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Silver nanoparticles in complex with bovine submaxillary 1
mucin possess strong antibacterial activity and protect against 2
seedling infection 3
Running title: Antimicrobial activity of mucin-silver nanoparticles 4
5
Daria Makarovsky,a* Ludmila Fadeev,b Bolaji Babajide Salam,a* Einat Zelinger,c Ofra 6
Matan,a Jacob Inbar,d Edouard Jurkevitch,a Michael Gozin,b Saul Burdmana# 7
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Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of 9
Agriculture, Food and Environment, The Hebrew University of Jerusalem, 10
Rehovot, Israela; School of Chemistry, Faculty of Exact Sciences, Tel Aviv 11
University, Tel Aviv, Israelb; Interdepartmental Core Facility, The Robert H. Smith 12
Faculty of Agriculture, Food and Environment, The Hebrew University of 13
Jerusalem, Rehovot, Israelc; Department of Economics and Business 14
Management, Faculty of Social Sciences and Humanities, Ariel University, Ariel, 15
Israeld 16
17
*Daria Makarovsky is currently at the Goldschleger Eye Institute, Sheba Medical 18
Center, Tel Hashomer, Israel. Bolaji Babajide Salam is currently at the 19
Department of Postharvest Science of Fresh Produce, The Volcani Center, 20
Agricultural Research Organization, Bet Dagan, Israel. 21
22
Address correspondence to Saul Burdman, [email protected]. 23
AEM Accepted Manuscript Posted Online 27 November 2017Appl. Environ. Microbiol. doi:10.1128/AEM.02212-17Copyright © 2017 American Society for Microbiology. All Rights Reserved.
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ABSTRACT A simple method for synthesis of nanoparticles (NPs) of silver (Ag) in a 24
matrix of bovine submaxillary mucin (BSM) was previously reported by some of the 25
authors of this study. Based on mucin characteristics such as long-lasting stability, 26
water solubility, and surfactant and adhesiveness characteristics, we hypothesized 27
that this compound, named BSM-Ag NPs, may possess favorable properties as a 28
potent antimicrobial agent. The goal of this study was to assess whether BSM-Ag NPs 29
possess antibacterial activity focusing on important plant pathogenic bacterial 30
strains representing both Gram-negative (Acidovorax and Xanthomonas) and Gram- 31
positive (Clavibacter) genera. Growth inhibition and bactericidal assays as well as 32
electron microscopy observations demonstrate that BSM-Ag NPs, at relatively low 33
concentrations of silver, exert strong antimicrobial effects. Moreover, we show that 34
treatment of melon seeds with BSM-Ag NPs effectively prevents seed-to-seedling 35
transmission of Acidovorax citrulli, one of the most threatening pathogens of 36
cucurbit production worldwide. Overall, our findings demonstrate strong 37
antimicrobial activity of BSM-Ag NPs and their potential application for reducing 38
spread and establishment of devastating bacterial plant diseases in agriculture. 39
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IMPORTANCE Bacterial plant diseases challenge agricultural production and the 40
means available to manage them are limited. Importantly, many plant pathogenic 41
bacteria have the ability to colonize seeds, and seed-to-seedling transmission is a 42
critical route by which bacterial plant diseases spread to new regions and countries. 43
The significance of our study resides on the following aspects: i) the simplicity of the 44
method of BSM-Ag nanoparticles’ (NPs) synthesis; ii) the advantageous chemical 45
properties of the BSM-Ag NPs; iii) their strong antibacterial activity at relatively low 46
concentrations of silver; and iv) the fact that, in contrast to most studies on effects 47
of metal NPs on plant pathogens, the proof-of-concept of the novel compound is 48
supported by in planta assays. Application of this technology is not limited to 49
agriculture. BSM-Ag NPs could be potentially exploited as a potent antimicrobial 50
agent in a wide range of industrial areas including medicine, veterinary, cosmetics, 51
textile and household. 52
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KEYWORDS silver, metal nanoparticles, mucin, bacterial plant diseases, Acidovorax 54
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INTRODUCTION 55
Plant pathogenic microorganisms are a major cause of crop yield losses and 56
agricultural intensification has been possible through the use of chemical pesticides 57
to cope with them. However, despite the intensive use of antimicrobial compounds, 58
estimations of world crop yield losses due to plant disease range from 15 to 20% (1- 59
3). Plant pathogenic bacteria are among the biotic agents causing significant losses in 60
crop production. Almost every important crop suffers from at least one or several 61
important bacterial diseases, which are often among the most significant diseases of 62
the given crop (3). Moreover, the strategies available to manage bacterial plant 63
diseases are generally limited, including the lack of efficient chemical bactericides for 64
disease control (4). 65
Importantly, many plant pathogenic bacteria are seedborne, namely, they 66
can survive in the seed and be transmitted via contaminated seeds to new fields, 67
regions and countries (4, 5). In a globalized world, in which a huge amount of plant 68
material (mainly seeds) is transferred from one country to the other, inadvertent 69
distribution of contaminated commercial seeds is one of the main ways by which 70
bacterial plant diseases are spread worldwide (4). Therefore, new strategies to 71
manage bacterial plant diseases are highly demanded in general, and in particular, to 72
prevent or reduce seed transmission of bacterial pathogens. 73
The aim of this study was to assess the antimicrobial activity of silver (Ag) 74
nanoparticles (NPs) in complex with bovine submaxillary mucin (BSM), focusing on 75
plant pathogenic bacteria. Silver has been extensively used to control microbial 76
infections since ancient times (6, 7). Silver-based medical products have been shown 77
to be effective in reducing and preventing bacterial infections (8). Silver ions are 78
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highly reactive exerting a broad range of antimicrobial activities. They are able to 79
bind to and damage proteins and DNA leading to disruption of disulfide bonds in 80
proteins, structural changes in the cytoplasmic membrane and in the cell wall, 81
altered membrane permeability, cell distortion and inhibition of replication and 82
respiratory activity, leading eventually to cell death (9, 10). In recent years, the 83
development of nanotechnologies has brought about a growing interest in the 84
industrial and medical fields in generation of bioactive biomaterials that combine the 85
relevant antibacterial properties of metals with the peculiar performance of the 86
biomaterial. A variety of nanosilver compounds have been developed and tested for 87
their antimicrobial properties (9, 11). Some nanosilver compounds were also 88
generated and evaluated for their potential application in agriculture. However, this 89
has been rather limited and very few studies were conducted to assess antibacterial 90
or antifungal activities of the compounds in planta (12-16). 91
Mucins are large, extracellular glycoproteins found as the main components 92
of mucus in almost all animals (17, 18). With molecular weights ranging from 0.5 to 93
20 MDa, mucins are highly glycosylated consisting of approximately 80% 94
carbohydrates. Among their chemical properties, mucins may act as moisture 95
holders, adhesins and solubilizers as well as reducing and surfactant agents (18). 96
Some of us have shown that BSM, a representative natural mucin, is capable of 97
binding and solubilizing various types of polycyclic aromatic hydrocarbons in 98
aqueous solutions, leading to an increase of their antimicrobial activity (19). Further, 99
a simple method was developed to generate Ag NPs inside a BSM matrix (20). 100
Synthesis of the BSM-Ag NP compound was carried out in an aqueous solution 101
without the need of an external reducing agent, exploiting the natural solubilizing, 102
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reducing and stabilizing properties of mucin (20). The generated complex, named 103
BSM-Ag NPs, is highly soluble in water and biodegradable, thus having the potential 104
to be active at very low concentrations, constituting a potential for powerful and 105
environment friendly tool for crop protection. 106
In the present study, growth inhibition and bactericidal assays as well as 107
electron microscopy observations demonstrate strong antibacterial effects of BSM- 108
Ag NPs. Moreover, seed transmission assays reveal that BSM-Ag NPs effectively 109
prevents seed-to-seedling transmission of Acidovorax citrulli, one of the most 110
threatening pathogens of cucurbit production worldwide (21). Overall, our findings 111
support the potential of BSM-Ag NPs as an efficient crop protection agent. 112
113
RESULTS 114
BSM-Ag NPs inhibit bacterial growth. BSM-Ag NPs tested in this report were 115
produced as described earlier (20; see Materials and Methods). The size of the Ag 116
NPs was found to be in the range of 5-20 nm, with an average diameter of about 10 117
nm (20). The concentrated complexes carried Ag NPs at concentrations ranging 118
between ~400 and 1000 mg l-1. We first assessed the antimicrobial activity of BSM- 119
Ag NPs by determining the effects of various Ag concentrations on growth of three 120
strains representing important seedborne plant pathogenic bacteria: the gram- 121
negative Acidovorax citrulli M6 [bacterial fruit blotch (BFB) of cucurbits (21)] and 122
Xanthomonas euvesicatoria 85-10 (bacterial spot disease of pepper and tomato 123
(22)], and the gram-positive Clavibacter michiganensis subsp. michiganensis NCPPB 124
382 (Cmm 382; causal agent of bacterial canker and wilt of tomato (23)]. 125
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BSM-Ag NPs exerted strong growth inhibition effects on all tested strains at 126
very low Ag concentrations. Representative growth curve experiments in nutrient 127
broth (NB) are shown in Fig. 1A for A. citrulli M6 and in Fig. S1 (see supplemental 128
material) for Cmm 382 and X. euvesicatoria 85-10. A delay in the exponential growth 129
phase of all strains was achieved at Ag concentrations as low as 0.13 mg l-1. Much 130
stronger inhibition effects were observed in the range of 0.67 to 2.68 mg Ag l-1. At 131
these concentrations bacterial growth was delayed by 24 to 40 h relative to controls 132
exposed to BSM alone. At Ag concentrations of 6.7 mg l-1 and higher, no growth 133
could be detected for A. citrulli M6 and Cmm 382 after 168 h (one week) of 134
incubation (Fig. 1A and Fig. S1A). In the case of X. euvesicatoria 85-10, substantially 135
delayed growth occurred at 6.7 mg Ag l-1, but no growth occurred at 13.4 mg Ag l-1 136
(Fig. S1B). In these experiments, we were not able to isolate bacteria following 137
dilution plating of samples in which no growth was observed, indicating that under 138
tested conditions, BSM-Ag NPs at relatively low Ag concentrations of 6.7 and 13.4 139
mg l-1 have bactericidal effects on A. citrulli/Cmm and X. euvesicatoria, respectively. 140
Confirmation of bactericidal activity of BSM-Ag NPs. We further verified the 141
bactericidal activity of the BSM-Ag-NPs against A. citrulli. We selected this pathogen 142
for further experiments because BFB disease caused by this bacterium is considered 143
as one of the most serious threats to the cucurbit industry worldwide, mainly to 144
melon and watermelon (21). Moreover, seed transmission has been responsible for 145
the extraordinarily fast global spread of BFB, thus making of this disease a great 146
concern to the seed production sector (21). 147
Bacterial suspensions of A. citrulli M6 (107 CFU ml-1) in 100 mM phosphate 148
buffer (pH 7.0) were exposed to different concentrations of BSM-Ag NPs for 24 h at 149
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25C. Then the suspensions were serially diluted and dilution samples were plated 150
onto nutrient agar (NA) plates to determine live bacterial concentrations at the end 151
of the experiment. Results of these experiments confirmed the strong bactericidal 152
activity of BSM-Ag NPs. Under these conditions, exposure of A. citrulli cells to BSM- 153
Ag NPs at 0.4 mg Ag l-1 strongly reduced bacterial numbers by two orders of 154
magnitude, and no bacteria could be retrieved after exposure to BSM-Ag NPs at 2.2 155
mg Ag l-1 (Fig. 1B). 156
BSM-Ag NPs damage bacterial cells. Scanning electron microscopy (SEM) 157
was used to observe morphological effects of BSM-Ag NPs on A. citrulli M6 cells. 158
While cells treated with BSM alone (without Ag NPs) possessed a typical rod-like 159
shape and a well-defined cell wall (Figs. 2A and C), clear morphological alterations 160
were observed in cells exposed to BSM-Ag NPs containing 10 mg Ag l-1: the latter 161
cells had a disorganized and irregular shape, looked damaged and surface vesicles 162
were detected on the surface of some of these cells (Fig. 2B and D). Backscattering 163
analysis of non-coated samples with in-lens detector revealed that BSM-Ag NP- 164
treated cells (Fig. 2D) but not BSM-treated cells (Fig. 2C) were covered with a high 165
atomic dense material (yellow spots in Fig. 2D), supporting association of Ag NPs 166
with the bacterial surface. Transmission electron microscopy (TEM) supported the 167
aforementioned alterations caused to the bacterial cell wall by BSM-Ag NPs (Fig. 2F) 168
as compared with cells exposed to BSM alone (Fig. 2E). Moreover, Ag NPs were also 169
detected in TEM observations (Figs. 2F and G). 170
BSM-Ag NPs prevent seed-to-seedling transmission of A. citrulli. We asked 171
whether BSM-Ag NPs have the potential to reduce seed-to-seedling transmission of 172
A. citrulli in melon. To answer this question, we carried out seed transmission assays 173
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following two forms of applications of the compound: 1) treatment with BSM-Ag NPs 174
of seeds that were previously inoculated with A. citrulli M6 (“treatment”), or 2) 175
pretreatment of the seeds with BSM-Ag NPs followed by bacterial inoculation 176
(“pretreatment”). BSM-Ag NPs were tested using three concentrations of Ag: 5, 10 177
and 22 mg l-1. As controls, seeds were non-inoculated, inoculated only, or inoculated 178
and treated/pretreated with BSM alone (without Ag NPs). Additional controls 179
included treatment and pretreatment with a known bacterial disinfectant, sodium 180
hypochlorite (NaClO), at a concentration of 0.6%. 181
Three independent experiments with similar results revealed that BSM-Ag 182
NPs, given either as seed treatment or as pretreatment, protected the emerging 183
seedlings in all tested concentrations of Ag (Figs. 3, 4, and S2 and S3 in supplemental 184
material). No significant differences in disease severity were observed among seed 185
treatments or pretreatments with BSM-Ag NPs at Ag concentrations of 10 and 22 mg 186
l-1 and these treatments did not significantly differ from non-inoculated (healthy) 187
controls and from treatment with 0.6% NaClO (Fig. 3). Treatment and pretreatment 188
of seeds with BSM-Ag NPs at an Ag concentration of 5 mg l-1 were slightly but 189
significantly (p<0.05) less effective than treatment/pretreatment with the highest 190
concentrations of Ag. However, these treatments significantly (p<0.05) reduced 191
disease severity as compared with inoculated controls (Fig. 3). 192
In melon-A. citrulli seed transmission assays, disease severity negatively 193
correlates with plant growth parameters like seedling weight (24). In agreement with 194
the disease severity scores, all seedlings emerging from BSM-Ag NP-pretreated or 195
treated seeds had significantly (p<0.05) higher foliage weights in comparison with 196
inoculated, non-treated/non-pretreated controls (Fig. S2). In agreement with in vitro 197
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experiments, seed pretreatment and treatment with BSM alone (without Ag NPs), 198
did not protect the seedlings (Figs. 3, S2 and S3). In contrast to the effective 199
protection exerted by the different pretreatments with BSM-Ag NPs, pretreatment 200
with 0.6% NaClO did not protect the seedlings at all (Figs. 3, S2 and S3). Importantly, 201
seedlings emerging from BSM-Ag NPs treated/pretreated seeds did not show visible 202
phytotoxicity symptoms. 203
Young seedlings of melon are highly sensitive to A. citrulli, with seed 204
inoculation at relatively high bacterial concentrations generally leading to seedling 205
blight and death (24, 25). In seed transmission experiments we recorded the number 206
of dead seedlings per treatment every day (Fig. 4). While most (above 90%) of the 207
control (inoculated, non-treated/pretreated) seedlings died at 8 days after sowing, 208
no seedling emerging from treated or pretreated seed with BSM-Ag NPs died in 209
these experiments, except for a relative low percentage (less than 20%) of seedlings 210
resulting from seeds treated with the lowest Ag concentration (5 mg l-1) (Fig. 4). As 211
similar as inoculated controls, most seedlings emerging from seeds treated or 212
pretreated with BSM without Ag and from seeds pretreated with NaClO, died in 213
these experiments, although the progress of seedling death was slightly delayed 214
relative to untreated plants (Fig. 4). Representative pictures of selected treatments 215
of seed transmission assays are shown in Fig. S3 (see supplemental material). 216
Verification of antibacterial activity of BSM-Ag NPs in seeds. To further 217
assess the antibacterial activity of BSM-Ag NP treatment or pretreatment on seeds, 218
A. citrulli M6-inoculated and treated or pretreated melon seeds were put to 219
germinate in NA plates at room temperature. Representative pictures from some of 220
the treatments are shown in Fig. 5. As expected, no A. citrulli colonies developed 221
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around non-inoculated seeds (Fig. 5A). In agreement with results from seed 222
transmission assays, we observed conspicuous A. citrulli M6 colony growth around 223
germinating seeds that were either inoculated and untreated (Fig. 5B), or treated 224
(Fig. 5C)/pretreated (not shown) with BSM alone. In contrast, inoculated, treated or 225
pretreated seeds with BSM-Ag NPs at 10 or 22 mg Ag l-1 were void of colonies around 226
germinating seeds (shown in Figs. 5D and 5E for 22 mg Ag l-1). Similarly, no A. citrulli 227
colonies were seen around NaClO-surface treated seeds (Fig. 5F); however, and in 228
agreement with results from seed transmission experiments, pretreatment with 229
NaClO did not prevent bacterial growth around the seeds (Fig. 5G). 230
231
DISCUSSION 232
In the last decade, silver nanoparticles (NPs) are gaining great interest in 233
many industrial fields including medicine, veterinary and agriculture due to their 234
antibacterial action, and to the assumption that Ag NPs are less toxic to eukaryotic 235
organisms than Ag cations (26). With that said, only very few studies have been done 236
to characterize the potential of metal NPs in general, and of Ag NPs in particular, in 237
true in-planta systems. There are several limitations for utilization of Ag NPs against 238
pathogens, including the tendency of Ag sols to coagulate, the action of electrolytes 239
and their ability to attach to the target protection niche (27). Several organic 240
compounds, either synthetic or from natural sources are therefore tested to stabilize 241
nanoparticle dispersions (27, 28). Here we used mucin for this purpose. 242
Mucins are widespread in nature and comprise major glycoprotein 243
components of the mucous present in the surface of cells of various tissues of almost 244
all animals. They protect epithelial cells from infection, dehydration, and physical as 245
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well as chemical injury (29). Some of us reported a simple and fast method to 246
produce stable, chiral Ag NPs using bovine submaxillary mucin (BSM) (20). The 247
reducing properties of mucin were exploited to produce stable Ag NPs without the 248
need of using additional reducing agents. Moreover, the typical dendritic structure 249
of mucin promotes stabilization, water solubility and dispersion of the Ag NPs (20). 250
Here we show that BSM-Ag NPs possess strong antibacterial activity against 251
several plant pathogenic bacteria, including representatives of both Gram-negative 252
and Gram-positive species. Further, we strengthened the proof-of-concept for 253
potential application of this compound for crop protection by in planta experiments 254
involving the melon-Acidovorax citrulli pathosystem. A. citrulli causes bacterial fruit 255
blotch (BFB) disease, one of the most threatening diseases of cucurbits worldwide, 256
and mainly of melon and watermelon (21, 30). A. citrulli is seedborne and seed 257
transmitted, and fast global dissemination of BFB has occurred due to 258
commercialization of contaminated cucurbit seeds (30). Moreover, to date, there are 259
no BFB resistance sources in the cucurbit germplasm and the methods available to 260
eradicate the bacterium from seeds and to control the disease in the field are highly 261
limited (21). While most significant losses caused by A. citrulli are due to fruit 262
infection, A. citrulli-infected seeds and plantlets are the main source of BFB 263
dissemination, and young seedlings are highly sensitive to the bacterium. Therefore, 264
seed contamination and seed-to-seedling transmission are a serious concern to seed 265
and nursery companies (21). 266
In the particular case of BFB, several seed treatments have been proposed 267
including fermentation of seeds with watermelon juice, or treatment with several 268
chemicals including sodium hypochlorite, calcium hypochlorite, hydrogen chloride or 269
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peroxyacetic acid (31, 32). While these treatments are able to reduce the bacterial 270
inoculum, none eradicates the pathogen and prevents seed transmission. Seed 271
transmission experiments carried out in this study showed that treatment or 272
pretreatment of A. citrulli-infested seeds with BSM-Ag NPs prevented or significantly 273
reduced disease of emerging seedlings, depending on the concentrations of Ag NPs: 274
prevention at 10 or 22 mg l-1 and significant reduction at 5 mg l-1. Importantly, even 275
the highest tested concentration is relatively low, taking into account the applied 276
concentrations of active compounds in agricultural pesticides, which are commonly 277
used in the order of hundreds of milligrams per liter leading to pesticide loads of 278
several kilograms per hectare (33). 279
In contrast to the high efficiency of BSM-Ag NPs given as pretreatment, 280
pretreatment of seeds with sodium hypochlorite did not prevent or reduce disease 281
severity of seedlings emerging from inoculated seeds. This result infers that while 282
sodium hypochlorite was washed away from the seed surface during the inoculation 283
procedure and/or after sowing in the soil, significant portions of the BSM-Ag NP 284
complex remained attached to the seed surface thus providing long-term protection 285
to the emerging seedlings. This could be explained by the adhesive and surfactant 286
characteristics of mucin (18, 29). These characteristics are of particular importance 287
for crop protection applications since in these cases, the applied antimicrobial 288
compounds might be washed away from the plant organ by rain or irrigation. 289
As mentioned above, silver-based products are effective in preventing and/or 290
reducing bacterial infections as they exert a broad range of antimicrobial activities, 291
including damage to the cell wall, to the cytoplasmic membrane, and to proteins and 292
nucleic acids (8-10). Scanning and transmission electron microscopy (SEM and TEM, 293
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respectively) observations carried out in this study revealed substantial damage 294
exerted by BSM-Ag NPs to the bacterial cells. Ag NPs could be detected in BSM-Ag 295
NP-treated cells, despite the fact that cells were thoroughly washed before electron 296
microscopy observations, suggesting that at least part of the NPs penetrated the 297
bacterial cells. The ability of Ag NPs to penetrate bacterial cells leading to damage of 298
intracellular functions was inferred in several other studies with Ag NPs of similar, 299
small sizes of those used in our study (in the range of 5-20 nm) (8-10, 34). 300
In our study we did not observe visible phytotoxicity effects on melon 301
seedlings emerging from BSM-Ag NP-treated or pretreated seeds. Today, there are 302
hundreds of pesticides that contain silver due to its antimicrobial properties (12). 303
However, phytotoxicity and toxicity of nanosilver and nanometals in general to 304
ecosystem and human is a major concern (12, 35). We hypothesize that, due to the 305
highly efficient antibacterial activity of BSM-Ag NPs at very low concentrations of Ag, 306
application of this technology for seed treatment, is not dangerous to the 307
environment and human health. Moreover, any mean leading to successful 308
prevention of disease transmission from infected seeds is expected to contribute 309
substantially to reduce much higher application of chemical pesticides in the field. 310
With that said, more research is needed to investigate possible adverse effects of 311
BSM Ag-NPs on non-target organisms. In addition, further studies are needed to 312
understand the antibacterial mode of action of this compound in the seed, including 313
how it affects bacterial survival in a more detailed and quantitative manner. 314
In conclusion, the unique BSM-Ag NP complex possesses strong bactericidal 315
properties at very low concentrations of Ag, and this was demonstrated in in planta 316
experiments. Most plant pathogenic bacteria are seed-transmitted and in many 317
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cases, this form of dissemination is the main source of primary inoculum in the field. 318
This study demonstrates that BSM-Ag NPs, which can be easily generated and 319
possess high stability, can substantially contribute for the management of BFB as 320
well as of other bacterial plant diseases. In addition, this new compound has the 321
potential to provide better protection and preservation of the environment by 322
reducing chemical loads and washouts from the crops into the soil and underground 323
water. Last, while in the present study we focused on plant pathogenic bacteria, 324
application of this technology is not limited to the agricultural field since BSM-Ag NPs 325
could be potentially exploited as potent antimicrobial agent in other industrial areas 326
such as medicine, veterinary, cosmetics, textile and household. 327
328
MATERIALS AND METHODS 329
Generation and characterization of BSM-Ag NPs. Bovine submaxillary mucin 330
(BSM)-silver (Ag) nanoparticles (BSM-Ag NPs) were synthesized as previously 331
described (20). Briefly, the complexes were generated by stirring an aqueous 332
solution containing 1 mg ml-1 AgNO3 and 10 mg ml-1 BSM type I (Sigma-Aldrich) at 333
room temperature for 24 h. The resulted complex was purified by size exclusion 334
chromatography (Sephadex G-25) and then treated with 50 mM sodium borate 335
buffer (pH 10.0). The reaction mixture was stirred until the solution acquired a 336
brownish color. Finally, the mixture was filtered through a 0.45 mm filter. The Ag 337
concentration of the synthesized complex was determined using an End-On-Plasma 338
ICP-AES model ‘ARCOS’ (Spectro GmbH) as described (20). 339
Bacterial strains and growth conditions. The bacterial strains used in this 340
study were Acidovorax citrulli M6 (36), Xanthomonas euvesicatoria 85-10 (37) and 341
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Clavibacter michiganensis subsp. michiganensis NCPPB 382 (38). All strains were 342
grown in nutrient agar (NA; Difco Laboratories) plates at 28°C for 48 h unless stated 343
otherwise. Colonies were resuspended in nutrient broth (NB; Difco Laboratories) or 344
100 mM phosphate buffer (pH 7.0), depending on the assay. The optical density at 345
600 nm (OD600nm) was adjusted to 0.2 [about 108 colony forming units (CFU) ml-1] 346
using a Helios Gama spectrophotometer (Thermo Fischer Scientific), and then the 347
suspensions were diluted to the desired concentrations that were verified by plating 348
of serially-diluted samples on NA. 349
Growth inhibition assays. Bacterial cell suspensions containing 107 CFU ml-1 350
were prepared as described above in NB containing BSM alone or BSM-Ag NPs at 351
different concentrations of Ag, ranging from 0.13 to 33.5 mg l-1. The suspensions 352
were dispensed in triplicates of 500 μl in Nunc 48-well polystyrene plates (Thermo 353
Scientific), which were incubated in an Infinite F200 plate reader (Tecan) at 28oC for 354
168 h. Bacterial growth was measured every hour as an increase in absorbance at 355
OD600nm. Negative controls were NB containing BSA and BSA-Ag NPs at different 356
concentrations but without bacterial inoculum. These controls were included as 357
blanks, to subtract the values from the corresponding treatments with bacteria and 358
to verify the lack of contamination. In some of the experiments, samples were taken 359
at several times for assessment of CFU by dilution plating. 360
Assessment of bactericidal activity. Cell suspensions containing 107 CFU ml-1 361
were prepared as described above in 100 mM phosphate buffer (pH 7.0) containing 362
BSM-Ag NPs at various concentrations (from 0 to 2.2 mg Ag l-1). The suspensions 363
were maintained for 24 h at 25C, after which bacterial concentrations were 364
determined following plating of serial dilution on NA plates. 365
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Scanning electron microscopy (SEM). Acidovorax citrulli M6 were grown for 366
48 h on NA plates as described above and cell suspensions of 107 CFU ml-1 were 367
prepared in NB. Loopfols from these suspensions were collected with a platinum 368
loop and placed onto 5 mm2 slices of grade GF/F Whatman glass microfiber filters 369
(Sigma-Aldrich) previously coated with a thin layer (~0.5 mm) of an autoclaved 370
solution containing 3% NA and 0.1% gelatin. The inoculated slices were placed onto 371
NA plates that were incubated at 28°C for 24 h. Then, 5 μl of a solution containing 372
BSM or BSM-Ag NPs with 10 mg Ag ml-1 were added to the top of the slices (in 373
controls no solution was added) and the plates were incubated for additional 24 h at 374
28°C. The slices were then fixed in in 4% glutaraldehyde in 100 mM phosphate buffer 375
(pH 7.0) for 1 h, washed three times with the same buffer and dehydrated using 376
increasing ethanol concentrations of 25%, 50%, 75%, 95% and 100% (10 min each). 377
The blocks were then dried in a CPD 030 Critical Point Dryer (Bal-Tec), displacing the 378
alcohol with liquid CO2, and finally dried by releasing CO2. The dried blocks were 379
mounted on the brass blocks, coated with carbon (Edvards) and visualized in a high- 380
resolution Ultra 55 FEG scanning electron microscope (Zeiss). Backscattering analysis 381
was performed on non-coated samples with an in-lens detector. 382
Transmission electron microscopy (TEM). Bacterial suspensions (108 CFU ml- 383
1) of A. citrulli M6 in NB with BSM or BSM Ag NPs at 10 mg Ag ml-1 were shaken at 384
28°C for 24 h. The cells were then collected by centrifugation (5000 g, 10 min, 4°C), 385
washed with 100 mM phosphate buffer (pH 7.0) and fixed with 0.2 M cacodylate 386
buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde for 2 h at 387
room temperature, after which the samples were kept at 4°C for 16 h. The bacteria 388
were then rinsed four times with 0.2 M cacodylate buffer (10 min each time) and 389
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post-fixed and stained with a solution containing 1% osmium tetroxide and 1.5% 390
potassium ferricyanide in 0.1 M cacodylate buffer for 1 h. Bacteria were then 391
washed 4 times in 0.2 M cacodylate buffer followed by dehydration in increasing 392
concentrations of ethanol consisting of 30%, 50%, 70%, 80%, 90% and 95% (10 min 393
each), followed by three times with 100% anhydrous ethanol (20 min each), and two 394
times with propylene oxide (10 min each). Then the samples were infiltrated with 395
increasing concentrations of agar 100 resin (Agar Scientific) in propylene oxide, 396
consisting of 25, 50, 75 and 100% resin for 16 h each step. Bacteria were then 397
embedded in fresh resin and let to polymerize in an oven at 60°C for 48 h. Embedded 398
bacteria in blocks were sectioned with a diamond knife on an LKB 3 microtome and 399
ultrathin sections (80 nm) were collected onto 200 mesh, thin bar copper grids. The 400
sections on grids were sequentially stained with uranyl acetate and lead citrate for 3 401
min each and viewed with a FEI T12 transmission electron microscope (Phillips) 402
equipped with an Olympus SIS MegaView III-mounted CCD camera. 403
Seed transmission assays. The effect of BSM-Ag NPs on protection of 404
emerging seedlings against A. citrulli was assessed by seed transmission assays (24) 405
with few modifications. The effects were assessed as both treatments of preinfested 406
seeds with BSM-Ag NPs (“treatment”), and as pretreatment with BSM-Ag NPs 407
followed by bacterial inoculation (“pretreatment”). In “treatments”, melon seeds 408
(cultivar Ophir, Zerahim Gedera) were placed in 107 CFU ml-1 bacterial suspensions of 409
A. citrulli M6 in 100 mM phosphate buffer (pH 7.0) and incubated at room 410
temperature for 2 h with gentle agitation. Seeds were then passed through a 411
strainer, and dried under a laminar flow hood for 2 h. Subsequently, seeds were 412
placed in solutions containing BSM-Ag NPs at three concentrations of Ag: 5, 10 and 413
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22 mg l-1. As controls, inoculated seeds were incubated in a solution containing BSM 414
alone. Seeds and BSM/BSM-Ag NP solutions were placed on a shaker with gentle 415
agitation for 2 h at room temperature. As an additional control for seed disinfection, 416
seeds were treated for 50 s with 0.6% sodium hypochlorite (NaClO, Biolab Ltd.), then 417
washed twice with sterile distilled water. Treated seeds were passed through a 418
strainer and dried under a laminar flow hood for 2 h. For “pretreatments”, seeds 419
were first placed in solutions containing BSM, BSM-Ag NPs (at the same 420
concentrations as described above), or 0.6% NaClO at similar conditions as described 421
for “treatments”. Then the seeds were collected and allowed to dry for 2 h in a 422
laminar flow hood before being inoculated with 107 CFU ml-1 of bacteria as described 423
above. Non-treated/pretreated and non-inoculated controls were included. Seeds 424
were sown in small sized 11 cm diameter poly-pots containing brown 602-moss peat 425
(Klasman). Emerging seedlings were grown in a greenhouse (28+2oC) for 10 days, and 426
then disease severity was scored on a 0 to 5 scale: 0, healthy plants; 1, slight necrosis 427
in one or two cotyledons; 2, wide necrosis in both cotyledons; 3, necrosis and 428
deformation in cotyledons; 4, necrosis and deformation in cotyledons and significant 429
damage to the seedling; 5, dead seedling. The number of dead seedlings was 430
recorded every day and the foliage weight of the seedlings was determined at the 431
end of the experiment. The experiment was carried out three times with 13 to 15 432
replicates per treatment, and the data were analysed by Tukey-Kramer HSD test 433
using JMP software (SAS Institute Inc.). In two additional experiments, melon seeds 434
treated as described above were put to germinate on NA plates. The plates were 435
wrapped in aluminum foil and incubated at 25C for 5 days after which presence or 436
absence of A. citrulli colonies around the germinating seeds were determined. The 437
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identity of the colonies was verified by colony-PCR using the A. citrulli-spefic primer 438
set BX-S, as described (39). 439
440
SUPPLEMENTAL MATERIAL 441
Supplemental material for this article including supplemental figures S1 to S3 442
may be found at https://doi.org/XX.XXXX/AEM.XXXXX.XX. 443
444
ACKNOWLEDGMENTS 445
This work was supported by a start-up grant from Yissum, the Hebrew 446
University of Jerusalem. TEM observations were conducted at the Bio-Imaging Unit, 447
The Alexander Silberman Institute of Life Science of the Hebrew University of 448
Jerusalem. SEM observations were conducted at the Irving and Cherna Moskowitz 449
Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science. 450
451
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FIGURE LEGENDS 569
FIG 1 Effects of bovine submaxillary mucin-silver nanoparticles (BSM-Ag NPs) on 570
Acidovorax citrulli. (A) Growth of A. citrulli strain M6 exposed to different 571
concentrations of BSM-Ag NPs. Bacteria were grown for 168 h at 28°C in ELISA plates 572
in nutrient broth (NB) medium containing BSM-Ag NPs at different concentrations of 573
Ag (control was BSM without Ag). The initial concentration of bacterial cells was 107 574
colony forming units (CFU) ml-1. Growth was measured by optical density at 600 nm. 575
Averages of 3 replicates per treatment of a representative experiment (out of three 576
with similar results) are shown. (B) Killing effect of BSM-Ag NPs on A. citrulli M6. 577
Bacterial suspensions containing 107 CFU ml-1 were treated with BSM-Ag NPs 578
solutions containing different Ag concentrations at 25C. After 24 h, the suspensions 579
were serially diluted and plated on nutrient agar (NA) plates for bacterial counting. 580
The graph represents bacterial concentrations (CFU ml-1) at the end of the 581
experiment. Averages of 3 replicates per treatment of a representative experiment 582
(out of two with similar results) are shown. 583
584
FIG 2 Electron microscopy (EM) images of A. citrulli M6 cells exposed to BSM (A, C 585
and E) or BSM-Ag NPs (B, D, F and G). Cells were grown on nutrient agar (NA) at 28°C 586
for 48 h. Then, 5-μL drops of BSM or BSM-Ag NPs (with 10 mg Ag l-1) were put on the 587
top of the colonies for 2 min followed by preparation for EM observation. A and B, 588
scanning electron microscopy (SEM, Ultra 55 FEG, Zeiss); C and D, Ultra 55 FEG SEM 589
with backscatter electron detector; E and F, transmission electron microscopy (TEM, 590
Tecnai T12 electron microscope). Bars: A and B, 400 nm; C and D, 1 μm E, 200 nm; F, 591
100 nm. 592
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593
FIG 3 Effects of seed treatment and pretreatment with BSM-Ag NPs on disease 594
severity in seed transmission experiments of melon (cv. Ophir) with A. citrulli M6. 595
Seed inoculation, and treatments (TRE)/pretreatments (PRE) are described in 596
Materials and Methods. Plants were grown in a greenhouse at 282°C and disease 597
severity scores were determined from 13 to 15 replicates per treatment 10 days 598
after sowing. Different letters indicate significant differences (p<0.05) among 599
treatments by Tukey-Kramer HSD test and ANOVA. Controls included inoculated 600
seeds, non-inoculated seeds, and treatments/pretreatments with BSM (without Ag 601
NPs) or 0.6% NaClO. Data (mean and standard errors) are from one representative 602
experiment out of three experiments with similar results. 603
604
FIG 4 Effects of seed treatment and pretreatment of BSM-Ag NPs on seedling 605
survival in seed transmission experiments of melon (cv. Ophir) with A. citrulli M6. 606
Seed inoculation, and treatments/pretreatments are described in Material and 607
Methods. Plants were grown in a greenhouse at 282°C and the percentage of 608
seedling survival was recorded every day. Data from one representative experiment 609
out of three with similar results are shown. Controls included inoculated seeds, non- 610
inoculated seeds, and treatments/pretreatments with BSM alone or 0.6% NaClO. To 611
allow distinguishing among the curves, pretreatments (PRE) and treatments (TRE) 612
were split into two plots (A and B, respectively). Inoculated and non-inoculated 613
controls are included in both plots. The following treatments did not lead to seedling 614
death, therefore their curves overlap at 100% survival: non-inoculated control and 615
pretreatments with BSM-Ag NPs containing 5, 10 and 22 mg Ag l-1 in A, and non- 616
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inoculated control, treatments with BSM-Ag NPs containing 10 and 22 mg l-1 Ag, and 617
treatment with 0.6% NaClO in B. 618
619
FIG 5 Assessment of effects of BSM-Ag NPs on melon seed infestation with A. citrulli 620
M6. Seeds were inoculated and treated or pretreated as described for seed 621
transmission assays. They were then placed onto NA plates that were wrapped in 622
aluminum foil. Germination and infestation were observed in two replicates (plates) 623
per treatment (each plate containing 4 seeds). The pictures are representative of 624
three experiments with similar results. A. non-inoculated/untreated control; B. 625
inoculated/untreated control; C. inoculated/treated with BSM alone; D. 626
inoculated/treated with BSM-Ag NPs (at 22 mg Ag l-1); E. pretreated with BSM-Ag 627
NPs (22 mg Ag l-1)/inoculated; F. inoculated/treated with 0.6% NaClO; G. pretreated 628
with 0.6% NaClO/inoculated. 629
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