characterization of the nanoparticles was followed by evaluation of zeta potential, pH, size and polydispersion. The techniques employed included transmission electron microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy. The formulation presented a size distribution of 635 +/- 12 nm, polydispersion of 0.518, zeta potential of -22.8 +/- 2.3 mV and association efficiency of 74.2%. There were significant differences between the release profiles of free paraquat and the herbicide associated with the alginate/chitosan nanoparticles. Tests showed that soil sorption of paraquat, either free or associated with the nanoparticles. was dependent on the quantity of organic matter present. The results presented in this work show that association of paraquat with alginate/chitosan nanoparticles alters the release profile of the herbicide, as well as its interaction with the soil, indicating that this system could be an effective means of reducing negative impacts caused by paraquat. (C) 2011 Elsevier B.V. All rights reserved.

Análise da utilização do pesticida Paraquat na agricultura do campo experimental de Bom Jardim, RJ.

O trabalho apresenta possíveis consequências da utilização do pesticida Paraquat na agricultura a partir de resultados da execução em laboratório de ensaios de batelada (adsorção e dessorção) e de coluna em um solo (Cambissolo) de Bom Jardim, RJ. A avaliação dos parâmetros de transporte (fator de retardamento e dispersão hidrodinâmica), obtidos pelos ensaios, mostrou que o Paraquat se comportou como sendo de elevada capacidade de adsorção ao solo estudado. Embora a taxa de dessorção tenha sido pequena, a mesma não pode ser desconsiderada tendo em vista os processos de erosão hídrica. Conclui-se, portanto, que o Paraquat apresenta baixo risco de contaminação de águas subterrâneas por lixiviação, sendo provavelmente o maior risco de contaminação via escoamento superficial, devendo tal ser mais investigado visando subsidiar o uso sustentável da área.

Determination of paraquat and diquat: LC-MS method optimization and validation.

2016

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Understanding and management of resistance to Group M, L and I Herbicides

This project will develop better understanding of resistance to glyphosate, paraquat and Group I herbicides to better inform weed management. The project will develop a range of tools for farm advisors to improve their confidence in decision making with respect to reducing the risk of glyphosate, Group I and paraquat resistance. These will include risk assessments, case studies and scenario exploring tools. The project will discuss with commercial providers the potential for new herbicide registrations. The project will establish farm advisor learning groups to work on the application of the research in local areas where resistance is already a major problem and to improve adoption of research outcomes from this and other projects.

Improving management of summer weeds in dryland cropping systems with Cotton

In 2001 a scoping study (phase I) was commissioned to determine and prioritise the weed issues of cropping systems with dryland cotton. The main findings were that the weed flora was diverse, cropping systems complex, and weeds had a major financial and economical impact. Phase II 'Best weed management strategies for dryland cropping systems with cotton' focused on improved management of the key weeds, bladder ketmia, sowthistle, fleabane, barnyard grass and liverseed grass.In Phase III 'Improving management of summer weeds in dryland cropping systems with cotton', more information on the seed-bank dynamics of key weeds was gained in six pot and field studies. The studies found that these characteristics differed between species, and even climate in the case of bladder ketmia. Species such as sowthistle, fleabane and barnyard grass emerged predominately from the surface soil. Sweet summer grass was also in this category but also had a significant proportion emerging from 5 cm depth. Bladder ketmia in central Queensland emerged mainly from the top 2 cm, whereas in southern Queensland it emerged mainly from 5 cm. Liverseed grass had its highest emergence from 5 cm below the surface. In all cases the persistence of seed increased with increasing soil depth. Fleabane was also found to be sensitive to soil type with no seedlings emerging in the self-mulching black vertisol soil. A strategic tillage trial showed that burial of fleabane seed, using a disc or chisel plough, to a depth of greater than 2 cm can significantly reduce subsequent fleabane emergence. In contrast, tillage increased barnyard grass emergence and tended to decrease persistence. This research showed that weed management plans can not be blanketed across all weed species, rather they need to be targeted for each main weed species.This project has also resulted in an increased knowledge of how to manage fleabane from the eight experiments; one in wheat, two in sorghum, one in cotton and three in fallow on double knock. For summer crops, the best option is to apply a highly effective fallow treatment prior to sowing the crops. For winter crops, the strategy is the integration of competitive crops, residual herbicide followed by a knockdown to control survivors. This project explored further the usefulness of the double knock tactic for weed control and preventing seed set. Two field and one pot experiments have shown that this tactic was highly effective for fleabane control. Paraquat products provided good control when followed by glyphosate. When 2, 4-D was added in a tank mix with glyphosate and followed by paraquat products, 99-100% control was achieved in all cases. The ideal follow-up times for paraquat products after glyphosate were 5-7 days. The preferred follow-up times for 2, 4-D after glyphosate were on the same day and one day later. The pot trial, which compared a population from a cropping field with previous glyphosate exposure and a population from a non-cropping area with no previous glyphosate herbicide exposure, showed that the pervious herbicide exposure affected the response of fleabane to herbicidal control measures. The web-based brochure on managing fleabane has been updated.Knowledge on management of summer grasses and safe use of residual herbicides was derived from eight field and pot experiments. Residual grass and broadleaf weed control was excellent with atrazine pre-plant and at-planting treatments, provided rain was received within a short interval after application. Highly effective fallow treatments (cultivation and double knock), not only gave excellent grass control in the fallow, also gave very good control in the following cotton. In the five re-cropping experiments, there were no adverse impacts on cotton from atrazine, metolachlor, metsulfuron and chlorsulfuron residues following use in previous sorghum, wheat and fallows. However, imazapic residues did reduce cotton growth.The development of strategies to reduce the heavy reliance on glyphosate in our cropping systems, and therefore minimise the risk of glyphosate resistance development, was a key factor in the research undertaken. This work included identifying suitable tactics for summer grass control, such as double knock with glyphosate followed by paraquat and tillage. Research on fleabane also concentrated on minimising emergence through tillage, and applying the double knock tactic. Our studies have shown that these strategies can be used to prevent seed set with the goal of driving down the seed bank. Utilisation of the strategies will also reduce the reliance on glyphosate, and therefore reduce the risk of glyphosate resistance developing in our cropping systems.Information from this research, including ecological and management data were collected from an additional eight paddock monitoring sites, was also incorporated into the Weeds CRC seed bank model "Weed Seed Wizard", which will be able to predict the impact of different management options on weed populations in cotton and grain farming systems. Extensive communication activities were undertaken throughout this project to ensure adoption of the new strategies for improved weed management and reduced risk for glyphosate resistance.

Control by glyphosate and its alternatives of glyphosate-susceptible and glyphosate-resistant Echinochloa colona in the fallow phase of crop rotations in subtropical Australia

Echinochloa colona is the most common grass weed of summer fallows in the grain-cropping systems of the subtropical region of Australia. Glyphosate is the most commonly used herbicide for summer grass control in fallows in this region. The world's first population of glyphosate-resistant E. colona was confirmed in Australia in 2007 and, since then, >70 populations have been confirmed to be resistant in the subtropical region. The efficacy of alternative herbicides on glyphosate-susceptible populations was evaluated in three field experiments and on both glyphosate-susceptible and glyphosate-resistant populations in two pot experiments. The treatments were knockdown and pre-emergence herbicides that were applied as a single application (alone or in a mixture) or as part of a sequential application to weeds at different growth stages. Glyphosate at 720 g ai ha−1 provided good control of small glyphosate-susceptible plants (pre- to early tillering), but was not always effective on larger susceptible plants. Paraquat was effective and the most reliable when applied at 500 g ai ha−1 on small plants, irrespective of the glyphosate resistance status. The sequential application of glyphosate followed by paraquat provided 96–100% control across all experiments, irrespective of the growth stage, and the addition of metolachlor and metolachlor + atrazine to glyphosate or paraquat significantly reduced subsequent emergence. Herbicide treatments have been identified that provide excellent control of small E. colona plants, irrespective of their glyphosate resistance status. These tactics of knockdown herbicides, sequential applications and pre-emergence herbicides should be incorporated into an integrated weed management strategy in order to greatly improve E. colona control, reduce seed production by the sprayed survivors and to minimize the risk of the further development of glyphosate resistance.