This is the accepted version of the article: Alonso-Díaz, Alejandro; Floriach Clark, Jordi; Fuentes, Judit; [et al.]. «Enhanc- ing Localized Pesticide Action through Plant Foliage by Silver-Cellulose Hybrid Patches». ACS biomaterials science & engineering, Vol. 5, Issue 2 (February 2019), p. 413-419. DOI 10.1021/acsbiomaterials.8b01171 This version is avaible at https://ddd.uab.cat/record/205853 under the terms of the license Subscriber access provided by UNIV AUTONOMA DE BARCELONA Letter Enhancing localized pesticide action through the plant foliage by silver-cellulose hybrid patches Alejandro Alonso-Diaz, jordi Floriach-Clark, Judit fuentes, Montserrat Capellades, nuria sanchez-coll, and Anna Laromaine ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01171 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. 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Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts. is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 16 ACS Biomaterials Science & Engineering 1 2 3 Enhancing localized pesticide action through the plant foliage by silver- 4 cellulose hybrid patches. 5 6 7 Alejandro Alonso-Díaz1, Jordi Floriach-Clark2, Judit Fuentes2, Montserrat 8 Capellades1, Núria S. Coll1*, Anna Laromaine2* 9 10 11 1Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, 12 Campus UAB, Bellaterra, Barcelona, 08193, Spain. 13 14 2 Institute of Material Science of Barcelona (ICMAB), CSIC, Carrer dels Til.lers, 15 16 Campus UAB, Bellaterra, Barcelona, 08193, Spain. 17 *Correspondingauthors: alaromaine@icmab.es, nuria.sanchez- 18 19 coll@cragenomica.es 20 21 KEYWORDS bacterial cellulose, silver nanoparticles, preventing infection, 22 Nicotiana benthamiana, pseudomonas. 23 24 25 ABSTRACT: 26 Efficacy and efficiency of pesticide application in the field through the foliage still 27 faces many challenges. There exists a mismatch between the hydrophobic 28 character of the leaf and the active molecule, low dispersion of the pesticides on 29 30 the leaves’ surface, runoff loss and rolling down of the active molecules to the field, 31 decreasing their efficacy and increasing their accumulation to the soil. We 32 produced bacterial cellulose-silver nanoparticles hybrid patches by in situ thermal 33 reduction under microwave irradiation in a scalable manner and obtaining AgNPs 34 strongly anchored to the BC. Those hybrids increase the interaction of the pesticide 35 36 (AgNPs) with the foliage and avoids runoff loss and rolling down of the 37 nanoparticles. The positive anti-bacterial and anti-fungal properties were assessed 38 in vitro against the bacteria Escherichia coli and two agro-economically relevant 39 pathogens: the bacterium Pseudomonas syringae and the fungus Botrytis cinerea. 40 41 We showed in vivo inhibition of the infection in Nicotiana benthamiana and 42 tomato leaves, as proven by the suppression of the expression of defense molecular 43 markers and reactive oxygen species production. The hydrogel-like character of 44 the bacterial cellulose matrix increases the adherence to the foliage of the patches. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 2 of 16 1 2 3 Food security and the need to increase sustainably crop yields for a rapidly growing 4 5 world population are among the greatest social and economic challenges of our 6 century. The most extended agricultural practice to enhance crop yield is to 7 increase host plant density, which in turn tends to increase the severity of plant 8 diseases.1 Most plant diseases occurring in agriculture are caused by fungal 9 pathogens2. Diseases caused by pathogenic bacteria are less prevalent; but their 10 11 effects are also devastating.3 12 To fight against pathogens, pesticides are effectively used in agriculture, securing 13 a stable crop yield.4 However, the efficacy and efficiency of the pesticides 14 application in the field still faces many challenges. Pesticides sprayed through the 15 16 leaves experience a mismatch of their hydrophobic character between the foliar 17 tissue and the active molecule, which promotes low dispersion of the pesticides on 18 the leaves’ surface, a runoff loss and rolling down of the pesticides to the field, 19 decreasing their efficacy and increasing their accumulation in the soil. 20 Several active ingredients with anti-pathogenic and anti-bacterial properties, such 21 22 as several metal ions, have been explored in crops.5 Currently, novel smart-based 23 nanomaterials for pesticides have been developed since they can improve the low 24 solubility issues of the active ingredients in water and its dispersions. The small 25 size, big surface area and target modified properties of nanomaterials holds 26 promise as nano-based pesticides.6 Silver compounds have been commonly used 27 28 as active ingredients in commercial pesticides7, despite their toxic properties 29 including DNA damage, inhibition of key enzyme activities or disruption of the 30 bacterial membrane.5,8,9 Silver compounds prepared as nanoparticles showed an 31 increased efficacy and specificity as anti-bacterial and antimicrobial agents.5,10 32 33 Price of those smart pesticides are not comparable to common bulk products 34 currently used; however they suit high value applications such as vineyards, fruit 35 trees or rare/valuable tree specimens. 36 We present a hybrid anti-bacterial and anti-fungal patch, which exploits the 37 potential of smart-based nanomaterials; silver nanoparticles (AgNPs) as anti- 38 39 bacterial active component. AgNPs are anchored to the bacterial cellulose matrix 40 by in situ thermal reduction under microwave irradiation which prevents the 41 release of the NPs to the environment and their runoff loss during application; 42 improving the efficiency and environmental sustainability of this patch. 43 44 45 Cellulose is the most abundant biopolymer11 and is the main constituent of the cell 46 wall of plants.12 However, cellulose can also be produced by different 47 microorganisms, including bacterial species such as Komagataeibacter xylinus 48 (Kx). Kx produces cellulose film as a sub-product of its metabolism, which has a 49 50 hydrogel texture holding up to 90 times its weight on water.13,14 Bacterial cellulose 51 (BC) is obtained as an ultra-fine and highly pure tridimensional network exhibiting 52 high water holding capacity, gel-like formulation13 and biocompatibility, thus 53 displaying high potential in regeneration and wound healing applications, Figure 54 55 S1.15 The bacterial cellulose matrix has similar chemical composition to the plant 56 cellulose present in leaves but shows higher purity, crystallinity and water 57 absorbance.16,13 We took advantage of the hydrogel-like nature of the bacterial 58 cellulose matrix to in situ synthesize and embedded AgNPs in order to confer anti- 59 pathogenic properties to the patches. Different authors exploited the combination 60 2 ACS Paragon Plus Environment Page 3 of 16 ACS Biomaterials Science & Engineering 1 2 3 of cellulose with silver nanoparticles, from impregnation17,18 to in situ synthesis in 4 5 cellulose, oxidized cellulose or combination with other materials19–24 and evaluated 6 them as anti-bacterial materials. However, the release of the NPs was commonly 7 observed on those materials or not evaluated, even though its environmental 8 hurdles. To avoid any runoff loss to the environment, we exploited the synthesis 9 by in situ thermal reduction under microwave irradiation25,26,27, in order to strongly 10 11 anchor the AgNPs to the bacterial cellulose matrix. 12 13 Briefly, BC films were immersed in an AgNO3 and PVP solution to ensure a 14 homogeneous distribution of the precursor inside the cellulose network. After 10 15 16 min of microwave radiation, we observed the change of color of the BC films from 17 translucent white to dark brown indicating the incorporation of the AgNPs into 18 the cellulose scaffold (Figure 1a). SEM of BC-AgNPs hybrid films confirmed the 19 homogeneous distribution of AgNPs within the film (Figure 1b) and by TEM we 20 measured the diameter width of spherical shape metallic silver nanoparticles to 21 22 13±4 nm (Figure 1c). We analyzed the leaching process of BC-AgNPs hybrid films, 23 immersing them in water for 14 days in gentle stirring (<60 rpm). We did not detect 24 any change in the size, shape or color of the films. We semi-quantitatively 25 measured BC-AgNPs leaching by analyzing the color change of the hybrid film by 26 ImageJ software (grey scale) and we could not appreciate any change of color 27 28 indicative of detachment of the particles from the hybrid films (Figure 1e). The 29 content of silver on those films upon immersion was quantitatively measured by 30 Coupled Plasma – Mass Spectroscopy (ICP-MS) and we did not observe any 31 change. ICP-MS data confirmed that the release to the solution was of 32 33 approximately 57 ppb in 14 days. Together, these data confirms the strong 34 attachment of AgNPs to the BC matrix similar to what has been previously reported 35 with iron oxide nanoparticles.26 In addition, the release and degradation of the 36 nanoparticles from the BC matrix is slow. The combination of the anti-pathogenic 37 properties of the BC-AgNPs films and the slow release contributes to obtain a 38 39 material that could be safer for the environment and more sustainable than most 40 common pesticides used nowadays. 41 In order to obtain commercial products, it is important to have good 42 reproducibility and scalability of the materials produced. We confirmed the 43 44 reproducibility of 30 films produced in 5 different batches and synthesized by 45 different users using different techniques such as thermogravimetric analysis, 46 TEM, SEM and color analysis. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 4 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Figure 1. (a) Production of the AgNP-BC hybrid film by reduction of AgNO3 28 assisted by microwave radiation; (b) SEM BSE image of AgNP-BC surface 29 indicating high homogeneity; (c) TEM image and SEAD of AgNP-BC composite; 30 (d) Qualitative measurement of the amount of Ag-NPs of the hybrids upon 31 32 leaching. (e) Images of BC-AgNPs hybrids produced in the same batch. (f) 33 Qualitative measurement of the reproducibility and amount of Ag-NPs in the BC- 34 AgNPs hybrids from five batches. Average gray value is 69 (dashed line); ±1σ range 35 is shaded; σ=19; n=30. 36 37 38 We analyzed the in vitro growth inhibitory effects of BC-AgNPs against a range of 39 bacteria and fungi. We choose Escherichia coli as a reference species because of its 40 known sensitivity against a wide range of antibiotics and Pseudomonas syringae pv 41 tomato DC3000, causing the bacterial speck disease in tomato and a model plant 42 43 pathogen. Growth inhibition of E. coli could be observed as a halo around a 44 particular treatment (Figure 2a) and was quantified by measuring its diameter 45 (1,2mm). In contrast, wet BC (BC-wet) film without AgNPs did not show any 46 obvious inhibitory effect on bacterial growth. SEM observed higher bacterial 47 densities in the positive control and BC-wet in comparison to the BC-AgNP 48 49 treatment as shown in Figure 2b. As expected, gentamicin and AgNO3 caused a 50 transparent halo on the culture media. Figure 2c also shows that the BC-AgNPs 51 film resulted in a transparent halo in P. syringae DC3000, demonstrating that this 52 composite is also toxic for phytopathogenic bacteria. Even though BC-AgNPs films 53 54 showed a low leaching and slow release of silver, we observed those films have anti- 55 bacterial properties. 56 57 58 59 60 4 ACS Paragon Plus Environment Page 5 of 16 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Figure 2: Toxicity assay of BC-AgNPs in bacteria (E. coli and P. syringae). (a) 27 Toxicity assay using E. coli. strain OP50. Bacteria was growth in LB plate at 37ºC, 28 and different treatments were applied: BC-wet (piece of 6 mm diameter), 5 µl 29 AgNO3 (50 mg/ml), 5 µl AgNO3 (5 mg/ml), BC-AgNPs (piece of 6 mm diameter), 5 30 31 µl sterile miliQ water and 5 µl Gentamicin (10 mg/ml). (b) SEM images of BC-wet, 32 BC-AgNPs and sterile miliQ water-treated bacteria were taken from areas pointed 33 by white arrows. (c) Toxicity assay using P. syringae (DC3000). Bacteria were 34 growth in KB plate at 28ºC, and different treatments were applied: BC-wet (piece 35 36 of 6 mm diameter), 5 µl AgNO3 (50 mg/ml), 5 µl AgNO3 (5 mg/ml), BC-AgNPs 37 (piece of 6 mm diameter), 5 µl sterile miliQ water and 5 µl Gentamicin (10 mg/ml). 38 All photos were taken at 24 hours after treatment. Scale bar: 5 µm and 6 mm. 39 40 Potentially toxic effects of hybrid BC with silver nanoparticles are commonly tested 41 42 only in bacteria and not assessed using fungal phytopathogens.18,28–33 Previous 43 reports showed that bacterial cellulose-copper oxide nanocomposites had anti- 44 microbial activity against bacteria and yeast.34 Therefore, to determine whether 45 hybrid films were only toxic for bacteria or their effect could be extended to 46 47 unrelated phytopathogens, we tested the patches against the plant pathogenic 48 fungus Botrytis cinerea (B05.10 strain), the causal agent of gray mold disease and 49 one of the most important fruit postharvest pathogens35. The fungal mycelium was 50 allowed to develop for 9 days (Figure 3a) and after that, spores were extracted and 51 quantified separately from a fixed area surrounding each treatment (Figure 3b). As 52 53 expected, the strongest effect was caused by the AgNPs alone due to their ability 54 to diffuse in the medium, which inhibited fungal colonization and spore 55 production, as it has been reported in previous publications36,37. The BC-AgNPs 56 also inhibited spore production (4.75·105 spores/ml) compared to the control 57 58 treatment (3.6·106 spores/ml), and resulted in reduced fungal colonization, similar 59 to what has been previously reported.38 BC-wet alone did not significantly inhibit 60 spore formation, although fungal colonization was slightly reduced. The growth 5 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 6 of 16 1 2 3 inhibition and toxicity of BC-AgNPs on the fungal mycelium can be clearly 4 5 observed in the images shown in Figure 3c. The 5 mm2 area adjacent to each 6 different treatment (BC-AgNPs, BC-wet and untreated) was visualized using SEM. 7 Untreated and BC-wet-treated mycelia showed no growth defects. In contrast, 8 mycelia treated with BC-AgNPs had obvious signs of damage and cell death, with 9 an apparent loss of turgor, deformation of the chitin cell walls and no spore 10 11 production, indicating the toxicity reaction caused by the patch. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Figure 3: Toxicity assay of BC-AgNPs in B. cinerea. (a) Toxicity assay in PDA 40 41 medium. 7,5 µl B. cinerea concentration 105 sp/ml inoculum were add in the center 42 of the PDA plate: Top image corresponds to 15 µl AgNPs (0.53mg/ml), right image 43 corresponds to BC-wet, left image corresponds to 15 µl sterile miliQ water and 44 below image corresponds to BC-AgNPs. (b) Spore count using a toxicity assay 45 46 plate. A piece of PDA plate of 2.7 cm diameter (marked with a circle) was excised 47 for each treatment; BC-AgNPs, BC, AgNPs, untreated and sterile miliQ water. (c) 48 SEM images were taken 9 days post infection (dpi) of a portion of the mycelia close 49 to each treatment (marked with a square); BC-AgNPs, BC and untreated. 50 51 52 Then we evaluated the in vivo effects of BC-AgNPs; by infecting leaves of the model 53 plant Nicothiana benthamiana with the bacterial pathogen P. syringae DC3000 on 54 well-delimited 1-cm diameter areas. Then, the infected areas were left uncovered 55 or covered for 6 days with BC film alone, or with BC-AgNPs film. P. syringae 56 57 DC3000 caused a necrotic reaction on N. benthamiana in the uncovered control 58 (Figure 4b and 4c). This reaction was mostly inhibited in the leaf area covered with 59 BC-AgNPs, indicating that the film had a strong anti-bacterial effect on leaves. 60 Interestingly, the BC film alone caused a slight inhibition of necrosis caused by 6 ACS Paragon Plus Environment Page 7 of 16 ACS Biomaterials Science & Engineering 1 2 3 bacterial infection. This effect might be the result of additive factors including an 4 5 altered environment for the bacteria (lower oxygen availability and reduced light 6 intensity) and the material properties (porosity, thickness and roughness) added 7 to the possibility that Kx-secreted molecules may have remained embedded in the 8 BC matrix, providing supplementary anti-bacterial properties. 9 The reduction of necrosis correlated with a decrease of bacterial growth in the 10 11 areas covered with BC-AgNPs and BC alone (Figure 4d). As expected, this 12 inhibition of bacterial growth was more dramatic in BC-AgNPs than in BC-covered 13 samples, in which only a minor, although significant, decrease of bacterial growth 14 was observed. The anti-bacterial effect of BC-AgNPs was not restricted to N. 15 16 benthamiana plants. We performed a P. syringae infection experiment using 17 tomato, a crop of high agricultural value, observing similar effects (Figure S3). 18 Curiously, in tomato we did not observe a significant decrease of bacterial growth 19 in BC-treated samples. 20 21 22 Importantly, BC and BC-AgNP films strongly adhered when placed wet on the 23 surface of leaves. These films stayed attached for long periods (more than 7 days) 24 on the leaf surface under normal growth conditions (Figure 4a). In contrast, an 25 analogous patch made of wet plant cellulose (filter paper) only stayed 1 day adhered 26 to the leaves. As previously described, BC has high water absorbance and its 27 28 composites retain this property. Therefore, we believe that the hydrogel 29 consistency of the BC-AgNP film allows it to dry slowly; while it dries, it embeds 30 the leaf trichomes (hair-like cells on the leaf adaxial surface) within the BC matrix, 31 favoring the adhesion of the patches to the leaves. This strong adherence of BC 32 33 may significantly contribute to the anti-pathogenic effects of the BC-AgNPs hybrid 34 film, as it provides a stable, strongly anchored matrix in which the AgNPs can exert 35 their toxic effect on the bacteria in close contact to the leaf, without detaching with 36 time. 37 38 39 Anti-fungal activity of the BC-AgNPs and BC-wet alone in plants were tested on N. 40 benthamiana leaves inoculated with a droplet of B. cinerea spore solution. Twenty- 41 four hours after, the infection sites were covered with BC-AgNPs, BC-wet alone or 42 left uncovered. After 6 days of treatment, effects were evaluated by taking pictures 43 44 of the infected leaves (Figure 4e) and lesion size was measured (Figure 4f). The 45 typical symptom caused by B. cinerea on N. benthamiana leaves is tissue necrosis, 46 as it could be clearly observed in the uncovered control. Strikingly, the BC-AgNPs 47 film totally inhibited the development of any visual fungal infection symptoms. In 48 contrast, both BC-wet and uncovered treatment did not block disease progression. 49 50 This indicates that the BC-AgNPs hybrid film has also a strong anti-fungal effect 51 on plants. 52 53 54 55 56 57 58 59 60 7 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 8 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Figure 4: Anti-bacterial and anti-fungal activity of BC-AgNPs in N. 42 benthamiana against P. syringae and B. cinerea (a) BC-AgNPs adhered to a N. 43 benthamiana leaf (b) Three week-old N. benthamiana plants were infiltrated with 44 45 P. syringae (DC3000) at an O.D.600 of 0.0008. After infiltration infected areas were 46 covered with different treatments: BC-AgNPs, BC or left uncovered. Photos were 47 taken 6dpi. (c) The area of necrotic tissue was measured with the ImageJ software 48 at 6dpi d) Bacterial growth within a 1 cm-diameter infected area was measured as 49 50 the average of colony forming units (CFUs) (logarithmic scale) per square 51 millimeter of infected tissue. (e) Three week-old N. benthamiana leaves were 52 infected with a 5 µl drop of 105 B. cinerea spores/ml and kept under 100 % humidity. 53 After 24 h infected areas were covered with BC-AgNPs, BC-wet or left uncovered. 54 After 6dpi the treatments were removed and digital pictures were taken. f) The 55 56 area corresponding to the necrotic tissue was measured with ImageJ software at 57 6dpi. 58 59 60 8 ACS Paragon Plus Environment Page 9 of 16 ACS Biomaterials Science & Engineering 1 2 3 The anti-pathogenic effect of BC-AgNPs could occur as a result of pathogen growth 4 5 inhibition or by an enhancement of the plant defenses. To test a potential immune 6 boosting effect of BC-AgNPs on bacterial infection, we quantified the expression 7 of PATHOGENESIS-RELATED 1a (PR1a), a plant defense marker gene 39 PR1a was 8 expressed in BC-AgNPs -treated infected tissue, although to very low levels, 18 9 times lower than the uncovered control (Figure 5a). The BC-AgNPs patch strongly 10 11 inhibited pathogenic bacteria perception and the subsequent immune response of 12 the plant. BC-wet film alone also caused partial inhibition of PR1a expression to 13 approximately 1.6 times lower than untreated leaf levels. This result correlates with 14 the decreased extent of pathogen-triggered necrosis observed upon infection in 15 16 BC-AgNPs-covered leaf areas when compared to the untreated control (Figure 5a 17 and 5e). 18 19 In parallel, we analyzed gene expression profiles of HOMEOBOX 1 (HB1) and 20 HARPIN-INDUCED GENE (HIN1) after fungal infection with B. cinerea. HBI is a 21 22 gene which is expressed in pathogen-induced cell death and its expression is 23 dependent on jasmonic acid signaling, which is activated upon necrotrophic fungal 24 infection40. On the other hand, HIN1 is a marker of pathogen-triggered cell death41. 25 Figure 5b shows that these genes are upregulated in inoculated tissue that was 26 either uncovered or covered with tape, indicating pathogen perception and 27 28 response by the plant. In contrast, BC-AgNPs-covered tissue shows minor 29 upregulation of the two marker genes, suggesting that BC-AgNPs may directly 30 inhibit B. cinerea infection, previous to the activation of the plant immune system. 31 This indicates that fungal growth arrest may occur at very early stages of infection. 32 33 34 Recognition of a pathogen by a plant results in a rapid burst of reactive oxygen 35 species (ROS), which can be measured as an output of plant defense responses.42 36 Previous reports showed also an increase of ROS upon exposure of AgNPs to some 37 plants.43 Production of hydrogen peroxide (H2O2), one of the main ROS produced 38 39 by the plant upon pathogen infection, was visualized in leaf areas infected with P. 40 syringae DC3000 using 3, 3 –diaminobenzidine (DAB) staining. Figure 5c-d clearly 41 shows that infection results in accumulation of H2O2 in uncovered tissue, when 42 compared to uninfected or mock-inoculated leaf tissue (MgCl2). In contrast, P. 43 44 syringae-inoculated tissue covered with BC-AgNPs displays a drastic reduction on 45 H2O2 accumulation. This observation corroborates the previous findings indicating 46 that defense responses are not induced in inoculated tissue covered with BC- 47 AgNPs. Together, these data corroborate that the anti-bacterial effect of BC-AgNPs 48 does not result from enhanced plant defense responses. BC-wet film alone did not 49 50 prevent ROS accumulation in the inoculated tissue, which is not surprising, 51 considering that the extent of bacterial growth inhibition caused by this film is 52 minor when compared to BC-AgNPs. Previous reports indicated an increase of ROS 53 caused by nanoparticle exposure,43 potentially harmful for living cells and tissues. 54 55 The fact that the leaf treatment with the BC-AgNPs hybrid does not result in ROS 56 production might constitute another indication that AgNPs are not released from 57 the BC matrix, avoiding any effects derived from AgNPs treatment alone and 58 preventing their release to the environment”. 59 60 9 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 10 of 16 1 2 3 ROS produced as a result of B. cinerea infection were absent in the BC-AgNPs- 4 5 covered sample; in contrast to inoculated samples covered with BC-wet which 6 showed ROS production levels comparable to those of the uncovered control 7 (Figure 5e and 5f).These data corroborate our previous observation indicating that 8 BC-AgNPs completely blocks fungal invasion at very early stages. Thanks to this 9 rapid elimination of the pathogen, the plant does not even perceive it and thus, 10 11 unnecessary defense reactions are prevented. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Figure 5: Defense molecular markers and reactive oxygen species (ROS) (a- 45 b) Quantitative PCR (qPCR) analysis of defense marker genes. Three week-old N. 46 benthamiana plants were infiltrated with (a)P. syringae DC3000 at an O.D.600 of 47 48 0.001, (b)Three week-old plants were sprayed with either 105 spores/ml of B. 49 cinerea and keep for 24 hours under 100% humidity.Then, the infected zones were 50 covered with the different treatment (BC-AgNPs, BC-wet and uncovered) during 51 12 hours and then RNA was extracted. qPCR was performed with specific primers 52 53 for N. benthamiana (a) PR1a, (b) HIN1, HBI and Tubulin (control) genes as 54 described in Material and Methods. (c) to (f) ROS production analysis. (c) Three 55 week-old N. benthamiana leaves were infected with P. syringae DC3000 (avrRpm1) 56 at an O.D.600 of 0.01 and then covered with: BC-AgNPs and BC or left uncovered. 57 As a negative control uninfected tissue was used. After 12 hpi the infected zones 58 59 with different treatments were stained using the H2O2 stain 3,3´-diaminobenzidine 60 (DAB). Stained leaves were imaged using a digital camera. (d) Quantification of 10 ACS Paragon Plus Environment Page 11 of 16 ACS Biomaterials Science & Engineering 1 2 3 the images shown in (c) as the percentage of DAB stain present using the Image J 4 5 software. (e) Three week-old N. benthamiana leaves were infected with B. cinerea 6 at 105 spores/ml concentration and keep for 24 hours under 100% humidity. Then, 7 the infected zones were covered with BC-AgNPs, BC-wet and uncovered and after 8 12 hpi. The infected zones and uninfected control were stained using DAB and 9 images were taken using a digital camera. Previously staining leaves were 10 11 processed with ImageJ. (f) Quantification of the images shown in (e) as the 12 percentage of DAB stain present using the ImageJ software. 13 14 In summary, we have developed environmentally friendly nanocomposite patches 15 based on bacterial cellulose and silver nanoparticles in a reproducible and scalable 16 17 manner; with positive results against the bacteria Escherichia coli and 18 Pseudomonas syringae and the fungus Botritis cinerea. Thanks to their hydrogel- 19 like consistency, we obtained a strong adherence to N. benthamiana leaves 20 inoculated B. cinerea spore solution. We confirmed the pathogen inhibitory 21 22 properties of the material by quantitative gene expression profiling of various plant 23 defense marker genes, and by reactive oxygen species quantification in the 24 inoculated tissue, showing no defense response activation probably due to an early 25 neutralization of the pathogen on site by the nanocomposite. The slow release of 26 silver nanoparticles from the bacterial cellulose makes this material potentially 27 28 safer for the environment than most current pesticides. Thus, this bacterial 29 cellulose silver nanoparticle composite shows promise as a new generation 30 pesticide, potentially overcoming bottlenecks to increase efficacy and efficiency of 31 current foliar pesticides. 32 33 34 Supporting Information 35 Materials and Methods for the production of BC and BC hybrids and their 36 characterization. Experimental protocols for the evaluation of the BC hybrids on 37 pathogens and plants. 38 39 40 Conflicts of interest 41 There are no conflicts of interest to declare. 42 43 44 Acknowledgements 45 We acknowledge financial support from the Spanish Ministry of Economy and 46 Competitiveness (MINECO) with grants 2016-78002-R (AGL) and RyC 2014-16158 47 (NSC), MAT2015-64442-R, FIP and through the “Severo Ochoa Programme for 48 Centres of Excellence in R&D” (SEV-2015-0533 and SEV-2015-0496). This work was 49 50 also supported by the CERCA Programme / Generalitat de Catalunya, the 51 Generalitat de Catalunya for the 2017SGR765 project. The authors would like to 52 thank all members of the “bacterial plant diseases and cell death” lab for helpful 53 comments. 54 55 56 57 58 59 60 11 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 12 of 16 1 2 3 References 4 5 6 7 (1) Sundström, J. 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Coll1*, Anna Laromaine2* 9 10 11 1Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, 12 Campus UAB, Bellaterra, Barcelona, 08193, Spain. 13 14 2 Instituteof Material Science of Barcelona (ICMAB), CSIC, Campus UAB, 15 16 Bellaterra, Barcelona, 08193, Spain. 17 *Correspondingauthors: alaromaine@icmab.es, nuria.sanchez- 18 19 coll@cragenomica.es 20 21 22 23 TOC 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 16 ACS Paragon Plus Environment