Application of Copper-Chitosan Nanoparticles Stimulate Growth and

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Application of Copper-chitosan nanoparticles stimulate growth and induce resistance in finger millet (Eleusine coracana Gaertn.) plants against blast disease Muthukrishnan Sathiyabama, and Appu Manikandan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05921 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 16, 2018

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Journal of Agricultural and Food Chemistry

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Application of Copper-chitosan nanoparticles stimulate growth and induce resistance in finger millet (Eleusine coracana Gaertn.) plants against blast disease

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Muthukrishnan Sathiyabama*, Appu Manikandan

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Department of Botany, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India

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* Corresponding Author Phone: +91 431 2407061; Fax: +91 431 2407045;

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e-mail: [email protected]

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ABSTRACT

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Copper-chitosan nanoparticle (CuChNp) was synthesized and used to study its effect on finger

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millet plant as a model plant system. Our objective was to explore the efficacy of CuChNp

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application to control blast disease of finger millet. CuChNp was applied to finger millet either

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as a foliar spray or as a combined application (involving seed coat and foliar spray). Both the

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application methods enhanced growth profile of finger millet plants and increased yield. The

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increased yield was nearly 89% in combined application method. Treated finger millet plants

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challenged with Pyricularia grisea showed suppression of blast disease development when

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compared to control. Nearly 75% protection was observed in the combined application of

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CuChNp to finger millet plants. In CuChNp treated finger millet plants, a significant increase in

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defense enzymes which was detected both qualitatively and quantitatively. The suppression of

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blast disease correlates well with increased defense enzymes in CuChNp treated finger millet

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plants.

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Key words: defense enzymes; suppression of blast disease; finger millet; copper-chitosan nanoparticle; Pyricularia grisea

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INTRODUCTION

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Finger millet (Eleusine coracana Gaertn.) is one of the important cereals which possesses a high

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content of calcium among all cereals and has the nutritional qualities better than that of other

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prominent cereals such as wheat, rice etc.1 Chandra et al. reported that the regular use of finger

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millet helps in managing different disorders of the body. 2 Finger millet is adapted to a wide

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range of environment and can withstand harsh climatic conditions,3 however its production is

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subjected to biotic factors. Among the biotic factors, finger millet blast caused by Pyricularia

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grisea is a major disease capable of devastating the unprotected finger millet, which results in

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reduction of physiological maturity, biomass and yield of the crop.4 The pathogen affects the

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crop at all growth stages from seedling stage, causing lesions and premature drying of young

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leaves, to flowering stage affecting the panicle causing neck and or finger blast5. It has been

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reported that the average yield loss due to finger millet blast is around 28% and it is as high as

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80-90% in endemic areas.5 P. grisea is also known to infect other cereal crops world wide and

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reduce crop yield and quality.6 Therefore, it is necessary to identify an effective control measure

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to protect cereal crops from this devastating pathogen to increase the yield. To date, satisfactory

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yield is obtained by the application of fungicides. The challenge posed by evolving adaptability

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of phyto-pathogens due to the uncontrolled use of synthetic fungicides/chemicals,7 have led to

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the exploration of alternative crop protection strategies in recent years. The search for such

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alternative disease management strategies supported by the advancement of nanotechnology,

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have paved the way for the application of nanomaterials as a potential candidate for disease

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control in plants.8,

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control of fungal pathogens.10,11

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The use of biopolymer-based nanomaterials holds great promise in the

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Copper has been used traditionally as major components of many agrochemicals for crop

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protection and improvement.10,12 Copper is relatively non-toxic to mammals and is toxic towards

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harmful microbes which offer it as an antimicrobial agent.13,14 Copper is one of the metal ions

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that can easily coordinate with chitosan.13 In plant system, chitosan has been reported to induce

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multifaceted disease resistance and enhance plant innate immunity.9 This property can be further

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enhanced by using it in the form of nanoparticles. Chitosan can form various chemical bonds

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with metal components, thus enhancing the stability of the nanoparticles.15 Nanoparticles are 3 ACS Paragon Plus Environment


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much more effective than its bulk form.16 Metal-based chitosan nanomaterials play a dual role

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as a plant growth promoter and plant protection agent.16 Copper-chitosan based nanomaterial

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has been synthesized and applied in various fields.13 We have reported the synthesis of copper-

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chitosan nanoparticle (CuChNP) with antibacterial activity.17 Previous reports indicated that

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application of Copper-chitosan nanoparticle could protect tomato plants from blight and wilt

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pathogen.16 However, the effect of CuChNp on blast disease of cereals particularly finger millet

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is not clear. In this study, the effect of application of CuChNp on the plant growth profile was

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evaluated. The biochemical approaches were made to investigate the potential of CuChNp to

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control blast disease of finger millet.

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MATERIALS AND METHODS

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Biological material.

Seeds of finger millet (susceptible to blast) were obtained from

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ICRISAT, Pantancheru, India. Pyricularia grisea was obtained from Tamil Nadu Agricultural

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University, Coimbatore, Tamil Nadu and maintained on PDA slants at 4oC.

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Preparation of CuChNP.

Synthesis of CuChNP was done as described previously.17

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Synthesized nanoparticles were characterized for physicochemical analyses, which showed

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similar characteristic details as reported earlier.17 Well-formed CuChNps were used to study the

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effect on finger millet plants.

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Effect of CuChNp on plant growth and yield related parameters. Seeds of finger millet

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were surface sterilized with sodium hypochlorite (0.01%, w/v) solution and washed thoroughly

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in sterile distilled water. The seeds (5 seeds/pot) were sown in clay pot (27cm diameter; 26 cm

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height) containing alluvial soil and grown under greenhouse condition. Preliminary experiments

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were conducted using various concentrations (0.01%, 0.05%, 0.1%, 0.15%) of CuChNp on

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growth of finger millet plants. Based on the growth profile, 0.1% (w/v) concentration was

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identified as optimum concentration and this was used for further studies. CuChNp was applied

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either as a foliar spray or as a combined application by combining seed coating and foliar spray.

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For foliar spray, the seedlings at 20 days age level were sprayed (foliar spray) with (0.1%, w/v)

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CuChNp (5ml/plant). This was repeated twice with 10 days interval up to 40 days. Water

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sprayed plants served as control.

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For a combined application (seed coating + foliar spray) method, the surface sterilized

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seeds were initially placed in a sterile petri plate containing CuChNp solution (0.1%, w/v) and

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kept in a rocker for 12 hours and then sown in pot as above. Seeds placed in water served as

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control. 20-Day-old seedlings (treated) were further sprayed with 0.01% CuChNp as described

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earlier. Control seedlings received water spray.

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were used (with triplicates) and all the experiments were repeated thrice.

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For each treatment, approximately 50 plants

Different parameters such as number of leaves, leaf length and shoot length, fresh and dry

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weight were recorded at different age level.

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described.18 Treated and untreated plants were monitored for the onset of flowering, number of

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inflorescence/plant, total number of fingers/inflorescence, total number of grains/finger. Total

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grain yield was recorded at maturity.

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Assessment of Cu Content.

Total chlorophyll content was determined as

Determination of copper content in grains harvested from

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CuChNp treated and untreated finger millet plants were done using Inductively Coupled Plasma-

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Optical Emission Spectrometry (Perkin-Elmer Optima 5300 DV ICP-OES). Grains of finger

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millet were washed with deionized water and dried at 60oC for 6 hours. The dried samples were

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ground to a fine powder using mortar and pestle. About 0.5g of powdered sample was mixed

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with 4 ml of HNO3: HCl (1:3). The samples were digested at 100oC for 60 mins. The digested

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samples were allowed to cool at room temperature, filtered and used for determination of copper.

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Spore germination assay. P. grisea spore suspension (1 x 105spores/ml) was prepared in half

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strength Czapek’s Dox Broth (CDB). Equal amount of conidial suspension and CuChNp (0.1%)

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was added and incubated in a shaker at 28oC with 120 rpm for seven days. Conidial suspension

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without the addition of nanoparticles was used as a control. Optical readings were taken at

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600nm at every 12h interval up to 7 days. Experiments were done in triplicate and average was

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calculated.

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In-vitro antifungal assay. To check whether the synthesized CuChNp have any effect on

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growth of P. grisea, CuChNp was amended to the CDA medium at various concentrations (0.01,

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0.05, 0.1 mg/ml). A 10 mm disc from seven days old P. grisea culture was kept upside down

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position and incubated at 27±1oC for fourteen days and radial growth was measured. Growth

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inhibition was expressed as the % inhibition of radial growth relative to the control.11 5 ACS Paragon Plus Environment


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Evaluation of blast disease incidence. The leaves of 30-day old treated (foliar, seed + foliar)

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and untreated seedlings were predisposed to nearly 95% humidity for 12hour. They were then

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challenge inoculated with P. grisea spore suspension (1 x 105spores/ml). The plants were

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monitored daily for first visible symptom appearance and for further development for next 50

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days. Disease incidence was determined on the basis of disease score, an estimate of the area

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affected using a scale (0-5) as follows: 0 = No symptoms on the leaves; 1 = small brown specks

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of pinhead size to slightly elongate (less than 20% affected tissue); 2 = a typical blast lesion

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elliptical 5-10 mm long (20-40% affected tissue); 3 = a typical blast lesion elliptical 1-2 cm long

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(40-60% affected tissue); 4 = 60-80% leaf area affected; 5 = complete blast. The % of blast

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disease incidence was calculated using the formula: Blast incidence = ([Scale x Number of plants

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infected] / [Highest scale x Total number of plants]) x 100.

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Protein extraction and estimation. Leaves (1mg/2ml) of treated and untreated plants were

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extracted with 0.01M potassium phosphate buffer pH 7.0 at 4oC using a pre-cooled mortar and

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pestle. The extract was centrifuged at 12,000 g for 15 minutes (Eppendorf, Germany). The

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supernatant was used for estimation of protein and for enzyme assays. Total protein content was

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determined by the dye binding method with BSA as a standard.19

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Enzyme assays.

Chitinase, Chitosanase, β-1,3 glucanase, Peroxidase, Polyphenol

oxidase and Protease Inhibitor activity were assayed as described previously.20, 21 In gel activity assay.

Chitinase, Chitosanase and Protease inhibitor were localized on

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substrates containing SDS-PAGE gel. Localization of β-1,3 glucanase, Peroxidase, Polyphenol

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oxidase were done on native PAGE containing their respective substrates.

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Statistical analysis. All the data were subjected to one-way analysis of variance to

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determine the significance of individual differences at p < 0.01 and 0.05 levels. All statistical

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analyses were conducted using SPSS 16 software support.

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RESULTS AND DISCUSSION

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New approaches are needed to increase agricultural productivity without damaging the

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ecosystem. In recent years, nanotechnology has acquired great influence in agriculture due to its 6 ACS Paragon Plus Environment

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ability in abiotic and biotic stress management.22, 23 Application of nanoparticles in agriculture

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enhances the efficiency and sustainability of agricultural practices by requiring less input than

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conventional products.8,24 The unique size and properties of nanoparticles result in enhanced

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performance in biological systems when compared to its bulk materials.25,26

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Effect of CuChNp on plant growth and yield. CuChNp treated plants showed positive

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morphological effects. There was an increase in the number of leaves were observed in CuChNp

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treated finger millet plants when compared to control (Fig. 1a). The increase was nearly 22%,

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33% in foliar spray and combined application respectively (Fig. 1a). Also CuChNp treated

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plants showed an increase in the average leaf area (data not shown). Leaf number as well as leaf

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area is regulated by a complex interaction of various genes.27 Significantly higher values of leaf

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length, shoot height were recorded in CuChNp treated plants than in control (Fig. 1b, c). The

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increase in leaf length was around 85%, 100% in foliar and combined application respectively.

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Nearly 36% increase in shoot height was observed in foliar spray whereas in combined

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application the increase was 46%. There was a significant difference in fresh and dry weight of

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the plants was recorded in CuChNp treated plants (Fig 1d, e). However, the plants received a

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combined application showed enhanced fresh and dry weight. The increase in fresh weight was

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found to be 14% in foliar treatment and 82% in combined application. There was nearly 152%

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increase in dry weight was observed in foliar treatment and in combined application it was

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297%. These results imply that CuChNp treatment interferes with the action of endogenous

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plant hormones, and induces changes in the growth profile of treated finger millet plants. The

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enhanced difference in growth parameters by combined application method when compared to

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foliar spray may be due to the increased vigor index observed after seed coating. Saharan et al.

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reported that application of Cu-chitosan nanoparticle to maize enhanced growth of seedlings. 28

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Chlorophyll content is considered as an index of the total amount of light harvesting

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complex and the electron transport components present in chloroplast membranes.29,30 Total

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chlorophyll content increased by 32%, 84% in finger millet plants treated with CuChNp by foliar

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and combined application respectively (Fig. 1f). Increase in total chlorophyll content leads to an

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increase in the photosynthase produced.31

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induced high chlorophyll contents in Asparagus and Sorghum.30,32 Uptake of CuChNp by finger

It has been reported that nanoparticle treatment



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millet plants analyzed using EDX showed an increase in Cu content in leaves of treated plants

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(data not shown) when compared to control plants.

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Early onset of flowering was noticed in CuChNp treated plants (Table 1). Treatment of

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finger millet plants with CuChNp also brought about a significant increase in the number of

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fingers/plant which also translated into an increase in average grain yield/plant (Table 1, Fig. 2).

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A significant increase in yield with nearly 42% and 89% was observed in finger millet plants

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treated with CuChNp by foliar application and combined application respectively. The enhanced

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growth rate in treated plants might have resulted in increased grain yield. More research is

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needed to understand how CuChNp promoted yield in finger millet. Improvement of agronomic

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traits with increased pod weight and grain yield in soybean by exposure to nano-iron oxide has

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been documented.8

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Cu content in grains. The grains obtained from CuChNp treated and untreated finger millet

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plants showed no detectable increase in copper content (Fig. 3). This indicates that CuChNp has

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been completely metabolized by the plant system.

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Effect of CuChNp on spore germination and antifungal activity. In this study, a typical

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inhibition of spore germination (up to 80%) of P. grisea was found in CuChNp amended

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medium when compared to control (Fig. 4a). The radial growth of P. grisea was found to be

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inhibited in CuChNp amended (0.1mg/ml) plates and the inhibition was nearly 80% when

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compared to that of the control plates (Fig. 4b). These results show that CuChNp exhibit

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antifungal property towards P. grisea. The antifungal properties of copper-chitosan nanoaprticle

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towards some phytopathogenic fungi under in-vitro condition have been reported.26, 33

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Effect of CuChNp on blast disease incidence. Typical symptoms of dark brown lesions were

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developed on the leaves after 15 days of challenge inoculation in control plants which progress

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rapidly and complete blast (100%) was observed on 50 days after challenge inoculation in

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control (untreated) plants (Fig. 5a). CuChNp application delayed the blast symptom appearance

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in finger millet plants (Fig.5a). First visible symptom appearance was on the 25th day in foliar

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application, whereas it was on the 30th day in combined application. On the 50th day after

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challenge inoculation only 25-28% of blast incidence was observed in CuChNp treated finger

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millet plants (Fig. 5a, b). These results clearly indicate that CuChNp application suppress the 8 ACS Paragon Plus Environment

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blast disease development on finger millet plants. The use of CuChNp to suppress the blast

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disease caused by P. grisea is a novel strategy for control of this devastating pathogen. The

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disease suppression may be due to the unique size and enhanced properties of CuChNp. No

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evidence of phytotoxicity was observed in these trials. The development of host resistance is the

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most successful strategy for plant disease control.34 Nanoparticles by themselves can negotiate

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cell walls and membranes far more effective compared to the core molecules,9,35 which results in

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better immune response. P. grisea infect other cereal crops worldwide and cause significant

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yield loss6. Therefore, this strategy could be employed for validation and exploitation in other

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cereal crops for control of P. grisea and for augmentation of yield. Nanoparticles of CuO were

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reported to increase growth and yield of tomatoes and eggplants when grown in pathogen-

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infested soils.36

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Effect of CuChNp on defense enzymes. Application of CuChNp to finger millet plants

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produced significant improvement in the innate immune response through induction of defense

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enzyme activity, including antioxidant enzymes both qualitatively and quantitatively (Table 2,

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Fig. 6).

Nearly 2 fold increase in chitinase and chitosanase was observed in CuChNp treated

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plants.

Activities of protease inhibitors, β-1,3 glucanase, peroxidase, polyphenol oxidase

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increased by 1.4 – 1.8 fold (Table 2). Chitinase, chitosanase, β-1,3 glucanase are reported to be

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the markers of the plant defense response.28 Though there was a significant difference in

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induction of defense enzymes in treated plants, the combined application method showed higher

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induction when compared to foliar spray (Table 2).

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reflected on the PAGE, which showed the appearance of various new polypeptides/isoforms in

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CuChNp treated finger millet plants when compared to untreated (control) plants (Fig. 6).

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Chitinase activity on SDS-PAGE showed only two polypeptides in control, whereas in treated

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plants apart from the constitutive polypeptides three new polypeptides of molecular mass 19.3,

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24, and 38 kDa was observed. In combined application polypeptides of molecular mass 8, 19.3,

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27, 32 kDa were observed apart from the constitutive (Fig. 6a).

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localized on SDS-PAGE. In control plants only one chitosanase polypeptide with molecular

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mass 28.5 kDa was localized. In foliar treatment two new polypeptides of molecular mass 12,

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22 kDa was observed apart from the constitutive band. In combined application chitosanase

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polypeptides of molecular mass 18.2, 19.3 were observed (Fig. 6b).

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localized on SDS-PAGE showed nearly five polypeptides in combined application (Fig.6c). β-

The enhanced enzyme activities also


Fig. 6b showed chitosanase

Protease inhibitors


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1,3 glucanase localized on native PAGE showed two isoenzymes in control, whereas in treated

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plants three new isoforms were observed (Fig. 6d). In peroxidase also two new isoenzymes

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(PO2, PO3) were localized in combined application whereas in foliar spray only one new

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isoform (PO2) was observed (Fig. 6e).

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PPO4, PPO5, PPO6) were observed in combined application (Fig. 6f).

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only PPO4, PPO5, PPO6 were observed. Chandra et al.9 reported that chitosan nanoparticle

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produced a significantly high defense response in Camellia sinensis by increasing the activity of

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defense enzymes such as peroxidase, polyphenol oxidase, β-1,3 glucanase etc. The observed

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suppression of blast disease in CuChNp treated finger millet plants may be due to the enhanced

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activities of defense enzymes. These results imply that application of CuChNp protected finger

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millet plants against the blast pathogen invasion by reinforcing the defense mechanism through

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enhancing the activities of defense enzymes. The strategy of induced resistance by stimulating

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plant’s immune system represents a sustainable approach to protect crop plants from

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phytopathogens.7

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Several new isoforms of polyphenoloxidase (PPO3, In foliar application

In conclusion, application of CuChNP to finger millet plants suppressed the blast disease

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incidence.

The treated plants also showed enhanced defense enzymes.

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enhanced defense enzyme activities might have played a role in blast disease suppression in

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CuChNP treated finger millet plants. Seed treatment along with foliar application showed

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significant protection against blast disease when compared to foliar application alone. The

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treated plants also improved the growth, leading to an increase in the net productivity in terms of

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grain yield. The results indicate that the prepared CuChNp play a dual role in enhancing the

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growth as well as protecting finger millet plants from blast fungus. However, this has to be

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evaluated under field condition.

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Notes The authors declare no competing financial interest.

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Acknowledgements The authors thank the University Authorities for providing the facilities and

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SAIF, IITM, Chennai for ICP-OES analysis. AM thanks the Bharathidasan University for

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providing University Research Fellowship.

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Figure Captions

387

Fig. 1. Effect of CuChNp on plant growth parameter

388 389

Urbonaviciute, A.; Samuoliene, G.; Sakalauskaite, J.; Duchovshis, P.; Brazaityte, A.;

Nair, R.; Varghese S. H.; Nair, B. G.; Maekawa T.; Yoshida, Y.; Kumar, D. S.

a) Number of leaves/plant b) Leaf length c) Shoot height weight f) Total chlorophyll

d) Fresh weight e) Dry

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Fig. 2. Finger millet Inflorescence (on 70th day) showing grain set

391

Fig. 3. Copper content in grains harvested from CuChNp treated finger millet plants. 14 ACS Paragon Plus Environment

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392 393 394 395 396


Fig. 4. Effect of CuChNp on spore germination (a); on radial growth of P. grisea (b) under in vitro condition. Fig. 5. Effect of CuChNp on blast disease incidence in finger millet plants (a); leaf showing blast symptoms (b). Fig. 6. Localization of defense enzymes of control and CuChNp treated (on 50th day) finger

397

millet plants on SDS- PAGE a) chitinase b) chitosanase c) protease inhibitors; on

398

Native PAGE d) β-1,3 glucanase e) peroxidase f) polyphenol oxidase.

399

Lanes: M – Marker; 1- control; 2- foliar spray; 3- combined application (seed coat+

400

foliar spray).

401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416



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Table: 1 Effect of CuChNP application on the onset of inflorescence and yield components

419

in finger millet plants

Treatment

Onset of Inflorescence ( days)

No. of Inflorescence/ Plant

No.of Fingers/ Inflorescence

No. of Grains/ Finger

Grain weight (g/ Plant)

Control

50

2.00 ± 0.57

4.00 ± 0.32

152 ± 4.16

2.12 ± 0.15

Foliar Spray

45

3.33 ± 0.57

5.33 ± 0.57

193 ± 3.05

3.02 ± 0.10

42.45

Seed coat + foliar spray

44

3.33 ± 0.57

6.33 ± 0.57

205 ± 4.44

4.029 ± 0.06

89.6

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 16 ACS Paragon Plus Environment

Increase in yield (%) _

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435 436 437

Table: 2 Effect of CuChNp application on the defense enzymes in finger millet plants

438

Days

30

40

50

Sample

Chitinase (Units/g protein)

Chitosanas e (Units/g protein)

Protease Inhibitors (Units/g protein)

β-1,3glucanase (Units/g protein)

Peroxidase (∆430 min/ g protein)

Polyphenol oxidase (∆495 min/ g protein)

Control

0.83 ± 0.04

12.7 ± 0.63

32.4 ± 0.62

22.3 ± 0.57

0.06 ± 0.13

2.01 ± 0.10

Foliar spray

2.44 ± 0.12

17.4 ± 0.87

68.0 ± 0.40

36.7 ± 0.83

0.22 ± 0.14

2.53 ± 0.12


2.88 ± 0.14

37.3 ± 0.82

69.2 ± 0.64

37.3 ± 0.86

0.35 ± 0.11

3.72 ± 0.18

Control

0.85 ± 0.04

14.5 ± 0.72

51.1 ± 0.55

24.1 ± 0.62

0.36 ± 0.18

2.29 ± 0.11

Foliar spray

6.06 ± 0.30

41.0 ± 0.54

70.6 ± 0.53

41.0 ± 0.54

0.71 ± 0.35

2.83 ± 0.14


4.70 ± 0.23

46.7 ± 0.33

72.3 ± 0.61

42.0 ± 0.95

0.85 ± 0.10

3.96 ± 0.19

Control

3.69 ± 0.18

30.5 ± 0.52

58.1 ± 0.42

29.9 ± 0.49

0.57 ± 0.25

2.54 ± 0.12

Foliar spray

7.49 ± 0.37

62.4 ± 0.52

84.5 ± 0.90

52.2 ± 0.61

0.84 ± 0.42

3.53 ± 0.17


7.50 ± 0.48

69.0 ± 0.45

103.0 ± 0.5

51.1 ± 0.92

1.53 ± 0.76

4.83 ± 0.24

439 440 441 442 443 444 445



446

Fig. 1

447

448 449 18 ACS Paragon Plus Environment

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450


Fig. 2

451

452 453 454

Fig. 3

455 456 457 19 ACS Paragon Plus Environment


458

Fig. 4

459

460 461 462

Fig. 5

463 464 465 466 467 20 ACS Paragon Plus Environment

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Fig. 6

470

471 472 473 474 475 476 477 478 21 ACS Paragon Plus Environment


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TOC graphics

483

484 485 486 487


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Application of Copper-Chitosan Nanoparticles Stimulate Growth and

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