Silver Nanoparticles Complexed with Bovine SubmaxillaryMucin Possess Strong Antibacterial Activity and Protectagainst Seedling Infection

Daria Makarovsky,a* Ludmila Fadeev,b Bolaji Babajide Salam,a* Einat Zelinger,c Ofra Matan,a Jacob Inbar,d

Edouard Jurkevitch,a Michael Gozin,b Saul Burdmana

aDepartment of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food, andEnvironment, The Hebrew University of Jerusalem, Rehovot, Israel

bSchool of Chemistry, Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, IsraelcInterdepartmental Core Facility, The Robert H. Smith Faculty of Agriculture, Food, and Environment, TheHebrew University of Jerusalem, Rehovot, Israel

dDepartment of Economics and Business Management, Faculty of Social Sciences and Humanities, ArielUniversity, Ariel, Israel

ABSTRACT A simple method for the synthesis of nanoparticles (NPs) of silver (Ag)in a matrix of bovine submaxillary mucin (BSM) was reported previously by some ofthe authors of this study. Based on mucin characteristics such as long-lasting stabil-ity, water solubility, and surfactant and adhesive characteristics, we hypothesizedthat these compounds, named BSM-Ag NPs, may possess favorable properties as po-tent antimicrobial agents. The goal of this study was to assess whether BSM-AgNPs possess antibacterial activity, focusing on important plant-pathogenic bacterialstrains representing both Gram-negative (Acidovorax and Xanthomonas) and Gram-positive (Clavibacter) genera. Growth inhibition and bactericidal assays, as well aselectron microscopic observations, demonstrate that BSM-Ag NPs, at relatively lowconcentrations of silver, exert strong antimicrobial effects. Moreover, we show thattreatment of melon seeds with BSM-Ag NPs effectively prevents seed-to-seedlingtransmission of Acidovorax citrulli, one of the most threatening pathogens of cucur-bit production worldwide. Overall, our findings demonstrate strong antimicrobial ac-tivity of BSM-Ag NPs and their potential application for reducing the spread and es-tablishment of devastating bacterial plant diseases in agriculture.

IMPORTANCE Bacterial plant diseases challenge agricultural production, and the meansavailable to manage them are limited. Importantly, many plant-pathogenic bacteria havethe ability to colonize seeds, and seed-to-seedling transmission is a critical route bywhich bacterial plant diseases spread to new regions and countries. The significance ofour study resides in the following aspects: (i) the simplicity of the method of BSM-Ag NPsynthesis, (ii) the advantageous chemical properties of BSM-Ag NPs, (iii) the strong anti-bacterial activity of BSM-Ag NPs at relatively low concentrations of silver, and (iv) thefact that, in contrast to most studies on the effects of metal NPs on plant pathogens,the proof of concept for the novel compound is supported by in planta assays. Applica-tion of this technology is not limited to agriculture; BSM-Ag NPs potentially could be ex-ploited as a potent antimicrobial agent in a wide range of industrial areas, includingmedicine, veterinary medicine, cosmetics, textiles, and household products.

KEYWORDS Acidovorax, bacterial plant diseases, metal nanoparticles, mucin, silver

Plant-pathogenic microorganisms are a major cause of crop yield losses, and agri-cultural intensification has been possible through the use of chemical pesticides to

cope with such microorganisms. However, despite the intensive use of antimicrobial

Received 5 October 2017 Accepted 17November 2017

Accepted manuscript posted online 27November 2017

Citation Makarovsky D, Fadeev L, Salam BB,Zelinger E, Matan O, Inbar J, Jurkevitch E, GozinM, Burdman S. 2018. Silver nanoparticlescomplexed with bovine submaxillary mucinpossess strong antibacterial activity andprotect against seedling infection. ApplEnviron Microbiol 84:e02212-17. https://doi.org/10.1128/AEM.02212-17.

Editor Eric V. Stabb, University of Georgia

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Saul Burdman,saul.burdman@mail.huji.ac.il.

* Present address: Daria Makarovsky,Goldschleger Eye Institute, Sheba MedicalCenter, Tel Hashomer, Israel; Bolaji BabajideSalam, Department of Postharvest Science ofFresh Produce, The Volcani Center, AgriculturalResearch Organization, Bet Dagan, Israel.

PLANT MICROBIOLOGY

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compounds, estimations of world crop yield losses due to plant diseases range from 15to 20% (1–3). Plant-pathogenic bacteria are among the biotic agents causing significantlosses in crop production. Almost every important crop suffers from one or severalimportant bacterial diseases, which often are among the most significant diseases forthe given crop (3). Moreover, the strategies available to manage bacterial plant diseasesare generally limited, including the lack of efficient chemical bactericides for diseasecontrol (4).

Importantly, many plant-pathogenic bacteria are seedborne, that is, they can survivein the seeds and be transmitted via contaminated seeds to new fields, regions, andcountries (4, 5). In a globalized world, in which a huge amount of plant material (mainlyseeds) is transferred from one country to another, inadvertent distribution of contam-inated commercial seeds is one of the main ways in which bacterial plant diseases arespread worldwide (4). Therefore, new strategies to manage bacterial plant diseases arehighly demanded in general and are needed to prevent or to reduce seed transmissionof bacterial pathogens in particular.

The aim of this study was to assess the antimicrobial activity of silver (Ag) nano-particles (NPs) in complex with bovine submaxillary mucin (BSM), focusing on plant-pathogenic bacteria. Silver has been used extensively to control microbial infectionssince ancient times (6, 7). Silver-based medical products have been shown to beeffective in reducing and preventing bacterial infections (8). Silver ions are highlyreactive, exerting a broad range of antimicrobial activities. They are able to bind to andto damage proteins and DNA, leading to disruption of disulfide bonds in proteins,structural changes in the cytoplasmic membrane and in the cell wall, altered membranepermeability, cell distortion, and inhibition of replication and respiratory activity,eventually leading to cell death (9, 10). In recent years, the development of nanotech-nologies has brought about growing interest in the industrial and medical fields in thegeneration of bioactive biomaterials that combine the relevant antibacterial propertiesof metals with the peculiar performance of the biomaterial. A variety of nanosilvercompounds have been developed and tested for their antimicrobial properties (9, 11).Some nanosilver compounds were also generated and evaluated for their potentialapplication in agriculture; this was rather limited, however, and very few studies wereconducted to assess antibacterial or antifungal activities of the compounds in planta(12–16).

Mucins are large extracellular glycoproteins found as the main components ofmucus in almost all animals (17, 18). With molecular masses ranging from 0.5 to 20MDa, mucins are highly glycosylated, consisting of approximately 80% carbohy-drates. Among their chemical properties, mucins may act as moisture holders,adhesins, and solubilizers, as well as reducing agents and surfactants (18). Some ofus have shown that BSM, a representative natural mucin, is capable of binding andsolubilizing various types of polycyclic aromatic hydrocarbons in aqueous solutions,leading to increased antimicrobial activity (19). Further, a simple method wasdeveloped to generate Ag NPs inside a BSM matrix (20). Synthesis of the BSM-Ag NPcompound was carried out in an aqueous solution without the need for an externalreducing agent, exploiting the natural solubilizing, reducing, and stabilizing prop-erties of mucin (20). The generated complex, named BSM-Ag NPs, is highly solublein water and biodegradable, with the potential to be active at very low concentra-tions, representing a potentially powerful and environmentally friendly tool for cropprotection.

In the present study, growth inhibition and bactericidal assays and electron micro-scopic observations demonstrate strong antibacterial effects of BSM-Ag NPs. Moreover,seed transmission assays reveal that BSM-Ag NPs effectively prevent seed-to-seedlingtransmission of Acidovorax citrulli, one of the most threatening pathogens of cucurbitproduction worldwide (21). Overall, our findings support the potential of BSM-Ag NPsas an efficient crop protection agent.

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RESULTSBSM-Ag NPs inhibit bacterial growth. BSM-Ag NPs tested in this report were

produced as described previously (20) (see Materials and Methods). The size of the AgNPs was found to be in the range of 5 to 20 nm, with an average diameter of �10 nm(20). The concentrated complexes carried Ag NPs at concentrations ranging between�400 and 1,000 mg liter�1. We first assessed the antimicrobial activity of BSM-Ag NPsby determining the effects of various Ag concentrations on the growth of three strains,representing important seedborne plant-pathogenic bacteria, i.e., Gram-negative Aci-dovorax citrulli M6 (bacterial fruit blotch [BFB] of cucurbits [21]) and Xanthomonaseuvesicatoria 85-10 (bacterial spot disease of peppers and tomatoes [22]) and Gram-positive Clavibacter michiganensis subsp. michiganensis NCPPB 382 (bacterial cankerand wilt of tomatoes [23]).

BSM-Ag NPs exerted strong growth-inhibitory effects on all tested strains at very lowAg concentrations. Representative growth curve experiments in nutrient broth (NB) areshown in Fig. 1A for A. citrulli M6 and in Fig. S1 in the supplemental material for C.michiganensis subsp. michiganensis NCPPB 382 and X. euvesicatoria 85-10. A delay in theexponential growth phase of all strains was achieved with Ag concentrations as low as0.13 mg liter�1. Much stronger inhibitory effects were observed in the range of 0.67 to2.68 mg Ag liter�1. With these concentrations, bacterial growth was delayed 24 to 40h, relative to controls exposed to BSM alone. With Ag concentrations of �6.7 mgliter�1, no growth could be detected for A. citrulli M6 and C. michiganensis subsp.michiganensis NCPPB 382 after 168 h (1 week) of incubation (Fig. 1A; also see Fig. S1A).In the case of X. euvesicatoria 85-10, substantially delayed growth occurred with 6.7 mgAg liter�1, but no growth occurred with 13.4 mg Ag liter�1 (Fig. S1B). In theseexperiments, we were not able to isolate bacteria following dilution plating of samples

FIG 1 Effects of BSM-Ag NPs on Acidovorax citrulli. (A) Growth of A. citrulli strain M6 exposed to differentconcentrations of BSM-Ag NPs. Bacteria were grown for 168 h at 28°C in enzyme-linked immunosorbentassay (ELISA) plates in NB containing BSM-Ag NPs at different concentrations of Ag (the control was BSMwithout Ag). The initial concentration of bacterial cells was 107 CFU ml�1. Growth was measured as theoptical density at 600 nm. Averages of 3 replicates per treatment from a representative experiment (ofthree with similar results) are shown. (B) Killing effect of BSM-Ag NPs on A. citrulli M6. Bacterialsuspensions containing 107 CFU ml�1 were treated at 25°C with BSM-Ag NP solutions containingdifferent Ag concentrations. After 24 h, the suspensions were serially diluted and plated on NA plates forbacterial counting. The graph represents bacterial concentrations at the end of the experiment. Averagesof 3 replicates per treatment from a representative experiment (of two with similar results) are shown.

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in which no growth was observed, indicating that, under the tested conditions, BSM-AgNPs at relatively low Ag concentrations of 6.7 and 13.4 mg liter�1 had bactericidaleffects on A. citrulli/C. michiganensis subsp. michiganensis and X. euvesicatoria, respec-tively.

Confirmation of bactericidal activity of BSM-Ag NPs. We further verified thebactericidal activity of the BSM-Ag-NPs against A. citrulli. We selected this pathogen forfurther experiments because BFB caused by this bacterium is considered one of themost serious threats to the cucurbit industry (mainly to melons and watermelons)worldwide (21). Moreover, seed transmission has been responsible for the extraordi-narily fast global spread of BFB, thus making this disease of great concern to the seedproduction sector (21).

Bacterial suspensions of A. citrulli M6 (107 CFU ml�1) in 100 mM phosphate buffer(pH 7.0) were exposed to different concentrations of BSM-Ag NPs for 24 h at 25°C. Thesuspensions were then serially diluted, and dilution samples were plated on nutrientagar (NA) plates to determine live bacterial concentrations at the end of the experi-ment. The results of these experiments confirmed the strong bactericidal activity ofBSM-Ag NPs. Under these conditions, exposure of A. citrulli cells to BSM-Ag NPs at 0.4mg Ag liter�1 reduced bacterial numbers by 2 orders of magnitude, and no bacteriacould be retrieved after exposure to BSM-Ag NPs at 2.2 mg Ag liter�1 (Fig. 1B).

BSM-Ag NPs damage bacterial cells. Scanning electron microscopy (SEM) wasused to observe the morphological effects of BSM-Ag NPs on A. citrulli M6 cells. Whilecells treated with BSM alone (without Ag NPs) possessed a typical rod-like shape anda well-defined cell wall (Fig. 2A and C), clear morphological alterations were observedin cells exposed to BSM-Ag NPs containing 10 mg Ag liter�1; the latter cells had adisorganized and irregular shape and looked damaged, and surface vesicles weredetected on the surface of some of the cells (Fig. 2B and D). Backscattering analysis ofnoncoated samples with an in-lens detector revealed that BSM-Ag-NP-treated cells (Fig.2D) but not BSM-treated cells (Fig. 2C) were covered with a material of high atomicdensity (yellow spots in Fig. 2D), supporting the association of Ag NPs with the bacterialsurface. Transmission electron microscopy (TEM) supported the aforementioned alter-ations to the bacterial cell wall caused by BSM-Ag NPs (Fig. 2F), compared with cellsexposed to BSM alone (Fig. 2E). Moreover, Ag NPs were also detected in TEM obser-vations (Fig. 2F).

BSM-Ag NPs prevent seed-to-seedling transmission of A. citrulli. We askedwhether BSM-Ag NPs have the potential to reduce seed-to-seedling transmission of A.citrulli in melons. To answer this question, we carried out seed transmission assaysfollowing two forms of application of the compound, namely, (i) treatment withBSM-Ag NPs of seeds that had been inoculated previously with A. citrulli M6 (“treat-ment”) or (ii) pretreatment of seeds with BSM-Ag NPs followed by bacterial inoculation(“pretreatment”). BSM-Ag NPs were tested using three concentrations of Ag (5, 10, and22 mg liter�1). As controls, seeds were noninoculated, inoculated only, or inoculatedand treated or pretreated with BSM alone (without Ag NPs). Additional controlsincluded treatment and pretreatment with a known bacterial disinfectant, sodiumhypochlorite (NaClO), at a concentration of 0.6%.

Three independent experiments with similar results revealed that BSM-Ag NPs,given either as seed treatment or as pretreatment, protected the emerging seedlings atall tested concentrations of Ag (Fig. 3 and 4; also see Fig. S2 and S3 in the supplementalmaterial). No significant differences in disease severity were observed among seedtreatments or pretreatments with BSM-Ag NPs at Ag concentrations of 10 and 22 mgliter�1, and the results of these treatments did not differ significantly from noninocu-lated (healthy) controls or from treatment with 0.6% NaClO (Fig. 3). Treatment andpretreatment of seeds with BSM-Ag NPs at an Ag concentration of 5 mg liter�1 wereslightly but significantly (P � 0.05) less effective than treatment and pretreatment withthe highest concentrations of Ag. However, these treatments significantly (P � 0.05)reduced disease severity, compared with inoculated controls (Fig. 3).

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In melon-A. citrulli seed transmission assays, disease severity negatively correlateswith plant growth parameters such as seedling weight (24). In agreement with thedisease severity scores, all seedlings emerging from BSM-Ag NP-pretreated or -treatedseeds had significantly (P � 0.05) higher foliage weights, in comparison with inocu-lated, nontreated/nonpretreated controls (Fig. S2). In agreement with in vitro experi-ments, seed pretreatment and treatment with BSM alone (without Ag NPs) did notprotect the seedlings (Fig. 3; also see Fig. S2 and S3). In contrast to the effectiveprotection exerted by the different pretreatments with BSM-Ag NPs, pretreatment with0.6% NaClO did not protect the seedlings at all (Fig. 3; also see Fig. S2 and S3).Importantly, seedlings emerging from BSM-Ag-NP-treated/pretreated seeds did notshow visible phytotoxicity symptoms.

FIG 2 Electron microscopic images of A. citrulli M6 cells exposed to BSM (A, C, and E) or BSM-Ag NPs (B, D, and F). Cells were grownon NA at 28°C for 48 h. Then, 5-�l drops of BSM or BSM-Ag NPs (with 10 mg Ag liter�1) were placed on the top of the colonies for2 min, followed by preparation for electron microscopic observations. (A and B) SEM with an Ultra 55 FEG scanning electronmicroscope (Zeiss). (C and D) SEM with an Ultra 55 FEG scanning electron microscope with a backscatter electron detector. (E and F)TEM with an FEI T12 electron microscope. Bars, 400 nm (A and B), 1 �m (C and D), 200 nm (E), and 100 nm (F).

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Young melon seedlings are highly sensitive to A. citrulli, with seed inoculation atrelatively high bacterial concentrations generally leading to seedling blight and death(24, 25). In seed transmission experiments, we recorded the number of dead seedlingsfor each treatment every day (Fig. 4). While most (�90%) of the control (inoculated,nontreated/nonpretreated) seedlings died 8 days after sowing, no seedlings emergingfrom seeds treated or pretreated with BSM-Ag NPs died in these experiments, exceptfor a relative low percentage (�20%) of seedlings emerging from seeds treated withthe lowest Ag concentration (5 mg liter�1) (Fig. 4). Similar to inoculated controls, mostseedlings emerging from seeds treated or pretreated with BSM without Ag or fromseeds pretreated with NaClO died in these experiments, although the progress ofseedling death was slightly delayed, relative to untreated plants (Fig. 4). Representativepictures of selected treatments of seed transmission assays are shown in Fig. S3 in thesupplemental material.

Verification of antibacterial activity of BSM-Ag NPs in seeds. To further assessthe antibacterial activity of BSM-Ag NP treatment or pretreatment of seeds, A. citrulliM6-inoculated and treated or pretreated melon seeds were put to germinate in NAplates at room temperature. Representative pictures for some of the treatments areshown in Fig. 5. As expected, no A. citrulli colonies developed around noninoculatedseeds (Fig. 5A). In agreement with results from seed transmission assays, we observedconspicuous A. citrulli M6 colony growth around germinating seeds that either wereinoculated and not treated (Fig. 5B) or were treated (Fig. 5C) or pretreated (data notshown) with BSM alone. In contrast, inoculated seeds treated or pretreated withBSM-Ag NPs at 10 or 22 mg Ag liter�1 demonstrated no colonies around germinatingseeds (shown in Fig. 5D and E for 22 mg Ag liter�1). Similarly, no A. citrulli colonies wereseen around NaClO-surface-treated seeds (Fig. 5F); however, in agreement with resultsfrom seed transmission experiments, pretreatment with NaClO did not prevent bacte-rial growth around the seeds (Fig. 5G).

DISCUSSION

In the past decade, silver nanoparticles have stimulated great interest in manyindustrial fields, including medicine, veterinary medicine, and agriculture, due to their

FIG 3 Effects of seed treatment and pretreatment with BSM-Ag NPs on disease severity in melon (cultivarOphir) seed transmission experiments with A. citrulli M6. Seed inoculation, treatments (TRE), andpretreatments (PRE) are described in Materials and Methods. Plants were grown in a greenhouse at 28 �2°C, and disease severity scores were determined from 13 to 15 replicates per treatment, 10 days aftersowing. Different letters indicate significant differences (P � 0.05) among treatments, by the Tukey-Kramer HSD test and analysis of variance (ANOVA). Controls (CON) included inoculated seeds (inoc.),noninoculated seeds (non-inoc.), and treatments/pretreatments with BSM (without Ag NPs) or 0.6%NaClO. Data (means and standard errors) are from one representative experiment (of three with similarresults).

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antibacterial action and to the assumption that Ag NPs are less toxic to eukaryoticorganisms than are Ag cations (26). However, only a very few studies have beenperformed to characterize the potential of metal NPs in general, and of Ag NPs inparticular, in true in planta systems. There are several limitations to the utilization of AgNPs against pathogens, including the tendency of Ag sols to coagulate and the actionof electrolytes and their ability to attach to the target protection niche (27). Therefore,several organic compounds, either synthetic or from natural sources, have been testedto stabilize nanoparticle dispersions (27, 28). Here we used mucin for this purpose.

Mucins are widespread in nature and constitute major glycoprotein componentsof the mucus present on the surface of cells of various tissues of almost all animals.They protect epithelial cells from infection, dehydration, and physical and chemicalinjury (29). Some of us reported a simple fast method to produce stable chiral AgNPs using BSM (20). The reducing properties of mucin were exploited to producestable Ag NPs without the need to use additional reducing agents. Moreover, thetypical dendritic structure of mucin promotes stabilization, water solubility, anddispersion of the Ag NPs (20).

Here we show that BSM-Ag NPs possess strong antibacterial activity against severalplant-pathogenic bacteria, including representatives of both Gram-negative and Gram-

FIG 4 Effects of seed treatment and pretreatment of BSM-Ag NPs on seedling survival in melon (cultivarOphir) seed transmission experiments with A. citrulli M6. Seed inoculation, treatments, and pretreatmentsare described in Materials and Methods. Plants were grown in a greenhouse at 28 � 2°C, and thepercentage of surviving seedlings was recorded every day. Data from one representative experiment (ofthree with similar results) are shown. Controls (CON) included inoculated seeds (inoc.), noninoculatedseeds (non-inoc.), and treatments/pretreatments with BSM alone or 0.6% NaClO. To distinguish amongthe curves, pretreatments (PRE) and treatments (TRE) were split into two plots (A and B, respectively).Inoculated and noninoculated controls are included in both plots. The following treatments did not leadto seedling death, and their curves overlap at 100% survival: noninoculated control and pretreatmentswith BSM-Ag NPs containing 5, 10, and 22 mg Ag liter�1 (A) and noninoculated control, treatments withBSM-Ag NPs containing 10 and 22 mg liter�1 Ag, and treatment with 0.6% NaClO (B).

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positive species. Further, we strengthened the proof of concept for potential applica-tion of this compound for crop protection by in planta experiments involving themelon-Acidovorax citrulli pathosystem. A. citrulli causes BFB, one of the most threaten-ing diseases of cucurbits (mainly melons and watermelons) worldwide (21, 30). A. citrulliis seedborne and seed transmitted, and fast global dissemination of BFB has occurreddue to the commercialization of contaminated cucurbit seeds (30). Moreover, to date,there are no BFB resistance sources in the cucurbit germplasm, and the methodsavailable to eradicate the bacterium from seeds and to control the disease in the fieldare quite limited (21). While most significant losses caused by A. citrulli are due to fruitinfection, A. citrulli-infected seeds and plantlets are the main source of BFB dissemina-tion, and young seedlings are highly sensitive to the bacterium. Therefore, seedcontamination and seed-to-seedling transmission are serious concerns for seed andnursery companies (21).

In the particular case of BFB, several seed treatments, including fermentation ofseeds with watermelon juice and treatment with several chemicals, including sodiumhypochlorite, calcium hypochlorite, hydrogen chloride, and peroxyacetic acid, havebeen proposed (31, 32). While these treatments are able to reduce the bacterialinoculum, none eradicates the pathogen and prevents seed transmission. Seed trans-mission experiments carried out in this study showed that treatment or pretreatmentof A. citrulli-infested seeds with BSM-Ag NPs prevented or significantly reduced diseasein emerging seedlings, depending on the concentrations of Ag NPs, with prevention at10 or 22 mg liter�1 and significant reduction at 5 mg liter�1. Importantly, even thehighest tested concentration was relatively low, taking into account the appliedconcentrations of active compounds in agricultural pesticides, which are commonly

FIG 5 Assessment of effects of BSM-Ag NPs on melon seed infestation with A. citrulli M6. Seeds were inoculatedand treated or pretreated as described for seed transmission assays. The seeds were then placed on NA plateswrapped in aluminum foil. Germination and infestation were observed in two replicates (plates) per treatment(with each plate containing 4 seeds). The pictures are representative of three experiments with similar results. (A)Noninoculated and untreated (control). (B) Inoculated and untreated (control). (C) Inoculated and treated with BSMalone. (D) Inoculated and treated with BSM-Ag NPs (at 22 mg Ag liter�1). (E) Pretreated with BSM-Ag NPs (22 mgAg liter�1) and inoculated. (F) Inoculated and treated with 0.6% NaClO. (G) Pretreated with 0.6% NaClO andinoculated. Arrows indicate development of bacterial colonies.

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used on the order of hundreds of milligrams per liter, leading to pesticide loads ofseveral kilograms per hectare (33).

In contrast to the high efficiency of BSM-Ag NPs given as pretreatment, pretreat-ment of seeds with sodium hypochlorite did not prevent or reduce disease severity forseedlings emerging from inoculated seeds. This result suggests that, while sodiumhypochlorite was washed away from the seed surface during the inoculation procedureand/or after sowing in the soil, significant portions of the BSM-Ag NP complex re-mained attached to the seed surface, thus providing long-term protection to theemerging seedlings. This could be explained by the adhesive and surfactant charac-teristics of mucin (18, 29). These characteristics are of particular importance for cropprotection applications, since the applied antimicrobial compounds might be washedaway from the plant organ by rain or irrigation in such cases.

As mentioned above, silver-based products are effective in preventing and/orreducing bacterial infections, as they exert a broad range of antimicrobial activities,including damage to the cell wall, to the cytoplasmic membrane, and to proteins andnucleic acids (8–10). Scanning and transmission electron microscopic observationscarried out in this study revealed substantial damage to the bacterial cells produced byBSM-Ag NPs. Ag NPs could be detected in BSM-Ag-NP-treated cells, despite the factthat cells were thoroughly washed before electron microscopy, suggesting that at leastsome of the NPs penetrated the bacterial cells. The ability of Ag NPs to penetratebacterial cells, leading to damage of intracellular functions, was inferred in several otherstudies with Ag NPs of small sizes similar to those used in our study (in the range of 5to 20 nm) (8–10, 34).

In our study, we did not observe visible phytotoxic effects on melon seedlingsemerging from BSM-Ag-NP-treated or pretreated seeds. Today, there are hundreds ofpesticides that contain silver, due to its antimicrobial properties (12). However, phyto-toxicity and toxicity of nanosilver and nanometals in general to ecosystems andhumans are major concerns (12, 35). We hypothesize that, due to the highly efficientantibacterial activity of BSM-Ag NPs at very low concentrations of Ag, the applicationof this technology for seed treatment would not be dangerous to the environment orhuman health. Moreover, any means leading to successful prevention of diseasetransmission from infected seeds is expected to contribute substantially to reducingmuch greater application of chemical pesticides in the field. However, more research isneeded to investigate possible adverse effects of BSM Ag-NPs on nontarget organisms.In addition, further studies are needed to understand the antibacterial mode of actionof this compound in the seeds, including how it affects bacterial survival, in a moredetailed and quantitative manner.

In conclusion, the unique BSM-Ag NP complex possesses strong bactericidal prop-erties at very low concentrations of Ag, and this was demonstrated in in plantaexperiments. Most plant-pathogenic bacteria are seed transmitted and, in many cases,this form of dissemination is the main source of primary inocula in the field. This studydemonstrates that BSM-Ag NPs, which can be easily generated and possess high levelsof stability, can contribute substantially to the management of BFB and other bacterialplant diseases. In addition, this new compound has the potential to provide betterprotection and preservation of the environment by reducing chemical loads andwashouts from crops into the soil and underground water. Lastly, while we focused onplant-pathogenic bacteria in the present study, application of this technology is notlimited to the agricultural field, since BSM-Ag NPs could be potentially exploited as apotent antimicrobial agent in other industrial areas, such as medicine, veterinarymedicine, cosmetics, textiles, and household products.

MATERIALS AND METHODSGeneration and characterization of BSM-Ag NPs. Bovine submaxillary mucin-silver nanoparticles

(BSM-Ag NPs) were synthesized as described previously (20). Briefly, the complexes were generated bystirring an aqueous solution containing 1 mg ml�1 AgNO3 and 10 mg ml�1 BSM type I (Sigma-Aldrich)for 24 h at room temperature. The resulting complex was purified by size exclusion chromatography(Sephadex G-25) and then treated with 50 mM sodium borate buffer (pH 10.0). The reaction mixture was

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stirred until the solution acquired a brownish color. Finally, the mixture was filtered through a 0.45-�mfilter. The Ag concentration of the synthesized complex was determined using end-on-plasma inductivelycoupled plasma atomic emission spectroscopy (ICP-AES) (Arcos system; Spectro GmbH), as describedpreviously (20).

Bacterial strains and growth conditions. The bacterial strains used in this study were Acidovoraxcitrulli M6 (36), Xanthomonas euvesicatoria 85-10 (37), and Clavibacter michiganensis subsp. michiganensisNCPPB 382 (38). All strains were grown in nutrient agar (NA) (Difco Laboratories) plates at 28°C for 48 hunless stated otherwise. Colonies were resuspended in nutrient broth (NB) (Difco Laboratories) or 100mM phosphate buffer (pH 7.0), depending on the assay. The optical density at 600 nm (OD600) wasadjusted to 0.2 (�108 CFU ml�1) using a Helios Gamma spectrophotometer (Thermo Fisher Scientific),and then the suspensions were diluted to the desired concentrations, which were verified by plating ofserially diluted samples on NA.

Growth inhibition assays. Bacterial cell suspensions containing 107 CFU ml�1 were prepared asdescribed above, in NB containing BSM alone or BSM-Ag NPs at different concentrations of Ag, rangingfrom 0.13 to 33.5 mg liter�1. The suspensions were dispensed in 500-�l triplicates in Nunc 48-wellpolystyrene plates (Thermo Scientific), which were incubated in an Infinite F200 plate reader (Tecan) at28°C for 168 h. Bacterial growth was measured every hour, as an increase in the OD600. Negative controlswere NB containing BSM and BSM-Ag NPs at different concentrations but without bacterial inocula.These controls were included as blanks, for subtraction of the values from those for the correspondingtreatments with bacteria and for verification of the lack of contamination. In some experiments, sampleswere taken at several times for assessment of CFU by dilution plating.

Assessment of bactericidal activity. Cell suspensions containing 107 CFU ml�1 were prepared asdescribed above, in 100 mM phosphate buffer (pH 7.0) containing BSM-Ag NPs at various concentrations(from 0 to 2.2 mg Ag liter�1). The suspensions were maintained at 25°C for 24 h, after which bacterialconcentrations were determined by plating of serial dilutions on NA plates.

Scanning electron microscopy. Acidovorax citrulli M6 was grown for 48 h on NA plates as describedabove, and cell suspensions of 107 CFU ml�1 were prepared in NB. Loopfuls of these suspensions werecollected with a platinum loop and placed on 5-mm2 slices of Whatman glass microfiber filters (gradeGF/F; Sigma-Aldrich) that had been coated previously with a thin layer (�0.5 mm) of an autoclavedsolution containing 3% NA and 0.1% gelatin. The inoculated slices were placed on NA plates, which wereincubated at 28°C for 24 h. Then, 5 �l of a solution containing BSM or BSM-Ag NPs with 10 mg Ag liter�1

was added to the top of the slices (no solution was added for controls), and the plates were incubatedfor additional 24 h at 28°C. The slices were then fixed for 1 h in 4% glutaraldehyde in 100 mM phosphatebuffer (pH 7.0), washed three times with the same buffer, and dehydrated using increasing ethanolconcentrations of 25%, 50%, 75%, 95%, and 100% (10 min each). The blocks were then dried in a CPD030 critical point dryer (Bal-Tec), displacing the alcohol with liquid CO2, and finally were dried byreleasing CO2. The dried blocks were mounted on brass blocks, coated with carbon (Edvards), andvisualized with a high-resolution Ultra 55 field-emission gun (FEG) scanning electron microscope (Zeiss).Backscattering analysis was performed with noncoated samples, with an in-lens detector.

Transmission electron microscopy. Bacterial suspensions (108 CFU ml�1) of A. citrulli M6 in NB withBSM or BSM Ag NPs at 10 mg Ag liter�1 were shaken at 28°C for 24 h. The cells were then collected bycentrifugation (5,000 � g for 10 min at 4°C), washed with 100 mM phosphate buffer (pH 7.0), and fixedfor 2 h at room temperature with 0.2 M cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and2% paraformaldehyde, after which the samples were kept at 4°C for 16 h. The bacteria were then rinsedfour times with 0.2 M cacodylate buffer (10 min each time) and postfixed and stained for 1 h with asolution containing 1% osmium tetroxide and 1.5% potassium ferricyanide in 0.1 M cacodylate buffer.Bacteria were then washed four times in 0.2 M cacodylate buffer, followed by dehydration withincreasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 95%, 10 min each) and then 100%anhydrous ethanol (three times, 20 min each) and propylene oxide (two times, 10 min each). The sampleswere then infiltrated with increasing concentrations of agar 100 resin (Agar Scientific) in propylene oxide(25%, 50%, 75%, and 100% resin, for 16 h at each step). Bacteria were then embedded in fresh resin andallowed to polymerize for 48 h at 60°C in an oven. Embedded bacteria in blocks were sectioned with adiamond knife on an LKB 3 microtome, and ultrathin sections (80 nm) were collected onto 200-mesh,thin-bar copper grids. The sections on grids were sequentially stained with uranyl acetate and lead citrate(3 min each) and viewed with a FEI T12 transmission electron microscope (Phillips) equipped with anOlympus SIS MegaView III-mounted charge-coupled device (CCD) camera.

Seed transmission assays. The effect of BSM-Ag NPs in the protection of emerging seedlings againstA. citrulli was assessed by seed transmission assays (24), with a few modifications. The effects wereassessed both with treatment of preinoculated seeds with BSM-Ag NPs (treatment) and with pretreat-ment with BSM-Ag NPs followed by bacterial inoculation (pretreatment). For treatments, melon seeds(cultivar Ophir; Zeraim Gedera) were placed in suspensions of A. citrulli M6 (107 CFU ml�1) in 100 mMphosphate buffer (pH 7.0) and incubated at room temperature for 2 h, with gentle agitation. Seeds werethen passed through a strainer and dried under a laminar flow hood for 2 h. Subsequently, seeds wereplaced in solutions containing BSM-Ag NPs at three concentrations of Ag, 5, 10, and 22 mg liter�1. Ascontrols, inoculated seeds were incubated in a solution containing BSM alone. Seeds and BSM/BSM-AgNP solutions were placed on a shaker, with gentle agitation, for 2 h at room temperature. As an additionalcontrol for seed disinfection, seeds were treated for 50 s with 0.6% NaClO (Biolab Ltd.) and then washedtwice with sterile distilled water. Treated seeds were passed through a strainer and dried under a laminarflow hood for 2 h. For pretreatments, seeds were first placed in solutions containing BSM, BSM-Ag NPs(at the same concentrations as described above), or 0.6% NaClO, under conditions similar to those

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described for treatments. The seeds were then collected and allowed to dry in a laminar flow hood for2 h before being inoculated with bacteria (107 CFU ml�1) as described above. Nontreated/nonpretreatedand noninoculated controls were included. Seeds were sown in small poly pots (11-cm diameter)containing brown medium peat (TS4 medium; Klasmann). Emerging seedlings were grown in a green-house (28 � 2°C) for 10 days, and then disease severity was scored on a scale of 0 to 5, as follows: 0,healthy plants; 1, slight necrosis in one or two cotyledons; 2, wide necrosis in both cotyledons; 3, necrosisand deformation in cotyledons; 4, necrosis and deformation in cotyledons and significant damage to theseedling; 5, dead seedling. The number of dead seedlings was recorded every day, and the foliage weightof the seedlings was determined at the end of the experiment. The experiment was carried out threetimes with 13 to 15 replicates per treatment, and the data were analyzed by the Tukey-Kramer honestlysignificant difference (HSD) test, using JMP software (SAS Institute Inc.). In two additional experiments,melon seeds treated as described above were put to germinate on NA plates. The plates were wrappedin aluminum foil and incubated at 25°C for 5 days, after which the presence or absence of A. citrullicolonies around the germinating seeds was determined. The identity of the colonies was verified bycolony PCR using the A. citrulli-specific primer set BX-S, as described previously (39).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02212-17.

SUPPLEMENTAL FILE 1, PDF file, 0.7 MB.

ACKNOWLEDGMENTSThis work was supported by a start-up grant from Yissum, the Hebrew University of

Jerusalem. TEM observations were conducted at the Bio-Imaging Unit, The AlexanderSilberman Institute of Life Science of the Hebrew University of Jerusalem. SEM obser-vations were conducted at the Irving and Cherna Moskowitz Center for Nano andBio-Nano Imaging at the Weizmann Institute of Science.

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