Perspective: root exudation of herbicides as a novel mode of herbicide resistance in weeds

The evolution of resistance to herbicides in weeds has become a great challenge for global agricultural production. Weeds have evolved resistance to herbicides through many different physiological mechanisms. Some weed species are known to secrete herbicide molecules from roots into the rhizosphere upon being treated. However, root exudation of herbicides as a mechanism of resistance has only recently been identified in two weed species. Root exudation pathways have been investigated in Arabidopsis, and this work suggested that ATP‐binding cassette (ABC) and multidrug and toxic compound extrusion (MATE) transporters play a role in the secretion of primary and secondary plant products from roots. We hypothesize that the mechanisms involved in root exudation of herbicides that result in resistance are mediated by overactive or overexpressed transporters, probably similar to those found for the exudation of primary and secondary compounds from roots. Elucidating the molecular and physiological basis of root exudation in herbicide‐resistant weeds would improve our understanding of the pathways involved in herbicide root secretion mediated by transporters in plants. © 2020 Society of Chemical Industry     

Root exudation of diclofop-methyl and triasulfuron from foliar-treated durum wheat and ryegrass

In this study, we evaluated the release of diclofop-methyl and triasulfuron from the roots of foliar-treated ryegrass and wheat. The study with ¹⁴C-diclofop-methyl indicated a basipetal translocation of foliar-applied herbicide in wheat and ryegrass. No root exudation from ¹⁴C-diclofop-methyl-treated wheat plants was observed, while 20 days after treatment (DAT) 0.2–0.9% of radioactivity absorbed by ryegrass was found exuded in the growing medium. Root exudation was stimulated three to six times by the presence of untreated wheat or ryegrass sharing the growing medium with diclofop-methyl-treated ryegrass. No subsequent uptake of exuded radiolabel by untreated plants (ryegrass or wheat) in the same pot with ¹⁴C-diclofop-methyl-treated ryegrass was observed. The study with ¹⁴C-triasulfuron indicated a basipetal translocation of foliar-applied herbicide in wheat and ryegrass and also into the growing medium. By 20 DAT, 0.5–4.2% of radioactivity absorbed by wheat or ryegrass was found exuded in the growing medium. The presence of untreated plants (wheat or ryegrass) in the same pot as triasulfuron-treated ryegrass or wheat induced exudation seven to 32 times more. The study also revealed a subsequent uptake of exuded compounds by untreated wheat or ryegrass sharing the medium of ¹⁴C-triasulfuron-treated plants. This study has demonstrated for the first time that the root exudation of exogenous compounds can be related to plant arrangement in pots. The implication is that herbicide root exudation and transfer, a form of allelopathy, could be significant in the field. A precise estimation of environmental fate, unexpected ecological side effects and residual activity of herbicides may require quantification of such exudation.     

Increased herbicidal activity of triazine herbicides by piperonyl butoxide

Piperonyl butoxide (PB) is a known Synergist which enhances the activity of insecticides by inhibiting their biotransformation to less active products. We have evaluated the possible use of PB as a herbicide synergist using triazine herbicides in sensitive, tolerant, and resistant plants. The effects of PB, triazine herbicides, and their combinations were examined in whole plants as well as in chloroplasts isolated from triazine-sensitive (S) and -resistant (R) weed biotypes. PB itself, applied postemergence (0.1–0.5%, v/v), was slightly toxic to the plants tested. However, foliar application of PB combined with atrazine, terbutryn or prometryn to maize seedlings significantly increased the phytotoxicity of the herbicides. Low rates of atrazine, prometryn, and terbutryn in a tank-mixture with PB, effectively controlled Solatium nigrum L. and Abutilon theophrasli Medik. PB enhanced atrazine efficacy in both S and R biotypes of Lolium rigidum Gaud. The synergistic effect of PB was evident also in vitro when atrazine and methabenzthiazuron were used to inhibit photosystem II electron transport in chloroplasts isolated from resistant weeds. These data demonstrate the potential of PB as a herbicide synergist and its possible utilization as an aid for improving the activity of triazine herbicides in sensitive, tolerant and resistant plants.     

Differential tolerance of peas to prometryne and terbutryn

The phytotoxicity of 2,4-bis(isopropyl)-6-(methylthio)-s-triazine (prometryne) and 2-(tert-butylamino)-4-(ethylamino)-6-(methylthio)-s-triazine (terbutryn) to peas (Pisum sativum L. var. Perfection 3040) was studied. No differences were found when the herbicides were applied to the roots of intact plants in nutrient solution or directly to leaf discs. However, prometryne was much more toxic when uptake was from soil. Absorption and translocation of ¹⁴C-labeled prometryne and terbutryn showed that the majority of terbutryn accumulated in the roots, whereas prometryne was uniformly distributed between the roots and the shoot. Thin layer chromatography of extracts from prometryne-treated peas showed that only 20% of the absorbed compound was metabolized to produce one breakdown product. Extracts of terbutryn-treated plants contained three different metabolites. After 120 hr of exposure to terbutryn, about half of the absorbed herbicide was metabolized. The results show that the main factors responsible for the differential toxicity of the herbicides to peas were availability from the soil, translocation pattern and initial detoxication.     

甲嘧磺隆等3种除草剂对云南黄馨、金钟扦插苗成苗及生长的影响

阔叶杂草是苗圃中重要的危害性杂草,然而控制苗圃阔叶杂草的有效化学方法却很少,主要原因是能防除阔叶草的除草剂往往对苗木不安全或缺少对其安全性研究。选择甲嘧磺隆、乙氧氟草醚和扑草净3种杀草谱较广的除草剂,通过2015年田间小区试验,研究不同用量下这3种除草剂对苗圃常见苗木云南黄馨、金钟扦插苗成苗、发芽、生根、株高等因素的影响。结果显示:用药时雨水、土壤湿度可严重影响植物生长状况,雨水多、湿度大,则发根、出叶少;用药时间影响药剂效应,扦插前用药,药剂影响大于扦插后用药,这可能是扦插前、扦插后苗在遇药时的叶芽状态及水分不同所造成的。研究还发现,药剂对苗木成苗无不良影响,7.5 g a.i./hm~275%甲嘧磺隆WDG处理对苗木出叶、株高无影响;而在生根方面,在药后30 d,只有对照的71.1%,用药后100 d,与对照相当,乙氧氟草醚144、288 g a.i./hm~2,扑草净450、900 g a.i./hm~24个处理的结果与甲嘧磺隆7.5 g a.i./hm~2处理相同;对于甲嘧磺隆15 g a.i./hm~2处理,在前期对苗木出叶、生根都有较大影响,3个月后影响逐渐减少甚至消失;甲嘧磺隆22.5 g a.i./hm~2处理对苗木出叶、生根、株高的抑制可持续3个月以上。     

精噁唑禾草灵、甲基二磺隆、扑草净处理后日本看麦娘残存植株的生长和结实能力

以采自江苏省扬州市小麦田的3个日本看麦娘种群(AJ191、AJ193、AJ195)为例,研究了供试种群对精噁唑禾草灵、甲基二磺隆、扑草净3种不同作用机理的代表性麦田除草剂的敏感性及药剂处理后残存植株的生长和结实能力.结果 表明,在推荐剂量处理下,精噁唑禾草灵对3个种群的控制效果均不理想,扑草净对3个种群均有高活性,甲基...     

Comparison of trifluralin,oryzalin, pronamide,propham, and colchicine treatments on microtubules

The mode of action of trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine), oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide), pronamide(N-(1,1-dimethylpropynyl) 3,5-dichlorobenzamide), and propham (isopropyl carbanilate) on purified microtubules from pig brains and on the ultrastructure of wheat (Triticum aestivum L. “Mediterranean,” C. I. 5303) and corn (Zea mays L. “yellow dent, U. S. 13”) roots was compared with that known for colchicine. Colchicine disrupts the in vivo cortical and spindle microtubules of root cells. Like colchicine, the herbicides trifluralin, oryzalin, and pronamide caused the loss of both cortical and spindle microtubules of root cells. The rate of microtubule disappearance depended on the type of herbicide and length of exposure of roots to the herbicide. Unlike colchicine, cortical microtubules were present in propham-treated roots but they were disoriented within the cell.In vitro polymerization studies with pig brain microtubules (Sus scrofa) showed that the herbicides failed to inhibit the assembly of purified microtubular protein into microtubules and that radioactively labeled herbicides did not bind to the microtubular protein. Colchicine inhibited the polymerization of microtubular protein and readily bound to the microtubular subunits. These results indicate that the mode of action of the herbicides is not similar to that of colchicine and that the loss of microtubules from root tip cells treated with trifluralin, oryzalin, and pronamide may be caused by these herbicides interfering with synthesis of microtubular protein or metabolism of endoplasmic reticulum membranes involved in microtubule assembly. The mode of action of propham appears to be on the microtubular organizing centers rather than on microtubules per se.     

DIFFERENTIAL PHYTOTOXICITY OF ATRAZINE AND AMETRYNE TO BANANAS*

Summary. In field screening trials for bananas (Musa acuminata var. Dwarf Cavendish) in Hawaii, ametryne (2-methylthio-4-ethylamino-6-isopropylamino-s-triazine) was less phytotoxic to bananas than atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine). Sand culture experiments showed that both herbicides were equally injurious to banana plants. Differential degradation of the herbicides by the plants did not account for the phytotoxicity observed. Both herbicides were partly metabolized by the plant to their common hydroxyl derivative (hydroxyatrazine) and two other unidentified metabolites after 3 and 7 days of exposure to nutrient solution containing ¹⁴C-labelled ametryne and atrazine. Phytotoxicity was directly related to leachability of the herbicides and negatively related to adsorption capacity of each soil for the herbicides. Organic matter content seemed to be correlated to the response observed. It was postulated that phytotoxicity in the field may have been attributed to differential location of the herbicide in relation to the roots.     

Metabolic fate of bioxone in cotton

The metabolic fate of the ¹⁴C-labeled herbicide, 2-(3,4-dichlorophenyl)-4-methyl-1,2,4-oxadiazolidine-3,5-dione (bioxone), in cotton (Gossypium hirsutum L. “Acala 4-42-77”) was studied using thin-layer chromatography, autoradiography, and counting. Bioxone-¹⁴C was readily metabolized by cotton tissue to 1-(3,4-dichlorophenyl)-3-methylurea (DCPMU) and 1-(3,4-dichlorophenyl)urea (DCPU). Leaf discs metabolized bioxone-¹⁴C rapidly; 12 hr posttreatment, 65% of the ¹⁴C in methanol extracts was in forms other than intact herbicide. Excised leaves treated through the petiole with either heterocyclic ring-labeled or phenyl ring-labeled herbicide contained little bioxone-¹⁴C after 1 day; DCPMU was formed early then decreased with time. DCPU accounted for 55–70% of the ¹⁴C in excised leaves 3 days posttreatment. In intact plants treated via the roots, the herbicide was rapidly metabolized in the roots to DCPMU and DCPU; little or no intact herbicide was translocated to the leaves. Little radioactivity accumulated in the roots with time; the radioactivity in the leaves accounted for 80–90% of the methanol-soluble ¹⁴C 47 days posttreatment. Most of the ¹⁴C in the leaves was recovered as DCPU (50–60%) and unidentified polar metabolite(s) which remained at the origin of the thin-layer plates (30–40%). The percentage of radioactivity which remained in cotton residue after methanol extraction increased with time. Digestion of the plant residues with the proteolytic enzyme pronase indicated that some of the nonextractable ¹⁴C may be DCPMU and DCPU complexed with proteins. Similar metabolic patterns were noted after treatment with either heterocyclic ring-labeled or phenyl ring-labeled bioxone-¹⁴C. Generally, bioxone was metabolized to DCPMU which in turn was demethylated to DCPU. The herbicide and DCPMU were 20 times as toxic as DCPU to oat (Avena sativa L.), a susceptible species.     

Carrot microtubules are dinitroaniline resistant. I. Cytological and cross-resistance studies

Treatment of both tolerant and susceptible species with dinitroaniline herbicides results in root swelling typical of mitotic disrupters, however, no root swelling was observed in carrots treated with saturated solutions of all the dinitroaniline herbicides tested, with the exception of oryzalin treatments at ≥ 10^?5 M. These levels of treatment are 100–1000 times the levels that caused root-tip swelling, even in tolerant plants. This properly classifies carrot as dinitroaniline-resistant rather than merely tolerant. Cross-resistance was noted to the structurally related mitotic disrupter, hexanitrodiphenylamine, and to the structurally unrelated herbicide, amiprophos-methyl. No resistance was noted to a number of other mitotic-disrupting herbicides. Both immunofluorescence and electron microscopy indicate that the microtubules of carrot roots were unaffected by dinitroaniline treatment. All organized microtubule configurations (cortical, pre-prophase bands, spindle, and phragmoplast) were observed in the treated material.     

STUDIES OF THE RESPONSE OF PLANTS TO ROOT-APPLIED HERBICIDES

Summary. Applications of several herbicides -were made to roots and to the bases of shoots of peas, cucumber, mustard and barley grown in soil, sand or water culture.
Localized applications (variation horizontal) of atratone and MCPA to roots of peas and barley in soil produced effects similar to those observed in water euluire, described in Pan I. Airatone killed the plants whether available to the whole or to only a portion of the root system whereas MCPA affected only the roots with which it was in direct contact, and growth continued when a portion of the root system was in herbicide-free environment.
In water culture, MCPA was more effective when applied to the lower (younger) roots with the upper (older) roots kept dry than when twice the concentration was applied evenly to the whole root system in water. When all the roots were kept wet the effect of application to the upper roots was greater than the effect of application to the lower roots. The response of plants to atratone was not appreciably altered whether applications were made to the upper or lower parts of the root system in water culture. Variations in water level had little effect.
Even when the herbicide solution was confined to the stem or hypocotyl, atratone and DNOC were little, if any, less effective than when applied to roots. MCPA, both as ester and sodium salt, was significantly less effective.
Partial replacement of solution in the root zone by sand and air did not reduce the activity of atratone at a given concentration. Similar replacement in the zone of the stem or hypocotyl greatly reduced the effectiveness of all herbicides. When sand of low water content was used, atratone and MCPA-sodium became quite ineffective via the stem, but DNOC and MCPA-ethyl ester remained active.
Études sur les réactions de certaines plantes á des herbicides appliqués aux racines II. Observations nouvelles sur l'effet de l'application localisée.     

Propanil metabolism in rice: A comparison of propanil amidase activities in rice plants and callus cultures

Propanil amidase is an enzyme found in rice, which hydrolyzes the herbicide 3,4-dichloropropionanilide, propanil. The activity of the enzyme as measured in rice plants was found to be two to four times greater in plants with four leaves than in plants with fewer than four leaves. The higher amidase activity of the four-leaf plants was localized in the unexpanded leaves.Rice root callus suspension in cultures also demonstrated propanil amidase activity. In vivo experiments indicated that the herbicide was metabolized by the tissue culture. Propanil amidase activity as determined in vitro, however, was detected only after the culture had developed to late stationary phase.     

Ameliorative effects of Mesorhizobium sp. MRC4 on chickpea yield and yield components under different doses of herbicide stress

The present study was conducted to assess the plant growth promoting activities of Mesorhizobium sp. in the presence of technical grade herbicides and its ameliorating effects on herbicide toxicity to chickpea grown in herbicide treated soils. The quizalafop-p-ethyl and clodinafop-tolerant Mesorhizobium isolate MRC4 recovered from the nodules of chickpea plants significantly produced IAA, siderophores, hydrogen cyanide and ammonia in medium amended with or without technical grade quizalafop-p-ethyl and clodinafop. Quizalafop-p-ethyl at 40, 80 and 120 μg kg⁻¹ soil and clodinafop at 400, 800 and 1200 μg kg⁻¹ soil in general, decreased the growth attributes of chickpea plants inoculated with Mesorhizobium MRC4 and un-inoculated chickpeas. The three concentrations of quizalafop-p-ethyl were comparatively more toxic and substantially decreased biomass, nodulation and leghaemoglobin content, nutrient uptake, seed yield and grain protein over the un-inoculated chickpea. Interestingly, Mesorhizobium isolate MRC4 with any concentration of the two herbicides significantly increased the measured parameters when compared to the plants grown in soils treated solely (without inoculant) with similar concentration of each herbicide. Conclusively, Mesorhizobium isolate MRC4 could be exploited as bio-inoculant for facilitating chickpea growth under herbicide stress.     

Use of Myxococcus xanthus protoporphyrinogen oxidase as a selectable marker for transformation of rice

Protoporphyrinogen oxidase (PPO) is the target enzyme of peroxidizing herbicides. The overexpression of Myxococcus xanthus PPO (Mx PPO) confers a high level of herbicide resistance in rice. Among the peroxidizing herbicides, butafenacil has an efficiency ∼1000-fold that of oxadiazon, as judged by calli susceptibility tests upon herbicide treatment. Butafenacil (0.1 μM) was used to select transgenic rice plants expressing Mx PPO under the control of the constitutive maize ubiquitin promoter. The ectopic expression of the Mx PPO transgene was investigated in the T₀ generation by Northern blot and Western blot analysis. The T₀ transgenic plants expressing the Mx PPO gene were resistant to butafenacil based on in vitro leaf disk and in vivo foliar spray tests.     

Synthesis of fluorescently labeled pyrazole derivative induceing a triple response in Arabidopsis seedlings

A fluorescent labeled pyrazole derivative with a dansyl moiety (EH-DF) was synthesized. Design of EH-DF was carried out by using a dansyl moiety to substitute the naphthalene moiety of the parent compound (EH-1). At a concentration of 30 µM, EH-DF displayed biological activity on inducing a triple response in Arabidopsis seedlings. Compared with the non-chemical treated control, the hypocotyl length of EH-DF-treated Arabidopsis seedlings was reduced from approximately 9.2±0.7 mm to 2.4±0.2 mm. The length of the roots was reduced from 1.7±0.1 mm to 1.0±0.1 mm, and the curvature of the hook of Arabidopsis seedlings increased from 60±16 degrees to 245±35 degrees. The maxim excitation wavelength and emission wavelength of EH-DF were 350 and 535 nm, respectively. Data obtained via fluorescent microscope analysis indicated that intensive fluorescent signals of EH-DF were observed in the shoot of Arabidopsis seedlings.     

Effects of the herbicide prometryn on the physicochemical properties of artificial model membranes

Several derivatives of 1,3,5-triazine (eg atrazine, prometryn) are effective herbicides and have been extensively used in agriculture. A study of prometryn (Prom)-phospholipid (PL), with different charges, was carried out by means of calorimetric and spectroscopic techniques. The Prom-dipalmitoylphosphatidylcholine (DPPC) and Prom-dipalmitoylphosphatidic acid (DPPA) water suspensions systems at pH values ranging from 5 to 9 were investigated. The results show that Prom does not significantly perturb the hydrophobic core of the lipid bilayer and suggest that the herbicide localizes near the glycerol backbone of the lipid, perturbing the environment of the carbonyls of the two ester groups. At pH 5 the vibrational band attributable to N–H stretch of Prom disappears thus suggesting that the absorption into the bilayer modifies the physicochemical properties of the herbicide. The two PL considered behave similarly and the effect of the charge is not noticeable. The strength of the Prom-PL interaction decreases with decreasing pH. © 1999 Society of Chemical Industry     

A TECHNIQUE FOR STUDYING THE RATE OF ROOT GROWTH OF INTACT SEEDLINGS IN A HERBICIDE MEDIUM

Summary. Time-lapse cine photography was used to record intact seedling root growth of pea and barley during separate exposure of root, shoot + seed, or entire needling to herbicides. The shoot + seed and the root zones were isolated in two square Petri dishes fixed edge to edge, and separately treated with moistened herbicide-treated sand. The seated dishes were placed at an angle of 30° in a photographic chamber. Photographs of roots were automatically recorded at 10-min intervals on 16 mm high speed reversal film over 72 h. Root length images on film were measured using an ocular micrometer. Root growth of pea and barley seedlings was normal when the shoot + seed zone was treated with 2,4-D at 1 and 10 ppm, respectively. In similar treatment of roots growth inhibition occurred after approximately 20 h in both plants, and root growth ceased alter 32 h in peas, and 57 h in barley. These results indicate the inherent tolerance of barley roots to 2,4-D.
Technique pour l'étude du taux de croissance des racines intactes de plantules dans un milieu herbicide     

Tolerance to acetolactate synthase and acetyl-coenzyme A carboxylase inhibiting herbicides in Vulpia bromoides is conferred by two co-existing resistance mechanisms

Vulpia bromoides is a grass species naturally tolerant to acetolactate synthase (ALS) and acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides. The mechanism of tolerance to ALS herbicides was determined as cytochrome P450-monooxygenase mediated metabolic detoxification. The ALS enzyme extract partially purified from V. bromoides shoot tissue was found to be as sensitive as that of herbicide susceptible Lolium rigidum to ALS-inhibiting sulfonylurea (SU), triazolopyrimidine (TP), and imidazolinone (IM) herbicides. Furthermore, phytotoxicity of the wheat-selective SU herbicide chlorsulfuron was significantly enhanced in vivo in the presence of the known P450 inhibitor malathion. In contract, the biochemical basis of tolerance to ACCase inhibiting herbicides was established as an insensitive ACCase. In vitro ACCase inhibition assays showed that, compared to a herbicide susceptible L. rigidum, the V. bromoides ACCase was moderately (4.5- to 9.5-fold) insensitive to the aryloxyphenoxypropionate (APP) herbicides diclofop, fluazifop, and haloxyfop and highly insensitive (20- to >71-fold) to the cyclohexanedione (CHD) herbicides sethoxydim and tralkoxydim. No differential absorption or de-esterification of fluazifop-P-butyl was observed between the two species at 48 h after herbicide application, and furthermore V. bromoides did not detoxify fluazifop acid as rapidly as susceptible L. rigidum. It is concluded that two co-existing resistance mechanisms, i.e., an enhanced metabolism of ALS herbicides and an insensitive target ACCase, endow natural tolerance to ALS and ACCase inhibiting herbicides in V. bromoides.     

A SPLIT-ROOT TETRAZOLIUM METHOD FOR EVALUATING EFFECTIVENESS AND PHYTOTOXICITY OF ROOT-ACTIVE HERBICIDES

Summary. Copper sulphate (GUSO₄.5H₂O) and sodium hydroxide (NaOH) were used to test the performance of a split-root method for evaluating the effectiveness and phytotoxicity of root-active herbicides. This method involves a vertical separation of the root system of weeping willow ( Salix babylonica L.) cuttings. One section is treated with the herbicide dissolved in half-strength Hewitt's medium while the other remains in medium without herbicide during the test period. Roots are assayed with 2,3,5-triphenyttetra-zolium chloride 24 h after treatment to determine their viability. Both herbicides satisfactorily reduced the viability of the treated roots as measured by the quantity of formazan extracted. Copper sulphate was systemic and injured untreated plant parts; sodium hydroxide affected only treated roots. The split-root tetrazolium method is relatively rapid and does not require sophisticated laboratory equipment.
Méthode au tétrazolium sur portions de racines pour l'évaluation de l'efficacité et de la phytotoxicité des herbicides agissant par les racines     

Use of corn root protoplasts in herbicide absorption studies

Intact and viable protoplasts were enzymatically isolated from corn (Zea mays L.) seedling roots and collected for herbicide absorption studies by differential centrifugation and flotation through a Ficoll density gradient. A method was developed for terminating herbicide absorption by rapid centrifugation of protoplasts out of solutions without washing the protoplasts. Within 10 sec, atrazine [2-chloro-4-ethylamino-6-isopropylamino-s-triazine] accumulated in protoplasts to a concentration 36% greater than the external concentration; no further absorption occurred through 30 min. However, 2,4-D [2,4-dichlorophenoxyacetic acid] accumulated to twice and 16 times the external concentration at pH 6.5 and 4.5, respectively. Calculations of theoretical 2,4-D concentrations in protoplasts also predicted greater accumulation at pH 4.5 than at pH 6.5. Both atrazine and 2,4-D absorption were consistent with previous measurements of absorption by plant tissues. Thus, corn root protoplasts are feasible experimental material for studying absorption of herbicides at the cellular level.