**2. Restriction of herbicide movement in resistant weeds**

As long as a plant biotype is susceptible to an herbicide, the biological activity resulting from the pulverization of the herbicide in the plant is dependant of the absorption and translocation of that herbicide in the plant.

Translocation is a desirable attribute because it allows the herbicide to reach both treated and untreated parts of the plant [5]. It is especially important when used for controlling plants that are able to regenerate themselves through structures such as bulbs, rhizomes, stolons, and tubers. If, for some reason, the herbicide fails to reach these structures due to restriction of movement, the plants are not going to be controlled and will therefore be resistant.

**1. Introduction**

160 Herbicide Resistance in Weeds and Crops

tion of these to the site of action [1].

the effectiveness of the product [4].

tion of that herbicide in the plant.

with susceptible weeds are lacking in the literature.

Herbicides can penetrate plants through their aerial structures (leaves and stems), subterraneous (root, rhizome, stolon, and tuber), and young structures such as radicles and caulicles. The main route of penetration of the herbicides in the plant is a function of a series of intrinsic and extrinsic (environmental) factors. Absorption of herbicides by roots or leaves is influenced by the availability of the products at the sites of absorption and environmental factors (temperature, light, relative humidity, and soil moisture), which also influences the transloca-

Among the biochemical and physiological mechanisms, the change in the absorption, translocation, or metabolism of resistant weed biotypes has been reported on several species for different herbicides. These resistance mechanisms have been studied over the last years, allowing the development and improvement of analytical techniques to diagnose this type of resistance [2]. However, an up-to-date, organized description and standardization of research procedures and methodology on the use of radioisotopes for detection of resistant weeds, through different mechanisms of absorption, translocation, and metabolism in comparison

Radioisotopes are used on several research areas, such as for the metabolism of drugs and pesticides, environmental studies to determine biological routes and mass balance studies for organic compounds, and the ones that are most frequently used are tritium and 14C. The method for using radiolabeled herbicides may be quantitative or qualitative, allowing associating the resistance to the reduced absorption and/or translocation, and/or to the accelerated metabolism in several weed species [3]. Therefore, it is important to understand concepts and measurement units of the main analytical techniques that use labeled molecules with 14C to study the biochemical and physiological resistance mechanisms to herbicides, as well as for studies that evaluate the destination of these molecules on the environment. Understanding these mechanisms is fundamental for management alternatives to be planned or to improve

Considering the above, the objective of this chapter was to conduct a description of the research procedures and the methodology related for detection of resistant weeds using 14C-herbicide

As long as a plant biotype is susceptible to an herbicide, the biological activity resulting from the pulverization of the herbicide in the plant is dependant of the absorption and transloca-

Translocation is a desirable attribute because it allows the herbicide to reach both treated and untreated parts of the plant [5]. It is especially important when used for controlling plants that are able to regenerate themselves through structures such as bulbs, rhizomes, stolons, and tubers. If, for some reason, the herbicide fails to reach these structures due

absorption, translocation, and metabolism compared with susceptible weeds.

**2. Restriction of herbicide movement in resistant weeds**

Weed Science Society of America (WSSA) defines herbicide resistance as the inheritable ability of a plant biotype to survive and reproduce following exposure to an herbicide dose that would normally be lethal to the wild type [2].

Resistance conferred by the restriction of herbicide movement mechanism is classified as non-target-site resistance (NTSR). Weeds that are resistant due to this mechanism commonly show higher foliar retention and reduced translocation, reducing the amount of herbicide that reaches the target, making it insufficient to exercise control over the weed.

Goggin et al. [6] employed 14C-labeled 2,4-dichlorophenoxyacetic acid (2,4-D) to study resistance in two wild radish (*Raphanus raphanistrum* L.) biotypes from Australia. When comparing with a susceptible population, results showed that the resistance is due to an inability to translocate 2,4-D out of the treated leaf. Further investigation is necessary, but the authors suggest that the restriction of herbicide movement could be due to an alteration in the activity of a plasma membrane ABCB-type auxin transporter responsible for facilitating long-distance transport of 2,4-D.

Reduced translocation was reported as the cause of resistance to paraquat in two populations of *Hordeum leporinum* [7]. The inability to translocate paraquat out of the treated leaves was verified with the use of <sup>14</sup>C-labeled paraquat comparing the two resistant populations with a susceptible one, all from Australia.

Riar et al. [8] studied three barnyardgrass (*Echinochloa crus-galli*) biotypes from the United States with cross-resistance to imazamox, imazethapyr, penoxsulam, and bispyribac-sodium. The authors concluded that reduced translocation could contribute to imazamox and bispyribac-sodium resistance for two out of three biotypes.

Regarding glyphosate, the world's most important and widely used herbicide, NTSR has been reported as one of the most widespread type of resistance [9].

Glyphosate is a foliar applied herbicide which follows a source-to-sink pattern and kills plants through the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which are most highly expressed in the meristems and flowers of plants [10]. Since it is applied on the shoots, it must traverse the non-living structures of the leaf cuticle and the cell walls of the epidermis, apoplast, and mesophyll prior to accessing the phloem for transport to sink tissues [11]. Glyphosate's great ability to translocation in the plant reaching vital areas such as the roots and shoot meristems is one of the characteristics that makes it so important and efficient, but it also makes it highly dependent on herbicide movement.

Ferreira et al. [12] reported an increase in foliar retention in hairy fleabane (*Conyza bonariensis*) resistant to glyphosate. Reduced translocation was reported to be one of the mechanism conferring resistance to glyphosate in rigid ryegrass (*Lolium rigidum*) [13] and perennial ryegrass (*Lolium perenne*) [14].

The mechanism of glyphosate absorption into plant cells is not well understood. There appears to be two different mechanisms of absorption. One is an active system that pumps the herbicide into plant cells, possibly via a phosphate transporter, and operates at low concentrations. Other may be a passive mass flow system which is gradient dependent (**Figure 1**).

The exact mechanism that promotes the reduction of cellular absorption and translocation of glyphosate in resistant weeds is not clear yet. Shaner [10] described four potential mechanisms that may cause the restriction of glyphosate movement (**Figure 2**): (1) alteration in a putative phosphate transporter responsible for the active cellular absorption of glyphosate, in a way that the transporter is no longer present or no longer recognizes glyphosate, resulting in reduced absorption and translocation; (2) evolution of a new transporter that pumps glyphosate into the vacuole, thus sequestering the herbicide and preventing it from reaching either the chloroplast or the phloem; (3) evolution of a new transporter that actively pumps glyphosate out of the cell into the apoplast; or (4) evolution of a transporter at the chloroplast envelope that pumps glyphosate out of the chloroplast, preventing the herbicide from reaching its target site.

In order to study glyphosate resistance, 31P nuclear magnetic resonance (NMR) spectroscopy studies were employed to track glyphosate movement and metabolism in resistant and susceptible biotypes of horseweed (*Conyza canadensis*), and the results showed that the rate of vacuole accumulation of this herbicide is faster and occurs to a greater extent in the resistant biotype rather than in the susceptible [15].

These results have been confirmed in different glyphosate-resistant *Lolium* spp. biotypes collected on three different continents [16], pointing to vacuolar glyphosate sequestration as the primary mechanism of resistance in these biotypes.

**Figure 1.** Proposed mechanisms of glyphosate absorption into plant cells. G, glyphosate (the size of the letter indicates relative size of glyphosate pool). (1) Active absorption of glyphosate into cell. (2) Passive diffusion of glyphosate into the cell. Arrows indicate direction of movement of glyphosate pools into and out of the cell, chloroplast, and vacuole. Source: Shaner [10].

Procedures for Detection of Resistant Weeds Using 14C-Herbicide Absorption, Translocation, and Metabolism http://dx.doi.org/10.5772/68092 163

**Figure 2.** Potential mechanisms for reduced glyphosate cellular absorption in glyphosate-resistant (GR) biotypes. (1) Inhibition of active absorption by a modification of active transporter. (2) An active transporter that pumps glyphosate into the vacuole. (3) An active transporter that pumps glyphosate from the cell into the apoplast. (4) Inhibition of glyphosate absorption into the chloroplast by a transporter that pumps it out of the chloroplast. G, glyphosate. Source: Shaner [10].
