A dual-action pesticide carrier that continuously induces plant

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Agricultural and Environmental Chemistry

A dual-action pesticide carrier that continuously induces plant resistance enhances plant anti-TMV activity of plant and promotes plant growth Shunyu Xiang, Xing Lv, Linhai He, Huan Shi, Shuyue Liao, Changyun Liu, Qianqiao Huang, Xinyu Li, Xiaoting He, Haitao Chen, Daibin Wang, and Xianchao Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03484 • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 24, 2019

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Journal of Agricultural and Food Chemistry

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A dual-action pesticide carrier that continuously

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induces plant resistance enhances plant anti-

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TMV activity of plant and promotes plant growth

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Shunyu Xiang1#, Xing Lv1#, Linhai He2, Huan Shi1, Shuyue Liao1, Changyun Liu1, Qianqiao

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Huang1, Xinyu Li1, Xiaoting He1, Haitao Chen4, Daibin Wang3, Xianchao Sun1*

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1College of Plant Protection, Southwest University, Chongqing 400715, China

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2School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715,

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China

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3Chongqing Tobacco Science Research Institute, Chongqing, 400715, China.

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*Correspondence: Xianchao Sun Current address: College of Plant Protection, Southwest

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University, Beibei, Chongqing 400715, China. Email: [email protected]

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# The authors contributed equally to this work

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ACS Paragon Plus Environment

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Abstract: Improving plant resistance against systemic diseases remains a challenging research

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topic. In this study, we developed a dual-action pesticide-loaded hydrogel with the capacity to

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significantly induce plant resistance against TMV infection and promote plant growth. We

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produced an alginate-lentinan-amino-oligosaccharide hydrogel (ALA-hydrogel) by coating the

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surface of an alginate-lentinan drug-loaded hydrogel (AL-hydrogel) with amino-oligosaccharide

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using electrostatic action. We determined the formation of the amino-oligosaccharide film by

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using various approaches including Fourier transform infrared spectrometry (FTIR), the zeta

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potential test, scanning electron microscopy (SEM) and elemental analysis. It was found that the

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ALA-hydrogel exhibited stable sustained-release activity, and the release time was significantly

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longer than that of the AL-hydrogel. In addition, we demonstrated that the ALA-hydrogel was

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able to continuously and strongly induce plant resistance against TMV and increase the release

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of calcium ions to promote Nicotiana benthamiana (N. benthamiana) growth. Meanwhile, the

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ALA-hydrogel maintained an extremely high safety to organisms. Our findings provide an

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alternative to the traditional approach of applying pesticide for controlling plant viral diseases. In

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the future, this hydrogel with the simple synthesis method, green synthetic materials, and its

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efficiency in the induction of plant resistance will attract increasing attention and have a good

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potential to be employed in the plant protection and agricultural production.

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KEYWORDS: amino-alginate hydrogel, Ca-alginate hydrogel, lentinan, GFP-TMV, induced res

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istance

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1. INTRODUCTION

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Tobacco mosaic virus (TMV), an RNA virus, infects tobacco, tomato, potato and many other

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plants. It cause mosaicism, poor plant growth, and deformed leaves symptoms resulting in severe

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losses in agricultural production and have the name ‘‘plant cancer’’. TMV is a systemic plant

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viral pathogen and to date there are no effective control methods and treatments available to

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protect plants in response to TMV infection.1-6 In recent years, inducers of plant resistance have

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gained increasing attention of researchers.7-13 The emergence of these inducers has altered the

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traditional way, directly targeting or eliminating viruses, on controlling plant diseases. It has

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been documented that inducing plant resistance remains effective in the control of TMV in

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agricultural production.14-18 However, the conventional application method through spraying the

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attractant on leaves lacks the ability to maximally induce resistance to viruses, and it is not able

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to continuously induce plant resistance over a long period of time.19-22 Some natural products are

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not stable enough to tolerate the complex environment of farmland, which could impair their

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normal functions.23-24 In addition, the duration of the inducers activity is relatively short, while a

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plant virus can survive throughout the whole life cycle of a plant. The short-term inducer activity

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and low induction efficiency make effective control of viral diseases difficult. Therefore, how to

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improve the control efficiency of inducer and prolong the induction time of inducer is crucial.

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Hydrogels have been widely used as carriers to assist drug release.25-29 In recent years, various

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environment-sensitive hydrogels, such as pH-sensitive hydrogels, temperature-sensitive

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hydrogels, and light-sensitive hydrogels, have been reported.30-35 Of these hydrogels, the

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sustained-release drug-loaded hydrogel is a relatively common and simple hydrogel system and

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it has a wide range of applications and can release drugs in different environments. These

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characteristics make it suitable for use in agricultural production. However, compared with other


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drug-loaded hydrogels, it is more difficult to control the drug release, and it is prone to an

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uneven release rate and can even burst, releasing the drug all at once.36-37 In practical, improving

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the performance of the slow-release hydrogel is a crucial step to prolong drug efficacy.

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Lentinan (LNT) is a neutral polysaccharide extracted from the fruiting body of Lentinus

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edodes and has the superior antifungal and antibacterial activities.38 Recently, LNT was found to

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have anti-plant virus activity.39 LNT can stimulate plant to switch off the stomata virus infection

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sites, so as to inhibit the viral infection of the plants.40 In addition, lentinan can induce systemic

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acquired resistance of the plants to suppress virus proliferation.41-42 When the plants are treated

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with lentinan, the plants will rapidly produce H2O2, which has been described as a hallmark of

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successful activation of plant defenses. Subsequently, the expression and accumulation of

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defense-related genes (PR-3, PR-1) of plants were induced. At the same time, some protective

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enzyme (POD, SOD Etc.) activity can be also induced.43-44 In this study, we selected sodium

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alginate as a pesticide carrier and lentinan as an inducer to synthesize sodium alginate-lentinan

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hydrogel (AL-hydrogel). Amino-oligosaccharide was used to form the film on the surface of the

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AL-hydrogel to control the release of LNT, and regulate the release rate of LNT. Interestingly,

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we found that ALA-hydrogel in the soil exhibited the ability to induce plant defenses against

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TMV and promote plant growth. With this approach, we obtained the long-term induction of

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plant resistance against TMV by stabilizing lentinan in the complex soil environment.


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SCHEME 1: Schematic representation of the synthesis of ALA-hydrogel and the induction of plant resistance.

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2. EXPERIMENTAL SECTION

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2.1. Experimental materials and instruments. Sodium alginate (SA, viscosity 200±20

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mPa.s) was purchased from Aladdin (Shanghai Aladdin biochemical technology co., LTD,

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Shanghai, China). The following kits and reagents were used: Eastep Super Total RNA

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Extraction Kit LS1040 (Promega Biotechnology Co., Ltd. Shanghai, China), PrimeScript RT

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Reverse Transcription Kit 037a (Shanghai Titan technology co. LTD, Shanghai, China), and

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SYBR green fluorescent dye solution (Chongqing Shuguang Biotechnology Co., Ltd.

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Chongqing, China). The following equipments were used in this work: an UV-visible

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spectrophotometer (PGENERAL), a scanning electron microscope (SEM, su8020, Hitachi,

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Japan), Fourier infrared (Nicolet 6700, Madison, WI, USA, 400–4000 cm−1), and Zetasizer

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Nano ZS90 (Malvern Panalytical, Heracles Almelo, Netherlands), elemental analyzer (Vario EL

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cube, Elementar Analysensysteme, Frankfurt, Germany) Petri dishes, and an ultraclean

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workbench (AIRTECH).

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2.2. Synthesis and characterization of hydrogels. In total, 0.2 grams of sodium alginate were

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dissolved in 20 mL distilled water and stirred until completely dissolved. Next, 0.2 g LNT were


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dissolved in 5 mL distilled water and poured into the sodium alginate solution and stirred with a

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magnetic stirrer (400 r/min) for 5–10 minutes. The LNT and sodium alginate solution were

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absorbed by droppers and slowly dropped into 250 mL aqueous CaCl2 (30 g/L) solution (stirred

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at 400 r/min, 30min ) to form a spherical hydrogel immediately (loading efficiency=14.7%,

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Encapsulation percentage=87.3%). The LNT hydrogel (AL-hydrogel) was rinsed with distilled

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water for 3 times, and the surface was dried with filter paper. 10 g/L aqueous amino-

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oligosaccharide solution (amino-oligosaccharide dissolved in distilled water) was added to the

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surface of lentinan-sodium-alginate hydrogel and stirred for 2 h (400 r/min). The amino-

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oligosaccharide-sodium-alginate hydrogel (ALA-hydrogel) was then washed three times with

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distilled water, and the surface water was dried with absorbent paper. Zeta potential, elemental

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analysis and scanning electron microscopy (SEM) were used to analyze the formation of the

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amino-oligosaccharide film. The following formula (1) and (2) were used to calculate the loading

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efficiency (LE) and encapsulation efficiency (EE) of LNT:45 𝑀1

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LE (%) = 𝑀 x100%

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where M1 is the quality of loaded LNT, and M2 is the quality of the synthesized hydrogel.

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2

𝑀𝐿0

EE (%)= 𝑀 x100% 𝐿1

(1)

(2)

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where ML0 is the quality of the loaded LNT, and ML1 is the quality of the input LNT.

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2.3. Preparation of LNT standard curve. The standard curve of LNT was determined by

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UV-visible spectrophotometry. LNT solutions were prepared with water to obtain concentrations

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of 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, and 31.3 μg/mL, and their respective

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absorbances were measured. The standard curve was drawn in Excel with the concentration as

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the abscissa and the absorbance as the ordinate to obtain the regression equation.


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2.4. Release Behavior Investigation. To compare the sustained-release ability and rate of

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ALA-hydrogel and AL-hydrogel, 50 pieces of ALA-hydrogel or AL-hydrogel were put into 10

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mL deionized water (pH=5) at 20 ℃ and ultraviolet-visible spectrophotometer was used to

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measure the absorbance of LNT in solution at 24-336 h, respectively. Next, to investigate the

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effects of different temperature, different pH and different concentration of cationic on the

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sustained-release ability and rate of ALA-hydrogel. 50 pieces of ALA-hydrogel were put into

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10mL water with pH of 3.0, 5.0, 7.0, and 9.0 adjusted by Tris-HCl, respectively, at 20 ℃ and

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used ultraviolet-visible spectrophotometer to measure the absorbance of LNT in solution at 24-

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336 h, respectively. 50 pieces ALA-hydrogel were put into 10ml deionized water (pH=5.0) at

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10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60℃ and ultraviolet-visible spectrophotometer was used to

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measure the absorbance of LNT in solution at 24-336 h, respectively. 50 pieces of ALA-hydrogel

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were put into 10mL NaCl solution (the concentration was 0.1 mol/L, 0.2 mol/L, 0.4 mol/L, 0.8

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mol/L, respectively) with pH of 5.0 at 20 ℃ and ultraviolet-visible spectrophotometer was used

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to measure the absorbance of LNT in solution at 24-336 h, respectively. Afterward, The

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following formula (3) was used to calculate the cumulative release rate46 (CRR) of LNT： 𝑡

CRR=

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∑0𝐶𝑡𝑉𝑡𝑜𝑡𝑎𝑙 𝑀0

(3)

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where Ct (mg/mL) is the concentration of the solution measured at time t respectively. Vtotal (10

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mL) is the volume of the total solution, and M0(mg) is the practical loading amount of LNT.

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2.5. The release kinetics measurement of ALA-hydrogel and AL-hydrogel. The release

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kinetics of LNT from ALA-hydrogel and AL-hydrogel were analyzed using Higuchi and

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Korsmeyer−Peppas models:47

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Higuchi: Q= Kt1/2

(4)


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Korsmeyer−Peppas: Q=Ktn

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(5)

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where Q is the cumulative release rate of LNT until time t, K is the kinetic constant, and n is

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an index which reflects the following release mechanisms: Fickian diffusion (n < 0.43), non-

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Fickian or anomalous diffusion (0.43 < n < 0.85), and case II transport (n > 0.85).

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2.6. Antivirus test of ALA-hydrogel hydrogel. Thirty tobacco seedlings (approximately 6-

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leaf age) with the same growth trend were selected. ALA-hydrogel, AL-hydrogel, Pure sodium

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alginate hydrogel (A-hydrogel) and sodium alginate-amino oligosaccharide hydrogel (without

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LNT) (AA-hydrogel) (50 pieces per each) were buried in the soil (near the roots of the tobacco

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seedlings), respectively. Each treatment group was set for six repeats. The seedlings were given

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10 mL water every 24 h (watered for 14 days) and placed in a constant temperature culture room

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at 25 °C (12 hours of light and 12 hours of darkness). After 7 days, TMV labeled with GFP was

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inoculated by the rubbing method.48 The inoculation methods are as follows: 0.1 g infected

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leaves were supplemented with 2 mL PBS buffer and ground into a homogenate, which was then

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centrifuged at 5000 r/min for 5 min before the supernatant was removed. The fourth and fifth

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leaves of N. benthamiana were selected, the leaf surface was rubbed by quartz sand to form a

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micro wound, and then, 100 μL toxic supernatant was applied to each leaf. Virus proliferation

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throughout the plant was assessed based on the diffusion of fluorescence in tobacco leaves

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observed under a UV lamp (Blak-Ray B-100AP, Upland, CA) 2 days post-inoculation (dpi), and

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one inoculated leaf was removed from each tobacco seedling and stored at −80 °C to quantify

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virus accumulation. At 7 dpi, optical photographs of the viral infection were taken with a camera

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(Nikon, Japan) under an UV lamp. The investigated new leaves of tobacco seedlings were stored

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at −80 °C prior to quantifying virus accumulation.


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2.7. Viral quantification test. The total RNA extraction kit was used to extract RNA from

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inoculated leaves and new leaves, and then cDNA was obtained using the reverse transcription

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kit. The concentrations of cDNA were measured and the samples were diluted to the same

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concentrations with sterile water. The SYBR green method for real-time fluorescence

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quantitative analysis was used to compare the leaf virus content for each treatment group.

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2.8. Plant growth test. Eighteen tobacco seedlings (approximately 3-leaf age) with the same

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growth trend were selected. A-hydrogel and AA-hydrogel (50 pieces per each) were buried in the

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soil (near the roots of the tobacco seedlings), respectively. Each treatment group was set for six

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repeats. The seedlings were given 10 mL water every 24 h (watered for 14 days) and placed in a

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constant temperature culture room at 25 °C (12 hours of light and 12 hours of darkness). After 14

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days, the plant height, plant width, leaf width, fresh weight and dry weight of N. benthamiana

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were measured.

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2.9. Biosafety Evaluation. Ten crucian carp with a length of 3-4 cm were placed in a 1 L LNT

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solution (the concentrations were 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL, and 1.6 mg/mL) and 1 L

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tap water (pH=5.5-6.0) with ALA-hydrogel or AL-hydrogel (The number of ALA-hydrogel or

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AL-hydrogel pieces per 1 L of water was 50, 100, 200, and 400, respectively). Pure tap water

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was used as a blank control and each group consisted of three repeats. The carp death number

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were observed, and recorded at 24h, 48h, 72h, and 96h, respectively.49

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3. RESULTS AND DISCUSSION

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3.1. Hydrogel characterization. Sodium alginate hydrogels were coated with amino-

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oligosaccharides by electrostatic action. Fig 1a showed the FTIR spectra of A-hydrogel and AA-

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hydrogel (without LNT). The A-hydrogel and AA-hydrogel exhibited peaks located at

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approximately 3300 cm-1. However, compared to the A-hydrogel, the AA-hydrogel featured a


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wider peak shape, which was attributed to the overlap associated with the peaks of the -OH

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stretching vibration of sodium alginate and the -NH stretching vibration of the amino-

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oligosaccharide. The peak at 1600 cm-1 was the characteristic -COOH peak of the A-hydrogel,

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while the AA-hydrogel exhibited a peak at approximately 1588 cm-1, which was formed by the

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overlap of the -NH3+ anti-symmetric deformation vibration peak of the amino-oligosaccharide

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and the sodium carboxylate absorption peak of sodium alginate. Furthermore, the AA-hydrogel

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peaked at 2927 cm-1 was due to the C-H stretching vibration of amino-oligosaccharide (Figure

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S-1), confirming the presence of amino-oligosaccharides in the AA-hydrogel. As expected,

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sodium alginate did not contain N, and the surface was negatively charged. Elemental analysis

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suggested that there was no elemental N in the A-hydrogel (Table 1). The zeta potential also

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showed that the surface was negatively charged. However, there was some elemental N in the

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AA-hydrogel, and their potentials became positive, which was due to the neutralization of the

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negative charge of sodium alginate by the positive charge of amino-oligosaccharide (Table 1 and

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Figure 1b). These data confirmed that the hydrogel was successfully coated with amino-

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oligosaccharide. Next, the appearances of the AA-hydrogel and A-hydrogel were determined by

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SEM. Figure 1c (SEMs of hydrogels) showed that the surface of A-hydrogel collapsed after

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drying, but the AA-hydrogel was intact with plump surface (Figure 1g), which was due to the

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amino-oligosaccharide film supporting the overall structure of the hydrogel surface. The surface

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of the A-hydrogel was loose and had large pores; in contrast, the AA-hydrogel was dense,

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comprising a network structure with some small pores. The compact surface played a key role in

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hindering the release of LNT from ALA-hydrogel. This property confirmed the formation of an

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amino-oligosaccharide film on the surface of A-hydrogel.


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Figure 1: FTIR spectra of the A-hydrogel and AA-hydrogel (a), Zeta potential of the A-hydrogel

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and AA-hydrogel (b), SEM images of the A-hydrogel (c-f), and SEM images of AA-hydrogel (g-

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j).

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Table 1: Elemental analysis of the A-hydrogel and AA-hydrogel.

Hydrogel A-hydrogel AA-hydrogel

N (wt%) 0 0.19

C (wt%) 10.23 12.38

H (wt%) 8.42 9.81

S (wt%) 0.04 0.01

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3.2. LNT release test and mechanism investigation. The controlled-release ability and the

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release time of drug directly affects its final therapeutic effect.50-53 Polysaccharides have an


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ultraviolet absorption peak at 210 nm.54 In this work, as the LNT was dissolved in water, the 210

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nm peak in Figure S-2 can be considered as the absorption peak of LNT. Figure 2a and 2b

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showed the cumulative LNT release concentration and the release rate of AL-hydrogel and ALA-

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hydrogel over 14 days, respectively. The released concentrations of hydrogel LNT calculated

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from the regression equation Y = 0.0028X + 0.0316 (R2=0.9992, indicating a strong positive

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linear correlation between the LNT concentration and absorbance) were shown in Figure S-3.

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Figure 2a showed that a large amount of LNT was released from the AL-hydrogel after soaking

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for 24 hours. At 7-day after soaking, the maximum cumulative release amount of LNT was

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achieved. This amount of release was kept from 7 to 14 days (Figure 2a and 2b). However, the

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amount of LNT released from ALA-hydrogel was significantly lower compared to that of AL-

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hydrogel, which remained at a constant rate from the one to fourteen-day after soaking (Figure

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2a). In addition, Figure 2b clearly showed that the release rate of LNT from the AL-hydrogel

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was largely higher than that of ALA-hydrogel. At 14-day after soaking, the cumulative release

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rate of LNT from ALA-hydrogel was approximately two-thirds of that AL-hydrogel, suggesting

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that the maximum sustained-release LNT cycle of the ALA-hydrogel exceeded 14 days. Based

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on these observations, we concluded that the sustained-release time of the ALA-hydrogel is

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much longer than that of the AL-hydrogel. This might be associated with the dense network

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structure formed by sodium alginate and amino oligosaccharides to retard the release of LNT.

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To further investigate the mechanism of the sustained release of ALA-hydrogel and AL-

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hydrogel. The release kinetics of LNT from ALA-hydrogel and AL-hydrogel were analyzed

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using Higuchi and Korsmeyer−Peppas models. Based on the Higuchi model fitting curve

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(Figure 2d), we found that there was a high linear correlation between the cumulative release

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rate and the square root of time during the release of LNT from the ALA-hydrogel (R2=0.9819)


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and AL-hydrogel (R2=0.9122). Similarly, a clear linear correlation between the logarithm of

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cumulative release rate and the logarithm of time in the process of LNT release from ALA-

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hydrogel (R2=0.9781) and AL-hydrogel (R2=0.9496) was observed in the Korsmeyer−Peppas

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model fitting curve (Figure 2c). In addition, the corresponding dispersion coefficients n of ALA-

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hydrogel and AL-hydrogel were 0.635 (0.43 < n < 0.85 )and 0.656 (0.43 < n < 0.85),

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respectively (Table 2), indicating that the release of LNT from ALA-hydrogel and AL-hydrogel

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under these conditions followed nonFick diffusion mechanism. This result strongly indicated that

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the amino-oligosaccharide film has no impact on the pesticide release mechanism of AL-

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hydrogel.

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Figure 2: Cumulative release of LNT from the ALA-hydrogel and AL-hydrogel (a). Cumulative

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release LNT rate from the AL-hydrogel and ALA-hydrogel (b). The fitting curves for the

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Korsmeyer-Peppas model (c) and Higuchi model. ** indicated separation among ALA-hydrogel

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and AL-hydrogel at the same amount by Duncan multiple comparison ( **: p < 0.01). vertical

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bars indicate standard deviations (n = 3) ± S.E.

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Table 2: Kinetic parameters associated with LNT released from the AL-hydrogel and ALAhydrogel.

Higuchi model

Korsmeyer-Peppas model

hydrogel

R

K

R

K

n

AL-hydrogel

0.9122

0.057

0.9496

0.08

0.656

ALA-hydrogel

0.9819

0.043

0.9781

0.06

0.635

2

2

257 258

3.3. Effect of pH, temperature and cation on LNT release from ALA-hydrogel. In the field,

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the soil environmental factors, such as pH, temperature, ion concentration, can be influenced by

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the microbial, rain, and fertilizer, etc.55-56 To determine the effects of soil environmental changes

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on the release of LNT from ALA-hydrogel, we measured the release of LNT from ALA-

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hydrogel at different pH, temperature and cation concentration. As shown in Figure 3a, the

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release rate of LNT from ALA-hydrogel was highly increased from 7.08 to 37.35 % when the pH

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increased from 3 to 9. This was attributed to that, in the acidic environment (low pH), most of

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the carboxyl groups on alginate was deionized, and could form strong hydrogen bonds with other

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functional groups such as hydroxyl groups and carboxyl groups, resulting in a tight shrinkage of

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the calcium alginate pellet to hinder the ALA-hydrogel from releasing LNT.57 Figure 3b showed

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that the cumulative LNT release rate increased with an increase in temperature from 10 ℃ to

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40 ℃. This might be caused by the increase of hydrogel swelling degree during the temperature


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increase, which could promote the release rate of LNT. In addition, we found that when the

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concentration of Na+ in the solution increased from 0 M to 0.8 M, the cumulative release rate of

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LNT increased from 28.1 to 98.5% (Figure 3c). This is the result of the ion exchange between

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Na+ in the solution and Ca2+ in the hydrogel (Table S-1), which led to the enhancement of

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electrostatics repulsion between alginate ions originated from −COO− and thus the improvement

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of swelling performance of hydrogel to stimulate the release of LNT.58

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Figure 3: The cumulative LNT release rate from ALA-hydrogel in different temperature (a), pH

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(b), and Na+ concentration (c). vertical bars indicate standard deviations (n = 3) ± S.E.

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3.4. Anti-TMV test of the ALA-hydrogel and AL-hydrogel. Previous studies reported that

280

hydrogel-controlled fertilizer release can effectively improve fertilizer utilization in the soil,

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suggesting that it can be utilized for improving pesticide utilization and efficacy.59-60 To assess

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the anti-virus activity of the ALA-hydrogel and AL-hydrogel, we visualized the spread of the

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TMV-GFP virus throughout the plant under an UV lamp. As shown in Figure 4a, at 2 dpi, the

284

number of green fluorescent spots in the inoculated leaves of N. benthamiana treated with two

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hydrogels was largely lower than that of the water control group. The number of green

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fluorescent spots in the AL-hydrogel-treated N. benthamiana was significantly lower than that of

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the ALA-hydrogel (Figure 4a) and the inhibition rate of AL-hydrogel is largely higher than that

288

of ALA-hydrogel (Figure 4b), indicating that the AL-hydrogel was more effective at inhibiting


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TMV-RNA spread than that of the AL-hydrogel. Furthermore, the number of TMV-GFP

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transcripts was quantified by qPCR and Figure 4c showed that the relative expression levels of

291

TMV RNA were significantly lower in the AL-hydrogel and ALA-hydrogel groups than that in

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the water control group. In contrast, At the same time, A-hydrogel and AA-hydrogel did not

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display any inhibition effect on the viral infection as compared with water control (Figure 4).

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Combined with pesticide-release analysis, we conclude that the AL-hydrogel released a large

295

amount of LNT in the early stage to induce plant resistance. The amount of LNT released from

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the ALA-hydrogel before 7-day after soaking was much lower than that of the AL-hydrogel,

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which resulted in a reduced ability to induce plant resistance against TMV compared with the

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AL-hydrogel in the early stage of LNT release.

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300

Figure 4: Comparisons of the TMV inhibition between different hydrogels at 2-day post TMV

301

inoculation. (a). The green fluorescent signals represented the viral replication in Nicotiana

302

benthamiana plants and the plants inoculated with Agrobacterium tumefaciens carrying TMV-

303

GFP constructs were photographed at 2-day post inoculation. The inhibition rate of TMV in each

304

treated plant (b). qPCR analysis showing the transcripts levels of TMV-GFP in Nicotiana

305

benthamiana plants. Expression levels were normalized to actin gene and three independent

306

experiments are presented (c). Mean values displayed in each bar followed by different letters

307

are significantly different according to the Duncan's multiple range test (p < 0.05). vertical bars

308

indicate standard deviations (n = 3) ± S.E.

309

3.5. ALA-hydrogel and AL-hydrogel delayed the spread of TMV in N. benthamiana. To furt

310

her examine the ability of both hydrogels to continuously induce plant resistance for a prolonged

311

period, we observed the spread of TMV in plants at 7 dpi. As shown in Figure 5a, at 7 dpi, pron

312

ounced green fluorescence signals were observed in the infiltrated and young leaves of water con

313

trol plant in comparison with the ALA-hydrogel and AL-hydrogel treated plants. Furthermore, t

314

he relative expression of TMV-GFP RNA in the water and treated plants was quantified by qPC

315

R and Figure 5b showed that the expression level of GFP in the ALA-hydrogel treated plant is si

316

gnificantly lower than that in other plants, indicating that ALA-hydrogel treated plant displayed

317

the strongest inhibition of TMV replication compared with other plants. This might be caused by

318

the stopped release of LNT from the AL-hydrogel almost stopped after the 7th day (Figure 2). T

319

herefore, we can conclude that the AL-hydrogel was not able to continuously induce plant resista

320

nce against TMV infection. Conversely, the ALA-hydrogel was able to continue to release LNT f

321

rom the 7th day to the 14th day, resulting in the continuously inducing plant resistance at 7 dpi to

322

inhibit the movement of the TMV in the plant treated with ALA-hydrogel. The result also sugges


17


Page 18 of 33

323

ted that the AA-hydrogel and the A-hydrogel were not able to inhibit the spread of TMV-GFP in

324

the host plants.

325 326

Figure 5: Comparisons of the TMV inhibition between different hydrogels at 7-day post TMV

327

inoculation. The green fluorescent signals represented the viral replication in Nicotiana

328

benthamiana plants and the plants inoculated with Agrobacterium tumefaciens carrying TMV-

329

GFP constructs were photographed at 7-day post inoculation. (a). qPCR analysis showing the

330

transcripts levels of TMV-GFP in Nicotiana benthamiana plants. Expression levels were


18

Page 19 of 33


331

normalized to actin gene and three independent experiments are presented (b). Mean values

332

displayed in each bar followed by different letters are significantly different according to the

333

Duncan's multiple range test (p < 0.05). vertical bars indicate standard deviations (n = 3) ± S.E.

334

3.6. The AA-hydrogel and A-hydrogel promote plant growth. Calcium ions (Ca2+), as a plant

335

nutrient, can promote the plant growth, improve photosynthesis and increase the synthesis and

336

accumulation of organic matter.61-64 In our study, hydrogels were formed by cross-linking Ca2+

337

with sodium alginate. As the LNT was released from the hydrogel, some of the Ca2+ were also

338

released (Figure S-4). To assess whether the hydrogel can stimulate plant growth, we measured

339

the plant growth including height, leaf width, dry weight and fresh weight of N. benthamiana

340

after the AA-hydrogel and A-hydrogel had been buried in the soil for 14 days. The AA-hydrogel

341

and A-hydrogel treated plants displayed significant increase in plant growth compared with that

342

of the water treated plant (Figure 6a and b, Table 3). The AA-hydrogel treated plant showed

343

higher increases in the plant growth measurements than the A-hydrogel, which was mainly

344

related to the concentration of the calcium ions released from the hydrogels (Figure 6 and Table

345

3). This implicated that the amino-oligosaccharide film is able to control the release rate of LNT

346

as well as promoting the release of calcium ions. This is because the cations in the solution can

347

exchange the Ca2+ in the sodium alginate-Ca2+ hydrogel, and the release amount of Ca2+ from

348

sodium alginate-Ca2+ hydrogel increased with increasing the concentration of cations in the

349

solution (Table S-1).58 In this work, the amino oligosaccharide is a polycation component, and

350

sodium alginate is a polyanion component. The strong electrostatic interaction between sodium

351

alginate and amino-oligosaccharides in the formation of the amino oligosaccharide film can

352

replace the Ca2+ on the surface of the sodium alginate-Ca2+ hydrogel to promote the Ca2+ release


19


Page 20 of 33

353

of ALA-hydrogel. Therefore, the ALA-hydrogel achieved a continuous increase in plant

354

resistance while also promoting plant growth.

355 356

Figure 6: Measurement of hydrogel in stimulating plant growth. Comparison of plant heights

357

between the AA-hydrogel, A-hydrogel, and water treated plants (a). Comparison of leaf widths

358

of the AA-hydrogel, A-hydrogel, and water treated plants (b).

359

Table 3: Effect of AA-hydrogel and A-hydrogel on the growth of N. benthamiana.

Measurement

CK

A-hydrogel

AA-hydrogel

Plant height (cm)

4.4±0.18 a

5.8±0.12 b

6.6±0.23 c

Plant width (cm)

8.2±0.14 a

10.2±0.30 b

11.3±0.24 c


20

Page 21 of 33


Leaf width (cm)

3.1±0.12 a

3.9±0.09 b

4.7±0.15 c

Fresh weight (g)

0.98±0.06 a

1.12±0.07 a

1.39±0.04 b

Dry weight (mg) 360

55.7±3.2 a 69.7±2.6 b 85.7±3.8 c Mean values displayed in each bar followed by different letters are significantly different

361

according to the Duncan's multiple range test (p < 0.05). vertical bars indicate standard

362

deviations (n = 3) ± S.E.

363

3.7. Biosafety investigation. Pesticide residues have been reported to seriously damage the

364

natural environment including polluting water bodies and causing aquatic organisms to die.49,65

365

Therefore, it is extremely important to evaluate the biosafety of new pesticides. To evaluate the

366

biosafety of ALA-hydrogel and AL-hydrogel, the toxic effects of series of concentrations of

367

ALA-hydrogel and AL-hydrogel on fish were examined. The LNT solution with different

368

concentration and pure tap water were used as the control groups. The result showed that the

369

LC50 of LNT to crucian carp at 24h, 48h, 72h, 96h was about 1.6 mg/mL, 0.8 mg/mL, 0.4 mg/mL

370

and 0.4 mg/mL, respectively (Table 4). According to the current pesticide toxicity classification

371

standards for fish,66 these data were significantly higher than 0.01 mg/mL. Therefore, LNT was

372

regarded as a low toxic pesticide with high safety. Interestingly, the results of ALA-hydrogel and

373

AL-hydrogel were the same as the water as no fish died in the ALA-hydrogel and AL-hydrogel

374

groups. Therefore, these hydrogels formed by the sodium alginate and LNT maintained an

375

extremely high safety to organisms.

376 377 378


21


Page 22 of 33

379 380

Table 4: Toxicity of LNT, AL-hydrogel, and ALA-hydrogel to crucian carp

Number of deaths Sample

Water LNT LNT LNT LNT AL-hydrogel AL-hydrogel AL-hydrogel AL-hydrogel ALA-hydrogel ALA-hydrogel ALA-hydrogel ALA-hydrogel

LNT content (1 L water) 0

24h

48h

72h

96h

0

0

0

1

200 mg

0

1

3

3

400 mg

1

2

5

5

800 mg

2

5

6

6

1600 mg

5

7

9

10

50 pieces (LNT:42 mg)

0

0

0

0


0

0

0

0

200pieces (LNT:168mg)

0

0

0

0

400 pieces (LNT:336mg)

0

0

0

0


0

0

0

0


0

0

0

1

200pieces (LNT:168mg)

0

0

0

0

400 pieces (LNT:336mg)

0

0

0

1

381 382

Taken together, we designed a green and simple dual-action pesticide hydrogel based on the

383

superior ability of LNT to induce plant resistance to TMV. The AL-hydrogel guaranteed that the

384

anti-viral effect of LNT maintained in the soil for a long time and achieved the sustained release

385

of LNT to induce plant resistance. More importantly, the formation of the amino-oligosaccharide

386

film further enhanced the slow-release ability of the AL-hydrogel, prolonged the sustained

387

release time, and achieved long-term induction of plant resistance. Furthermore, the film

388

improved the release of Ca2+ into the soil to promote N. benthamiana growth. These findings can


22

Page 23 of 33


389

significantly increase plant resistance against TMV. This strategy differs with the traditional

390

method of using antiviral agents, resulting in the strong improvement of preventing and

391

controlling viral diseases. Our study will provide new ideas and directions for the prevention and

392

control of systemic plant diseases.

393

SUPPORTING INFORMATION FTIR spectra of amino-oligosaccharide, ultraviolet spectrum

394

of lentinan, standard curve of lentinan, A-hydrogel and AA-hydrogel calcium ion release

395

statistics, and the Ca2+ cumulative release amount of ALA-hydrogel at different Na+

396

concentrations solution within 14 days.

397

ACKNOWLEDGMENTS

398

This study was partly supported by the National Natural Science Foundation of China

399

(31670148, 31870147), the Fundamental Research Funds for the Central Universities

400

(XDJK2016A009, XDJK2017C015) and the science and technology projects of Chongqing

401

Company

402

NY20180401070001, NY20180401070008).

403

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TOC graphic:

597


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A dual-action pesticide carrier that continuously induces plant

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