Herbicidal Spectrum, Absorption and Transportation, and

Jul 12, 2017 - ABSTRACT: Berberine is a natural herbicidal alkaloid from Coptis chinensis Franch. Here we characterized its herbicidal spectrum and ...
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Herbicidal spectrum, absorption and transportation, physiological effect to Bidens pilosa of natural alkaloid, berberine Jiao Wu, Jing-jing Ma, Bo Liu, Lun Huang, Xiao-qing Sang, and Li-juan Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01259 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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Journal of Agricultural and Food Chemistry

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Herbicidal spectrum, absorption and transportation, physiological effect to Bidens pilosa of natural alkaloid, berberine

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Jiao Wu,|| Jing-Jing Ma,|| Bo Liu, Lun Huang, Xiao-Qing Sang, and Li-Juan Zhou*

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Key Lab of Natural Pesticides & Chemical Biology, Ministry of Education,

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Department of Pesticide Science, South China Agricultural University, Guangzhou,

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Guangdong, China, 510642

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* Correspondence to:

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Li-Juan Zhou

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Key Lab. of Natural Pesticides & Chemical Biology, Ministry of Education

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South China Agricultural University, Guangzhou, Guangdong, China,

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Phone: 86 15917363981

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E-mail: [email protected]

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ABSTRACT

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Berberine is a natural herbicidal alkaloid from Coptis chinensis Franch. Here we

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characterized its herbicidal spectrum, absorption and transportation in plant, and the

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possible mechanism. Berberine showed no effect on the germination of the 10 tested

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plants. The IC50 values of berberine on the primary root length and fresh weight of the

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10 tested plants ranged from 2.91 to 9.79 mg L−1 and 5.76 to 35.07 mg L−1,

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respectively. Berberine showed a similar herbicidal effect to Bidens pilosa as the

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commercial naturally derived herbicide cinmethylin. HPLC and fluorescence analysis

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revealed that berberine was mainly absorbed by B. pilosa root and transported through

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vascular bundle acropetally. Enzyme activity studies, GC-MS analysis, SEM and

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TEM observations indicated that berberine might firstly function on cell membrane

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indicated by variation of the IUFA percent and then cause POD, PPO, and SOD

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activity changes and cellular structure deformity, which was eventually expressed as

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the decrease of cell adaptation ability and abnormal cell function and may even result

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in cell death. Environmental safety evaluation tests revealed that berberine was low in

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toxicity to Brachydanio rerio. These indicate that berberine has potential to be a

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bioherbicide and/or a lead molecule for new herbicides.

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KEYWORDS: berberine, Bidens pilosa L., herbicidal spectrum, absorption,

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transportation, physiological effect, mechanism 2

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INTRODUCTION

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Weeds are plants whose undesirable qualities outweigh their good points, at

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least according to humans. Weeds compete with crops for space, nutrients, water and

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light, and therefore cause substantial economic loss. Traditionally, synthetic

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herbicides are widely used and have become a major control method for weed

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management. Herbicides provide a convenient, economical, and effective way to

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help manage weeds. However, the continuous use of traditional synthetic herbicides

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leads to various negative impacts such as weed resistance to herbicides.1 Therefore,

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there is an urgent need for the development of alternatives to traditional synthetic

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herbicides which are much safer and with new mode of actions. Generally, natural

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products are particularly attractive as templates because they occupy a wider

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chemical space with greater structural diversity than traditional synthetic

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compounds.2

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Bioherbicides are biologically-based control agent for weeds. They can be used

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directly and have been used as a model for the development of several successful

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herbicides (glufosinate and the triketone herbicides) that introduced new mechanisms

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of action, which are greatly needed to overcome the acquired resistance to synthetic

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herbicides in agricultural production. There is, therefore, a great interest in exploring

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natural products to develop new bioherbicides.3 Herbicidal effects of many natural

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chemicals have been studied. These chemicals include quinones (e.g., strigol,

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sorgoleone, juglone), flavonoids (e.g., catechin, ceratiolin, chalcones, kulkulkanin B

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and heliannones), phenols (e.g., heliannuols, ellagic acid, usnic acid), ethers (e.g.,

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cyperin, heliespirone A, helianane), terpenoids (e.g., 1,4-cineole, 1,8-cineole, geranial,

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citral, solstitiolide, messagenic acids, melilotigenins, breviones, germacranolides,

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guaianolides, parthenolide, costunolide, ailanthone, artemisinin, trichocaranes,

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chaparrine, ailanthinol B), coumarins (e.g., coumarin, psoralen), benzoxazinoids (e.g.,

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hydroxamic acids), glucoinolates (e.g., glucohirsutin, hirsutin, arabin), fatty acids (e.g.,

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pelargonic acid), amides (e.g., sarmentine), alkaloids (e.g., fagomine), some amino

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acids such as meta-tyrosine and some essential oils such as clove oil and lemongrass

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oil.4-6

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Included in these chemicals are alkaloids, which are naturally-occurring

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nitrogen-containing biologically active heterocyclic compounds. Over the last few

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years, a large number of biologically important alkaloids with antiviral, antibacterial,

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anti-inflammatory, antimalarial, antioxidant and anticancer activities have been

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isolated from various biosources.7 However, there are few assessments of the

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herbicidal activities of natural alkaloids.8

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Berberine (chemical formula: C20H18NO4. Refer to Fig. 1), a benzylisoquinoline

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alkaloid, is a major component present in numerous medicinal plants which are used

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for treating diarrhea and other gastrointestinal diseases in Asian countries. Berberine

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is known to have a wide range of biological activities such as antioxidant,

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antibacterial, antifungal, antiviral, anti-inflammatory, anti-tumor, anti-diarrhoeal,

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anti-dyslipidemic and anti-diabetic properties.9 In addition to its pharmaceutical

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activities, berberine is also used in agriculture as insecticide and fungicide.10-11

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Interestingly, it was also found to possess herbicidal activity as a result of a

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bioactivity-guided isolation of the herbicidal active components from the rhizomes

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of Coptis chinensis Franch.12 We previously reported that extracts of C. chinensis

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exhibited significant inhibition of root growth in some weeds through the active

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component berberine.13 In this study, we further explored the herbicidal activities of

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berbine on monocot and dicot weeds and characterized the mode of inhibition by

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examining its absorption site, transport pattern and the physiological effects and

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evaluating its ecological safety. Here we show that berberine possessed non-selective

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herbicidal activity and mainly was absorbed by roots and transported from stem

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vascular bundle acropetally to leaves and showed low toxicity to Brachydanio rerio.

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MATERIALS AND METHODS

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Herbicidal activities of berberine on some monocot and dicot plants.

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The herbicidal effects of berberine (Sigma, CAS:141433-60-5) were determined

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using dicot and monocot plants. The seeds of weeds were collected on the campus of

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South China Agricultural University. These species are important components of the

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herbaceous vegetation in crop fields and forests, especially, Eucalyptus forests, of

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southern provinces in China.

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Fungal and bacterial control was accomplished by treating the plant seeds (5 dicots:

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Ageratum conyzoides, Mikania micrantha, Celosia argentea, Amaranthus retroflexus and

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Bidens pilosa; 5 monocots: Echinochloa crusgalli, Dactyloctenium willd, Eleusine indica,

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Triticum aestivum and Oryza sativa) with 1.0% (v/v) sodium hypochlorite for 10 min.

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Then the seeds were washed 2-3 times by the tapping water. Berberine was applied in

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distilled water at concentrations from 2 to 32 mg L−1 to glass beakers containing 20 seeds

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of a plant species underlain by Whatman #2 filter paper. Berberine solutions (8 mL) were

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introduced into the glass beakers. Then, the glass beakers, sealed with polyethylene

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wrapping film with several little holes for ventilation, were placed in a growth chamber

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calibrated to provide 12 h light/12 h darkness at 25±2 °C. Distilled water only was

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introduced as a control. A completely randomized design was selected and all treatments

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were replicated at least three times. The germination rate was calculated each day after

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the treatment. The primary root length and fresh weight were measured at 7 d after the

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treatment. Root inhibition rate was calculated by the following formula: root inhibition

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rate (%) = ((root length of control-root length of treatment) / root length of control ) ×

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100.14

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The herbicidal effects of berberine at 10 mg L−1 on duckweed (Lemna minor)

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were determined by the method described by Iwasa (2000).15 Hogland nutrient

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solution was used as a nutrient-supplemented liquid medium. Berberine was applied

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to the plastic bowls containing 30 L. minor plants in Hoagland’s media at final

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concentrations 10 mg L−1 and placed in an environmental growth chamber calibrated

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to provide 12 h light/12 h darkness at 25±2 °C. The fresh weight of each replicate was

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investigated before the treatment. Eighteen replicates in each treatment. Three

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randomly selected replicates in each treatment were used to evaluate the fresh weight,

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thallus number, contents of chlorophyll a, chlorophyll b and total chlorophyll 3, 5, 7,

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and 9 d after the treatment. Visual results were presented in a 6 -well cell culture plate.

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Glyphosate, cinmethylin, sulcotrione and 2,4-D were used as contrast herbicides

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to show the herbicidal effect of the natural product berberine. Achenes of B. pilosa

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were pretreated as above. Berberine and glyphosate (Dikma Technologies, CAS:

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1071-83-6, 95.5%) were dissolved in water while 2,4-D (Sigma, CAS: 94-75-7, 98%)

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were dissolved in ethanol to make 5000 mg L−1 stock solution and then diluted with

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water to get series of concentrations from 2 to 32 mg L−1. Cinmethylin (Sigma, CAS:

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87818-31-3, 98.7%) and sulcotrione (Sigma, CAS: 99105-77-8, 98.8%) were

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dissolved in acetone to make 2000 mg L−1 stock solution and then diluted with water

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to get series of concentrations from 2 to 32 mg L−1. A similar set of experiments were

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conducted for the seeds treated with sterile water only for berberine or glyphosate, or

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treated with ethanol (0.64%) for 2,4-D to serve as controls. The primary root length

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and fresh weight were investigated at 14 d after incubation.16 All experiments were

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repeated at least three times.

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Absorption and transport of berberine in B. pilosa seedling

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Main absorption site was determined by applying berberine on B. pilosa

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seedlings (4-leaf stage) at a final concentration of 32 mg L−1 by root incubation,

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stem-coating and leaf-dipping. Primary root length, plant height, whole plant fresh

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weight and root fresh weight were measured at 1, 2, 3, 5 and 7 d after incubation.

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Transport pattern of berberine in B. pilosa was determined by the method

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described previously by Kamal et al.17 Specifically, B. pilosa seedlings (4-6-leaf stage)

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were treated by root incubation with 32 mg L−1 berberine or by leaf-painting with

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1000 mg L−1 berberine (40 µL on four new leaves of each B. pilosa, nine plants in

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each pot). Then the content of berberine in stem, leaf, root and the incubation solution

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was detected by HPLC (Agilent 1100, Santa Clara, CA) at different time intervals (0.5,

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1, 2, 3, 5 and 7 d after the treatment). Chromatographic analysis was investigated on a

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Spherisorb 5HC-C18, 250 mm×4.6 mm (Agilent) column. Detection was performed

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with a UV detector. Acetonitrile + 0.05mol L-1 sodium dihydrogen phosphate (25 + 75

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by volume) was used as the mobile phase. The injection volume of sample was 10.00

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µL and the flow rate was kept at 1 mL min-1. Column temperature was

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30 °C.Berberine has an excitation wavelength of 405 nm and emission at 520 nm.18

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Transport of berberine was further confirmed in B. pilosa by fluorescence microscope.

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Visualization of uptake and transportation of berberine was performed on 20 d old B.

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pilosa seedlings. Specifically, roots, stems and leafs were collected at 0.5, 1, 2, 3 and

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5 d after root incubation treatment with 32 mg L−1 berberine and washed thoroughly

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with water to remove the residue. Free-hand cross-sections of root, stem and petiole

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were prepared.19 Then sections were examined using fluorescence microscopy (Nikon

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DS-Ri1). The presence of fluorescence at the transportation site is positive for

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berberine. All experiments were repeated at least three times.

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Analysis of B. pilosa root treated with berberine.

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The sterilized seeds were planted in 10×10 cm Petri dish containing Hoagland's

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media. Berberine was added to the Hoagland's media at final concentrations ranging

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from 2 to 32 mg L−1. Growth conditions were described as above.

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Fresh root samples (0.2 g) were ground in 1 mL phosphate buffer solution (0.1

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mol L-1, pH7.8) in a mortar precooled to -20 °C, and then centrifuged at 12000 rpm for

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15 min at 4 °C. The supernatant was used to detect the relative factors.

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The root vigor of B. pilosa root was measured using triphenyl tetrazolium

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chloride (TTC) as described by Ruf et al.20 The activities of superoxide dismutase

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(SOD), polyphenol oxidase (PPO) and peroxidase (POD) were measured according to

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the methods by Zhang et al.,21 Thipyapong et al.,22 and Zipor et al.23

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Oxidative damage to root lipids, resulting from berberine, was estimated by the

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content of total 2-thiobarbituric acid reactive substances (TBARS) expressed as

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equivalents of malondialdehyde (MDA). The TBARS content was estimated by the

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method of Hajlaoui et al.24 with some modifications. Fresh root samples (0.5 g) were

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ground in 5 mL of 10% (w/v) trichloroacetic acid (TCA). Following the centrifugation

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at 4000 rpm for 10 min, an aliquot of 1 mL from the supernatant was added to 1 mL

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of 0.6% (w/v) thiobarbituric acid (TBA). The samples were heated at 100 °C for 15

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min. Thereafter, the reaction was stopped by cooling down in an ice water bath. After

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centrifugation, the absorbance of the supernatant was read at 532 nm on a

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spectrophotometer (Model Camspec M330 UV/Vis) and corrected for non-specific

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turbidity by subtracting the absorbance at 600 nm. The concentration of

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malondialdehyde was calculated using the extinction coefficient of 155 mm cm-1.

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Fatty acid (FA) composition was determined by gas chromatography-mass

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spectrometry (GC-MS). Total fatty acids were converted into their methyl esters using

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sodium methylate, according to the method described by Bettaieb et al.25 Individual

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FA were separated and quantified by GC-MS using a model Agilent Technologies

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6890N network GC system equipped with an Agilent Technologies 5975 inert XL

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mass selective detector. The test columns all had nominal dimensions of 30 m × 250

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µm × 0.5 µm film thickness. The temperature was programmed to increase from 120

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to 180 °C at a rate of 12 °C per min and continues to keep 1 min, after the

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programmed to increase from 180 to 250 °C at a rate of 8 °C per min and continues to

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keep 15 min. FA in samples was identified by library searching of NIST. The relative

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amount of each FA was calculated from their peak areas as compared with that of

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standards.

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Analysis of root tip surface and root meristem of B. pilosa treated with

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berberine.

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Scanning electron microscopy (SEM) was performed in order to examine the

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effect of berberine on the root surface of 7 d old B. pilosa seedlings, treated with

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berbeine at 3.2 mg L−1 and 32 mg L−1 respectively. The SEM samples were processed

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as previously described by Cao et al.26 Root tip samples were visualized by SEM (FEI

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XL-30, Netherlands).

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Transmission electron microscopy (TEM) was performed to examine the effect

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of berberine on root meristem cells of 7 d old B. pilosa seedlings, treated berberine at

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3.2 mg L−1 and 32 mg L−1. The TEM samples were processed as previously described

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by Houot et al.27 Root tip samples were examined by TEM (FEI Tecnai 12,

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Netherlands).

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Safety evaluation of berberine on sensitive organism, Brachydanio rerio

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Brachydania rerio was domesticated in a tank of dechlorinated water for 1 week

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before the start of the experiment. During domestication, B. rerio were fed once per

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day. Fasting was forced upon them before the day of experiment. The death rate was

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below 1% during the domestication period. The B. rerio length was 3.05±0.26 cm,

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and the corresponding weight was 0.26±0.06 g. The acute toxicity was measured by

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the method of Botelho et al.28

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Statistical analysis

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The inhibition rates of the fresh weight or the root lengths of tested weeds were

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analyzed using SPSS 19.0 with the probit analysis. Then, the estimation of the median

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inhibition concentration (IC50) or the median lethal concentration (LC50) and its 95%

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confidence limits were obtained. All quantitative data were presented as the mean ±

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SD of at least three independent experiments using the Duncan's multiple range test or

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Student’s t test for group differences. A p < 0.05 was considered as statistically

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significant.

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RESULTS AND DISCUSSION

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Herbicidal activity of berberine

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Berberine displayed broad spectrum herbicidal activities against a variety of

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weeds including monocots and dicots. However, berberine at 2-32 mg L−1 showed no

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effect on the germination of these tested plants (Table 1).

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The IC50 values of berberine on the primary root length of dicot plants, Ageratum

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conyzoides, Mikania micrantha, Celosia argentea Amaranthus retroflexus and Bidens

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pilosa 7 d after the treatment, were 2.91, 2.36, 2.39 3.76 and 3.03 mg L−1, respectively,

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and the IC50 values of berberine on fresh weight of Ageratum conyzoides, Mikania

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micrantha, Celosia argentea Amaranthus retroflexus and Bidens pilosa were 5.76,

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6.42, 5.83, 4.57 and 6.45 mg L−1, respectively. In parallel, the IC50 values of berberine

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on root lengths of monocot plants, Echinochloa crusgalli, Dactyloctenium willd,

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Eleusine indica, Triticum aestivum and Oryza sativa 7 d after the treatment were 6.71,

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5.44, 4.83, 9.79 and 6.79 mg L−1, respectively, and the IC50 values of berberine on

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fresh weight of Echinochloa crusgalli, Dactyloctenium willd, Eleusine indica,

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Triticum aestivum and Oryza sativa 7 d after the treatment were 16.89, 18.83, 17.32,

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28.36 and 35.07 mg L−1, respectively, which were only slightly higher than those of

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dicot plants, indicating that berberine has non-selective herbicidal activities among

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monocot and dicot weeds (Table2, Table 3). The selectivity of berberine is similar to

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other non-selective bioherbicides.29

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The herbicidal activity of berberine on L. minor was examined with 10 mg L−1

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berberine. Between 3 to 14 d, the biomass of untreated L. minor increased 17.32 times

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(181.90 mg /10.50 mg), and meanwhile their thallus number also increased 5.17 times

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((131.67-21.33) /21.33). By contrast, the treated L. minor decreased 6.22 times

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(-16.8/-2.7) in biomass, and produced no new thallus (Table 4). Especially, 3 d after

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treatment, L. minor turned to yellow; when the treatment was extended to 9 d, all of

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the L. minor plants have rotted completely (Fig.2). The yellowish phenotype in treated

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L. minor suggested that chlorophyll synthesis could be inhibited. Indeed, after 3 d,

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treatment, chlorophyll a and b were reduced to 52.24% and 50.53% of untreated L.

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minor, respectively. After 7 d, the synthesis of chlorophyll a, chlorophyll b and the

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total chlorophyll were all completely abolished (Table 5, Table 6). These results

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indicated that 10 mg L−1 berberine possessed significant herbicidal activity on L.

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minor, which is not consistent with what was reported by Iwasa (2000), who showed

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that 10 mg L−1 berbeine only resulted in a 75% inhibition rate and therefore berbeine

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has no potential for agrochemical use (Iwasa, 2000).15 This inconsistency could be

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resulted from the different growth potentials of different L. minor populations and the

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different test temperatures.

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In a 14-d treatment, berberine reduced fresh weight of B. pilosa by 80.91% at 16

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mg L−1; and by 84.84% at 32 mg L−1. Similar reduction was observed with

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cinmethylin treatment (82.62% at 16 mg L−1; and 86.24% at 32 mg L−1). Fresh weight

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loss caused by glyphosate in the similar condition was 93.66% at 16 mg L−1; and

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95.56% at 32 mg L−1, significantly higher than that caused by either berberine or

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cinmethylin (Table 7). In the concentration between 4 to 32 mg L−1, berberine

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inhibited B. pilosa primary root growth with a rate similar to that of cinmethylin and

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there were no significant differences. However, generally, glyphosate showed better

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efficacy to B. pilosa those of berberine and cinmethylin (Table 7). This is very active

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for a natural product and QSAR of berberine and its possible derivatives need to be

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further studied.

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Sulcotrione and 2,4-D both showed the highest herbicidal activity on B. pilosa

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among the five weeds we tested here. They were found to inhibit the germination

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process, as shown in figure 3, therefore the inhibition of fresh weight and the primary

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root were not calculated.

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More often, natural herbicidal compounds are considered as lead compound due

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to their less potent activity compared with their synthetic counterparts (glufosinate

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and the triketone herbicides).3 Nevertheless, they still have great potential in their

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possible new mode of action, a consideration needed in order to deal with the

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evolution of resistance to herbicides in agricultural production. For example, the

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optimal concentration of sarmentine, isolated from Piper longum, for excellent control

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of barnyard grass was 5 mg/mL.4 Still, sarmentine was attractive due to its multiple

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herbicide mechanisms of action.30 Surely scientists have found some plant-derived

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compounds with distinctive herbicidal activity such as patented m-tyrosine.31 In our

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study, 7 d after the treatment, the IC50 of berberine, glyphosate and 2,4-D on the root

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growth of B. pilosa were 3.27, 3.45 and 0.23 mg L−1, respectively, whereas the IC50

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values of berberine, glyphosate and 2,4-D on the fresh weight of B. pilosa were 6.11,

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7.72 and 0.92 mg L−1, respectively.

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In conclusion, berberine possessed broad spectrum herbicidal activities on both

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moncots and dicots and showed similar effect on B. pilosa to the commercial naturally

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derived herbicide cinmethylin. These results indicated that berberine could be used as

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a new bioherbicide directly or serve as a template compound for a new type of

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herbicides.

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Absorption and transport of berberine in B. pilosa.

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Berberine treatment caused a continuous increase in the inhibition rates (the

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inhibition rate of root elongation, whole plant height, fresh root weight and fresh plant

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weight) in root incubation treatment during the time intervals from 1 to 7 d. However,

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the inhibition rates of root elongation, plant height, fresh root weight and fresh plant

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weight caused by both stem-coating and leaf-dipping methods were not significant,

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which indicated that berberine was mainly absorbed by root (Fig.4).

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To examine the absorption and transport pattern of berberine in the plant, B.

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pilosa seedlings (incubated in Hoagland solution for 50 d) were treated by root

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incubation in a water solution of berberine at a concentration of 32 mg L−1, or by stem

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coating with berberine at a concentration of 1000 mg L−1, or by leaf painting with

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berberine at a concentration of 1000 mg L−1.

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After berberine treatment (32 mg L−1) by root incubation, the accumulation of

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berberine in stem, leaf and root were detected by HPLC at different time points.

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During the time periods of 0.5, 1, 2, 3, 5 and 7 d after treatment, the concentrations of

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berberine in roots were 51.69, 63.59, 76.74, 50.45, 38.33 and 27.38 µg g-1,

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respectively; and were 6.41, 8.05, 12.33, 19.23, 14.58 and 9.09 µg g-1, respectively in

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stems; and were 0, 0, 2.76, 3.17, 3.14 and 2.63 µg g-1, respectively in leaves (Fig. 5A).

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These results indicate that berberine was gradually absorbed by root and was

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transported upward to stem and leaf. As it was shown (Fig. 5A), berberine

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concentration in root reached peak at 2 d after the treatment due to the absorption and

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accumulation of berberine at the first 2 d and decreased after 2 d, suggesting that

322

berberine degradation occurred 2 d after the treatment. Berberine concentration in the

323

root reached a peak at 3 d after the treatment in stem and leaf and decreased

324

afterwards (Fig 5A), possibly due to the relatively long distance of transport from root

325

to leaf.

326

The rate of degradation of berberine in the incubation solution with or without B.

327

pilosa were different. Without B. pilosa, 0.5, 1, and 2 d after the treatment, berberine

328

concentrations remaining in the solution were 31.99, 31.53 and 25.54 mg L−1,

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respectively; no berberine could be detected after 3 d of the treatment. Interestingly,

330

with B. pilosa, 0.5, 1, 2 and 3 d after the treatment, berberine remaining in the

331

solution were 30.96, 28.96, 26.69 and 13.73 mg L−1, while berberine could not be

332

detected 5 d after the treatment. The results indicated that berberine was degraded

333

faster in the control than in the treatment. The difference in the degradation rates

334

between the control and the treatment might be due to the effect of existence of the

335

root exudate of B. pilosa on berberine (Fig.5B).

336

After berberine treatment (1000 mg L−1) by leaf painting, berberine

337

concentrations in leaves during the time periods of 0.5, 1, 2, 3, 5 and 7 d, were 14.01,

338

18.07, 28.31, 18.86, 13.94 and 9.30 µg g-1, respectively (Fig. 5C). However, berberine

339

was not detectable in stems and roots (Fig. 5C). This result suggested that berberine

340

was transported acropetally, but not basipetally.

341

The uptake and transport of berberine within B. pilosa seedlings was visualized

342

under the fluorescence microscope. In roots, we observed that fluorescence was

343

present in endodermis, pericycle, phloem and protoxylem in the triarch root of B.

344

pilosa 0.5, 1, 2, 3 and 5 d after the treatment of berberine at 32 mg L−1. In the lower

345

part of the stem, with increasing times of treatment (0.5, 1, 2, 3 and 5 d) fluorescence

346

became more prominent in vascular bundles. From row 2 to 5, we observed quick

347

absorption and transport of berberine in root and the gradual transport and

348

accumulation of berberine through the vascular bundle in stem to leaves (Fig. 6).

349 350

In conclusion, berberine was mainly absorbed by plant root and was acropetally transported through vascular bundle in stem to leaves.

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Effects of berberine on physiological activities of B. pilosa.

352

Roots play an important role in the nutrient cycling of terrestrial ecosystems

353

because they constitute both sinks and sources for nutrients. Berberine caused the

354

increase of root vigor at concentrations ranging from 4 to 32 mg L−1, reaching a peak

355

at 16 mg L−1 berberine and the root vigor of which was 6.6 times of the control (Fig.

356

7A). This could be due to the increasing amount of berberine induced stress on B.

357

pilosa root and thus stimulated the metabolic activities in related root cells to degrade

358

the alien toxin, berberine. However, berberine at concentrations higher than 16 mg L−1

359

might cause an irreversible phytotoxic effect on root cells and therefore induced a

360

decrease of root vigor.

361

Environmental stresses, including herbicidal stress, induced the production of

362

reactive oxygen species (ROS), which can cause oxidative damage to plant cells.32

363

The responses of the antioxidant enzymes to berberine stress were different (Fig. 7).

364

The induction of POD activity in plants occurs in response to numerous biotic and

365

abiotic stimuli, including exposure to pathogens or elicitor preparations, chemical

366

oxidizing agents, red light, and mechanical stimuli. The roles that POD can play in

367

strengthening the cell wall and in the production of toxic secondary metabolites and

368

its simultaneous oxidant and anti-oxidant capabilities can make it an important factor

369

in the integrated defense response of plants to a variety of stresses.33 PPO catalyzes

370

the oxygen-dependent oxidation of mono- and o-diphenols to o-diquinones, highly

371

reactive intermediates, the secondary reactions of which are believed to be responsible

372

for the oxidative browning that occurs as a consequence of plant senescence,

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wounding and pathogen infection.34 PPOs have been known to biochemistry for a

374

century and several hypotheses regarding the function of PPO have been proposed,

375

including roles in the phenylpropanoid pathway and plant defense.35 A defensive role

376

for PPO has frequently been suggested due to the conspicuous appearance of PPO

377

reaction products upon wounding, pathogen infection, or insect infestation, and due to

378

the inducibility of PPO in response to various abiotic and biotic injuries or signaling

379

molecules.36 Berberine treatment caused almost a continuous increase in the POD and

380

PPO activities as berberine concentrations increased from 8 to 32 mg L−1 (Fig. 7B),

381

suggesting that the exposure to berberine caused an obvious oxidative damage to B.

382

pilosa. Berberine increased the POD and PPO activities more significantly at higher

383

concentrations. POD activities were 3.1, 2.6 and 11.4 times higher than the control

384

with the treatment of berberine at 8, 16 and 32 mg L−1, respectively, and PPO

385

activities were 3.1, 2.6 and 11.4 times higher than the control with the treatment of

386

berberine at 8, 16 and 32 mg L−1, respectively.

387

As antioxidative enzymes in plants, SOD plays important roles in scavenging

388

ROS produced under oxidative stress. The reduction of the enzyme activities can be

389

considered as indirect evidence for enhanced production of ROS.37 The activities of

390

SOD in B. pilosa (Fig. 7B) were obviously inhibited by berberine, and the inhibition

391

rates increased with increasing berberine concentrations. Berberine treatment had no

392

significant impact on SOD activity at the concentration of 4 mg L−1. However,

393

berberine at concentrations from 8 to 32 mg L−1 caused decreases in SOD activity.

394

Antioxidant enzymes are important for the metabolism of xenobiotics.38 These

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enzymes can be inactivated under a severe oxidative stress.39 We speculate that the

396

inhibition of SOD might be due to the accumulation of ROS induced by berberine.

397

The MDA accumulation is an indicator of lipid peroxidation in plant cells.40 We

398

measured MDA levels to quantify lipid peroxidation. Berberine treatments at

399

concentrations of 4 to 32 mg L−1 significantly increased the levels of MDA in B.

400

pilosa root (Fig. 7C). The MDA accumulation reached a maximum at berberine

401

concentration of 8 mg L−1 (about 48 nmol•gFW-1 in root) and remained at

402

concentrations of up to 32 mg L−1, indicating a higher degree of lipid peroxidation due

403

to berberine stress. Our results from the antioxidant enzyme activities and MDA

404

content indicated that berberine induced the oxidative stress and a higher

405

concentration of berberine could cause a stronger oxidative damage to B. pilosa.

the

406

Lipid peroxidation alters membrane properties and causes cell defects such as ion

407

leakage and cellular decompartmentation.41-42 Fatty acid composition can affect the

408

membrane fluidity, stability, and functions. The main roles of fatty acids in plant are

409

related to cell membrane functions and to metabolic processes. The degree of fatty

410

acid unsaturation is important in maintaining the fluidity of the membrane and in

411

providing the appropriate environment for membrane functions. The degree of

412

unsaturation of membrane fatty acids is also important in the process of plant

413

adaptation to growth environment.43 The computation of the index of unsaturated fatty

414

acids (IUFA) was calculated according to Liu et al.44 The results showed that the

415

IUFA first increased with berberine at 12.5 to 50 mg L−1 and then decreased with

416

berberine at 50 to 200 mg L−1 (Fig. 7D), suggesting lower concentration of berberine

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caused a positive reaction of B. pilosa to adapt to the stress by berberine treatment.

418

Nevertheless, higher concentrations of berberine from 50 to 200 mg L−1 caused

419

irreversible changes, revealed by the continuous decrease of IUFA percent. The

420

degree of fatty acid unsaturation is the more important factor intervening in the

421

maintaining of membrane fluidity and provides the appropriate environment for

422

membrane function and the processes of plant adaptation in the face of constraining

423

conditions. The plasma membrane is at the interface between the cell and its

424

environment, and serves to hold intact the entire cellular structure. Consequently,

425

destabilization of the lipid bilayer, often via reactive oxygen species (ROS)-induced

426

stress membrane peroxidation, results in uncontrolled electrolyte leakage and cell

427

death.45 Our results suggested that berberine disrupt the IUFA percent of cell

428

membrane and therefore affect membrane fluidity, which was eventually expressed as

429

decrease of adaptation ability of cell to stress environment and abnormal cell function

430

or even cell death.

431

Alteration of the root surface ultrastructure of B. pilosa by berberine

432

SEM images of the root surface of B. pilosa were acquired at magnifications of

433

800× and 12800× to study the effect of berberine on root inhibition of B. pilosa (Fig.

434

8).

435

Under a magnification of 800×, we observed regular longitudinal ridges on the

436

root surface in both control samples and samples treated with lower concentration of

437

berberine (3.2 mg L−1). However, the regular longitudinal ridges on the root surface

438

almost disappeared and irregular unevenness appeared with the treatment of higher

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concentration of berberine (32 mg L−1) (Fig. 8A, B, C).

440

Under higher magnification of 12800×, we observed smooth root surfaces in

441

control samples. By contrast, obvious shrinking occurred on the root surface with

442

berberine at 3.2 mg L−1. In addition to the shrinkage we also observed uneven

443

projections on the root surface when treated with berberine at 32 mg L−1 (Fig. 8D, E,

444

F).

445

These morphological changes indicated that berberine treatment might cause

446

damage in subcellular organization to root cells. Therefore, we conducted SEM

447

analysis to investigate the subcellular effect of berberine on root cells.

448

Longitudinal sections of root cells treated with 3.2 or 32 mg L−1 berberine for 7 d

449

were examined by conventional electron microscopy. TEM analysis of the

450

longitudinal sections of root cells showed chromatin condensation in nucleus,

451

nucleolus condensation, mitochondria collapse, and a slight increase in cell wall

452

opacity when plants were treated with 3.2 mg L−1 berberine. Severe damage was

453

observed in cell structure at higher berberine concentration 32 mg L−1. In this

454

treatment, plasmolysis occurred and damage to basic cell structure was severe, as

455

revealed by the fact that nucleolus condensation became more apparent, and as well

456

by damage to both the nuclear envelop and the plasma membrane and was

457

accompanied by increased cell wall opacity. As a contrast, intact nuclear envelope and

458

normally distributed chromatin were present in the control roots.

459

The appearance of invaginations of the nuclear envelope was also observed in

460

the treatment groups. Images of the nuclear bulge showed that these membrane

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profiles originated from the invaginations of the nuclear envelope. At these late stages

462

the nucleus showed a high condensation of the remaining chromatin and numerous

463

vesicles (Fig. 9E). These cells present anarchic structural changes and resemble

464

explosive necrosis.

465

apoptotic-like cell death.

466

Safety evaluation of berberine to aquatic organisms

27

Hence, berberine treatment appeared to induce necrosis and

467

The LC50 of dichromate to the tested batch of B. rerio was 241.32 mg L−1, which

468

met the required LC50 values of dichromate on the tested B. rerio at 24 h between 200

469

and 400 mg L−1. The LC50 value of berberine to B. rerio 96 h after the treatment were

470

126.38 mg L−1, which revealed that berberine was low toxic to B. rerio (Table 8). As a

471

contrast, the 96 h LC50 of commercially used herbicide atrazine to B. rerio is 29.06

472

mg L−1.46

473

Generally, berberine is considered to be a non-toxic alkaloid at doses used in

474

clinical situations.47 A dose up to 14,500 mg L−1 in rats and 7,000 mg L−1 in mice did

475

not cause any maternal deaths. The lowest observed adverse effect levels in maternal

476

rats and developmental toxicity LOAEL, based on reduced fetal body weight per litter

477

was 7,250 mg L−1 and 14,500 mg L−1, respectively. There were no fetal adverse

478

effects noted.48 Briefly, berberine was low toxic to the sensitive organism,

479

Brachydanio rerio. Therefore, the use of berberine as a safe, biocontrol agent on

480

weeds is promising.

481 482

AUTHOR INFORMATION 22

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Corresponding Author

484

*(L.-J.Z.)Phone: 86 15917363981. E-mail: [email protected].

485

ORCID

486

Jiao Wu: 0000-0003-1871-6034

487

Lijuan Zhou: 0000-0002-7720-4138

488

Author Contributions

489

||

490

Funding

491

This work was supported by Science and Technology Planning Programs of

492

Guangdong Province, China (2016A050502047 and 2015A020209151) and Science

493

and Technology Program of Guangzhou, China (201607010125)

494

Notes

495

The authors declare no competing financial interest.

J.W and JJ.M contributed equally to this work.

496

497

ABBREVIATIONS USED

498

ALS, acetolactate synthase; AR, analytical reagent; FA, fatty acid; GC-MS, gas

499

chromatography-mass spectrometry; IUFA, index of unsaturated fatty acids; MDA,

500

malondialdehyde; POD, peroxidase; PPO, polyphenol oxidase; ROS, reactive oxygen

501

species; SEM, scanning electron microscope; SOD, superoxide dismutase; TBA,

502

thiobarbituric acid; TBARS, 2-thiobarbituric acid reactive substances; TCA,

503

trichloroacetic acid; TEM, transmission electron microscopy; TTC, triphenyl

504

tetrazolium chloride.

505 506

ACKNOWLEDGMENT 23

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We thank all members of the department of Pesticide Science, South China

508

Agricultural University for the help with this project. This project was funded by

509

Science and Technology Planning Programs of Guangdong Province, China

510

(2016A050502047 and 2015A020209151) to L. Z. and Science and Technology

511

Program of Guangzhou, China (201607010125) to L. Z..

512 513

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(48) Jahnke, G. D.; Price, C. J.; Marr, M. C.; Myers, C. B.; George, J. D. Developmental toxicity

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Figure 1. Molecular structure of berberine.

654

Figure 2. Inhibitory effect of 10 mg L-1 berberine on L. minor. (A): Overall view of L. minor; (B):

655

Change of biomass; (C): Change of thallus number; (D) Chlorophyll a content; (E): Chlorophyll b

656

content; (F): Total chlorophyll content; (G): Inhibition rate of chlorophyll content. Letters represent

657

values that differed significantly in the Student’s t test (A,B,C,D,E,F) and Duncan's multiple range test

658

(G) (P < 0.05). Shown are the averages of 30 plants ± SE (n = 3)

659

Figure 3. Inhibitory effect of berberine, glyphosate, cinmethylin, sulcotrione and 2,4-D at different

660

concentration on B. pilosa. (A): Overall view of B. pilosa; (B): Inhibition rate of fresh weight; (C):

661

Inhibition rate of primary root length. Letters represent values that differed significantly in the

662

Duncan's multiple range test (P < 0.05). Shown are the averages of 30 plants ± SE (n = 3)

663

Figure 4. Inhibitory effect of berberine with different application methods (root incubation,

664

stem-coating and leaf-dipping ) on B. pilosa. (A): Root inhibition rate; (B): Plant height inhibition rate;

665

(C): Fresh root weight inhibition rate; (D): Total fresh plant weight inhibition rate. Letters represent

666

values that differed significantly in the Duncan's multiple range test (P < 0.05). Shown are the averages

667

of 30 plants ± SD (n = 3)

668

Figure 5. Uptake of berberine by root incubation and leaf coating obtained from B. pilosa

669

seedlings. Data are mean of 9 plant ± SE (n = 3). (A) Distribution of berberine in B. pilosa with

670

treatment of 32 mg L−1. (B) The remaining amount of berberine in water solution and control, shown

671

are the averages ± SE (n = 3). (C) Distribution of berberine with leaf coating at 1000 mg L−1

672

concentration of 40µL, each bar represents the mean of 9 plants ± SE, n = 3.

673

Figure 6. Representative fluorescence photographs of berberine in 6-d-old B. pilosa seedlings: The

674

roots were incubated in a solution containing 32 mg L−1 berberine. A1, B1, C1, D1, E1 and F1 was root;

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675

A2, B2, C2, D2, E2 and F2 was the lower part of the stem; A3, B3, C3, D3, E3 and F3 was the upper

676

stem; A4, B4, C4,D4, E4 and F4 was the first true leaf petiole; A5, B5, C5,D5, E5 and was the second

677

true leaf petiole; A (A1, A2, A3, A4 and A5), B (B1, B2, B3, B4 and B5), C (C1, C2, C3, C4 and C5),

678

D (D1, D2, D3, D4 and D5), E (E1, E2, E3, E4 and E5) and F (F1, F2, F3, F4 and F5) were control, 0.5,

679

1, 2, 3, 5 and 7 d, respectively. The presence of fluorescent berberine was indicated by arrowheads. P:

680

pith. VB: vascular bundle.

681

Figure 7. Physiological effects of berberine on B. pilosa. A: Effects of berberine on root vigor of B.

682

pilosa root; B: Effects of berberine on POD, PPO and SOD activity of B. pilosa root; C: Effects of

683

berberine on MDA of B. pilosa root; D: Effect of berberine on IUFA of B. pilosa. Letters represent

684

values that differed significantly in the Duncan's multiple range test (P < 0.05). Shown are the averages

685

± SD (n = 3). Index of unsaturated fatty acid (IUFA): {[16 : 1 %×1] + [18 : 1 % ×1] + [18 : 2 %×2]

686

+ [18 : 3 %×3]} ×100 % .

687 688

47

Figure 8. SEM analysis of B. pilosa root surface under control (A and D) and treatment (B, E and C, D) with berberine (3.2 and 32 mg L−1)

689

Figure 9. Ultrastructure changes observed in root cells treated with berberine. Root cells were

690

treated with 3.2 and 32 mg L−1 berberine for 7 d, observed by transmission electron microscopy. TEM

691

details of the effect of berberine. The control cells (A, B, C), treatment with berberine (D, E, F treated

692

with berberine 3.2 mg L−1 and G, H, I treated with berberine at 32 mg L−1).

693 694 695 696 697 698 699 700

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701

Table 1 The effect of berberine on the germination rates of some plants

Test plant

Ageratum conyzoides

Mikania micrantha

Germination rate (%)

Time (d)

Control

2 mg L-1

4 mg L-1

8 mg L-1

16 mg L-1

32 mg L-1

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

2

26.03±3.69a

21.98±0.55a

25.42±3.25a

27.40±1.29a

23.15±3.34a

27.04±5.96a

3

39.36±3.07a

38.83±4.03a

38.47±4.48a

36.99±3.90a

30.79±1.62a

30.37±4.82a

4

39.36±3.07a

38.83±4.03a

38.47±4.48a

36.99±3.90a

30.79±1.62a

30.37±4.82a

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

2

78.46±1.54a

83.65±2.56a

82.42±2.77a

81.84±2.77a

81.48±1.75a

82.65±1.38a

3

82.31±2.31a

86.03±0.32a

84.98±0.36a

83.80±1.00a

83.56±2.63a

84.87±0.97a

4

82.31±2.31a

86.03±0.32a

84.98±0.36a

83.80±1.00a

83.56±2.63a

84.87±0.97a

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

2

29.88±2.02a

24.95±0.81a

27.00±1.66a

30.54±2.60a

26.91±1.31a

29.79±1.10a

3

46.58±2.34a

42.35±0.75a

44.79±1.22a

42.97±3.12a

40.14±4.67a

43.75±2.53a

4

46.58±2.34a

42.35±0.75a

44.79±1.22a

42.97±3.12a

40.14±4.67a

43.75±2.53a

1

77.76±0.48a

72.15±1.07a

76.61±2.85a

77.25±3.81a

78.57±2.03a

74.25±1.80a

2

77.76±0.48a

72.15±1.07a

76.61±2.85a

77.25±3.81a

78.57±2.03a

74.25±1.80a

3

77.76±0.48a

72.15±1.07a

76.61±2.85a

77.25±3.81a

78.57±2.03a

74.25±1.80a

4

77.76±0.48a

72.15±1.07a

76.61±2.85a

77.25±3.81a

78.57±2.03a

74.25±1.80a

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

2

35.05±2.51a

33.73±4.37a

32.46±6.42a

33.23±1.84a

35.45±5.79a

36.46±4.64a

3

66.92±4.59a

69.52±6.67a

64.52±1.40a

66.54±3.40a

67.58±2.58a

64.88±3.96a

4

90.46±0.26a

91.90±1.67a

90.32±0.16a

93.72±1.41a

92.12±2.88a

91.79±1.66a

1

5.25±2.72a

3.71±1.87a

5.59±3.06a

4.04±2.02a

3.98±2.03a

3.42±1.71a

2

42.61±3.47a

39.34±1.30a

41.15±2.17a

40.81±1.81a

39.73±3.23a

42.00±1.76a

3

46.51±2.06a

43.06±2.09a

42.91±0.47a

42.89±0.86a

45.79±2.99a

47.27±1.61a

4

46.51±2.06a

43.06±2.09a

42.91±0.47a

42.89±0.86a

45.79±2.99a

47.27±1.61a

1

4.74±2.47a

8.23±1.77a

5.79±3.22a

5.36±3.04a

4.78±2.63a

2.97±1.48a

2

39.38±2.05a

39.56±1.08a

44.58±2.92a

43.34±2.46a

42.91±1.30a

43.62±2.13a

3

43.30±1.88a

41.78±2.66a

48.66±3.55a

45.19±1.11a

44.67±1.83a

45.13±1.66a

4

43.30±1.88a

41.78±2.66a

48.66±3.55a

45.19±1.11a

44.67±1.83a

45.13±1.66a

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

Celosia argentea

Amaranthus retroflexus

Bidens pilosa

Echinochloa crusgalli

Dactyloctenium willd

Eleusine indica

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Triticum aestivum

Page 32 of 51

2

41.35±0.83a

43.94±2.14a

43.47±1.93a

43.57±0.71a

42.60±0.75a

41.29±2.46a

3

41.35±0.83a

43.94±2.14a

43.47±1.93a

43.57±0.71a

42.60±0.75a

41.29±2.46a

4

41.35±0.83a

43.94±2.14a

43.47±1.93a

43.57±0.71a

42.60±0.75a

41.29±2.46a

1

78.57±3.91a

78.26±2.80a

75.93±1.85a

77.73±0.72a

77.78±2.78a

75.00±1.61a

2

82.27±2.35a

83.72±2.88a

79.63±1.85a

79.69±1.37a

79.86±2.50a

78.70±2.45a

3

82.27±2.35a

83.72±2.88a

79.63±1.85a

79.69±1.37a

79.86±2.50a

78.70±2.45a

4

82.27±2.35a

83.72±2.88a

79.63±1.85a

79.69±1.37a

79.86±2.50a

78.70±2.45a

1

89.10±2.89a

89.29±3.00a

89.93±2.66a

90.11±2.20a

88.85±2.79a

88.89±2.78a

2

89.10±2.89a

89.29±3.00a

89.93±2.66a

90.11±2.20a

88.85±2.79a

88.89±2.78a

3

89.10±2.89a

89.29±3.00a

89.93±2.66a

90.11±2.20a

88.85±2.79a

88.89±2.78a

4

89.10±2.89a

89.29±3.00a

89.93±2.66a

90.11±2.20a

88.85±2.79a

88.89±2.78a

Oryza sativa

702 703 704

The germination rates in the treatments did not change from 3 or 4 d to 7 d after the treatment. All data represent means ± SE. Values followed by different letters in the same line were significant differences (P < 0.05) according to Duncan’s multiple range test..

705 706 707 708 709 710 711 712 713 714 715 716 717

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718

Table 2 IC50 values of berberine on root length of some plants (7 d) r

IC50 (mg L−1) (95% CL)

y=3.9664+2.2271 x

0.9648

2.91(1.99-4.25)

Mikania micrantha

y=4.3693+1.6874x

0.9878

2.36 (1.64-3.40)

Celosia argentea

y=4.2976+1.8587x

0.9767

2.39 (1.85-3.07)

Amaranthus retroflexus

y=4.0338+1.6782x

0.9790

3.76 (2.68-5.28)

Bidens pilosa

y=4.3419+1.3676x

0.9799

3.03 (2.32-3.95)

Echinochloa crusgalli

y=3.7822+1.4726x

0.9958

6.71 (5.54-8.14)

Dactyloctenium willd

y=3.6580+1.8249x

0.9954

5.44 (4.64-6.37)

y=3.2961+2.4906x

0.9782

4.83 (4.18-5.58)

Triticum aestivum

y=2.8594+2.1602x

0.9882

9.79 (9.12-10.52)

Oryza sativa

y=3.7256+1.5320x

0.9980

6.79(6.08-7.58)

Test plants Ageratum conyzoides

Dicot

Monocot

719 720

Eleusine indica

Regression equation

All treatments were replicated at least three times and repeated at least three times. IC50 was the median inhibition rates of the root lengths of tested weeds.

721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746

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747

Table 3 IC50 values of berberine on fresh weight of some plants (7 d)

Dicot

Monocot

748 749

Page 34 of 51

Test plants

Regression equation

r

IC50 (mg L−1) (95% CL)

Ageratum conyzoides

y=3.9431+1.3904x

0.9637

5.76 (4.36-7.60)

Mikania micrantha

y=4.1526+1.0493x

0.9838

6.42 (5.15-8.01)

Celosia argentea

y=4.2069+1.0361x

0.9908

5.83 (4.99-6.80)

Amaranthus retroflexus

y=4.4723+0.7996x

0.9949

4.57 (2.97-7.04)

Bidens pilosa

y=4.0857+1.1297x

0.9958

6.45 (5.67-7.33)

Echinochloa crusgalli

y=3.4982+1.2234x

0.9807

16.89 (14.47-19.71)

Dactyloctenium willd

y=3.7437+0.9854x

0.9544

18.83 (14.48-24.49)

Eleusine indica

y=3.0383+1.5839x

0.9798

17.32 (12.98-23.11)

Triticum aestivum

y=3.2755+1.1871x

0.9844

28.36 (26.63-30.20)

Oryza sativa

y=3.2593+1.1267x

0.9683

35.07(31.55-38.97)

All treatments were replicated at least three times and repeated at least three times. IC50 was the median inhibition rates of the fresh weight of tested weeds.

750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775

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776

Table 4 Change of biomass and thallus number of L. minor

Time(d) 3 5 7 9 11 14 777 778

Change of biomass (mg) Control 10 mg L-1 10.50±2.35a 31.20±2.09a 62.23±3.93a 89.23±1.44a 100.47±3.15a 181.90±22.42a

Change of thallus number Control 10 mg L-1

-2.70±0.59b -3.23±2.39b -5.30 ±3.31b -5.97±1.51b -13.5±1.19b -16.8±2.08b

21.33±0.67a 38.33±4.33a 57.67±7.22a 79.33±3.93a 110.00±2.65a 131.67±9.33a

0.00±0.00b 0.00±0.00b 0.00±0.00b 0.00±0.00b 0.00±0.00b 0.00±0.00b

All data represent means ± SE. Values followed by different letters in the same line were significant differences (P < 0.05) according to Student's t test.

779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794

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Table 5 Change of chlorophyll content of L. minor (mg g-1)

795 Time (d)

796 797

Page 36 of 51

Chlorophyll a content (mg g-1)

Chlorophyll b content (mg g-1)

Total chlorophyll content (mg g-1)

Control

10 mg L-1

Control

10 mg L-1

Control

10 mg L-1

3

0.284±0.024a

0.120±0.019b

0.094±0.005a

0.047±0.007b

0.379±0.026a

0.166±0.026b

5

0.232±0.003a

0.030±0.002b

0.084±0.006a

0.013±0.002b

0.316±0.008a

0.043±0.004b

7

0.197±0.002a

0.000±0.000b

0.082±0.001a

0.000±0.000b

0.279±0.003a

0.000±0.000b

9

0.203±0.006a

0.000±0.000b

0.068±0.003a

0.000±0.000b

0.271±0.008a

0.000±0.000b

11

0.196±0.001a

0.000±0.000b

0.062±0.003a

0.000±0.000b

0.259±0.004a

0.000±0.000b

14

0.170±0.004a

0.000±0.000b

0.048±0.007a

0.001±0.001b

0.219±0.010a

0.007±0.004b

All data represent means ± SE. Values followed by different letters in the same line were significant differences (P < 0.05) according to Student's t test.

798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 36

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814 815

816 817

-1

Table 6 Inhibition rate of chlorophyll content of L. minor treated with 10 mg L berberine

Time (d)

Chlorophyll a (%)

Chlorophyll b (%)

Total chlorophyll (%)

3 5 7 9

52.24±7.45c 87.03±0.88b 100.00±0.00a 100.00±0.00a

50.53±7.70c 84.56±1.76b 100.00±0.00a 100.00±0.00a

56.05±6.78c 86.37±1.09b 100.00±0.00a 100.00±0.00a

All data represent means ± SE. Values followed by different letters in the same row were significant differences (P < 0.05) according to Duncan’s multiple range test.

818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 37

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Journal of Agricultural and Food Chemistry

835

Table 7 Inhibition rate of plant fresh weight and primary root length of B. pilosa (%) (14 d)

Concentration (mg L-1) 2 4 8 16 32 836 837

Page 38 of 51

Inhibition rate of plant fresh weight (%) berberine glyphosate cinmethylin

Inhibition rate of primary root (%) berberine glyphosate cinmethylin

44.08±1.94b 53.15±1.77c 64.57±1.82c 80.91±0.91b 84.84±0.44b

46.49±1.83b 75.52±1.56b 84.84±1.81b 88.48±0.43b 94.64±0.36ab

69.49±1.36a 86.67±0.63a 90.52±1.57a 93.66±0.15a 95.56±0.25a

70.59±0.81a 79.94±1.18b 80.99±0.17b 82.62±1.00b 86.24±1.52b

79.57±0.71a 88.03±0.26a 93.28±1.13a 93.86±0.25a 95.40±0.08a

82.85±1.71a 86.25±0.24b 88.56±1.02ab 90.26±1.40b 93.63±0.48b

All data represent means ± SE. Values followed by different letters in the same line were significant differences (P < 0.05) according to Duncan’s multiple range test.

838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855

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856

Table 8 The LC50 of berberine on the B. rerio (96 h) Toxicity regression equation

Correlation coefficient

LC50 (mg L−1) (95% CL)

y=-10.0319 + 7.1522 x

0.9311

126.38 (115.99-145.78)

857

All treatments were replicated at least three times and repeated at least three times.

858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896

LC50 was the median lethal concentration.

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Journal of Agricultural and Food Chemistry

897

Figure 1. Molecular structure of berberine

898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916

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A

917 918

C

B

D

E

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Journal of Agricultural and Food Chemistry

F

G

919 920

Figure 2. Inhibitory effect of 10 mg L-1 berberine on L. minor. (A): Overall view of L. minor; (B):

921

Change of biomass; (C): Change of thallus number; (D) Chlorophyll a content; (E): Chlorophyll b

922

content; (F): Total chlorophyll content; (G): Inhibition rate of chlorophyll content. Letters represent

923

values that differed significantly in the Student’s t test (B,C,D,E,F) and Duncan's multiple range test (G)

924

(P < 0.05). Shown are the averages of 30 plants ± SE (n = 3)

925 926 927 928

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A

929 43

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C

B

930 931

Figure 3. Inhibitory effect of berberine, glyphosate, cinmethylin, sulcotrione and 2,4-D at

932

different concentration on B. pilosa. (A): Overall view of B. pilosa; (B): Inhibition rate of fresh weight;

933

(C): Inhibition rate of primary root length. Letters represent values that differed significantly in the

934

Duncan's multiple range test (P < 0.05). Shown are the averages of 30 plants ± SE (n = 3)

935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 44

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Journal of Agricultural and Food Chemistry

18

a

B 18

1d

a

Inhibition rate of plants height (%)

Inhibition rate of roots elongation (%)

A

2d b

3d 5d

12

7d

c d a a a

6 a

a

a a

bb

b

2d

a a

3d

b

5d

12

7d c a ab

d

6

a ab ab

b c

0

bc c

c

0 root incubation

stem-coating

leaf-dipping

root incubation stem-coating

C

D 45

Inhibition rate of fresh plants weight (%)

Inhibition rate of fresh roots weight (%)

1d

1d

a

2d

a

3d 5d

b

30

7d

c a a

15

a

d

a

a a

a a

a

a

leaf-dipping

a

40

1d 2d 3d

b

30

5d c

20

7d

d a a

a

a

a a 10

a a e

a

b

0

0 root incubation

stem-coating

root incubation

leaf-dipping

stem-coating

leaf-dipping

961

Figure 4. Inhibitory effect of berberine with different application methods on (root incubation,

962

stem-coating and leaf-dipping ) B. pilosa. (A): Root inhibition rate; (B): Plant height inhibition rate;

963

(C): Fresh root weight inhibition rate; (D): Total fresh plant weight inhibition rate. Letters represent

964

values that differed significantly in the Duncan's multiple range test (P < 0.05). Shown are the averages

965

of 30 plants ± SD (n = 3)

966 967 968 969 970

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A

30

Concentration (µg g-1)

Journal of Agricultural and Food Chemistry

25

Page 46 of 51

leaf stem root

20 15 10 5 0 0.5d

1d

2d Time (d)

3d

5d

7d

971 40

C without B. pilosa

30

with B. pilosa

20 10 0 0.5d

1d

2d

3d

5d

Concentration (µg g-1)

Concentration (mg L-1)

B

7d

80

root

60

stem leaf

40 20 0 0.5d

1d

Time (d)

2d 3d Time (d)

5d

7d

972

Figure 5. Uptake of berberine by root incubation and leaf coating obtained from B. pilosa

973

seedlings. (A) Distribution of berberine in B. pilosa treated with 32 mg L−1 of berberine by root

974

incubation (B) The remaining amount of berberine in water solution with or without B. pilosa, B. pilosa

975

was treated as in (A); (C) Distribution of berberine with leaf coating (40 µL, 1000 mg L−1 ) Data are

976

means of 9 plants ± SE (n = 3).

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987

988

Figure 6. Representative fluorescence photographs of 6-d-old B. pilosa seedlings: The roots were

989

incubated in a solution containing 32 mg L−1 berberine. A1, B1, C1, D1, E1 and F1: roots; A2, B2, C2,

990

D2, E2 and F2: lower part of the stem; A3, B3, C3, D3, E3 and F3: upper stem; A4, B4, C4,D4, E4 and

991

F4: the first true leaf petiole; A5, B5, C5,D5 and E5: the second true leaf petiole; A (A1, A2, A3, A4

992

and A5), B (B1, B2, B3, B4 and B5), C (C1, C2, C3, C4 and C5), D (D1, D2, D3, D4 and D5), E (E1,

993

E2, E3, E4 and E5) and F (F1, F2, F3, F4 and F5) were control, 0.5, 1, 2, 3, 5 and 7 d, respectively. The

994

presence of fluorescent xenobiotics was indicated by arrowheads. P: pith. VB: vascular bundle.

995

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996 1200

a

800 b b 400 c d 0 0

4 8 16 Concentration (mg L−1)

32

PPO

c

c

4

a

0

6000

0

4

4000

8

16 32 b c

2000

e

d d

d

a b

c

0 0

4

8

16

32

1d 3d 5d 7d

a

b

40

20

a

SOD b

8 8000

POD

a

50

D

40 IUFA(%)

MDA concent (nmol•gFW-1)

a

a

Concentration (mg L−1)

60

C

12

10000

B Enzyme activity (U•gFW-1•min-1)

Root vigor (µg•g-1•h-1)

A

c

30

20

0 0

4 8 16 Concentration (mg L−1)

0

32

12.5

25

50

Concentration (mg

100 L−1)

997

Figure 7. Physiological effects of berberine on B. pilosa. A: Effects of berberine on root vigor of

998

B. pilosa; B:Effects of berberine on POD, PPO and SOD activity of B. pilosa root; C: Effects of

999

berberine on MDA of B. pilosa root; D: Effect of berberine on IUFA of B. pilosa. Letters represent

1000

values that differed significantly in the Duncan's multiple range test (P < 0.05). Data shown are the

1001

means ± SD (n = 3). Index of unsaturated fatty acid (IUFA): {[16 : 1 %×1] + [18 : 1 % ×1] + [18 :

1002

2 %×2] + [18 : 3 %×3]} ×100 %47.

1003 1004 1005 1006 1007 1008 1009 48

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1010 1011

1012 1013

A

B

C

D

E

F

Figure 8. SEM analysis of B. pilosa root surface under control (A and D) and treatment (B, E and C, F) with berberine (3.2 and 32 mg L−1)

1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029

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1030

1031

Figure 9. TEM analysis of the ultrastructure changes in root cells treated with berberine. Root

1032

cells were treated with 3.2 and 32 mg L−1 berberine for 7 d, A, B, C: root cells treated with water only ,

1033

D, E, F: root cells treated with 3.2 mg L−1 berberine ;G, H, I: root cells treated with 32 mg L−1

1034

berberine.

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