Enhancing cinnamon essential oil activity by nanoparticle encapsulation to control seed pathogens

Industrial Crops & Products 124 (2018) 755–764

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Enhancing cinnamon essential oil activity by nanoparticle encapsulation to control seed pathogens

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Marimar Bravo Cadenaa, , Gail M. Prestonb, Renier A.L. Van der Hoornb, Nicola A. Flanaganc, ⁎ Helen E. Townleya,d, Ian P. Thompsona, a

Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK Department of Plant Sciences, University of Oxford, S Parks Road, Oxford OX1 3RB, UK c Oxford Materials Characterisation Service, Department of Materials, University of Oxford, Begbroke Science Park, Begbroke Hill, Woodstock Road, Oxford OX5 1PF, UK d Nuffield Department of Women’s and Reproductive Health, University of Oxford, Level 3, Women’s Centre, John Radcliffe Hospital, Oxford, OX3 9DU, UK b

A R T I C LE I N FO

A B S T R A C T

Keywords: Essential oils Cinnamaldehyde Alginate Mesoporous silica nanoparticles Phytopathogens Crop protection

Natural biocides, such as cinnamon (Cinnamomum zeylanicum) bark essential oil, have enormous potential as antimicrobials but are limited by their volatility and rapid degradation. To counteract this and to prolong the efficacy of the biocide, cinnamaldehyde (CNAD, the main bioactive compound of cinnamon essential oil) was encapsulated into mesoporous silica nanoparticles (MSNPs). The synthesised CNAD-MSNPs can be used to tackle the issue of global crop loss; every year, more than 40% of global food production (estimated at $500 billion USD) is lost to diseases. This is despite the annual use of over two million tonnes of pesticides. To address seed borne diseases, CNAD-MSNPs were incorporated into a sodium alginate seed coating. As a proof of concept, this system was tested against Pseudomonas syringae pv. pisi, the causative agent of pea bacterial blight, and demonstrated to increase the number of symptomless plants by 143.58% twenty days after sowing. Additionally, the concentration of CNAD present in the alginate coating was estimated to be < 0.0000034% (v/v); up to 90,000-fold lower than concentrations of free cinnamon oil previously reported to control some bacterial diseases. Furthermore, alginate-treated seeds germinated faster than control plants, and were physiologically similar, demonstrating the dual benefit of this treatment. To the best of our knowledge, this is the first study to exploit the combined properties of essential oils, alginate and MSNPs as a seed treatment to control bacterial phytopathogens. Moreover, this study proved that the antimicrobial activity of plant products can be significantly enhanced by MSNP encapsulation, allowing volatile biocides, such as essential oils, to be used effectively at very low concentrations to treat and prevent microbial diseases in crops.

1. Introduction Essential oils (EOs) are naturally occurring, highly antimicrobial and biologically active compounds produced by secondary metabolism in aromatic plants. Many EOs are widely used in several industries, have GRAS (Generally Regarded as Safe) status and appear in the EAFUS (Everything Added to Food in the US) list. These compounds are safe, have low toxicity, are generally accepted by the public and have strong antimicrobial properties, making them promising candidates to substitute chemicals and antibiotics currently used in agriculture (Bajpai et al., 2011). However, their use as free compounds can be hindered by their inherent characteristics such as high volatility, or predisposition to premature degradation and poor miscibility in aqueous substances. The encapsulation of EOs into nanoparticles such as

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mesoporous silica nanoparticles (MSNPs), has been shown to prevent volatilisation of the oils while improving their stability, immiscibility in aqueous solutions and long-term antimicrobial effects (Bernardos et al., 2014; Bravo Cadena et al., 2018; Chan et al., 2017). MSNPs are composed of silicon dioxide (SiO2), also known as silica, a material of growing interest because of its properties including chemical stability, versatility and biocompatibility (Slowing et al., 2008). Also, it decomposes in the natural environment into relatively harmless silicic acid by-products (Diaconu et al., 2010). Silica nanoparticles can be easily functionalised and are more stable due to their SieO bonds (Liong et al., 2008). In addition, they have tuneable pore size and porosity (Trewyn et al., 2007), with simple and low-cost synthesis methods (Kwon et al., 2013). Furthermore, the size of MSNPs can be adjusted to control or prevent internalisation into plant systems; plants

Corresponding authors. E-mail addresses: [email protected] (M. Bravo Cadena), [email protected] (I.P. Thompson).

https://doi.org/10.1016/j.indcrop.2018.08.043 Received 1 June 2018; Received in revised form 14 August 2018; Accepted 16 August 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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enough to initiate an epidemic and destroy a crop under favourable conditions (Hollaway et al., 2007; Taylor and Dye, 1976). Therefore, it is important to develop effective treatment measures that will not present potential risks such as bacterial resistance or human and environmental hazards. Hence, essential oils are promising candidates for preventing bacterial diseases in agriculture. The objectives of this study were: (i) to enhance the antimicrobial activity of cinnamon essential oil by nanoparticle encapsulation, and (ii) to develop an alginate seed coating capable of incorporating CNADloaded MSNPs to be used as a seed treatments against plant pathogens. The results from this work demonstrated the nanoparticle-enhanced activity of cinnamon essential oil and the feasibility of this system as a potential seed treatment to prevent and control the incidence of microbial infections in crops.

walls typically have pores of 3–8 nm (Carpita and Gibeaut, 1993) so only MSNPs smaller than the largest pores are able to penetrate the cell wall into the plasma membrane. Silica also plays an important physiological role in plants (Solanki et al., 2015) and its characteristics and natural presence in the environment and plant systems make it a promising material for biocide delivery in agriculture. MSNPs have been reported to have mainly beneficial effects on plants (Haghighi et al., 2012; Lin et al., 2004a, 2004b; Lu et al., 2002; Nair et al., 2011; Shah and Belozerova, 2008; Siddiqui et al., 2014; Siddiqui and Al-Whaibi, 2014; Sun et al., 2014; Suriyaprabha et al., 2012), improving tolerance to abiotic stress, enhancing plant growth, development and yields (DeRosa et al., 2010). Their application in agriculture consequently presents comparatively low risks with respect to toxicity and safety to the environment and food chain introduction. The present work utilises cinnamaldehyde (CNAD, the main bioactive compound of cinnamon essential oil) loaded MSNPs, which we previously demonstrated can eliminate up to 99.99% of bacterial growth of five different bacterial strains with MSNPs encapsulation highly increasing its antimicrobial activity in vitro by 10-fold compared to free cinnamon EO (Bravo Cadena et al., 2018). In this study, an alginate seed coating was developed to incorporate CNAD-loaded MSNPS. Alginate, or alginic acid, is a natural polysaccharide widely present in brown algae, consisting of β- 1,4-linked Dmannuronic acid (M) and α- 1,4-linked L-guluronic acid (G) residues arranged in homopolymeric blocks (MM and GG) together with alternating sequence blocks (MG) (Joint FAO/WHO Expert Committee on Food Additives, 1997; Smidsrød, 1974). An alginate solution can form a hydrogel in the presence of divalent cations such as Ca+2 due to selective binding of the ions to the GG blocks (Simpson et al., 2004; Smidsrød, 1974). Calcium ions induce inter-chain association that constitute the junction zones responsible for gel formation (Morris et al., 1978). The formation of these junction zones in alginate gelation is described by the egg-box model (Grant et al., 1973). Alginate has been widely used in many industries, including agriculture, where it has been employed as fertilizer (Guo et al., 2006), biofertilizer (Ivanova et al., 2005), and soil conditioner (Abd El-Rehim, 2006), to produce artificial or synthetic seeds (Asmah et al., 2011; Khor et al., 1998; Pintos et al., 2008; Redenbaugh, 1983; Sakhanokho et al., 2013; Sharifeh et al., 2011; Slade et al., 1989) or to encapsulate biocontrol agents (Fravel et al., 1985). It is safe to use, biodegradable, and has GRAS status. Additionally, alginate and other natural polymers have been shown to promote plant activities such as germination, root and shoot elongation and growth (Abd El-Rehim, 2006; Luan et al., 2002), and a degraded product of alginate has been shown to act as a growth promoter and an enhancer of enzymatic activity (González et al., 2013; Idrees et al., 2011; Le et al., 2003; Naeem et al., 2011; Sarfaraz et al., 2011). As a proof of concept, the CNAD-MSNPs alginate coating was tested on common peas (Pisum sativum) against bacterial blight. This disease is caused by Pseudomonas syringae pv. pisi, which is a seed-borne and seedtransmitted pathogen. It has been reported in most pea growing areas worldwide and can cause devastating effects, significantly reducing yield and seed quality (Roberts, 1993). Pea bacterial blight is usually controlled by crop rotation and hygiene measures (disinfecting and cleaning equipment and machinery), sowing time, and employing disease-free seeds and resistant cultivars. P. syringae pv. pisi can remain viable in seeds for at least three years (Lawyer and Chun, 2001) and infected crops may result from sowing infected seeds and bacteria spreading between plants through contact or water splashing (Grondeau et al., 1991). In the UK, bacterial blight is mainly controlled through a seed certification scheme (The Plant Health (Great Britain) SI 1987/1758, 1987The Plant Health (Great Britain) SI /, 1987The Plant Health (Great Britain) SI 1987/1758, 1987The Plant Health (Great Britain) SI /, 1987The Plant Health (Great Britain) SI 1987/1758, 1987); however, a negative seed test result does not necessarily guarantee a seed batch is free of the pathogen and only one infected seed in 10,000 (0.01%) is

2. Materials and methods 2.1. Loading (immobilisation) of CNAD onto MSNPs Mesoporous silica nanoparticles, previously synthesised and characterised by our research group (see (Huang et al., 2014) for full characterisation details), were used throughout this study. Synthesised MSNPs were imaged via transmission electron microscope (TEM) using a JEOL JEM 2100 at 200 kV. Samples were prepared by dry deposition onto holey carbon-coated TEM copper grids (Agar Scientific, UK), and air-dried overnight. The immobilisation of CNAD onto mesoporous silica was achieved using a modified version of a previously described protocol (Ruiz-Rico et al., 2017). CNAD (2 ml) and 3-aminopropyltriethoxysilane (2.3 ml) were dissolved in 20 ml dichloromethane (Sigma Aldrich, UK) and stirred at 350 rpm for one hour. The mixture was added to 40 ml acetonitrile (Sigma Aldrich, UK) with 1 g MNSPs and stirred for 5.5 h at room temperature. The solution was centrifuged and washed with acetonitrile and water. The resulting loaded nanoparticles were dried at room temperature under vacuum for 36 h and collected. For more information about the loading and unloading of CNAD onto MSNPs, see our previous study (Bravo Cadena et al., 2018). 2.2. Development of an alginate seed coating Pea seeds (“Kelvedon Wonder” cultivar) were sterilised by immersing seeds in 70% ethanol (Sigma Aldrich, UK) in a sterile flask for 1 min before washing seeds with sterile water for an additional minute. Seeds were then immersed in a 2% sodium hypochlorite (Sigma Aldrich, UK) solution for 5 min and washed 10 times with sterile water (1 min per wash). They were then drained and placed in a sterile petri dish and air dried. Only seeds that remained wrinkled after sterilisation were used. Sterile, dry seeds were coated by immersing peas in a 1% or 3% (w/ v) stirred sodium alginate (Sigma Aldrich, UK) solution and swirled for 2–3 min to remove any bubbles on the seeds’ surface. They were transferred into a 0.1 M or 2 M CaCl2·2H2O solution (≥99.0% Sigma Aldrich, UK) at room temperature and left for 30 min, swirling occasionally, to allow the coat to form around the seeds. After coating, seeds were placed in a sterile petri dish and allowed to dry overnight. Fifteen pea seeds were coated with each of the four resulting combinations (1% or 3% alginate with 0.1 M or 2 M CaCl2) and with two additional formulations containing 25 mg/ml MSNPs. To evaluate the effects of the seed coating; germination and plant height were monitored and measured two weeks after sowing. Shoot and root emergence, root length and number of secondary roots, leaves and nodes were also measured (data not shown). For the infection experiments, a seed coating using 1% sodium alginate and 0.1 M CaCl2 was selected and 2 mg/ml CNAD-loaded MSNPs were dissolved in the sodium alginate solution before the seed coating protocol. Alginate-MSNPs-coated seeds were used as the test group 756

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After 20 days of incubation, symptomless plants were transferred to soil and incubated for a further four weeks. A mixture of Pot and Bedding Compost (Levington Advance, UK) and Vermiculite (Sinclair Pro, UK) with 0.28 g/L Imidasect (Sinclair Pro, UK) to control sciarid fly, was used to grow plants. Plants were watered three times a week and incubated under the same initial conditions.

while alginate-coated seeds without the addition of MSNPs were used as a control group. The experiment was performed in triplicate with 26 seeds per treatment in 3 experimental batches. 2.2.1. Atomic Force Microscope (AFM) Alginate layers (1% and 0.1 M; or 3% and 2 M sodium alginate with CaCl2) on glass slides were analysed using an Atomic Force Microscope (Agilent 5400 AFM/SPM; Agilent Technologies, UK) to evaluate the hardness of the different coating formulations. A hydrophobic marker was used to define an area in a glass slide to be covered with 100 μl sodium alginate before submerging the slide into CaCl2·2H2O for 30 min. The alginate coating was allowed to dry before microscopy analyses were carried out. Force-Distance (F-D) curves were made through the Spectroscopy feature of the instrument’s Contact mode using MikroMasch cantilevers (HQ:NSC35/Al BS) with resonance frequency of 150–300 kHz (or 4.5–14 N/m).

2.4. Evaluation of the long-term effects of MSNPs-alginate coating on peas against P. syringae pv. pisi Fifty days after the initial sowing, the growth and development of the remaining symptomless plants was analysed. All plants (15 alginate treated controls and 22 alginate-MSNPs treated) were removed from the soil and any loose soil was washed off. Plants were blotted gently to remove free surface moisture and weighted immediately to record wet mass. Numbers of leaves, flowers, pods, and nodes, as well as height and root length were measured. To evaluate chlorophyll content, two circular sections of leaves (5 mm diameter, ∼3 mg) were cut from all plants from representative leaves. Samples were frozen with liquid nitrogen and kept at -80 °C until analysis. One ml cold methanol was added and plant material was homogenised using a MM300 Tissuelyser (Qiagen Retsch, Germany) at 30 Hz for 1.5 min. Samples were centrifuged at 10,000 rpm and 4 °C for 10 min and supernatants were collected and analysed using a spectrophotometer (SmartSpec™ 3000, BioRad) at 665 and 652 nm, subtracting background readings at 750 nm. The content of chlorophyll a, chlorophyll b and total chlorophylls was calculated using the equations described by (Porra et al., 1989). All plants were dried in an oven at 40 °C for 14 h and then at 80 °C for 2.5 h. Plants that were not completely dried after this process were left at 40 °C overnight. Total dry mass, root mass and shoot mass were recorded for each sample.

2.3. Effect of alginate seed coating containing CNAD-MSNPs against pea bacterial blight in planta 2.3.1. Microorganism and growth conditions Pseudomonas syringae pv. pisi race 2 strain 203 (NCPPB 2585) was obtained from the Department of Plant Sciences, University of Oxford, UK. Bacterial stocks were prepared with 80% glycerol to a final glycerol concentration of 32% and maintained at −80 °C in 2 ml cryotubes. Test microorganism cultures were prepared from glycerol stocks, streaking onto Luria-Bertani Agar (LBA; Sigma Aldrich, UK) plates and incubating overnight at 28 °C before inoculating 25 ml of Luria-Bertani Broth (LB; Sigma Aldrich, UK) with one colony and incubating at 28 °C and 220 rpm overnight. Overnight cultures were used to inoculate 25 ml LB in 50 ml Falcon tubes, which were incubated until mid-exponential phase was achieved. Bacterial cells were harvested by centrifugation at 4000 rpm and 10 °C for 15 min and washed twice with phosphate-buffered saline (PBS; Sigma-Aldrich, UK). Turbidity was adjusted to the desired optical density for each test.

2.5. Statistical analyses Statistical analyses were performed using Minitab® version 18.1. To determine statistical significance of differences between samples, analysis of variance (ANOVA) and t-tests were undertaken. These were followed by Tukey’s HSD (honest significant difference) test or a Chisquared test when appropriate for quantitative or categorical data, respectively. P values of 0.05 and 0.01 were used and are specified for each analysis.

2.3.2. LD50 test To determine the bacterial concentration required to cause pea bacterial blight symptoms on 50% of the inoculated seeds, an LD50 test was performed. Seeds were immersed in 2-fold dilutions of P. syrinage pv. pisi bacterial cultures (OD600 = 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125) for 5 min and allowed to dry overnight before sowing in Murashige and Skoog (MS) media. Seeds without any bacterial inoculation and immersed in sterile water were used as negative controls. Germination, shoot emergence, secondary roots, appearance of bacterial blight signs and survival rate were evaluated for each treatment, three weeks after infection.

3. Results We previously demonstrated that the immobilisation of cinnamon essential oil onto MSNPs significantly enhanced the antimicrobial activity of this bioactive compound in vitro against five different bacterial strains (Bravo Cadena et al., 2018). The present study aims to demonstrate this enhanced activity can be used to treat and protect crops from bacterial infections. A TEM image of the MSNPs used throughout this study can be observed in Fig. 1.

2.3.3. Seed inoculation A total of 78 seeds coated with alginate (Alg controls) and 78 seeds coated with alginate-MSNPs were immersed in 50 ml P. syringae pv. pisi inoculum (OD600 = 0.025) for 5 min, transferred to sterile Petri dishes and left to air dry. For control purposes (data not shown), ten positive controls (without coating) were also inoculated; and ten negative controls (without coating) were immersed in sterile water. Dry seeds were stored for 5 days before sowing in MS media. The experiment was performed in triplicate with 26 seeds per replicate for each treatment.

3.1. Development of an alginate seed coating Sterile “Kelvedon Wonder” pea seeds were coated with sodium alginate with or without the addition of 2 mg/ml CNAD-immobilised MSNPs. The sodium alginate surrounding the seed formed a thin layer around the seed once immersed in the calcium chloride solution. The alginate coating did not affect the size or shape of seeds. A high molarity of CaCl2 (2 M) prevented the germination of some seeds by trapping rootlets and/or seedling inside the alginate coat (Fig. A1). However, seeds treated with a low molarity (100 mM) germinated faster than the control and produced significantly taller plants (p < 0.05). Furthermore, the addition of MSNPs to the alginate coating permitted the germination of seeds, even of those treated with 2 M CaCl2 (Fig. A2).

2.3.4. Seed incubation and growth MS media was prepared by dissolving 4.4 g MS basal salt mixture (Sigma Aldrich, UK), 10 g sucrose (Sigma Aldrich, UK) and 2.5 g Phytagel (Sigma Aldrich, UK) in 1 L of distilled water (DW). Seeds were individually placed in sterile culture tubes containing 25 ml of MS agar and incubated with a photoperiod of 16 h at 21/20 °C, under fluorescent light (80 μmol photons m−2s−1). Germination, shoot emergence, secondary roots and appearance of bacterial blight signs were monitored daily for 20 days. 757

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Fig. 3. LD50 test. Effect of serial concentrations of P. syringae pv. pisi bacterial culture on pea seedlings. Six different concentrations were evaluated ranging from OD600 = 0.0125 to OD600 = 0.4 and the percentage of symptomless plants was monitored during 21 days post-infection (dpi). n = 15 seeds for each treatment.

Fig. 1. TEM Imaging of MSNPs. Pores (A) and channels (B) of the MSNPs can be observed according to the nanoparticle orientation during imaging. CNADloaded and unloaded MSNPs appear similar under TEM imaging.

and 0.0125 were prepared and seeds were soaked for 5 min in the inoculum to allow time for infection. Germination, shoot emergence and appearance of secondary roots were mainly affected by the higher concentrations of bacterial inoculum. The highest concentration of bacteria only allowed 73% of seeds to germinate, 60% of shoot and 43% of secondary roots emergence, compared to 93%, 93% and 73% of the controls, respectively. The highest bacterial inoculum concentration infected half the seedlings in less than four days post-infection (dpi), with a final survival rate of only 13%. The three highest concentrations resulted in a higher rate of infection, while there was no observable difference between the effects of the three lowest concentrations (Fig. 3). A final OD600 = 0.025 resulted in a survival rate close to 50% three weeks after infection and was selected for future experiments.

Alginate layers on glass slides were prepared with 1% sodium alginate and 0.1 M CaCl2 and with 3% sodium alginate and 2 M CaCl2 to obtain what was hypothesised to be the softest and hardest (respectively) of the formulations. AFM analyses demonstrated that the coating preventing germination (2 M CaCl2) was indeed a softer but more flexible coating that would not break as easily and therefore trapped plant parts inside. In contrast, the alginate layer with 0.1 M CaCl2 produced a harder but more brittle coating that could be more easily broken by the roots and shoots (Fig. 2). Moreover, the addition of MSNPs to the alginate layer with 2 M CaCl2, modified the hardness of the alginate layer, making it even more brittle and harder than the layer with 0.1 M CaCl2, positively influencing germination.

3.2.2. Plant development and protection After determining the concentration of the bacterial inoculum required for a 50% survival rate, seeds coated with alginate (1% sodium alginate and 0.1 M CaCl2), with or without CNAD-MSNPs, were sown and incubated. Germination, shoot emergence, secondary roots and appearance of bacterial blight signs were monitored daily over 20 days to determine the effect of the CNAD-loaded nanoparticles on plant protection against pea bacterial blight. The appearance of pea bacterial blight symptoms was very characteristic (Fig. A3). The seeds usually presented no signs unless the level of infection was extremely severe. Signs of infection appeared at different stages of the plants’ growth and development but mainly during the second week after sowing. Germination, shoot emergence and number of seeds with secondary roots were similar in treated seeds and the controls. Treated seeds germinated significantly faster (p < 0.01) than control seeds. Three days after sowing, 87.87% of treated seeds had germinated compared to only 68.75% of the controls. However, after four days both treatments reached comparable germination percentages (Fig. 4a). A similar result was observed for shoot emergence where six days after sowing, MSNPtreated seeds showed a significantly greater (p < 0.05) percentage of seedlings with shoots than the control (Fig. 4b). The percentage of seedlings with secondary roots remained slightly greater for MSNPtreated seeds (84.28% after 20 days) than the control (76.89%) but the difference was not significant (Fig. 4c). The similar results between MSNP-treated and control seeds suggest that the addition of CNADMSNPs did not affect plant germination and growth. Nonetheless, statistical analyses demonstrated that there was a significant difference between the number of symptomless and infected plants for each treatment (p < 0.01) twenty days after sowing. Half of the control seeds coated with alginate but not protected by the

3.2. Effect of alginate seed coating containing CNAD-MSNPs against pea bacterial blight in planta 3.2.1. LD50 test An LD50 test was performed to determine the bacterial concentration required to achieve 50% survival of seedlings infected with P. syrinage pv. pisi. Bacterial cultures at OD600 = 0.4, 0.2, 0.1, 0.05, 0.025,

Fig. 2. AFM results. Force-Distance (F-D) curves of alginate layers. Each line represents one measurement at one specific point on the alginate layer. Percentage of sodium alginate and CaCl2 molarity are indicated, as well as the addition of nanoparticles (NPs). The calibration curve was constructed using a hard surface (glass slide). A higher molarity of CaCl2 produced a softer and more flexible alginate layer that was difficult to be penetrated by the root during germination. A lower molarity produced a harder and more brittle layer that allows for the correct germination of seeds. Addition of MSNPs altered the properties of the alginate coating, making it more brittle, allowing the root and shoot to penetrate the alginate coating. 758

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Fig. 5. Appearance of pea bacterial blight infection. Seeds were monitored for the appearance of pea bacterial blight signs and a statistically significant difference can be observed between MSNP-treated seeds and the control (p < 0.01). Only 28.21% of MSNP-treated plants showed signs of infection compared to 50% of control plants. Error bars represent ± standard deviation, n = 78 seeds per treatment (26 seeds per treatment for each of the three replicates).

blight (Fig. 5). 3.3. Evaluation of the long-term effects of MSNPs-alginate coating on peas against P. syringae pv. pisi After 20 days of incubation, plants that presented no signs of infection were transferred to soil and incubated for a further four weeks. The number of leaves, nodes, flowers and pods were recorded for each plant as well as height, wet and dry mass, root/shoot ratio and chlorophyll content. The total number of leaves and nodes was not significantly different between treatments. However, there was a significant difference (p < 0.01) with respect to the number of leaves and nodes that were not drying out and plants treated with nanoparticles developed healthier nodes and leaves (Fig. 6A&B). The number of flowers and pods was not statistically different between treatments; but the pods from plants whose seeds were treated with alginate/MSNPs were longer and developed faster than pods from seeds only treated with alginate (p < 0.1, Fig. 6C). Plant height was not significantly different between treatments (Fig. 6D), but the wet and dry mass of plants treated with MSNPs was slightly greater than the controls (p < 0.1, Fig. 6E) and their roots were significantly longer (p < 0.05, Fig. 6D). Additionally, seeds treated with alginate/MSNPs resulted in higher shoot dry mass than the controls (p < 0.1, Fig. 6F), whilst there was no significant difference in the root dry mass between treatments (Fig. 6G). Similarly, there was no significant difference in chlorophyll (Chl) a and b content between plants whose seeds were only coated with alginate and those coated with alginate containing CNAD-MSNPs (Fig. 6H).

Fig. 4. Seed germination and growth. A) Germination, B) shoot emergence, and C) appearance of secondary roots of CNAD-MSNPs-treated seeds and control seeds (alginate only coat with 1% sodium alginate and 0.1 M CaCl2). Data shows mean ± Standard Deviation, n = 78 seeds per treatment (26 seeds per treatment for each of the three replicates).

nanoparticles showed severe signs of bacterial infection (Fig. 5). In contrast, 71.79% of the plants that were protected by CNAD-MSNPs embedded in the alginate coating remained symptomless, demonstrating a significant decrease in infection and the efficacy of the seed coating to protect peas against the bacterial infection (Fig. 5). The results during the first week indicated that the CNAD-MSNPs were most effective during the first 6 days, when the biocide concentration was higher and able to inhibit bacterial growth almost completely. After 7 days, some bacterial growth was observed and a few seedlings started displaying signs of infection (12.82% of protected vs 30.77% of control). Still, there was a significant difference between both treatments (p < 0.01, calculated using a Chi-squared Test) demonstrating the effectiveness of the CNAD-MSNPs against pea bacterial

4. Discussion The aim of this study was to investigate the potential of nanoparticles as delivery vehicles to enhance the antimicrobial activity of essential oils by protecting them from evaporation or degradation and enabling the application of volatile biocides in agriculture to prevent microbial diseases. This study showed that an effective seed coating could be produced by embedding cinnamaldehyde-immobilised mesoporous silica nanoparticles (CNAD-MSNPs) into an alginate matrix. This was tested on the model plant Pisum sativum (common pea) since it is a well-studied organism with a short generation time, and ease of laboratory growth. We have previously demonstrated that the immobilisation of CNAD 759

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Fig. 6. Effect of MSNP-alginate coating on peas against pea bacterial blight. Number of A) leaves, B) nodes, C) flowers/pods and pod length, D) plant height and root length, E) wet and dry mass, F) shoot dry mass, G) root dry mass, and H) chlorophyll content of all pea plants were measured 50 days after initial sowing. Two treatments were compared, peas coated with alginate containing cinnamaldehyde-loaded mesoporous silica nanoparticles (Alg-NPs) and peas coated only with alginate (Alg). Data shows mean ± standard deviation of all replicas. Only symptomless plants that had survived 50 days after sowing were measured, n = 15 (Alg), 22 (Alg-NPs).

For that reason, an alginate seed coating was developed to incorporate antimicrobial-loaded nanoparticles onto the seeds’ surface to achieve protection against bacterial diseases. The deployment of alginate in agriculture has mainly been focused on the production of synthetic seeds or to encapsulate nutrients or microorganisms to be used as fertilisers. Sarrocco et al. (2004) evaluated the encapsulation of true seeds (cabbage, basil, radish and wheat) and showed that germination was similar to controls, with the exception of wheat (Sarrocco et al., 2004). Alginate is a plant growth promoter and its derivatives have been shown to trigger antimicrobial and antioxidant production, enhancing enzymatic activity and metabolism (González et al., 2013; Le et al., 2003; Naeem et al., 2011; Sarfaraz et al., 2011). Our results are in agreement; alginate-treated seeds germinated faster than control seeds producing taller plants. The selected coating formulation was 1% sodium alginate and 0.1 M CaCl2 since higher concentrations of CaCl2

onto MSNPs increased its antimicrobial activity by 10-fold compared to free cinnamon essential oil, and was able to reduce P. syringae pv. pisi growth by 99.99% in vitro. The incorporation of CNAD-MSNPs into an alginate coating that could potentially treat and protect peas from bacterial blight infection was also investigated. A coating was selected as a nanoparticle delivery method since seed treatment reduces the contact area with the product from 10,000 m2 (foliar products) or 500 m2 (furrow applications) to only 50 m2 (International Seed Federation, 2007), making it a more cost-effective and environmentally-friendly application for crop protection. Furthermore, the accumulation of nanoparticles on plant leaves has been shown to lead to over-heating and stomatal obstructions that can cause changes in cellular and physiological functions of the plant (Fernandez and Eichert, 2009) so a foliar application could cause a disruption of the cellular functions. 760

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released from 2 mg/ml loaded MSNPs (Bravo Cadena et al., 2018)). This resulted in a CNAD concentration of < 0.0000034% v/v present in the alginate coating, which is ∼90,000-fold lower than concentrations of free cinnamon oil (0.3%) previously reported to control certain bacterial phytopathogens (Van Der Wolf et al., 2008). The use of MSNPs was thus shown to permit much lower doses of biocide to be effective as antimicrobials. The CNAD-MSNP-alginate coating effectively decreased the percentage of plants infected by P. syringae pv. pisi from 50% of control plants to only 28.21% of treated plants displaying bacterial blight symptoms. Additionally, the treatment resulted in larger pods, greater wet and dry masses, and longer roots. It is important to mention that as the number of plants that died 20 days after infection was higher in the control; the difference between the treatments at day 50 was reduced, possibly underestimating its beneficial effect on the plants. Future experiments using slightly higher MSNPs concentrations could be carried out to determine the optimal treatment to further reduce bacterial infection. Also, this treatment could be applied to different crops and several other essential oils could be used to treat and prevent a vast array of microbial diseases. It is essential to mention that natural variation is an important source of divergence in biological experiments (Yarnes, 2013) and this intrinsic variability in terms of individual variation in germination, growth and rate of infection could explain the variance in Fig. 5. Additionally, this study utilised an extreme condition where seeds were soaked in a highly concentrated bacterial inoculum. Despite this, the treatment of seeds with CNADMSNPs-alginate resulted in a statistically significant (p < 0.01) protection of peas against the bacterial pathogen. In a more realistic scenario where seeds are infected through water-splashing or contact with infected debris/plants; the level of protection would be expected to be even greater. The treatment was particularly effective during the first week after sowing and efficacy was reduced over time, possibly due to the inevitable loss of the antimicrobial via evaporation or degradation. This treatment could therefore be highly effective as a first line of defence before germination. Moreover, the alginate-CNAD-MSNPs seed treatment not only provided protection from bacterial blight infection but also resulted in improved germination and plant development.

prevented seed germination and trapped the seedlings and/or rootlets inside the alginate coat. This could be due to greater concentration of CaCl2 during the gelation process creating a thicker “egg-box” model (Martinsen et al., 1989). AFM analyses of alginate layers demonstrated that the low CaCl2 molarity formulations were harder and more brittle than the high molarity formulations, which were softer and more flexible. It has been previously suggested that more elasticity or viscous gels can prevent the emergence of encapsulated embryos from alginate beads (Onishi et al., 1994). This was confirmed by AFM analysis, where the difference in elasticity was demonstrated to be CaCl2 concentration-dependent. Moreover, AFM analysis of an alginate coating containing MSNPs revealed that MSNPs altered the properties of the gel, making it harder and more brittle (similar to a gel prepared with less calcium chloride); enabling improved seed germination. This may have been due to the MSNPs interfering with the interaction of the sodium alginate coils and the calcium ions thus reducing the formation of ‘egg-box’ dimers, therefore creating a structure more similar to a gel produced with a lower concentration of CaCl2. Consequently, alginate could be used as a scaffold to deliver MSNPs to seeds while allowing an improved seed germination and plant development. Once the alginate layer was demonstrated to have only beneficial effects on plant development and growth, infection experiments were carried out. ‘Kelvedon wonder’ cultivar peas were selected for this study due to this cultivar’s susceptibility to all seven races of P. syringae pv. pisi (Taylor et al., 1989). Seeds treated with CNAD-MSNPs germinated and developed faster during the first week after sowing. Silica nanoparticles have been shown to improve germination, provide nutrient availability, enhance plant dry weight and levels of proteins, chlorophyll and phenols; improve seedlings growth and quality, and increase plant stress resistance (Haghighi et al., 2012; Janmohammadi and Sabaghnia, 2015; Lin et al., 2004a, 2004b; Shah and Belozerova, 2008; Siddiqui et al., 2014; Siddiqui and Al-Whaibi, 2014; Suriyaprabha et al., 2012). Silicon, present as SiO2 in soil, has an important physiological role in plants; it is deposited as hydrated amorphous silica in the endoplasmic reticulum, cell wall and intercellular spaces and its deficiency increases the plant’s susceptibility to lodging and infections (Solanki et al., 2015). Its natural role in plant systems make silica a low-risk candidate to be used in agriculture to deliver antimicrobial cargo. MSNPs have previously been used to deliver DNA and chemicals into plant cells (Torney et al., 2007) or to encapsulate pesticides such as validamycin (Liu et al., 2006) and 1-naphthylacetic acid (Ao et al., 2013) and the insecticide chlorfenapyr (Song et al., 2012). In addition to providing efficient delivery and cargo stability, MSNPs have been shown to be safe for plants and humans (Shin et al., 2007). For these reasons, MSNPs are suitable delivery vehicles for volatile biocides such as EOs. Other studies have previously evaluated EOs seed treatments, usually by soaking the seeds in a high concentration solution of EOs. Eucalyptus, rosemary and niaouli EOs at 2% concentration have been shown to reduce the incidence and severity of bacterial leaf spot disease caused by Xanthomonas perforans in tomato 21 days after sowing (Mbega et al., 2012). Moreover, eugenol (4 mg/ml) seed treatments of beans resulted in a significant reduction of Xanthomonas campestris pv. phaseoli var. fuscans, however, after 72 h of incubation a significant reduction of germination was observed (Lo Cantore et al., 2009). Cabbage seeds treated with thyme, oregano, cinnamon or clove oils had a reduced count of seed-associated bacteria, but relatively high concentrations (≥0.1%) were needed for a bactericidal effect. Additionally, concentrations of > 1% cinnamon oil had a negative effect on germination while > 3.3% resulted in significant seed damage (Van Der Wolf et al., 2008). Likewise, high doses of clove, and oregano oils (5%, 10%) could inhibit fungal growth on seeds but also totally inhibited seed germination (Karaca et al., 2017). The utilisation of MSNPs to deliver EOs such as CNAD allows the effective antimicrobial activity of the biocide at much lower doses thus avoiding a negative impact on the plant’s germination or development. The alginate solution utilised in this study contained 2 mg/ml MSNPs, equivalent to 0.0339 μlCNAD/mlalginate (35.63 mg/L CNAD are

5. Conclusions In conclusion, this is the first study to exploit the combined benefits of essential oils, MSNPs and alginate to enhance the antimicrobial activity of plant products. MSNPs protected the loaded EO from degradation, evaporation or run-off whilst enhancing the stability, and enabling a controlled delivery of the biocide. Furthermore, MSNPs permitted the incorporation of hydrophobic and volatile antimicrobials such as EOs into a seed coating formulation and enabled a much lower dose of EO (< 0.0000034% v/v CNAD) to be effective against the target pathogen, avoiding the use of higher doses that would otherwise negatively affect plant germination and development. The controlled slow-release of EOs from MSNPs resulted in a higher efficacy attained by increased solubility, stability and effectiveness, thereby showing a significant increase of symptomless plants. Declaration of interests None. Acknowledgements Marimar Bravo Cadena is funded by the National Council of Science and Technology, Conacyt, Mexico (grant no. 384507) and the Schlumberger Foundation (FFTF Award). Renier Van der Hoorn received funding from ERC Consolidator grant616449 ‘GreenProteases’. Helen E. Townley is grateful to the Williams Fund for ongoing support. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. 761

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Appendix A

Fig. A1. Alginate coating prepared using a high calcium chloride molarity (2 M) prevents correct germination of peas. A) Roots or B) shoots can get trapped inside the alginate coating. Arrows point towards trapped plant parts inside the alginate coat.

Fig. A2. Seed germination and growth of alginate-coated peas compared to control seeds. A) Seed germination per treatment. B) Average plant height (mm) fifteen days after sowing. Data shows mean ± standard deviation. Control used was uncoated pea seeds. Treatments refer to the % (m/v) of sodium alginate and the molarity of CaCl2 used for the seed coating. MSNPs = Mesoporous silica nanoparticles added to the alginate solution (25 mg/ml). Statistically significant differences between treatments were calculated using Tukey’s HSD test *p < 0.05, **p < 0.01, n = 15 seeds per treatment. 762

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Fig. A3. Signs of pea bacterial blight infection. A) Bacterial ooze covering seed with papery dead seedling showing severe infection and bacterial ooze B) Watersoaked lesion (WSL) on rootlet characteristic of bacterial blight infection C) WSL on leaves and rootlet with observable bacterial colonies D) WSL, bacterial colonies on leaves and wilting of plant E) Interveinal tissues drying out and becoming papery F) Bacterial ooze covering seed and plant G) Peas displaying signs of bacterial blight infection, some seeds die and are not able to germinate while other peas show signs at different growth stages. Scale bars = 1 cm.

interactions between polysaccharides and divalent cations: the egg-box model. FEBS Lett. 32, 195–198. https://doi.org/10.1016/0014-5793(73)80770-7. Grondeau, C., Samson, R., Poutier, F., 1991. Spread of Pseudomonas syringae pv. pisi : can prevailing winds help bacteria’s progression? 4th Int. Work. Gr. Pseudomonas syringae Pathovars’. pp. 439. Guo, M., Liu, M., Liang, R., Niu, A., 2006. Granular urea-formaldehyde slow-release fertilizer with superabsorbent and moisture preservation. J. Appl. Polym. Sci. 99, 3230–3235. https://doi.org/10.1002/app.22892. Haghighi, M., Afifipour, Z., Mozafarian, M., 2012. The effect of N-Si on tomato seed germination under salinity levels. J. Biol. Environ. Sci. 6, 87–90. Hollaway, G.J., Bretag, T.W., Price, T.V., 2007. The epidemiology and management of bacterial blight (Pseudomonas syringae pv. pisi) of field pea (Pisum sativum) in Australia: a review. Aust. J. Agric. Res. 58, 1086–1099. https://doi.org/10.1071/ AR06384. Huang, X., Young, N.P., Townley, H.E., 2014. Characterization and comparison of mesoporous silica particles for optimized drug delivery. Nanomater. Nanotechnol. 4. https://doi.org/10.5772/58290. Idrees, M., Naeem, M., Alam, M., Aftab, T., Hashmi, N., Khan, M.M.A., Moinuddin, Varshney, L., 2011. Utilizing the γ-irradiated sodium alginate as a plant growth promoter for enhancing the growth, physiological activities, and alkaloids production in Catharanthus roseus L. Agric. Sci. China 10, 1213–1221. https://doi.org/10.1016/ S1671-2927(11)60112-0. International Seed Federation, 2007. Seed Treatment. A Tool for Sustainable Agriculture. Nyon, Switzerland. . Ivanova, E., Teunou, E., Poncelet, D., 2005. Alginate based macrocapsules as inocula carriers for production of nitrogen biofertilizers. Proceedings of the Balkan Scientific Conference of Biology 90–108. Janmohammadi, M., Sabaghnia, N., 2015. Effect of pre-sowing seed treatments with silicon nanoparticles on germinability of sunflower (Helianthus annuus). Bot. Lith. 21, 13–21. Joint FAO/WHO Expert Committee on Food Additives, 1997. Compendium of Food Additive Specifications. Addendum 5. World Health Organization, Food and Agriculture Organization of the United Nations, Rome. Karaca, G., Bilginturan, M., Olgunsoy, P., 2017. Effects of some plant essential oils against fungi on wheat seeds. Indian J. Pharm. Educ. Res. 51. https://doi.org/10.5530/ijper. 51.3s.53. Khor, E., Ng, W.-F., Loh, C.-S., 1998. Two-coat systems for encapsulation of Spathoglottis plicata (Orchidaceae) seeds and protocorms. Biotechnol. Bioeng. 59, 635–639. https://doi.org/10.1002/(SICI)1097-0290(19980905)59:5<635::AID-BIT14>3.0. CO;2-8. Kwon, S., Singh, R.K., Perez, R.A., Abou Neel, E.A., Kim, H.-W., Chrzanowski, W., 2013. Silica-based mesoporous nanoparticles for controlled drug delivery. J. Tissue Eng. 4. https://doi.org/10.1177/2041731413503357. Lawyer, A., Chun, W., 2001. Foliar diseases caused by bacteria; bacterial blight. In: Kraft, J., Pfleger, F. (Eds.), Compendium of Pea Diseases and Pests. The American

References Abd El-Rehim, H.A., 2006. Characterization and possible agricultural application of polyacrylamide/sodium alginate crosslinked hydrogels prepared by ionizing radiation. J. Appl. Polym. Sci. 101, 3572–3580. https://doi.org/10.1002/app.22487. Ao, M., Zhu, Y., He, S., Li, D., Li, P., Li, J., Cao, Y., 2013. Preparation and characterization of 1-naphthylacetic acid-silica conjugated nanospheres for enhancement of controlled-release performance. Nanotechnology 24 (35601). https://doi.org/10.1088/ 0957-4484/24/3/035601. Asmah, N., Hasnida, N., Zaimah, N., Salmi, N., 2011. Synthetic seed technology for encapsulation and regrowth of in vitro-derived Acacia hyrid shoot and axillary buds. Afr. J. Biotechnol. 10, 7820–7824. https://doi.org/10.5897/AJB11.492. Bajpai, V.K., Kang, S.-R., Xu, H., Lee, S.-G., Baek, K.-H., Kang, S.-C., 2011. Potential roles of essential oils on controlling plant pathogenic bacteria Xanthomonas species: a review. Plant Pathol. J. 27, 207–224. https://doi.org/10.5423/PPJ.2011.27.3.207. Bernardos, A., Marina, T., Žáček, P., Pérez-Esteve, É., Martínez-Mañez, R., Lhotka, M., Kouřimská, L., Pulkrábek, J., Klouček, P., 2014. Antifungal effect of essential oil components against Aspergillus niger when loaded into silica mesoporous supports. J. Sci. Food Agric. https://doi.org/10.1002/jsfa.7022. Bravo Cadena, M., Preston, G.M., Van der Hoorn, R.A.L., Townley, H.E., Thompson, I.P., 2018. Species- specific antimicrobial activity of essential oils and enhancement by encapsulation in mesoporous silica nanoparticles. Ind. Crops Prod. 122, 582–590. https://doi.org/10.1016/j.indcrop.2018.05.081. Carpita, N.C., Gibeaut, D.M., 1993. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3, 1–30. Chan, A.C., Bravo Cadena, M., Townley, H.E., Fricker, M.D., Thompson, I.P., 2017. Effective delivery of volatile biocides employing mesoporous silicates for treating biofilms. J. R. Soc. Interface 14. https://doi.org/10.1098/rsif.2016.0650. DeRosa, M.C., Monreal, C., Schnitzer, M., Walsh, R., Sultan, Y., 2010. Nanotechnology in fertilizers. Nat. Nanotechnol. 5, 91. https://doi.org/10.1038/nnano.2010.2. Diaconu, M., Tache, A., Ana-Maria, S., Eremia, V., Gatea, F., Litescu, S., Radu, G.L., 2010. Structural characterization of chitosan coated silicon nanoparticles –a FT-IR approach. U.P.B. Sci. Bull. Ser. B Chem. Mater. Sci. 72, 115–122. Fernandez, V., Eichert, T., 2009. Uptake of hydrophilic solutes through plant leaves: current state of knowledge and perspectives of foliar fertilization. Crit. Rev. Plant Sci. 36–68. https://doi.org/10.1080/07352680902743069. Fravel, D.R., Marois, J.J., Lumsden, R.D., Connick Jr, W.J., 1985. Encapsulation of potential biocontrol agents in an alginate-clay matrix. Phytopathology 75, 774–777. González, A., Castro, J., Vera, J., Moenne, A., 2013. Seaweed oligosaccharides stimulate plant growth by enhancing carbon and nitrogen assimilation, basal metabolism, and cell division. J. Plant Growth Regul. 32, 443–448. https://doi.org/10.1007/s00344012-9309-1. Grant, G.T., Morris, E.R., Rees, D.A., Smith, P.J.C., Thom, D., 1973. Biological

763

Industrial Crops & Products 124 (2018) 755–764

M. Bravo Cadena et al.

alginate on the growth, physiological activities and essential oil production of fennel (Foeniculum vulgare Mill.). J. Med. Plants Res. 5, 15–21. Sarrocco, S., Raeta, R., Vannacci, G., 2004. Seeds encapsulation in calcium alginate pellets. Seed Sci. Technol. 32, 649–661. https://doi.org/10.15258/sst.2004.32.3.01. Shah, V., Belozerova, I., 2008. Influence of metal nanoparticles on the soil microbial community and germination of lettuce seeds. Water Air Soil Pollut. 197, 143–148. https://doi.org/10.1007/s11270-008-9797-6. Sharifeh, S., Katouzi, S., Majd, A., Fallahian, F., Bernard, F., 2011. Encapsulation of shoot tips in alginate beads containing salicylic acid for cold preservation and plant regeneration in sunflower (Helianthus annuus L.). AJCS 5, 1469–1474. Shin, J.H., Metzger, S.K., Schoenfisch, M.H., 2007. Synthesis of nitric oxide-releasing silica nanoparticles. J. Am. Chem. Soc. 129, 4612–4619. https://doi.org/10.1021/ ja0674338. Siddiqui, M.H., Al-Whaibi, M.H., 2014. Role of nano-SiO2 in germination of tomato (Lycopersicum esculentum seeds Mill.). Saudi J. Biol. Sci. 21, 13–17. https://doi.org/ 10.1016/j.sjbs.2013.04.005. Siddiqui, M.H., Al-Whaibi, M.H., Faisal, M., Al Sahli, A.A., 2014. Nano-silicon dioxide mitigates the adverse effects of salt stress on Cucurbita pepo L. Environ. Toxicol. Chem. 33, 2429–2437. https://doi.org/10.1002/etc.2697. Simpson, N.E., Stabler, C.L., Simpson, C.P., Sambanis, A., Constantinidis, I., 2004. The role of the CaCl2–guluronic acid interaction on alginate encapsulated βTC3 cells. Biomaterials 25, 2603–2610. https://doi.org/10.1016/j.biomaterials.2003.09.046. Slade, D., Fujii, J.A., Redenbaugh, K., 1989. Artificial seeds: a method for the encapsulation of somatic embryos. J. Tissue Cult. Methods 12, 179–183. https://doi. org/10.1007/BF01404446. Slowing, I.I., Vivero-Escoto, J.L., Wu, C.-W., Lin, V.S.-Y., 2008. Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv. Drug Deliv. Rev. 60, 1278–1288. https://doi.org/10.1016/j.addr.2008.03.012. Smidsrød, O., 1974. Molecular basis for some physical properties of alginates in the gel state. Faraday Discuss. Chem. Soc. 57, 263–274. https://doi.org/10.1039/ DC9745700263. Solanki, P., Bhargava, A., Chhipa, H., Jain, N., Panwar, J., 2015. Nano-fertilizers and their smart delivery system. In: Rai, M., Ribeiro, C., Mattoso, L., Duran, N. (Eds.), Nanotechnologies in Food and Agriculture. Springer, pp. 81–101. Song, M.-R., Cui, S.-M., Gao, F., Liu, Y.-R., Fan, C.-L., Lei, T.-Q., Liu, D.-C., 2012. Dispersible silica nanoparticles as carrier for enhanced bioactivity of chlorfenapyr. J. Pestic. Sci. 37, 258–260. https://doi.org/10.1584/jpestics.D12-027. Sun, D., Hussain, H.I., Yi, Z., Siegele, R., Cresswell, T., Kong, L., Cahill, D.M., 2014. Uptake and cellular distribution, in four plant species, of fluorescently labeled mesoporous silica nanoparticles. Plant Cell Rep. 33, 1389–1402. https://doi.org/10. 1007/s00299-014-1624-5. Suriyaprabha, R., Karunakaran, G., Yuvakkumar, R., Rajendran, V., Kannan, N., 2012. Silica nanoparticles for increased silica availability in maize (Zea mays L.) seeds under hydroponic conditions. Curr. Nanosci. 8, 902–908. https://doi.org/10.2174/ 157341312803989033. Taylor, J.D., Dye, D.W., 1976. Evaluation of streptomycin seed treatments for the control of bacterial blight of peas (Pseudomonas pisi Sackett 1916). N.Z. J. Agric. Res. 19, 91–95. Taylor, J.D., Bevan, J.R., Crute, I.R., Reader, S.L., 1989. Genetic relationship between races of Pseudomonas syringae pv. pisi and cultivars of Pisum sativum. Plant Pathol. 38, 364–375. https://doi.org/10.1111/j.1365-3059.1989.tb02155.x. The Plant Health (Great Britain) SI 1987/1758, 1987. The Plant Health (Great Britain) Order. 1987. UK. . Torney, F., Trewyn, B.G., Lin, V.S.-Y., Wang, K., 2007. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat. Nanotechnol. 2, 295–300. https://doi. org/10.1038/nnano.2007.108. Trewyn, B.G., Slowing, I.I., Giri, S., Chen, H.-T., Lin, V.S.-Y., 2007. Synthesis and functionalization of a mesoporous silica nanoparticle based on the sol-gel process and applications in controlled release. Acc. Chem. Res. 40, 846–853. https://doi.org/10. 1021/ar600032u. Van Der Wolf, J.M., Birnbaum, Y., Van Der Zouwen, P.S., Groot Van Der Wolf, S.P.C., Van Der Zouwen, Y., Groot, P.S., Groot, S.P.C., 2008. Disinfection of vegetable seed by treatment with essential oils, organic acids and plant extracts. Seed Sci. Technol. 36, 76–88. Yarnes, S.C., 2013. Introduction to Experimental Design [WWW Document]. URL. (Accessed 2 June 2018). http://articles.extension.org/pages/69752/introduction-toexperimental-design.

Phytopathological Society, St. Paul, Minn, pp. 22–23. Le, Q., Nguyen, Q., Nagasawa, N., Kume, T., Yoshii, F., Nakanishi, T.M., 2003. Biological effect of radiation-degraded alginate on flower plants in tissue culture. Biotechnol. Appl. Biochem. 38, 283. https://doi.org/10.1042/BA20030058. Lin, B.-S., Diao, S., Li, C., Fang, L., Qiao, S., Yu, M., 2004a. Effect of TMS (nanostructured silicon dioxide) on growth of Changbai larch seedlings. J. For. Res. 15, 138–140. https://doi.org/10.1007/BF02856749. Lin, B.-S., Fang, L., Qiao, S., Yu, M., 2004b. Application of TMS organic fertilizer in spruce seedlings. J. Beihua Univ. Nat. Sci. 553–556. Liong, M., Lu, J., Kovochich, M., Xia, T., Ruehm, S.G., Nel, A.E., Tamanoi, F., Zink, J.I., 2008. Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery. ACS Nano 2, 889–896. https://doi.org/10.1021/nn800072t. Liu, F., Wen, L.-X., Li, Z.-Z., Yu, W., Sun, H.-Y., Chen, J.-F., 2006. Porous hollow silica nanoparticles as controlled delivery system for water-soluble pesticide. Mater. Res. Bull. 41, 2268–2275. https://doi.org/10.1016/j.materresbull.2006.04.014. Lo Cantore, P., Shanmugaiah, V., Sante Iacobellis, N., 2009. Antibacterial activity of essential oil components and their potential use in seed disinfection. J. Agric. Food Chem. 57, 9454–9461. https://doi.org/10.1021/jf902333g. Lu, C., Zhang, C., Wen, J., Wu, G., Tao, M., 2002. Research of the effect of nanometer materials on germination and growth enhancement of Glycine max and its mechanism. Soybean Sci. 168–171. Luan, L.Q., Thi, V., Ha, T., Hai, L., Hien, N.Q., Hang, N.D., Tuong, N., Lan, L., Tu, L.H., 2002. Study on the Biological Effect of Radiation-Degraded Alginate and Chitosan on Plant in Tissue Culture. Vietnam. . Martinsen, A., Skjåk-Braek, G., Smidsrød, O., 1989. Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads. Biotechnol. Bioeng. 33, 79–89. https://doi.org/10.1002/bit.260330111. Mbega, E.R., Mabagala, R.B., Mortensen, C.N., Wulff, E.G., 2012. Evaluation of essential oils as Seed Treatment for the control of Xanthomonas spp. associated with the bacterial leaf spot of tomato in Tanzania. J. Plant Pathol. https://doi.org/10.4454/JPP. FA.2012.024. Morris, E.R., Rees, D.A., Thom, D., Boyd, J., 1978. Chiroptical and stoichiometric evidence of a specific, primary dimerisation process in alginate gelation. Carbohydr. Res. 66, 145–154. https://doi.org/10.1016/S0008-6215(00)83247-4. Naeem, M., Idrees, M., Aftab, T., Khan, M.M.A., Moinuddin, Varshney, L., 2011. Irradiated sodium alginate improves plant growth, physiological activities and active constituents in Mentha arvensis L. J. Appl. Pharm. Sci. 2, 28–35. Nair, R., Poulose, A.C., Nagaoka, Y., Yoshida, Y., Maekawa, T., Kumar, D.S., 2011. Uptake of FITC labeled silica nanoparticles and quantum dots by rice seedlings: effects on seed germination and their potential as biolabels for plants. J. Fluoresc. 21, 2057–2068. https://doi.org/10.1007/s10895-011-0904-5. Onishi, N., Sakamoto, Y., Hirosawa, T., 1994. Synthetic seeds as an application of mass production of somatic embryos. Plant Cell Tissue Organ Cult. 39, 137–145. https:// doi.org/10.1007/BF00033921. Pintos, B., Bueno, M.A., Cuenca, B., Manzanera, J.A., 2008. Synthetic seed production from encapsulated somatic embryos of cork oak (Quercus suber L.) and automated growth monitoring. Plant Cell Tissue Organ Cult. 95, 217–225. https://doi.org/10. 1007/s11240-008-9435-4. Porra, R.J., Thompson, W.A., Kriedemann, P.E., 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta Bioenergy 975, 384–394. https://doi.org/10.1016/S0005-2728(89)80347-0. Redenbaugh, M.K., 1983. Delivery system for meristematic tissue. Patent EP0141373 B1. US 4583320 A. Roberts, S.J., 1993. Effect of bacterial blight (Pseudomonas syringae pv. pisi) on the growth and yield of single pea (Pisum sativum) plants under glasshouse conditions. Plant Pathol. 42, 568–576. https://doi.org/10.1111/j.1365-3059.1993.tb01537.x. Ruiz-Rico, M., Pérez-Esteve, É., Bernardos, A., Sancenón, F., Martínez-Máñez, R., Marcos, M.D., Barat, J.M., 2017. Enhanced antimicrobial activity of essential oil components immobilized on silica particles. Food Chem. 233, 228–236. https://doi.org/10.1016/ j.foodchem.2017.04.118. Sakhanokho, H.F., Pounders, C.T., Blythe, E.K., 2013. Alginate encapsulation of Begonia microshoots for short-term storage and distribution. Sci. World J. 2013, 341568. https://doi.org/10.1155/2013/341568. Sarfaraz, A., Naeem, M., Nasir, S., Idrees, M., Aftab, T., Hashmi, N., Khan, M.M.A., Moinuddin, Varshney, L., 2011. An evaluation of the effects of irradiated sodium

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