Application History, Mode of Action and Resistance

## **Chapter 1**

## Liquid Chromatography Tandem Mass Spectrometry after the QuEChERS Method for Determining 20 Herbicide Residues in Wheat and Flour

*Islam R. Ghoniem*

### **Abstract**

Agriculture is the backbone of the economy and social structure, and it plays a critical part in each country's overall growth. Because of the significant food gap that exists in several vital crops, wars, and the continual expansion in the population, the role of agriculture products has recently become critical. The world is currently experiencing a severe food shortage, estimated to be over 60% of its strategic food requirements. As a result, there is a need to increase the area of farmed land in order to satisfy the growing population and raise food demand by eliminating weeds that can reduce agricultural output. Weed is an unwanted plant (one that grows in the incorrect area) that reduces crop output. Herbicides are a type of pesticides that are used to kill weeds and increase crop output. As a result, herbicide residues on food, particularly cereals, must be determined. In this study, the QuEChERS approach for determining herbicides in wheat and corn by direct injection to Exion HPLC coupled with a SciexQtrap API 6500+ LC–MS/MS system using an electrospray positive ionization (ESI+) at lower concentrations without utilizing acids or clean-up is evaluated, optimized, and validated in this work.

**Keywords:** QuEChERS, LC–MS/MS, agriculture, herbicides, cereals

### **1. Introduction**

As a result of the continuing expansion in the world's population, the use of pesticides in contemporary agriculture has become one of the most critical necessities for meeting society's food needs, and millions of tons of pesticides are used annually for this purpose [1]. Pesticides are one of the most commonly utilized substances on the planet. Despite their usefulness, pesticides are one of the most dangerous compounds that damage humans, animals, and surface water in particular [2]. When pesticides are used in large quantities in the environment, they have the potential to harm the environment, especially human health [3]. Weeds are any unwanted plants that grow in a field and threaten crops, animals, or human health. Herbicides are a type of pesticide that kills weeds to protect plants and boost crop output [4]. Herbicides are frequently employed in agriculture and turf management in the landscape. They account for almost 70% of all agricultural pesticide use worldwide [5]. Herbicides can cause everything from skin rashes, nausea, and weariness to headaches, chest pain, and even death in some cases.

Pesticides are used in roughly 2 million tons over the world, with 47.5% being herbicides, 29.5% being insecticides, 17.5% being fungicides, and 5.5% being other pesticides [6]. China, the United States, Argentina, Thailand, Brazil, Italy, France, Canada, Japan, and India are the top ten pesticide-using countries in the world [7]. Furthermore, it is predicted that by 2020, global pesticide usage will have increased to 3.5 million tons [8]. Africa's economy is heavily reliant on agriculture, with approximately 59% of the population relying on it for a living [9]. Despite this, the African continent contributes 2–4% of the global pesticide market share and has the lowest pesticide usage rate in the world [9]. Food demand is expected to rise rapidly in the next three decades as a result of the rising population, and demand for pesticides, herbicides, and fungicides are also expected to rise [10].

The quick, easy, cheap, effective, rugged, and safe (QuEChERS) approach was used to detect this chemical and estimate its concentration [11–18]. In terms of analysis costs and turnaround time, multiresidue methods are the most efficient way for herbicide analysis. The majorities of the procedures have multiple steps and use a lot of different solvents and reagents. In terms of good recovery, short duration of analysis, cheap cost, and safety, the QuEChERS approach combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) was determined to be the optimal combination for determining herbicides in some foods. Because of the more ionized herbicides, LC–MS/MS is now commonly employed [12–14, 19, 20].

Controlling herbicide residues in food items through monitoring and a maximum residue limit (MRL) setting is critical for consumer safety. The Codex Alimentarius Commission (CAC) and the European Commission determined MRLs based on residues in food that must be found at safe levels for consumers [21, 22]. In the European Union (EU) legislation, the lowest limit of analytical quantitation (LOQ ) is specified as the MRL that equals 0.01 mg/kg if the MRL obtained by different trials is not safe for consumers [22].

Yingying et al. [23] improved and validated a QuEChERS technique for determining florasulam and pyroxsulam residues in wheat grain and straw using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The approach was tested on cereals such as oat, millet, corn, and rice. Average recoveries ranged from 76 to 113%, with RSDs ranging from 2 to 15%. TAO et al. [24] developed an efficient method for determining various phenoxy acid herbicide residues in grains. The study of phenoxy acid herbicides in rice, corn, and wheat was optimized using a QuEChERS approach combined with high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Renata Raina et al. [25] developed pesticide residue testing procedures for a wide range of foods, including cereal-based foods, nutraceuticals and associated plant products, and infant feeds. Many processed consumer products are made from these grain, fruit, vegetable, and plant-based components. A modified QuEChERS approach has been applied for cereal and nutraceuticals, which are dry sample products, with additional steps to allow wetting of the dry sample matrix and subsequent cleanup using dispersive or cartridge format SPE to eliminate matrix effects.

*Liquid Chromatography Tandem Mass Spectrometry after the QuEChERS Method… DOI: http://dx.doi.org/10.5772/intechopen.104964*

Wheat is a widely cultivated crop whose seed is a grain that is consumed as a staple food all over the world. The most important wheat types are common wheat (*Triticum aestivum*), durum wheat (*Triticum durum*), and club wheat (*T. aestivum*) (*T. compactum*). Wheat is grown as a commercial crop because it generates a high yield per unit area, thrives in a temperate climate with a short growing season, and produces versatile, high-quality flour. Wheat flour is used to produce bread, pasta, cereal, pastries, cookies, crackers, muffins, tortillas, and pitas, among other things. Wheat is the second most widely grown cereal grain after maize, and its global trade volume exceeds that of all other crops combined. The total global wheat production in 2020 was 760 million tons. China, India, and Russia are the world's three greatest individual wheat producers, accounting for over 41% of global wheat production. Individually, the United States is the world's fourth-largest wheat producer. If the European Union were counted as a single entity, it would produce more wheat than any other country save China [26].

The current study's technique describes the examination of a mixture of herbicides in various matrices after extraction using the QuEChERS technology. The QuEChERS technique is evaluated, optimized, and validated for the determination of 20 herbicides in wheat and flour by direct injection to LC–MS/MS at lower concentrations without the use of acids or clean-up in this study. Exion HPLC paired with the SciexQtrap API 6500+ LC–MS/MS System was used to determine these chemicals utilizing electrospray positive ionization (ESI+).

#### **2. Experimental method**

#### **2.1 Instrumentation and analysis**


The injection volume was 2 μL and the column temperature was 40°C. The pesticides are separated using a Gradient mixing program of 10% 50 mM ammonium format in deionized water, which is mostly used for positive ionization mode, with 0.1% formic acid as eluent A and methanol as eluent B at 300 μL/min flow rate starting by A bottle 60% for 1 min, changed continuously till 11.5 min to be 10% for 0.5 min, changed progressively till 12 min to be 0% for 2 min and returned to 60% from A in min 14 for 2 min to be 16 min complete run time for every one of the 20 pesticides. Electrospray ionization in the positive ion mode with multiple reactions monitoring (MRM) mode was used to complete the MS/MS analysis.

The LC mobile phase stock solution was 50 mM ammonium formate solution in methanol/water (1:9), and the LC mobile phase was 10 mM ammonium formate solution in methanol/water (1:9), dilute 200 mL of LC mobile phase stock solution with 800 mL methanol/water (1:9), adjust the pH to about 3.78 ± 0.02 with ammonia solution (33%), and then add 100 mL methanol and LC mobile phase was 10 mM ammonium formate solution in methanol/water (1:9), dilute 200 mL of LC mobile phase stock solution with 800 mL methanol/water (1:9), the pH should be 4 ± 0.1, adjust as needed.

#### **2.2 Reagents and materials**

Atrazine (99%), clodinafop (free acid) (99%), clodinafop-propargyl ester (99%), cycloxydim (98.8%), diphenamid (99%), fenoxaprop-P-ethyl (R-enantiomer) (99%), haloxyfop-2-ethoxyethyl ester (99%), haloxyfop (free acid) (99%), imazamethabenz-methyl (97.4%), imazethapyr (99%), mesosulfuron-methyl (98%), metolachlor (98.5%), metribuzin (99.5%), metsulfuron-methyl (99.5%), pendimethalin (98.8%), quizalofop-ethyl (99.3%), quizalofop-P-ethyl (98.4%), simazine (98%), sulcotrione (99%), and triclopyr butotyl (99.1%) were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Methanol (99.9%) HPLC grade was purchased from J.T. Baker (PA, USA). Acetonitrile 99.9% -HPLC grade was purchased from J.T. Baker (Pennsylvania, USA). Deionized water (<18M\_cm resistivity) was performed in the laboratory using a Millipore (Billerica, MA, USA) MilliQ water purification system. Ammonia solution (33%) was purchased from Riedel-de Häen (Seelze, Germany). Formic acid (98–100%) was purchased from Riedel-de Häen. QuEChERS extraction kits.—5982–5650 was purchased from Agilent {Agilent QuEChERs salts and buffers are prepackaged in anhydrous packages(4 g MgSO4; 1 g NaCl; 1 g trisodium citrate dihydrate;0.5 g disodium citrate sesquihydrate)} (Santa Clara, CA, USA).

#### *2.2.1 Standard preparation*

Stock solutions (1000 μg/mL) of each pesticide standard were prepared by dissolving atrazine in toluene, clodinafop (free acid) in toluene, clodinafop-propargyl ester in toluene, cycloxydim in toluene, diphenamid in toluene, fenoxaprop-P-ethyl (R-enantiomer) in toluene, haloxyfop-2-ethoxyethyl ester in toluene, haloxyfop (free acid) in toluene, imazamethabenz-methyl in toluene, imazethapyr in methanol/ toluene (3:7 v/v), mesosulfuron-methyl in toluene/acetone (7:3 v/v), metolachlor in toluene, metribuzin in toluene, metsulfuron-methyl in toluene, pendimethalin in toluene, quizalofop-ethyl in toluene/acetone (8:2 v/v), quizalofop-P-ethyl in toluene, simazine in acetone, sulcotrione in toluene/acetone (9:1 v/v), and triclopyr butotyl in toluene/acetone (9:1 v/v). All stock solutions were prepared and kept at −20 ± 2°C. Working mixtures of the examined pesticides (5 g/mL each) and calibration mixtures of concentration levels 0.01, 0.05, 0.1, and 0.5 g/l were made by diluting suitable aliquots of the stock solutions with methanol kept at 4 ± 2°C.

#### *2.2.2 Spiked samples preparation*

The flour and wheat were purchased at the local market. The samples were thoroughly ground before being homogenized in an electric mill. In recovery experiments, wheat and flour samples were spiked with a suitable amount of working mixture standard solution.

#### **2.3 Extraction procedure**

Herbicide residues in wheat and flour were extracted using the QuEChERS technique for herbicide residue analysis. Initial single-phase extraction of 2 g of homogenized sample with deionized water in a 50 mL PFTE centrifuge tube, 10 mL deionized water added, tube closed and shaken vigorously by geno grinder at 500 rpm for 1 min, and then with acetonitrile in a 50 mL PFTE centrifuge tube, 10 mL acetonitrile added, tube closed and shaken vigorously by geno grinder at 500 rpm for 1 min.

*Liquid Chromatography Tandem Mass Spectrometry after the QuEChERS Method… DOI: http://dx.doi.org/10.5772/intechopen.104964*

After that, a mixture of Agilent QuEChERs salts and buffers is added to the tube, which is then closed and rapidly shaken for 1 min at 500 rpm with a geno grinder, then centrifuged for 5 min at 4000 rpm (3430 rcf). The cleaned extract is filtered using syringe filters (0.45 m) and transferred to a PP vial after centrifugation. Finally, the liquid sample was injected into a liquid chromatography-mass spectrometry (LC–MS/MS) apparatus.

#### **3. Result and discussion**

The analysis technique used in this study was created with the goal of detecting and quantifying as many herbicides as feasible in a single run. When deciding which herbicides to include, two criteria were used: (1) herbicides registered for crop protection by local authorities, and (2) searching the literature for commonly studied compounds. Acidification was used in this method in the form of buffer citrate salt (trisodium citrate dihydrate and disodium citrate sesquihydrate), which served two purposes: (1) improving extraction by converting conjugate of some herbicide to neutral form, thereby increasing recovery, and (2) adjusting pH 5–5.5, thereby increasing herbicide sensitivity. The herbicides were determined using LC–MS/MS with an ESI source and MRM mode, which offered a highly selective and sensitive technique. All of the target analytes were ionized to (M + H) + form in the positive mode, according to the physicochemical parameters of the target. The positive mode was chosen since it works well for the majority of analytes. Herbicides can be quantified directly using the LC–MS/MS approach, which does not require any derivatization and requires minimal cleaning. A QuEChERs approach was used to design the method for 20 herbicides. The chromatograms obtained for each compound, as shown in **Figure 1**, were determined with sufficient precision and accuracy. The approach was tested on a total of 20 herbicides, each with a distinct retention time of 16 min. Although an excellent summary of the LC–MS/MS methods used for herbicides was offered, it did not cover all herbicides discussed in this study, and only a few studies for determining several classes of herbicides in wheat and flour in a single multiresidue approach were published.

#### **Figure 1.**

*The approach was validated using chromatograms produced by liquid chromatography tandem mass spectrometry (LC–MS/ MS) with electrospray ionization (ESI) in positive mode and MRM mode for 20 herbicides used in the study.*

### **3.1 Mass spectrometry study of 20 herbicides**

To discover the best precursor, product ions, and operating conditions, 20 herbicides were injected directly into the LC–MS/MS system in 1:1 methanol at a concentration of 0.1 μg/mL. **Table 1** summarizes the precursor and product quantification and confirmation ion pairs, as well as the declustering potential and collision energies.

#### **3.2 Method validation**

The developed method was validated in compliance with the document's method validation standards SANTE/2020/12830 document [27].

#### *3.2.1 Linearity of calibration curves*

Plotting the detector response area ratio vs. the concentration of the analytical solutions at various concentration levels ranging from 0.001 to 0.1 μg/mL established the linearity of the calibration curve of 20 herbicides. The calibration curves were prepared using sex levels of calibration standards in the concentration ranges of 0.001, 0.002, 0.005, 0.01, 0.05, and 0.10 μg/mL. Plotting the peak area vs. concentration yielded a calibration curve. According to European guidelines, the analytes showed linear behavior in the studied concentration levels with a correlation


*Q1: Precursor ion, Q3: Product ion, DP = Decluster Potential [V], EP = Entrance Potential [V], CE = Collision Energy [V] and CXP = Collision Cell Exit Potential [V].*

#### **Table 1.**

*List of herbicides and MRM parameters in LC-MSMS-ESI positive mode.*

*Liquid Chromatography Tandem Mass Spectrometry after the QuEChERS Method… DOI: http://dx.doi.org/10.5772/intechopen.104964*


#### **Table 2.**

*R2 values for the 20 herbicides.*

coefficient (r2) greater than 0.99 as shown in **Table 2**, indicating that all analytes were within the acceptable range and the coefficient of variation (CV percent) for each calibration point was less than 20% [28].

#### *3.2.2 Matrix effect*

A matrix effect research was carried out on blank wheat and flour samples using a conventional herbicide mixture of 20 herbicides. To correct for matrix-induced suppression in LC–MS/MS, matrix-matched standard calculations were performed at 0.01, 0.05, and 0.1 mg/kg.

The following formula was used to make the calculations:

Matrix effect % = ((peak area STD in matrix/peak area STD in solvent) −1/100).

To compensate for the matrix effect suppression on the results, 450 μL of blank sample was fortified with 50 μL of 0.5 μ g/mL standard solutions to achieve 0.05 μg/mL concentration levels [29].

#### *3.2.3 Quantification limit (LOQ )*

The quantitation limit of all of the substances investigated was determined to be 0.01 mg/kg for all of them. The validity of this level has been established in accordance with the SANTE guidelines [28] and EU 396/2005 regulation [22].


**Table 3.** *Average recoveries and coefficient of variation (CV%), on wheat and flour samples were spiked at 3 different concentration levels 0.01, 0.05 and 0.1 mg/kg.*

#### *New Insights in Herbicide Science*

*Liquid Chromatography Tandem Mass Spectrometry after the QuEChERS Method… DOI: http://dx.doi.org/10.5772/intechopen.104964*

#### *3.2.4 Accuracy and precision*

Six replicate spiked wheat and flour samples were analyzed at three distinct levels (0.01, 0.05, and 0.1 mg/kg) to acquire accuracy and precision. The percentage of the money recovered ranged from 71–105%. The precision was based on the corresponding relative standard deviations, and the trueness was based on the mean recoveries (RSD). **Table 3** shows the recoveries, means, and RSD percent. Reproducibility (interday accuracy and precision) was tested over a two-month period at a fortification level of 0.05 mg/kg and found to be less than 12%.

#### **4. Conclusion**

The current study developed a multiresidue technique of testing for 20 herbicides with a limit of determination of 0.01 mg/kg, which meets the EU MRLs for wheat and flour farm goods. Two MRMs for quantification and conformation were chosen based on the optimal declustering potential and collision energy, and the mass spectrometric parameters were tuned to give the best sensitivity. In terms of approved recovery, short duration of analysis, cheap cost, and safety, the QuEChERS method followed by Exion HPLC and a SciexQtrap API 6500+ LC–MS/MS system using an electrospray positive ionization (ESI+) technology was shown to be the optimal combination for determining the 20 herbicides. Herbicides can be quantified directly using the LC–MS/ MS method, which does not require any derivatization and requires minimum cleanup with a total runtime of 16 min. The majority of the chemicals tested had recovery rates ranging from 71–105%, with relative standard deviations of less than 12%, indicating adequate precision. Recovery trials on six replicates of spiked blank wheat and flour samples at 0.01, 0.05, and 0.1 mg/kg were used to determine the method's precision and accuracy. The developed assay was linear over a concentration range of 0.01–0.5 μg/mL, with a correlation coefficient of more than 0.99 at the 0.01 μg/mL limit of quantification.

#### **Acknowledgements**

The author thanks the Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, which was awarded an international Accreditation Certificate in all analyses by the Finnish Branch (FINAS) of the European Accreditation Center for Laboratories (EAL).

### **Authors' contributions**

The study's inception and design were aided by the author. Islam R. Ghoniem was in charge of material preparation, data collecting, and analysis. Islam R. Ghoniem wrote the first draught of the manuscript and provided feedback on prior draughts. The final manuscript was read and approved by the author.

#### **Funding**

Not applicable. The author works at the Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, which supported this research.

## **Data availability**

On reasonable request, the corresponding author will provide the datasets used and/or analyzed during the current work.

## **Declarations**

The author declares no competing interests.

## **Author details**

Islam R. Ghoniem Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, Agricultural Research Center, Giza, Egypt

\*Address all correspondence to: islam\_refaat@qcap-egypt.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Liquid Chromatography Tandem Mass Spectrometry after the QuEChERS Method… DOI: http://dx.doi.org/10.5772/intechopen.104964*

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## **Chapter 2** Modes of Herbicide Action

*Suman Bagale*

### **Abstract**

Weed Management is one of the most important crop intervention practice to counter crop loss. Different physical, mechanical, biological, and chemical methods are employed for the successful management of weeds. Among these chemical weed management practices focus on managing weeds using several chemical formulations which are commonly known as herbicides. Herbicides control the weed species through interference, mitigation, and disruption of the biochemical and physical processes of a cell. When herbicides are applied to a plant, it gets absorbed through plant surfaces and gets translocated to the specific site of action where it produces toxicity in the physiological and biochemical processes and ultimately check the growth and development of plant species. The sequential process from the introduction of herbicides to till it kills a plant is known as herbicides modes of action. The herbicides modes of action can be studied on nine different headings where the chemical group present in each herbicide acts on specific sites and interferes with the normal functioning of such sites ultimately checking the growth and development of a plant. This chapter is aimed at decoding the specific herbicide action in relation to its chemical family, translocation, action mechanism, and injury produced in the weed species.

**Keywords:** chemicals, glyphosate, herbicides, herbicide resistance, novel modes

## **1. Introduction**

The global demand for food crops is rapidly increasing with the increase in the world's population, on other hand production of crops is constrained by several factors such as weeds pest, insects, and diseases, among these all weeds are one of the major factors that can cause loss of productivity of field crops. Weeds are any plants that are grown in undesirable places and compete with crops plants for nutrients, sunlight, moisture, and other growth factors. Anything that grows in unintended places are generally referred to as weeds. According to Gharde [1], weeds are notorious yield reducer than pests, disease, and insects, which are thought to cause an estimated loss of 11billion USD in 10 major crops, which causes 31.4% loss in soybean, 30.8% in green grams, 25.3% in maize, 21.4% in mustard, 18.6% in wheat and 21.4% in directseeded rice. Reduction of crop yield in crops is due to competition between crops and weeds for space and other growth factors. Yield loss of crops due to weeds depends on several factors such as weed emergence time, weed density, types of weeds, competition ability of crops, and if left uncontrolled, they can cause 100% loss in crop production [2]. The successful and strategic management of weeds can decrease the yield loss significantly, which can ensure more grain harvest. The management

of weeds has become one of the most researched aspects in the field of crop science. In small farm size, it can be managed through hand weeding or mechanical weeding machines like cono-weeder and weed-roller whereas its management in the large farms has become a problematic issue. Mostly in the case of commercial cultivation weeds species are mostly managed by using different pre-emergence and post-emergence herbicides. With the increase in the weed resistance towards these herbicides, there is a need for weed science research focusing on herbicide resistance and herbicide mode of action. In a study carried out by Heap [2], it was observed a total of 511 unique cases of herbicide resistance belonging to 266 weed species (153 dicot and 133 monocots) have been reported globally out of which major herbicide-resistance weed species were reported in wheat followed by maize, rice, soybean, spring barley, and cotton. Herbicide-resistant weed populations are rapidly evolving as the process of natural selection and development of traits by weeds to escape the action of herbicides. The graphs show that, herbicide resistance has been steeply increasing from 7 cases in 1975 to 509 cases at the end of 2020. The major herbicide resistant traits were observed in the weed family belonging to Poaceae or grass. The five major weed families Poaceae, Asteraceae, Brassicaceae, Amaranthaceae, and Chenopodiaceae account for 70% of total herbicide resistance cases though they only include 50% of total principal weeds [3]. More weed species are resistant to ALS inhibitors, with the reported 160 species, which is followed by Photosystem II inhibitors. Glyphosate one of the most common post-emergence herbicide used as broad broad-spectrum control of weeds has become less effective due to intensive use of herbicide leading to the quick emergence of glyphosate-resistant biotypes [4]. Mitigating herbicide resistance has become one of the most important things to consider during crop production. The herbicide resistance in plants can be somehow coped with by introducing different herbicides of the varied mode of action, crop rotation, and using integrated weed management practices in crops. The successful management of herbicide resistance in input-intensive agriculture can be combated by diversifying the herbicide products, cultivating crops with combined herbicide resistance, increasing reliance on preemergence herbicides than post-emergence herbicide, breeding weed-competitive crop cultivars, and advances in site-specific and precision weed management [5].

The advancement in the field of genetics, plant physiology, chemistry, and plant science has made open to many researchers to understand the basis and mechanism of herbicide resistance. Herbicide resistance mechanisms can be target site resistance, non-target site resistance, cross-resistance, and multiple resistance [6]. The target site herbicide resistance is due to the mutation in genes encoding herbicide enzymes, nontarget herbicide resistance is due to the reduced amount of herbicide active ingredients through reduced absorption or translocation. The cross-resistance is due to the use of several herbicides with the same mode of action and multiple resistance is due to two or more herbicide resistance mechanisms in response to a sequential selection of herbicides with a different mode of action. The herbicide mode of action explains how the active ingredients present in commercial herbicide formulation act on plants. The mode of action of herbicide is variable based on the chemical composition of their active ingredients and the weeds species in which they act on. Some herbicides act on plants through the root system, some act on photosynthesis and photosystem, and some herbicides are found to act on the cell membrane and enzymatic pathways. Understanding the mode of action of herbicides is important for the management, classification, organization, and hierarchy of the herbicides as it also provides an insight into herbicide resistance, which has become a problem in sustainable agricultural management [7].

#### *Modes of Herbicide Action DOI: http://dx.doi.org/10.5772/intechopen.105356*

Herbicide enters into a plant system through several different mechanisms. These acting mechanism differs in between the herbicide in relation to the chemical nature that is present in the active ingredients of the herbicide. The herbicide mode of action discusses on the sequence of events from the introduction of herbicide in the environment till its kills the plant through toxicity produced by the chemicals presents in the active ingredients of the herbicide, whereas the herbicide mechanism of action discusses on physiological and biochemical changes caused by the herbicide within plant system. Understanding the mode of herbicide action helps to relate the chemistry of herbicide and the physiology that exists within a plant. The knowledge of acting mechanisms helps to cope with the problem of herbicide resistance and helps to maximize the efficacy of herbicide during weed management. The study incorporates the parts of how herbicide gets absorbed in the plant surface and how they act on the physiology of weed plant and injure them to eliminate them from the competition with crop species. The knowledge on general chemistry, plant physiology, genetics, and plant science can help to decipher the roles that lie beyond the herbicide mode of action. In general herbicides are classified as pre-emergence and post-emergence herbicides. In pre-emergent herbicide the mode of action is principally through absorption from root zones, whereas in the case of post-emergent herbicide the mode of action is mainly through absorption from foliar parts. In general, to acts as an herbicide on a plant, it must pass through certain sequential stages of contact, absorption, movement, toxicity, and death of weed species and the mode of herbicide action to produce injury includes inhibition, disruption, interruption, and mitigation of regular growth of weed species [8]. The exploration of the mode of herbicide action is dynamic and new modes of action of herbicide has been constantly adding, which is helping for the discovery of new herbicide. Based on the site of action of herbicide and mode of action altogether 22 types of herbicide action have been developed. Therefore, understanding the mode of herbicide action can substantially help in understanding the mechanism of herbicide resistance and exploring new strategies to cope with herbicide resistance. So, the chapter focuses on different herbicide mode of action in relation to their chemical family, mechanism of action, translocation, and toxicity. In recent years, a perennial weed of *Roegneria* genus commonly known as wild rye, which is widely distributed in China has shown tolerance to ACCase inhibitor herbicides like fenoxaprop, clodinafop and pinoxaden [9]. It was observed that the ACCase activity were increased by 1.46 and 1.34-fold in wild rye and wheat plant after 72 hours of fenoxaprop treatment than at 0 hrs of treatment as shown in **Figure 1**. It was suggested that the enhanced activity of ACCase is due to enhanced metabolism of herbicide, leading to herbicide tolerance.

### **2. Lipids synthesis inhibitors**

These kinds of herbicides are those which cause disruption in lipid synthesis and check the growth of plants through rupture of the cell wall and cell oozing. The herbicide having group 1 site of action falls under these categories, where herbicide inhibits Acetyl CoA Carboxylase (ACCase) enzyme which is required for fatty acids synthesis that forms a part of phospholipid bilayer in the cell membrane of plant cells. The inhibition of (ACCase) enzymes restricts the formation of cell wall in meristematic regions and ultimately kills the plant cell. The (ACCase) inhibitors herbicides are used for the selective control of weed species, which are found to have resistance with glyphosate herbicide [10]. The mechanism begins when the herbicide comes in

#### **Figure 1.**

*Increase in the enzymatic activity of ACCase with increase in treatment time in wild oat and wheat plant depicting tolerance to fenoxaprop [9].*

contact with plant species and it gets translocated in the meristematic region through phloem where it inhibits the meristematic activity producing necrotic symptoms in the growing tissues after one week of application [10, 11]. The chemical family of this herbicide includes aryloxyphenoxylpropinate, cyclohexanedione, and phenylpyrazole which are applied as post-emergence herbicides to control grassy weeds in broadleaf crops [11]. The common herbicides include fenoxaprop, fluazifop, diclofop, quizalofop, clethodim, sethoxydim, and Pinoxaden. These groups of herbicides are applied through foliar spay and translocated through phloem in meristematic regions. The major injury includes plants turn brown, chlorotic symptoms can been seen in the leaves, and vein browning and purpling can be seen after 3-4 days of herbicide application. (ACCase) inhibitor herbicides are short-lived in soil, relatively low solubility in soil, and used relatively in low rates. They have low leaching potential and are found to be less hazardous to the environment. (ACCase) inhibitors herbicides are found to have resistance against 43 grass weeds species [12].

#### **3. Amino-acid synthesis inhibitors**

These kinds of herbicide are found to have two sites of action. They belong to group 2 Acetolactate Synthetases (ALS) that catalases the synthesis of branched-chain amino acids, such as leucine, isoleucine, and valine. The inhibition of these enzymes restricts the biosynthesis of these amino acids, which are the essential part of protein necessary for cell membrane formation. The ALS inhibitors are found to have effect on the reproduction of some plant species such as inducing male sterility and their potency, which can act extremely at low concentrations, and the rapid evolution of resistance to these herbicides in some plants [13, 14]. They are the largest group of herbicides that are post-emergence selective in nature. The chemical ingredients are of these herbicides are absorbed through roots and foliage and translocated through both xylem and phloem. The major injury of ALS inhibiting herbicide includes interveinal chlorosis, purpling and root pruning. The major chemical family includes sulphonyl urea, Imidazolinone, Sulfonylurea, and Triazolopyrimidine [15]. The common

#### **Figure 2.**

*The control of callus biomass is more effect in lower concentration than in higher concentration of amino acid inhibitor herbicide imazapic [16].*

herbicides include imazamox, imazapic, imazaquin, imazethapyr, nicosulfuron, metsulfuron, triasulfuron, chlorsulfuron, rimsulfuron, prosulfuron, pyroxsulam, diclosulam, and flumetsulam. In a study conducted by Dor [16], the tissue culture of broomrape was found to be more sensitive to imazapic in which a concentration of 0.05μM significantly decreased the biomass and a concentration of 10μM caused blackening of died callus, which suggests that free amino acid content increased with the increased in the concentration of imazapic as shown in **Figure 2**.

The second group of herbicides that causes amino-acid synthesis inhibition are group 9 herbicides, which causes blockade in the production of enzymes from 5- enoylpuruvyl Shikimate-3-phosphate (EPSP) pathway. The enzymes in EPSP pathway catalyze the biosynthesis of aromatic amino acids like phenylalanine, tyrosine, and tryptophan. These amino are essential for protein synthesis and the absence of them causes cell membrane disintegration. The broad-spectrum herbicide glyphosate belongs to the chemical family which checks the EPSP pathway [17]. Herbicides having this mode of action is non-selective and absorbed through phloem tissues. They produce major injury in foliage, causing foliage discolorations stunting and killing the plant ultimately. The growth and development of the plant is check right after the herbicide application; the major symptoms appear only after a few days of application.

#### **4. Growth regulators**

Growth regular mode of mechanisms of herbicide checks the growth of plant by modulating the balances of growth hormones and regulators within the plant system. The herbicide having this mode of action belongs to two different group of the site of action. Which consists of group 4 herbicides which are generally synthetic auxins such as 2,4-D. The synthetic auxins imbalance the Indole Acetic Acid level and causes growth abnormalities in plants and leading plants to ultimate death. The major chemical family includes Phenoxy, benzoic acids, and carboxylic acids. The common herbicides in use are 2,4-D, 2,4-DB, Dichlorprop, MCPA, MCPB, Dicamba,

**Figure 3.** *Distribution of Synthetic Auxin Research from 2011 to 2013. Adapted from Todd [19].*

Clopyralid, and Picloram. These herbicides are commonly used to control broadleaf weeds in plant species, having narrow leaves as post-emergence herbicides [18]. The action of herbicide is controlled by multiple factors rather than a single factor, which disturb the nucleic acid metabolism and cell wall integrity. During recent years, the herbicide efficacy and use of synthetic auxin herbicides has been decreased due to the problem of herbicide resistance and the evolution of other herbicides. **Figure 3** depicts the research status on synthetic auxin herbicides from 2011 to 2019 published by WSSA [19].

The group of herbicides belonging to the growth regulators mode of action are chemicals that check the transport of auxin in the meristematic regions. Through the mode of action of these herbicides remain elusive, the majority of these classes of herbicide are found to check the bi-directional flow of auxin by inhibiting vesicle trafficking in plants [20]. The major chemical family of this herbicide are semicarbazone which checks the growth of broadleaf weed in grass crops. The herbicide is absorbed through roots and foliage and they translocate through xylem and phloem. The application of these herbicide during pre-post emergence gives better control of broad leaf weeds. They produce injuries in growth and reproduction abnormalities, leaf malformation, cupping of leaves, abnormal outgrowths of tissues, brittleness in stem, and stalk.

#### **5. Photosynthesis inhibitors**

Photosynthesis inhibitors disturbs the process of photosynthesis by binding with the specific binding sites in photosystem II present in the chloroplast of plant cells. Inhibition of photosynthesis could result in slow starvation of the plant and cessation of starch translocation; however rapid death occurs perhaps from the production of secondary toxic substances. Herbicides of photosystem II belong to the following

#### *Modes of Herbicide Action DOI: http://dx.doi.org/10.5772/intechopen.105356*

chemical classes: s-triazines, triazinone, uracil, urea, phenyl carbamates, anilide, cyanophenols, dinitrophenol, which are classified into three different groups 5, 6, and 7 on the basis of site of action [21]. The commonly used herbicides having this mode of action are atrazine, simazine, metribuzin, hexazinone, terbacil, bromoxynil, bromacil, pyrazone, bentazon, diuron, and linuron. The group 5 herbicides inhibit photosynthesis by binding within serine in PSII and are absorbed through roots and shoots and translocated through xylem and phloem, group 6 herbicide inhibits photosynthesis by binding with histidine, these herbicide acts as post-emergence contact herbicide, so through spraying of herbicide is recommended. The group 7 herbicides bind with protein complex present in the thylakoid membrane, which checks the transport of electron in the Electron transport Chain. The blocking of electron causes reduced carbon dioxide fixation and production of ATP and NADPH2, which are known as energy packets of respirations. These herbicide controls both narrow and broadleaf weeds. The action of these herbicides is greater during the daytime when there is full sunlight as the herbicide gets activated in presence of light. The herbicides show symptoms of chlorosis and necrosis of leaf margins which progresses towards the base of the leaves after a few hours of application.

### **6. Nitrogen metabolism inhibitors**

This mode of mechanism belongs to herbicide of group 10 having site of action at glutamate synthesis pathway. These herbicide inhibits the production of glutamate syntheses enzymes, which is essential for the conversion of ammonia to other nitrogenous compounds [22]. The blocking causes the accumulation of ammonia ions in the plant leading to increase in PH of the surrounding tissues. This causes protein disintegration, breakage of fatty acids, rupturing of cells, and overall imbalance of ion within cell sap. The major chemical family of herbicides having this mode of action are Phosphorylated Amino Acids commonly traded in the chemical name of glufosinate. These are the broad-spectrum, postemergence herbicide having limited translocation within plant systems so that through spraying of this herbicide is recommended for maximum efficiency. The major injury produced by this herbicide is foliar injury in the plant. The injury symptoms are more prevalent in the younger leaves, in contrast to the deficiency symptoms and plant stress symptoms.

### **7. Pigment inhibitors**

Pigment inhibitors are those herbicides that cause blocking in pigment formation such as anthocyanins, carotene, retinol, and chlorophyll. These herbicides belong to group 12 site of action which blocks the enzymatic activity of 4-hydrooxy phenyl Pyruvate dehydrogenase (HPPD), which plays a role in the synthesis of pigments like chlorophyll, anthocyanin, and carotene. The another group comprises of herbicides from group 13, which causes inhibition of determine synthesis that causes inhibition of synthesis of retinol and degradation of phytin pigments [23]. The major chemical family of these herbicides includes Pyrazole, Pyrazolone, and Pyridazinone. The commonly used herbicides are amitrole, clomazone, isoxaflutole, and mesotrione. The level of pigments is highly reduced leading presence of unbound lipid radicles which causes lipid oxidation, make some protein dysfunctional, and ruptures of the cell membrane. The injury produced by these herbicides is prominent as they show white or bleaching

coloration right after the application. In prolonged symptoms expression of translucent leaves, and rapid wilting of weeds species can be seen in the applied area.

#### **8. Cell membrane disrupter**

These herbicides interfere with the cell membrane activity, causing them to distort in their structure and functions. The site of action of these herbicides comprises of herbicides belonging to group 14, which causes inhibition of Protoporphyrinogen Oxidase (PPO) enzymes which catalyzes the conversion of ProtoporphyrinogenIX (PPGIX) to Protoprophyrin (PPX). The accumulation of PPGIX causes interbonding to form triplet PPGIX which in the presence of light can disrupt the hydrogen bond, break the bond between fatty acids, and degradation of protein structures. Likewise, triplet PPGIX can obstruct the biosynthesis of chlorophyll and haeme pigments [24]. The chemical family under this group of herbicides are Diphenyl Ether, Thiadiazole, Triazolinone, and Trifluro Methyl Uracil, which includes commonly used herbicides like lactofen, oxyfluorfen, acifluorfen, fomesafen, flumiclorac, and sulfentrazone.

The other group of herbicides, which acts as cell membrane disrupters are chemical belonging to Group 22, which causes inhibition of Photosystem I during photosynthesis. The major chemical family of this group are bipyridylium, which comprises of commercialized herbicides such as diquat and paraquat. These chemical causes the diversion of the electron from the PSI and generate herbicide radicals, which on reacting with oxygen form hydrogen peroxide and hydroxyls radical that causes the breaking of unsaturated fatty acids, chlorophyll, lipids, and proteins in the cell membrane [25]. These herbicides are post-emergence herbicides that get activated under bright light and have a contact mode of action. These herbicides are found to control weeds well under the maturity period too. The major injury system appears in the plant after 1–2 hours of application with evident water-soaked foliage, browning, and necrosis.

#### **9. Seedling root growth inhibitors**

These group of herbicides belong to group 3 of the site of action which inhibits the root development in young seedlings by interfering with the cell wall microtubules. Due to this mode of action of herbicides they are commonly called microtubules inhibitors. These chemicals inhibit cell division and cause the blocking of root growth and extension due to the assembly of herbicide-tubulin complex inside microtubules. The complex inhibits the polymerization of microtubules disturbing root cell wall formation [26]. These herbicides are used as pre-emergence herbicides their application through direct soil incorporation gives the best result. The chemical family of these herbicides is dinitroaniline, which is commercialized in the chemical form of pendimethalin. Other commonly used herbicides include trifluralin, ethafluralin, cycloate, and butylate. The major injury of this herbicide is swollen coleoptile, swollen hypocotyl, callus formation, brittle stem, and formation of short secondary roots.

#### **10. Seedling shoot growth inhibitors**

Seedling shoot growth inhibitors are the herbicides belonging to group 8 site of action, which interfere with the activity of lipid synthesis through chemical

#### *Modes of Herbicide Action DOI: http://dx.doi.org/10.5772/intechopen.105356*

thiocarbamate. These chemical inhibits the biosynthesis of protein, fatty acids, flavonoids, and gibberellins. The other group of herbicides acting as seedling shoot growth inhibitors is long chain fatty acids inhibitors. These herbicide conjugates with Acetyl CoA to form thiocaramate sulfoxide which inhibits long-chain fatty acids during seedling seed growth [27]. The chemical family chloroacetamide, which comprises of chemical herbicides like alachlor, butachlor, and metolachlor are herbicides belonging to this mode of action. They are used as pre-emergence herbicides, soil incorporation of these herbicides give better efficiency. These herbicides are volatile in nature, which are absorbed through roots and emerging shoots and only translocated through xylem vessels in plants. The major injury produced by these groups of chemicals are stunting and enlarged cotyledons.

### **11. Some novel modes of herbicide action**

The problem of herbicide resistance has led the researcher to explore on new modes of herbicide action. Exploring herbicides with a new mode of action can potentially be effective for those weed species which are resistant to conventional herbicides. Several methods are being employed to explore herbicide that acts on new site of action. Major focus has been put on the exploration of phytotoxic primary and secondary metabolites such as protoporphyrin IX and sphingoid bases. The next approach commonly used for the study is identifying the potential site of action with very low-level enzyme level [28]. The herbicide with the target site, Dihydrodipiconitae Sythetase (DHDPS), which catalase the first and rate-limiting step in lysine synthesis is found to be effective in *Arabidopsis thaliana* conformed by using high throughout the chemical screen. The class of inhibitors are found to bind with the novel and unexplored packets within DHDPS, which produces the symptoms of retarded growth and germination [29]. Another novel herbicide with the chemical form tetflupyrolimet belonging to Weed Science Society of America (WSSA) group 28 is found to be effective against the control of long-season grass weeds in rice fields. The herbicide acts on homogentiosata solanesyltransferase (HST) and dihydroorotate dehydrogenase (DHODH) inhibition [30]. The use of tetflupyrolimet has been under research on several other crops like sugarcane, wheat, soybean, and corn. The herbicide is expected to launch commercial in the year 2023, especially recommended for transplanted and direct-seeded rice.

#### **12. Conclusion**

Herbicides have become one of the indispensable parts of commercial agriculture to control the weed species efficiently. The continuous and excessive use of herbicides has evolved the problem of herbicide resistance in many weed species. The new exploration of herbicide mode of action has provided new insights on the target action of herbicides and their acting mechanism along with providing solutions for herbicide resistance. A sound knowledge on the mode of herbicide action help farmer to select the herbicide based on degree of weed infestation, a suitable method for herbicide application, and understand the action mechanism involved to check the growth of weeds in field crops. The study of the mode of action interrelates with the study of the site of action, active chemical involved, and injury produced by such chemicals in the growth and development of weed crops which is equally useful for

herbicide formulation. Hence, from the discussions in this chapter, it is evident that different herbicides have their own mode of action to kill the field weeds. Knowing the herbicide mode of action can help for the tactical management of weed species and cope with the resistance trait that lies within plant species.

## **Acknowledgements**

The author acknowledges Mr. Roshan Subedi, Assistant Professor of the Institute of Agriculture and Animal Science, Tribhuvan University for his constant guidelines and support during the preparation of this book chapter.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Suman Bagale Institute of Agriculture and Animal Science, Tribhuvan University, Kirtipur, Nepal

\*Address all correspondence to: sumanbagale74@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 3**

## New Insight in Herbicides Science: Non-Target Site Resistance and Its Mechanisms

*Ermias Misganaw Amare*

#### **Abstract**

Managing weeds in crop production, whether in the field, or greenhouse, can be troublesome; however, it is essential to successful production. Weeds compete with the crop for nutrients, space, sunlight and also host plant pathogens and insect pests. The economic impacts of weeds include both monetary and non-monetary. In Australia, the overall cost of weeds to grain growers is estimated at AUD 3.3 billion annually. In India, weeds cost over USD 11 billion each year. In the USA, weeds cost USD 33 billion in lost crop production annually. Herbicide use is indispensable in agriculture as it offers tool for weed management; however, repeated applications of herbicides with the same mode of action resulted in the selection of herbicideresistant weed populations. Herbicide resistance is a rapidly growing worldwide problem that causes significant crop yield losses as well as increases in production costs. Non-target-site resistance to herbicides in weeds can be conferred as a result of the alteration of one or more physiological processes such as reduced herbicide translocation, increased herbicide metabolism, decreased rate of herbicide activation. Non-Target Site Resistance mechanisms are generally more complex and can impart cross-resistance to herbicides with different modes of action. To date, approximately 252 species have evolved resistance to 23 of the 26 known herbicide modes of action.

**Keywords:** non-target site resistance, absorption, translocation, metabolism

### **1. Introduction**

Weed is one of the main biotic factors that brings about a significant crop yield loss since the beginning of agriculture about 10,000 years ago. Weed will cause the highest potential yield loss to crops. In addition, weeds harbor insects pests, and pathogens, which attack crop plants. Weeds compete with crops for sunlight, water, nutrients, and space. Moreover, weeds infest and destroy native habitats, threatening native plants and grazing lands. Crop yield losses as a result of weeds depend on several factors including weed emergence time, weed density, type of weeds, type of crops, soil fertility, etc. Left uncontrolled, weeds can result in 100% yield loss. In Australia, the overall cost of weeds to grain growers is estimated at AUD 3.3 billion annually. In terms of yield losses, weed loss amounted to 2.7 million tons of grain at a national level [1]. In India, weeds cost over USD 11 billion each year [2]. In India, the yield losses because of weeds were estimated at 36% in peanut, 31% in soybean, 25% in maize, and 19% in wheat. In the USA, weeds cost USD 33 billion in lost crop production annually [2]. Hence, weed management is one of the most important components of cropping systems, which results in significant yield loss as well as increased cost of production. In the early 1950s, synthetic herbicides revolutionized agriculture and have been at the foundation of both weed science research and the intensification and expansion of industrialized agriculture [3].

In developing countries, where farm size is small, weeds management is carried out by hand removal however as a result of rising labor costs and it is being replaced by herbicide use. In most developed countries, herbicides are already widely used to control weeds. However, repeated application of herbicides with similar modes of action has resulted in the development of herbicide-resistant weeds. Currently, more than 500 unique cases of herbicide-resistant weeds have been documented across the globe [4]. The majority of herbicide-resistance weed cases were reported from the USA (more than 160) followed by Australia (over 90 cases) and the remaining cases are reported from Canada, China, and Brazil. The maximum number of herbicide-resistant weed species was reported in different crops, including wheat, maize, rice, soybean spring barley, canola, and cotton [4]. These crops are the most widely produced food crops as well as the important industrial crops. Glyphosate is the most traded herbicide across the globe and used for non-selective post-emergence control of both annual and perennial weeds [3]. This herbicide disrupts the activity of enzymes including 5-enolypyruvyshikimate-3-phospahate synthase [5, 6].

#### **2. Weed management methods**

Managing weeds in crop production, whether in the field, greenhouses, or outdoor containers, can be troublesome; however, it is essential to successful production. Weeds not only compete with the crop for plant nutrients, space, and sunlight but also serve as an alternative host to virulent plant pathogens and notorious insect pests. The economic impacts of weeds include both monetary and non-monetary. For example, blackberries restrict human and animal access, harbor pests, reduce pasture production, impede establishment of plants, and reduce naturalness and biodiversity [7]. Some of the common weed management practices are explained below. Weed management activities include preventive/ quarantine, use of cover crops, mowing, flaming, mulching, solarization, and herbicide application.

#### **2.1 Herbicides**

In modern agriculture, herbicide spray are very common and rapid weed management method in many crop production areas across the globe. By using herbicides before weeds emerge, weed competition with the crop can be reduced or eliminated, resulting in higher yield and fewer labor costs despite ecological disturbance and health hazards. Herbicides are generally classified according to time of application to the crops and weed growth stage. Preplant herbicides are applied before planting. These herbicides are used before the desirable plants are present because some can control both germinating seedlings and established plants. Pre-emergence herbicides kill weeds at the seed germination stage. These herbicides are applied before weeds

*New Insight in Herbicides Science: Non-Target Site Resistance and Its Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.105173*

emerge. Post-emergence herbicides are applied after the weeds have emerged. Preemergence and post-emergence herbicides may be applied before or after the crop is planted depending on the crop and the herbicide selected. However, their extensive utilization across the globe imposes strong selection pressure on resistant weed populations, threatening our ability to successfully manage weed populations. Herbicide resistance is a rapidly growing worldwide problem that causes significant crop yield and quality losses as well as increases production costs. To date, approximately 252 species have evolved resistance to 23 of the 26 known herbicide modes of action, representing over 161 different herbicides [8].

#### **3. Herbicide resistance**

The acquired inheritable trait of plants to survive and reproduce under herbicide exposure is defined as resistance. The Weed Science Society of America defines herbicide resistance as the inherited ability of a plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type. Under continuous selection pressure, that is, the repeated use of herbicides with the same mode of action, the resistant weed plants increase in frequency over time, resulting in the domination by individuals resistant to a particular herbicide. Biological and genetic factors of weed species, properties of herbicides, and agronomic practices play a significant role in the evolution and spread of herbicide resistance [8]. Biological characteristics of troublesome weeds, including prolific seed production, high germination percentage, seed dispersal, and longevity, help to maintain a high frequency of resistant individuals in the population. Genetic factors, such as natural mutations conferring herbicide resistance, inheritance of herbicide-resistant genes in the weed population, and fitness of resistance genes in the presence or absence of the herbicide, also play an important role in the evolution and spread of herbicide resistance [8].

Mechanisms of herbicide resistance in weeds can be broadly classified into two categories [8, 9] (i) modifications in the herbicide target enzyme (target-site resistance; TSR) and (ii) mechanisms not involving the target enzyme (non-target-site resistance; NTSR). TSR is typically conferred by single major-effect alleles, whereas NTSR is believed to be conferred by multiple small-effect alleles [9]. The TSR mechanisms largely involve mutation(s) in the target site of action of herbicide, resulting in an insensitive or less-sensitive target protein of the herbicide. In such cases, TSR is primarily determined by monogenic traits. Additionally, TSR can also evolve as a result of the over-expression or amplification of the target gene. TSR mechanisms alter the amino acid sequence and/or expression level of the target enzyme, reducing the herbicide's ability to inhibit the enzyme or requiring a greater herbicide concentration to achieve adequate inhibition [10]. TSR to acetolactate synthase (ALS) inhibitors and acetyl-CoA carboxylase (ACCase) inhibitors, two large classes of herbicides used to control grass weeds, is the most widely documented mechanism of resistance [4]. On the other hand, NTSR mechanisms include all mechanisms that reduce the concentration of active herbicide remaining available to interact with the target site protein, as well as mechanisms that allow the plant to cope with inhibition of the target site [10]. NTSR mechanisms include reduced herbicide uptake/translocation, increased herbicide metabolism, decreased rate of herbicide activation, and/or sequestration [10, 11] (**Figure 1**).

#### **Figure 1.**

*Weeds can evolve resistance to a herbicide by reducing its absorption, altering translocation and/or sequestration, or developing rapid necrosis of the foliage via degradation of the active ingredient. Source: [12].*

#### **3.1 Non-target site resistance (NTSR) mechanisms in weed species**

Mechanisms that can contribute to NTSR are complex and involve several different gene types and families. This molecular and genetic complexity makes the identification of particular genes involved in NTSR difficult. Recent advances in this area have identified putative NTSR genes contributing to enhanced herbicide metabolism [13].

#### *3.1.1 Metabolism-based NTSR*

Plants contain large numbers of genes encoding enzymes that perform biochemical reactions for the synthesis of secondary metabolites and for detoxifying xenobiotic compounds (e.g., herbicides) [14]. Herbicide metabolism is the degradation of herbicide molecules by endogenous plant enzymes. Metabolism-based NTSR involves increased the activities of enzyme complexes including esterases, cytochrome P450s (CYP450s), glutathione S-transferases (GSTs), and/or Uridine 5′-diphospho (UDP) glucosyl transferasesm [8]. NTSR, if it involves herbicide detoxification by these enzymes, is usually governed by multiple genes (polygenic) and may confer resistance to herbicides with completely different modes of action [15]. Enhanced rates of herbicide metabolism in NTSR are, in general, have a three-phase process [12, 16].

Phase I reactions increase the polarity of the herbicide and involve oxidation, reduction, or hydrolysis, which form free amino, hydroxyl, or carboxylic acid groups The most common phase I reactions are oxidation reactions carried out by cytochrome P450 monooxygenases (P450s). P450s are a large superfamily of enzymes and catalyze oxygen- and NADPH-dependent monooxygenase reactions [8, 12, 16].

Phase II reactions are commonly catalyzed by the glutathione S-transferase (GST) superfamily that is large and diverse. Higher plants have at least 10 different GST classes, of which the predominant phi and tau classes have broad substrate specificities and are primarily responsible for herbicide detoxification. GSTs conjugate glutathione to oxidized xenobiotics and individual GSTs of several classes are

*New Insight in Herbicides Science: Non-Target Site Resistance and Its Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.105173*

key players in NTSR to herbicides. The best-characterized role of GSTs in NTSR is for the "Peldon" MHR *Alopecurus myosuroides* populations that are resistant to the photosystem II inhibitor chlorotoluron and several ACCase-inhibiting herbicides. The glycosyltransferase enzyme family is also involved in phase II of herbicide metabolism. Specifically, glycosyltransferases conjugate herbicides directly or conjugate a sugar molecule to a variety of lipophilic molecules including xenobiotics. Glycosyl transferases have been shown to metabolize many herbicides and have important roles in conferring tolerance to other abiotic stresses such as salt, cold, and drought, by modifying anthocyanin accumulation [12, 16].

The third phase of herbicide metabolism involves compartmentalization and transportation of the conjugated herbicide into the vacuole or extracellular space [17]. The most common transporters in phase III are ABC transporters. ABC transporters have been shown to transport herbicide metabolites of primisulfuron, glutathioneconjugated herbicide metachlor, and have potential roles in conferring NTSR to glyphosate in *Conyza canadensis* (**Figure 2**).

#### *3.1.1.1 Acetyl CoA carboxylase (ACCase)-inhibitors*

Acetyl CoA carboxylase is a very crucial enzyme, which involves in the formation of malonyl CoA *via* the carboxylation of acetyl CoA [8]. Malonyl CoA is needed for *de novo* fatty acid biosynthesis, which is essential for plant survival. ACCase-inhibitors impair malonyl CoA formation in some grass species and ultimately lead to plant death [8]. Research results found metabolic resistance to ACCase-inhibiting herbicides has occurred on many weed plants including Asia minor bluegrass, barnyard grass, blackgrass, Italian ryegrass, Japanese foxtail, rigid ryegrass, and wild oat. In the majority of these cases, enhanced metabolism mediated by CYP450s was reported. For instance, rapid degradation of diclofop-methyl was observed in rigid ryegrass populations from Australia. Interestingly, exposure to low doses of diclofopmethyl acid application is rapidly selected for metabolic resistance in rigid ryegrass. Moreover, the metabolites produced in these resistant plants were found to be similar to those in wheat formed *via* ring hydroxylation and sugar conjugation. This result suggests that in resistant grasses, the metabolism of ACCase-inhibitors occurs through a wheat-like detoxification pathway mediated by CYP450s [8].

#### *3.1.1.2 Acetolactate synthase (ALS)-inhibitors*

Enhanced metabolism conferring resistance to ALS inhibitors has been documented in some grass and broadleaf weeds, such as barnyard grass, common waterhemp, Palmer amaranth, rice barnyard grass, rigid brome, short awn foxtail, and water chickweed [8]. Numerous studies have also elucidated the molecular basis of metabolic resistance to ALS inhibitors. Most of the studies have predominantly identified multiple CYP450 genes that are either constitutively expressed or upregulated. For example, the mechanism of mesosulfuron-methyl resistance in short-awn foxtail was studied and two CYP450 genes (CYP94A1 and CYP71A4) were overexpressed in the resistant plants. In a similar, two CYP450 genes, (CYP81A12 and CYP81A21) were identified as candidate genes conferring resistance to bensulfuron-methyl and penoxsulam in rice barnyard grass. Several CYP450 genes mediating NTSR to ALS inhibitors have been identified in water chickweed, ryegrass, flaxseed, and blackgrass. In addition to CYP450s, involvement of GSTs, GTs, and ATP-binding cassette (ABC) transporters has also been reported. For instance, in ALS-inhibitor-resistant

#### **Figure 2.**

*Herbicide metabolized in three phases. (a) Initially, herbicide is subjected to a redox reaction to increase its hydrophilicity (phase I). This metabolized herbicide is further processed into phase II. Metabolism may be concluded with the storage of metabolized compounds (phase III). Sources: [15].*

water chickweed, four genes including three CYP450s and an ABC transporter were highly expressed in all resistant plants [8].

Recently, a new resistance mechanism in weeds has been identified. Glyphosate resistance is possible through aldo-ketoreductase (AKR)-based metabolism [18], upregulation of an ABC membrane transporter pumping out glyphosate outside the cell [19], and programmed cell death causing rapid necrosis [20]. Similarly, 2,4-D resistance due to either CYP-450-based metabolism [21], a double point mutation [22] or 9-codon deletion in an auxin transcriptional repressor [23], or rapid necrosis [24] has also been reported. These recent findings depicted that herbicide selection for many survival mechanisms will occur and increase the chances for plants to harbor multiple resistance mechanisms.

#### *3.1.1.3 Photosystem II (PSII) inhibitors*

The PSII complex is located within the thylakoid membranes of chloroplasts and contains two proteins, D2 and D1 [25]. PS-II inhibitors act by competitively binding to the plastoquinone binding site (QB) on the D1 protein in the PS-II complex

#### *New Insight in Herbicides Science: Non-Target Site Resistance and Its Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.105173*

of the chloroplast. Once a PSII-inhibiting herbicide binds, it blocks the transfer of electrons from plastoquinone QA in D2 to plastoquinone QB in D1, which prevents CO2 fixation and production of ATP and NADPH. Blocking electron transport leads to the production of reactive oxygen species (ROS), which destroys cell integrity [15, 25]. Some herbicide chemical groups such as triazines, triazinones, and ureas inhibit Photosystem II [15]. Till now, 74 weed species have been reported to develop resistance to PS-II inhibitors across the globe, through both TSR and NTSR mechanisms [25]. NTSR to PS-II inhibitors have been reported in many weed species including bluegrass, common ragweed, common water hemp, Palmer amaranth, and wild radish. The metabolism of PS-II inhibitors was catalyzed by increased activity of GST enzymes and/or CYP450 enzymes [25].

#### *3.1.2 Reduced herbicide absorption*

To be effective, herbicides must be absorbed into cells of plants through the roots, in the case of soil-applied herbicides, or from the leaves in the case of foliar-applied herbicides. During herbicide application, herbicide droplets must land on the leaf surfaces and overcome a number of barriers before cellular uptake. This passive process largely depends on leaf surface characteristics, herbicide chemical properties, and their interactions. Herbicide absorption from cellular uptake, where absorption is the process of overcoming the physical barrier of leaves (i.e., cuticle) before the herbicide reaches the apoplast, and uptake is the movement of herbicide from the apoplast into plant cells. Herbicide-resistant weed populations exhibit reduced herbicide absorption, characterized by a reduction in the penetration *via* the cuticle before reaching the epidermis, whereas cell walls do not pose a significant resistance to cellular uptake. Reduced absorption is not a common NTSR mechanism; however, it has occurred in both dicots and monocots to some herbicide groups such as synthetic auxins and 5-enolpyruvylshikimate-3-phosphate synthase inhibitors [15].

Differences in root absorption of herbicides between species have been associated to root morphology differences. There are no cases of evolved resistance to soil-applied herbicides due to reduced root absorption [26]. Differences in foliar absorption of herbicides between weed plants have been highly associated with leaf anatomical structure than biochemical differences [10]. Differential foliar absorption of herbicides between species was directly linked to differences in cuticle thickness and/or composition; however, the number and/or structures of leaf features such as trichomes and hairs have also been involved. For instance, Hirsute leaves are covered with hairy trichomes that can retain spray droplets better than smooth, hairless, or glandless cuticles, hence facilitating absorption. Other leaves have lysigenous glands involved in the production and storage of oily secondary metabolites that can compartmentalize lipophilic herbicides, preventing them from reaching their site of action [26].

Decreased absorption is uncommon NTSR mechanism; however, it has been reported with the resistance of common sunflower to imazethapyr and chlorimuron, prickly lettuce to 2,4-D, annual bluegrass to atrazine, and *L. multiflorum* to glyphosate. No differences were found in cuticular wax amount per unit area of leaf surface between two biotypes of *L. multiflorum* with a threefold difference in glyphosate susceptibility and reduced absorption in the less sensitive biotype. When reduced absorption is implicated, it is most often only one contributing factor to the overall resistance mechanism. For example, resistance to glyphosate in *A. tuberculatus* biotypes was due to both reduced absorption and a herbicide resistance allele of the glyphosate enzyme target EPSPS [12].

#### *3.1.3 Reduced translocation and sequestration*

Many foliar-applied systemic herbicides rely on translocation through the phloem. These herbicides must overcome the cuticle barrier and enter the cells of mature source leaves (symplast). This transport can involve active and/or passive diffusion processes [12]. Once inside the symplast, systemic herbicides translocate from source leaves to younger sink leaves *via* the phloem [16]. Herbicide resistance due to reduced translocation occurs when the herbicide is contained in source leaves and prevented from translocating to young leaves. Mechanisms that trap the herbicide in source leaves (e.g., through sequestration within vacuoles of leaf trichomes) or prevent its normal movement to the growing points across membrane barriers (through altered activity of active membrane transporters) will reduce the total amount of herbicide translocated, thus conferring resistance [12]. Therefore, alterations of translocation patterns can lower herbicide efficacy. Herbicide resistance as a result of reduced translocation has been observed in grass weed species, such as *Lolium* spp. [15]. Reduced translocation of glyphosate is the most common type of NTSR mechanism [27]. In these plants, the amount of glyphosate delivered to the meristems is lower than what is essential to be toxic to the weed plant. Reduced glyphosate translocation was first recorded in glyphosate-resistant *Lolium rigidum*, less glyphosate translocated to the meristems, relative to glyphosate-susceptible *L. rigidum* [28]. Glyphosateresistant *C. canadensis* had reduced translocation [27]. This is due to differences in the cellular distribution of glyphosate and subsequent phloem loading and translocation. In these biotypes, glyphosate enters the source leaves normally; however, it cannot translocate to the meristems because it is rapidly sequestered within the vacuole [29]. Vacuole sequestration activity is temperature-dependent, with less sequestration observed in *C. canadensis* under lower temperatures (**Figure 3**) [30].

*New Insight in Herbicides Science: Non-Target Site Resistance and Its Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.105173*

### **4. Conclusion**

Managing weeds in crop production, whether infield, greenhouses, or containers, can be challenging and costly practice; however, it is essential to successful production. Weeds not only compete with the crop for plant nutrients and sunlight but also host plant pathogens. Herbicides are used in many crop production areas as an economical option to control weeds. By using herbicides before weeds emerge, weed competition with the crop can be reduced or eliminated, resulting in higher quality yield and less labor costs. However, their extensive utilization across the globe imposes strong selection pressure, which results in resistant weed population development. Herbicide resistance is a rapidly growing worldwide problem that causes significant crop yield loss and increases production costs. The most common herbicide resistance form is target-site resistance and non-target site resistance. Non-target site herbicide resistance is complex and involves several different gene types and families. This molecular and genetic complexity makes the identification of particular genes involved in NTSR difficult. Non-target site resistance mechanisms include reduced herbicide uptake/translocation, increased herbicide metabolism, decreased rate of herbicide activation, and/or sequestration. Lack of new herbicides in the market makes utilization of already available herbicides inevitable. Therefore, it is very imperative to integrate various weed management practices to curve a rapid increase in non-target site resistance development. It is equally important to reduce application of the same kind of herbicide over time to overcome resistance to weed population establishment.

## **Author details**

Ermias Misganaw Amare College of Agriculture and Environment Sciences, Gondar University, Gondar, Ethiopia

\*Address all correspondence to: ermiasamare2@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 2
