# Biogenic AgNPs—A Nano Weapon against Bacterial Canker of Tomato (BCT)

Authors: Asma Noshad; Mudassar Iqbal; Crispin Hetherington; Hassan Wahab
Journal: Advances in Agriculture
 (2020)

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## Abstract

Bacterial canker of tomato caused by the bacterial pathogenClavibacter michiganensissubsp. michiganensis(Cmm) is a major limiting factor for tomato production worldwide. Currently there exists no resistant variety of tomato to bacterial canker; only cultural and chemical controls are available. This study synthesized AgNPs (silver nanoparticles) via a green chemistry route and investigated their bactericidal potential against bacterial canker of tomato (BCT). AgNPs were prepared using mycellial aqueous extract of agriculturally beneficial fungi Pythium oligandrum.The formation of AgNPs was confirmed by using UV–Vis spectroscopy for the absorbance pattern while their morphology was investigated by the transmission electron microscopy (TEM). The X-ray diffraction profile for the biogenic AgNPs confirmed a crystalline structure with an average particle size of 12 nm. AgNPs treated seeds showed a normal germination rate with normal seedling growth. An in-vitro study found that the prepared AgNPs caused the maximum inhibition of the bacterial pathogen. In the greenhouse the introduction of AgNPs significantly prevents and inhibits the bacterial pathogen Cmm on tomato plants. These results suggest that this process is a strong candidate for industrial scale production of AgNPs. These particles act as an inhibitor and broad spectrum antibacterial agent against cmm, and hence offer a new and eco-friendly alternative in BCT control.


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## Body

## 1. Introduction

The development of multidrug resistant bacteria is becoming a severe problem, causing adverse effects on plants, animals, and public health [1–5], and resulting in millions of infections and thousands of deaths on a global scale [6]. Although a number of new antibiotics have been produced in the last couple of decades, the growth in antimicrobial resistance is faster than the current rate of developments in effective antibiotics production [7]. A significant decrease of over 50% in approved antibiotics in the last two decades has made it more alarming [8] and compels researchers to search for novel, appropriate, and efficient antimicrobial agents that are cost effective.BacteriumClavibacter michiganensissubsp. michiganensisis the causative agent of bacterial canker of tomato (BCT) [9, 10] and a major production restraint, causing significant economic losses worldwide [11–13]. Cmmstrains from different countries across the globe shows the same genetic profile [14]. Various biotic and abiotic stress factors contribute to this low yield however this study is focused on one of the biotic stress factors known as BCT. It is considered to be controlled by quarantine measures in Europe but is still prevalent in Pakistan and many other countries [15]. Possible reasons behind this could be the lack of appropriate quarantine measures and the use of infected seeds [16]. Lack of conventional resistance in tomato cultivars has made it very difficult to control.Research into conventional nanotechnology involves chemical and physical methods e.g. streptomycin has been used for decades as an antibacterial product [14] but there is a need to develop eco-friendly and cost-effective alternatives. Integration of a green chemistry approach by using microbes including bacteria, fungi, actinomycetes, yeast, and viruses for the synthesis of nanoparticles (NPs) could be a positive alternative [17]. Fungi in particular could play a crucial role through their ability to bioaccumulate metals. They are also preferred over other microbes for large scale production of NPs synthesis due to easy biomass handling [18]. Long before the pharmaceutical antibiotic revolution, silver has been used as antibacterial over a broad range of microorganisms [19–23] as silver ions are highly disruptive to bacterial integrity [24]. Bulk silver has been reported as an effective bactericide by many research studies [25, 26]; however, to boost the performance, nanotechnology could help by reducing bulk silver to the nano-scale [27–31]. Furthermore, AgNPs have recently attracted attention due to their antiviral effects against hepatitis B type virus and human immune deficiency virus (HIV-1) [32].Chemically synthesized NPs release silver in ionic form leading to the formation of reactive oxygen species ROS. These ROS on interaction with plants have shown adverse effects, not only by penetrating into the root but also causing physical damage to the plant surface. So this study aimed at synthesizing biogenic AgNPs using the nonpathogenic fungal strainP. oligandrum,and applying its antibacterial properties against BCT caused by Cmm. Very little work has been carried out concerning the prevalence of BCT and no research has been done on measures for controlling it. To our knowledge this is the first study to employ mycelial extract of P. oligandrum against BCT. The production of antimicrobial compounds by P. oligandrumand its ability to induce resistance in plants highlights its importance [33–35].


## 2. Experimental

### 2.1. Collection of Fungi and Growth Media

A pure culture ofP. oligandrum was obtained from the culture bank of the Department of Agricultural Chemistry, the University of Agriculture Peshawar, Pakistan. It was reidentified at the Department of Plant Pathology at the same University. For inoculation purposes, a pure culture of the same species was grown on a Potato Dextrose Agar (PDA) a general purpose growth medium PDA, incubated at 29 ± 2°C and then grown in a Potato Dextrose Broth (PDB) media for 7 days at 28°C to obtain the cell filtrate extract for biosynthesis studies. The mycelial cells were harvested after complete incubation of 7 days growth by filtering it twice through a Whatman filter paper followed by washing it trice with sterilized double distilled water. To get maximum extraction of the bioactive compounds, 50 gm of this biomass was brought into contact with 500 mL of sterile distilled water for 24 h followed by agitation and refiltration. The obtained filtrate was used for further biosynthesis of AgNPs.


### 2.2. Synthesis of AgNPs

The prepared mycelial filtrate ofP. oligandrumwas mixed with silver nitrate (AgNO3) solution in the ratio of 1 : 1. Three different concentrations 0.088 mg/L, 0.176 mg/L, and 0.44 mg/L were prepared by treating the AgNO3solution with the mycelial extract on a magnetic stirrer for 24 h at 29°C as illustrated in Figure 1(a). The biotransformation was monitored by following the color change from a light brown, Figure 1(b) to a pale white Figure 1(c) then to a dark brown after complete reduction in 24 h Figure 1(d). The formation of AgNPs was confirmed and quantified by spectrophotometric analysis. The AgNPs were separated by centrifugation (at 10,000 rpm for 15 min) after 24 h of the reaction. The supernatant was separated and the settled material was dried to obtain solid material for characterization purpose. No color change was observed in the control experiment when incubated under the same conditions. To check the stability of the reaction mixture, it was kept at room temperature for six months and the absorption was monitored regularly at 429 nm.Figure 1
(a) Silver nitrate solution. (b) Fungal mycelium extracts. (c) Mixture of solution and extract on magnetic stirrer for 24 h at 29°C. (d) Formation of AgNPs. Images reproduced with permission from Ref. [53] showing a typical progress of the synthesis of AgNPs using fungal extract (the actual material used was Trichoderma harzianum) as the starting material and silver nitrate salt solution as reducing agent.


### 2.3. Characterization of the AgNPs

For quantitative analysis of the synthesized AgNPs, a UV–Visible spectrophotometer with a split beam (model: Optima sp3000+, Japan) was operated at a resolution of 1 nm band. The absorbance spectrum was measured at wavelength ranges between 300 and 750 nm. To analyze the size, shape, and elemental composition of the synthesized silver nano particles, a High resolution Transmission Electron Microscope (HrTEM, JEOL 3000F) equipped with an energy dispersive X-ray (EDX) spectrometer was used. The biosynthesized AgNPs were subjected to a Powder X-ray diffraction PXRD (STOE Stadi MP) analysis for further insight into the phase variety and particle size and crystal structure. The machine was operated at a voltage of 40 kV and current of 40 mA with a scan rate of 0.01°/s. The coherently diffracting domain size of the AgNPs was calculated from the width of the XRD peaks using the Scherrer formula [36].


### 2.4. In-Vitro Assay

The synthesized AgNPs were subjected to antibacterial assay on PDA plates against Gram-positive bacterial strains includingXanthomonas campestris, Clavibacter michiganensis subsp. michiganensis, Streptococcus thermophilus,and Gram-negative bacterial strains including Bacillus subtilis, E. coli, Agrobacterium tumefaciensusing disk diffusion assay {Noshad, 2019 #153}. Each strain was swabbed uniformly onto individual plates; the sterile disks (5 mm; Oxide, UK) were loaded with AgNPs solution (0.088 mg/L, 0.176 mg/L, and 0.44 mg/L), aqueous extract, aqueous AgNO3 solution and a standard streptomycin as a positive control. Each plate was incubated at 29 ± 2°C for 24 h. The test was repeated twice and each treatment replicated three times. The zone of inhibition was measured in mm using calipers and recorded as mean ± S.E. (standard error).


### 2.5. Greenhouse Trials and Data Analysis

A screen house experiment was conducted to study the effect of biologically synthesized AgNPs fromP. oligandrumon controlling BCT. 6 weeks old plants were transplanted (1 seedling/pot) to 15 cm-diameter earthen pots. Each pot had 2 kg of standard commercial mix with slow release of fertilizer. Different doses, i.e., 0.088 mg/L, 0.176 mg/L, and 0.44 mg/L of AgNPs were mixed with the soil before transplanting seedlings. The potted plants were watered as needed. 6 weeks old plants were inoculated by clipping 3 inoculation sites per plant with scissors that had been dipped into a Cmm suspension. The sites are as follows: (1) a shoot, (2) one of the youngest actively growing leaves, and (3) the middle stem. The disease symptoms were assessed on the basis of severity index according to the scale of 0–4, where 0 = no canker symptoms, 1 = canker symptoms at the inoculation point only, 3 = canker expanding through the shoot, and 4 = canker affecting the shoot and other leaves down to the shoot. The experiment was terminated 6 weeks after transplanting and the data were recorded on disease severity, plant height (cm), tomato yield/plant (g), plant fresh biomass (g), number of shoots/plant, root weight (g), and plant dry biomass (g). The disease incidence and disease severity for each plant was calculated by using the following formula.(1)Diseaseincidence=Totalno.ofinfectedplantsTotalno.ofplantsassessed×100.The commercial chemical bactericide streptomycin was used as a positive control and distilled water was used as a negative control. Plants were monitored weekly and the status of observations was recorded. CRD with three replications was used for the experiment.


## 2.1. Collection of Fungi and Growth Media

A pure culture ofP. oligandrum was obtained from the culture bank of the Department of Agricultural Chemistry, the University of Agriculture Peshawar, Pakistan. It was reidentified at the Department of Plant Pathology at the same University. For inoculation purposes, a pure culture of the same species was grown on a Potato Dextrose Agar (PDA) a general purpose growth medium PDA, incubated at 29 ± 2°C and then grown in a Potato Dextrose Broth (PDB) media for 7 days at 28°C to obtain the cell filtrate extract for biosynthesis studies. The mycelial cells were harvested after complete incubation of 7 days growth by filtering it twice through a Whatman filter paper followed by washing it trice with sterilized double distilled water. To get maximum extraction of the bioactive compounds, 50 gm of this biomass was brought into contact with 500 mL of sterile distilled water for 24 h followed by agitation and refiltration. The obtained filtrate was used for further biosynthesis of AgNPs.


## 2.2. Synthesis of AgNPs

The prepared mycelial filtrate ofP. oligandrumwas mixed with silver nitrate (AgNO3) solution in the ratio of 1 : 1. Three different concentrations 0.088 mg/L, 0.176 mg/L, and 0.44 mg/L were prepared by treating the AgNO3solution with the mycelial extract on a magnetic stirrer for 24 h at 29°C as illustrated in Figure 1(a). The biotransformation was monitored by following the color change from a light brown, Figure 1(b) to a pale white Figure 1(c) then to a dark brown after complete reduction in 24 h Figure 1(d). The formation of AgNPs was confirmed and quantified by spectrophotometric analysis. The AgNPs were separated by centrifugation (at 10,000 rpm for 15 min) after 24 h of the reaction. The supernatant was separated and the settled material was dried to obtain solid material for characterization purpose. No color change was observed in the control experiment when incubated under the same conditions. To check the stability of the reaction mixture, it was kept at room temperature for six months and the absorption was monitored regularly at 429 nm.Figure 1
(a) Silver nitrate solution. (b) Fungal mycelium extracts. (c) Mixture of solution and extract on magnetic stirrer for 24 h at 29°C. (d) Formation of AgNPs. Images reproduced with permission from Ref. [53] showing a typical progress of the synthesis of AgNPs using fungal extract (the actual material used was Trichoderma harzianum) as the starting material and silver nitrate salt solution as reducing agent.


## 2.3. Characterization of the AgNPs

For quantitative analysis of the synthesized AgNPs, a UV–Visible spectrophotometer with a split beam (model: Optima sp3000+, Japan) was operated at a resolution of 1 nm band. The absorbance spectrum was measured at wavelength ranges between 300 and 750 nm. To analyze the size, shape, and elemental composition of the synthesized silver nano particles, a High resolution Transmission Electron Microscope (HrTEM, JEOL 3000F) equipped with an energy dispersive X-ray (EDX) spectrometer was used. The biosynthesized AgNPs were subjected to a Powder X-ray diffraction PXRD (STOE Stadi MP) analysis for further insight into the phase variety and particle size and crystal structure. The machine was operated at a voltage of 40 kV and current of 40 mA with a scan rate of 0.01°/s. The coherently diffracting domain size of the AgNPs was calculated from the width of the XRD peaks using the Scherrer formula [36].


## 2.4. In-Vitro Assay

The synthesized AgNPs were subjected to antibacterial assay on PDA plates against Gram-positive bacterial strains includingXanthomonas campestris, Clavibacter michiganensis subsp. michiganensis, Streptococcus thermophilus,and Gram-negative bacterial strains including Bacillus subtilis, E. coli, Agrobacterium tumefaciensusing disk diffusion assay {Noshad, 2019 #153}. Each strain was swabbed uniformly onto individual plates; the sterile disks (5 mm; Oxide, UK) were loaded with AgNPs solution (0.088 mg/L, 0.176 mg/L, and 0.44 mg/L), aqueous extract, aqueous AgNO3 solution and a standard streptomycin as a positive control. Each plate was incubated at 29 ± 2°C for 24 h. The test was repeated twice and each treatment replicated three times. The zone of inhibition was measured in mm using calipers and recorded as mean ± S.E. (standard error).


## 2.5. Greenhouse Trials and Data Analysis

A screen house experiment was conducted to study the effect of biologically synthesized AgNPs fromP. oligandrumon controlling BCT. 6 weeks old plants were transplanted (1 seedling/pot) to 15 cm-diameter earthen pots. Each pot had 2 kg of standard commercial mix with slow release of fertilizer. Different doses, i.e., 0.088 mg/L, 0.176 mg/L, and 0.44 mg/L of AgNPs were mixed with the soil before transplanting seedlings. The potted plants were watered as needed. 6 weeks old plants were inoculated by clipping 3 inoculation sites per plant with scissors that had been dipped into a Cmm suspension. The sites are as follows: (1) a shoot, (2) one of the youngest actively growing leaves, and (3) the middle stem. The disease symptoms were assessed on the basis of severity index according to the scale of 0–4, where 0 = no canker symptoms, 1 = canker symptoms at the inoculation point only, 3 = canker expanding through the shoot, and 4 = canker affecting the shoot and other leaves down to the shoot. The experiment was terminated 6 weeks after transplanting and the data were recorded on disease severity, plant height (cm), tomato yield/plant (g), plant fresh biomass (g), number of shoots/plant, root weight (g), and plant dry biomass (g). The disease incidence and disease severity for each plant was calculated by using the following formula.(1)Diseaseincidence=Totalno.ofinfectedplantsTotalno.ofplantsassessed×100.The commercial chemical bactericide streptomycin was used as a positive control and distilled water was used as a negative control. Plants were monitored weekly and the status of observations was recorded. CRD with three replications was used for the experiment.


## 3. Results

### 3.1. UV–Vis Spectroscopy

The production ofP. oligandrum mediated AgNPs was confirmed by observing the UV–Vis surface plasmon resonance peak (SPR) at 429 nm in the wavelength ranges of 350−800 nm as shown in Figure 2. The reduction of Ag+ into Ag0 was primarily observed through the color change of the suspension from yellowish to dark brown (see image inset in Figure 2). This is a robust indication for the formation of AgNPs. No color change for the control experiment suggested that the reducing agents released by the fungi are responsible for the reduction of Ag ions to AgNPs [37]. Previous work on AgNPs found that a SPR peak between 410–450 nm may be attributed to spherical AgNPs [38–40], and this was further confirmed by using TEM and EDX spectroscopic analysis. Fungal extract was taken as a control.Figure 2
A UV–Vis spectrum of AgNPs along with a photograph showing change in color during the formation of AgNPs. The photograph illustrates a typical color change during AgNPs formation “(the actual material used in this images wasAspergillus fumigatus)” The inserted image is used with permission from Ref. [40].


### 3.2. Is the Synthesis of AgNPs an Enzymatic Reaction?

To justify that the fabrication of AgNPs is an enzymatic reaction, enzymes in each of the fungal mycelia were denatured by boiling them for 15 minutes and were brought into contact with silver salt for reduction process. The solution mixture shows no color change along with no absorbance signifying no AgNPs production (Figure3(a)). A change in color from transparent to dark brown was observed when the fungal extract was exposed to AgNO3 salt solution in 1 : 1 ratio, signifying a reduction of Ag+into Ag0(Figure 3(b)) which was further confirmed by UV–Vis spectroscopic analysis. The absorbance peak at 430 nm for nonboiled biomass supports the hypothesis that enzymes secreted from fungi are responsible for reducing silver ions into AgNPs. The production of plant growth promoter auxin compounds e.g. tryptamine (TNH2) by P. oligandrumalso adds to its importance [41]. The obtained result is in complete agreement with previously published work [42–44].Figure 3
(a) Shows UV spectrum for the solution mixture of boiled fungal extract ofP. oligandrum and AgNO3 salt, showing no absorbance and hence no AgNPs production. (b) Represents untreated fungal extract exposed to AgNO3 salt solution showing absorbance peak centered at 430 nm.


### 3.3. Stability and Aggregation Pattern of AgNPs

Monodispersity and stability are considered very important features for NPs synthesis [15]. So the biogenic AgNPs were regularly monitored for six months in order to ensure their stability and agglomeration pattern. The obtained absorbance peaks 430 nm (Figure 4(b)) and TEM micrographs (Figure 4) showed the stability and well dispersed nature of the synthesized AgNPs.Figure 4
TEM images taken after six months of synthesis to show the nanoparticles stability. (a) A scanning TEM dark field image shows particles with white contrast, dispersed across the TEM support film. (b) A high resolution TEM image shows particles of sizes 6–12 nm. Internal structure such as twinning is visible. The lattice planes indicated by the arrow have a spacing of 0.22 nm which corresponds to the 111 type planes in silver.
(a)
(b)


### 3.4. Effect of pH

The pH for the reaction mixture was measured and recorded consecutively for 24 h, until a stable SPR peak was attained using UV spectroscopy. An increase in pH has been observed for all of the samples under study, and that higher value of pH stabilized within 24 h of incubation period as listed in Table1. At low pH, a pale white color was observed. The color then turned to dark brown with a subsequent increase in pH of the reaction which is in agreement with previous research studies [45, 46]. The production of smaller and regular round shape AgNPs has been observed for high pH, i.e. greater than a pH of 10 [46]. The pH values for the synthesized AgNPs on the selected PDB media were found to be in the range of 8.5–11.0.Table 1
Changes in pH for the reaction mixture during the synthesis of AgNPs.
Source
Reduction time (h)
UV–Vis peak (nm)
Color change
pH change
P. oligandrum
6
409.13
Pale yellow
8.5
12
413.15
Light brown
9.2
18
421.20
Brownish
10.0
24
429.01
Dark brown
10.9
24
429.01
Dark brown
11.0


### 3.5. Characterization

#### 3.5.1. TEM Analysis

TEM analysis revealed the size, shape, and agglomeration pattern of the biogenic AgNPs. Round shaped AgNPs with a range of particle size of around 2−13 nm were observed in this study (Figure5(a)). This outcome is in good agreement with our XRD analysis which suggested the presence of metallic crystalline AgNPs and gave an average crystallite size of 12 nm. To our knowledge, there has been no previous work on biogenic AgNPs from this fungal species so the results cannot be compared for consistency with previous reports. However mycelial extract of P. oligandrum has the potential to induce a defense response in plants and to suppress bacterial wilt in tomatoes. The observed particles were monodispersed with no agglomerated material.Figure 5
(a) Represents a dark field scanning TEM image of several AgNPs of different sizes sitting on the TEM support film. The circle drawn on the large particle indicates the approximate size of the probe from which the EDX spectrum in (b) is taken. The peak at 2.98 kV (black line) identifies the particle as silver. (Other peaks C, O, Si, and Cu come from the support film and specimen holder.)
(a)
(b)


#### 3.5.2. EDX Analysis

EDX analysis for AgNPs is presented in Figure5(b), showing energy in keV on the X-axis and number of counts on the Y-axis. The EDX spectrum contained a strong signal of silver (Ag). The presence of a strong signal for Ag indicates the high purity of silver. C, Cu, Si, and O signals come from the support film and the copper grid that are used for sample preparation for TEM analysis.


#### 3.5.3. XRD Analysis

The XRD profile for the biogenic AgNPs showed a face centered cubic (FCC) silver crystal structure (Figure6). The four diffraction peaks at 2θ values 38.11°, 46.19°, 64.44°, and 77.39° corresponding to lattice plane (1 1 1), (2 0 0), (2 0 2), and (3 1 1) originating from silver can be seen in Figure 6. The intense and sharp peak in the pattern confirms the crystalline nature of the AgNPs. An average crystallite size of 12 nm was calculated, and this figure is consistent with the TEM analysis that gave a particle size in the range of 2–13 nm.Figure 6
XRD profile for the synthesized AgNPs.


### 3.6. In-Vitro Antibacterial Assay

Figure7 displays the in-vitro inhibitory test for AgNPs using the disk diffusion method against Gram-positive and Gram-negative bacteria. High inhibitory activity was recorded for the biogenic AgNPs against all tested organisms. However, the highest zone of inhibition was observed against Cmm(sample number 1 in Figure 7) which was comparable with standard antibiotic. In comparison to control parameters of the experiment, statistically significant growth inhibition was observed even at the lower concentration of 0.088 mg/L. AgNPs with concentration of 0.176 mg/L and 0.44 mg/L showed comparable and higher level of inhibition as shown in Table 2.Figure 7
Antibacterial activity forP. oligandrum assisted with AgNPs against six bacterial strains.Table 2
Antibacterial activity of AgNPs synthesized by the aqueous extract ofP. oligandrum.
Indicator bacteria
AgNPs
AgNPs
AgNPs
Streptomycin
AgNO3
0.088 mg/L/disk
0.176 mg/L/disk
0.44 mg/L/disk
0.44 mg/L
0.44 mg/L
(1) Cmm
1.10 ± 0.10
1.14 ± 0.10
1.15 ± 0.10
1.16 ± 0.13
0.86 ± 0.08
(2) B. sub
0.86 ± 0.08
0.96 ± 0.10
0.98 ± 0.13
1.16 ± 0.13
0.86 ± 0.08
(3) Ag. tf
1.06 ± 0.03
1.10 ± 0.08
1.10 ± 0.00
1.25 ± 0.10
0.86 ± 0.08
(4) X. cmp
1.03 ± 0.13
1.03 ± 0.10
1.03 ± 0.11
1.36 ± 0.10
0.86 ± 0.08
(5) E. coli
0.96 ± 0.10
1.06 ± 0.10
1.06 ± 0.08
1.13 ± 0.11
0.86 ± 0.08
(6) Stp. Slv
1.13 ± 0.10
1.16 ± 0.10
1.16 ± 0.03
1.26 ± 0.10
0.86 ± 0.08
Note: zone of inhibition was measured in mm after incubation period of 24 h at 29°C in Agar medium and each value represents the average of three replicates. ± Values are the standard error. Cmm = Clavibacter michiganensis subsp. michiganensis, B. sub = Bacillus subtilis subsp. subtilis, Ag. tf = Agrobacterium tumefaciens subsp. tumefaciens, X. cmp = Xanthomonas campestris subsp. campestris, E. coli = Escherichia coli, Stp. Slv = Streptococcus thermophilus subsp. salivarius, S.E. = standard error, SN = silver nitrate.


### 3.7. Impact of Biogenic AgNPs on Germination of Seeds

As the synthesized AgNPs exhibited excellent inhibitory potential againstCmm,our work could proceed to experiments conducted on plants in a green house. Before this step, it was necessary to investigate in the laboratory possible effects of AgNPs on the germination rate of seeds. Both positive and negative impacts of AgNPs on seed germination have been reported [47, 48]. Previous studies reported that the toxicity of AgNPs could be specific to plant species, concentration, size of NPs, and exposure conditions [47, 49–51]. Figure 8 shows the germination rate measured on tomato seeds in this work. The germination rate was higher than the control (only water), even at the lowest concentration of 0.088 mg/L, suggesting that this concentration promotes seed germination. On the other hand, exposure to AgNO3solution resulted in a reduced germination rate while the fungal extract had no measurable effect on the germination rate. We suppose that the stability of AgNPs is enhanced by the fungal extract of P. oligandrumand that the toxicity is reduced, and this was supported by a previous study [48].Figure 8
A graph presenting the effect ofP. oligandrum assisted AgNPs on the germination of tomato seeds.


### 3.8. Greenhouse Trial

The germinated seeds were transferred to the greenhouse on the 12th day of germination. They were planted in pots and left for the next 6 weeks. On the 7th week of growth, the plants were exposed to AgNPs once after the symptoms appeared. Several plant growth parameters including plant height, fresh, and dry biomass, three shoots per plant and the disease incidence were measured during the experiment and the results are listed in (Table 3). Exposure to AgNPs inhibited the growth of Cmmwhile in the case of the control treatment Cmm appeared with clear symptoms on the leaves, shoots, and stem of the plant as shown in Figure 9. All of the growth parameters under consideration showed normal healthy growth while reduced growth was observed in case of control (AgNO3) treatments (Table 3). Significant increases in the plant growth parameters showed a beneficial effect of biogenic AgNPs against the harmful effects of bacterial pathogen Cmm. A large in-vitro inhibitory effect against Cmmwas observed for all concentrations of AgNPs (2−13 nm) and the application of the same concentrations in in the green house inhibited the growth of Cmm and enhanced plant growth as compared to control treatments. A comparable effect was observed for standard antibiotic and biogenic AgNPs application in the green house where as the control experiment did not reduce in bacterial pathogen Cmm. Importantly, the synthesized AgNPs appeared not to produce any undesirable effects on the tomato plant during the entire experiment. The in-vitro and screen house experiments were successful against BCT and showed similar pattern of the results as observed in seed germination.Table 3
Impact of AgNPs on disease incidence percentage, plant height, fresh biomass, dry biomass, and the shoot per plant grown under greenhouse conditions.
Treatment
Plant height cm
Fresh biomass (g)
Dry biomass (g)
Shoot/plant
Disease incidence (%)
0.088 mg/L
65.2
284.38
70.79
5
28.57
0.176 mg/L
66
308.32
75
5
23.8
0.44 mg/L
66.8
315.28
75.07
6
23.8
Streptomycin
72.8
381.6
95.2
7.6
20
AgNO3
39
105.2
39.5
2.4
100
Water
36.8
159.4
27.88
2.4
100Figure 9
Taken during greenhouse experiment, showing infestation of tomato plants withCmm culture, BCT symptoms, and normal healthy growth of tomato plants.


## 3.1. UV–Vis Spectroscopy

The production ofP. oligandrum mediated AgNPs was confirmed by observing the UV–Vis surface plasmon resonance peak (SPR) at 429 nm in the wavelength ranges of 350−800 nm as shown in Figure 2. The reduction of Ag+ into Ag0 was primarily observed through the color change of the suspension from yellowish to dark brown (see image inset in Figure 2). This is a robust indication for the formation of AgNPs. No color change for the control experiment suggested that the reducing agents released by the fungi are responsible for the reduction of Ag ions to AgNPs [37]. Previous work on AgNPs found that a SPR peak between 410–450 nm may be attributed to spherical AgNPs [38–40], and this was further confirmed by using TEM and EDX spectroscopic analysis. Fungal extract was taken as a control.Figure 2
A UV–Vis spectrum of AgNPs along with a photograph showing change in color during the formation of AgNPs. The photograph illustrates a typical color change during AgNPs formation “(the actual material used in this images wasAspergillus fumigatus)” The inserted image is used with permission from Ref. [40].


## 3.2. Is the Synthesis of AgNPs an Enzymatic Reaction?

To justify that the fabrication of AgNPs is an enzymatic reaction, enzymes in each of the fungal mycelia were denatured by boiling them for 15 minutes and were brought into contact with silver salt for reduction process. The solution mixture shows no color change along with no absorbance signifying no AgNPs production (Figure3(a)). A change in color from transparent to dark brown was observed when the fungal extract was exposed to AgNO3 salt solution in 1 : 1 ratio, signifying a reduction of Ag+into Ag0(Figure 3(b)) which was further confirmed by UV–Vis spectroscopic analysis. The absorbance peak at 430 nm for nonboiled biomass supports the hypothesis that enzymes secreted from fungi are responsible for reducing silver ions into AgNPs. The production of plant growth promoter auxin compounds e.g. tryptamine (TNH2) by P. oligandrumalso adds to its importance [41]. The obtained result is in complete agreement with previously published work [42–44].Figure 3
(a) Shows UV spectrum for the solution mixture of boiled fungal extract ofP. oligandrum and AgNO3 salt, showing no absorbance and hence no AgNPs production. (b) Represents untreated fungal extract exposed to AgNO3 salt solution showing absorbance peak centered at 430 nm.


## 3.3. Stability and Aggregation Pattern of AgNPs

Monodispersity and stability are considered very important features for NPs synthesis [15]. So the biogenic AgNPs were regularly monitored for six months in order to ensure their stability and agglomeration pattern. The obtained absorbance peaks 430 nm (Figure 4(b)) and TEM micrographs (Figure 4) showed the stability and well dispersed nature of the synthesized AgNPs.Figure 4
TEM images taken after six months of synthesis to show the nanoparticles stability. (a) A scanning TEM dark field image shows particles with white contrast, dispersed across the TEM support film. (b) A high resolution TEM image shows particles of sizes 6–12 nm. Internal structure such as twinning is visible. The lattice planes indicated by the arrow have a spacing of 0.22 nm which corresponds to the 111 type planes in silver.
(a)
(b)


## 3.4. Effect of pH

The pH for the reaction mixture was measured and recorded consecutively for 24 h, until a stable SPR peak was attained using UV spectroscopy. An increase in pH has been observed for all of the samples under study, and that higher value of pH stabilized within 24 h of incubation period as listed in Table1. At low pH, a pale white color was observed. The color then turned to dark brown with a subsequent increase in pH of the reaction which is in agreement with previous research studies [45, 46]. The production of smaller and regular round shape AgNPs has been observed for high pH, i.e. greater than a pH of 10 [46]. The pH values for the synthesized AgNPs on the selected PDB media were found to be in the range of 8.5–11.0.Table 1
Changes in pH for the reaction mixture during the synthesis of AgNPs.
Source
Reduction time (h)
UV–Vis peak (nm)
Color change
pH change
P. oligandrum
6
409.13
Pale yellow
8.5
12
413.15
Light brown
9.2
18
421.20
Brownish
10.0
24
429.01
Dark brown
10.9
24
429.01
Dark brown
11.0


## 3.5. Characterization

### 3.5.1. TEM Analysis

TEM analysis revealed the size, shape, and agglomeration pattern of the biogenic AgNPs. Round shaped AgNPs with a range of particle size of around 2−13 nm were observed in this study (Figure5(a)). This outcome is in good agreement with our XRD analysis which suggested the presence of metallic crystalline AgNPs and gave an average crystallite size of 12 nm. To our knowledge, there has been no previous work on biogenic AgNPs from this fungal species so the results cannot be compared for consistency with previous reports. However mycelial extract of P. oligandrum has the potential to induce a defense response in plants and to suppress bacterial wilt in tomatoes. The observed particles were monodispersed with no agglomerated material.Figure 5
(a) Represents a dark field scanning TEM image of several AgNPs of different sizes sitting on the TEM support film. The circle drawn on the large particle indicates the approximate size of the probe from which the EDX spectrum in (b) is taken. The peak at 2.98 kV (black line) identifies the particle as silver. (Other peaks C, O, Si, and Cu come from the support film and specimen holder.)
(a)
(b)


### 3.5.2. EDX Analysis

EDX analysis for AgNPs is presented in Figure5(b), showing energy in keV on the X-axis and number of counts on the Y-axis. The EDX spectrum contained a strong signal of silver (Ag). The presence of a strong signal for Ag indicates the high purity of silver. C, Cu, Si, and O signals come from the support film and the copper grid that are used for sample preparation for TEM analysis.


### 3.5.3. XRD Analysis

The XRD profile for the biogenic AgNPs showed a face centered cubic (FCC) silver crystal structure (Figure6). The four diffraction peaks at 2θ values 38.11°, 46.19°, 64.44°, and 77.39° corresponding to lattice plane (1 1 1), (2 0 0), (2 0 2), and (3 1 1) originating from silver can be seen in Figure 6. The intense and sharp peak in the pattern confirms the crystalline nature of the AgNPs. An average crystallite size of 12 nm was calculated, and this figure is consistent with the TEM analysis that gave a particle size in the range of 2–13 nm.Figure 6
XRD profile for the synthesized AgNPs.


## 3.5.1. TEM Analysis

TEM analysis revealed the size, shape, and agglomeration pattern of the biogenic AgNPs. Round shaped AgNPs with a range of particle size of around 2−13 nm were observed in this study (Figure5(a)). This outcome is in good agreement with our XRD analysis which suggested the presence of metallic crystalline AgNPs and gave an average crystallite size of 12 nm. To our knowledge, there has been no previous work on biogenic AgNPs from this fungal species so the results cannot be compared for consistency with previous reports. However mycelial extract of P. oligandrum has the potential to induce a defense response in plants and to suppress bacterial wilt in tomatoes. The observed particles were monodispersed with no agglomerated material.Figure 5
(a) Represents a dark field scanning TEM image of several AgNPs of different sizes sitting on the TEM support film. The circle drawn on the large particle indicates the approximate size of the probe from which the EDX spectrum in (b) is taken. The peak at 2.98 kV (black line) identifies the particle as silver. (Other peaks C, O, Si, and Cu come from the support film and specimen holder.)
(a)
(b)


## 3.5.2. EDX Analysis

EDX analysis for AgNPs is presented in Figure5(b), showing energy in keV on the X-axis and number of counts on the Y-axis. The EDX spectrum contained a strong signal of silver (Ag). The presence of a strong signal for Ag indicates the high purity of silver. C, Cu, Si, and O signals come from the support film and the copper grid that are used for sample preparation for TEM analysis.


## 3.5.3. XRD Analysis

The XRD profile for the biogenic AgNPs showed a face centered cubic (FCC) silver crystal structure (Figure6). The four diffraction peaks at 2θ values 38.11°, 46.19°, 64.44°, and 77.39° corresponding to lattice plane (1 1 1), (2 0 0), (2 0 2), and (3 1 1) originating from silver can be seen in Figure 6. The intense and sharp peak in the pattern confirms the crystalline nature of the AgNPs. An average crystallite size of 12 nm was calculated, and this figure is consistent with the TEM analysis that gave a particle size in the range of 2–13 nm.Figure 6
XRD profile for the synthesized AgNPs.


## 3.6. In-Vitro Antibacterial Assay

Figure7 displays the in-vitro inhibitory test for AgNPs using the disk diffusion method against Gram-positive and Gram-negative bacteria. High inhibitory activity was recorded for the biogenic AgNPs against all tested organisms. However, the highest zone of inhibition was observed against Cmm(sample number 1 in Figure 7) which was comparable with standard antibiotic. In comparison to control parameters of the experiment, statistically significant growth inhibition was observed even at the lower concentration of 0.088 mg/L. AgNPs with concentration of 0.176 mg/L and 0.44 mg/L showed comparable and higher level of inhibition as shown in Table 2.Figure 7
Antibacterial activity forP. oligandrum assisted with AgNPs against six bacterial strains.Table 2
Antibacterial activity of AgNPs synthesized by the aqueous extract ofP. oligandrum.
Indicator bacteria
AgNPs
AgNPs
AgNPs
Streptomycin
AgNO3
0.088 mg/L/disk
0.176 mg/L/disk
0.44 mg/L/disk
0.44 mg/L
0.44 mg/L
(1) Cmm
1.10 ± 0.10
1.14 ± 0.10
1.15 ± 0.10
1.16 ± 0.13
0.86 ± 0.08
(2) B. sub
0.86 ± 0.08
0.96 ± 0.10
0.98 ± 0.13
1.16 ± 0.13
0.86 ± 0.08
(3) Ag. tf
1.06 ± 0.03
1.10 ± 0.08
1.10 ± 0.00
1.25 ± 0.10
0.86 ± 0.08
(4) X. cmp
1.03 ± 0.13
1.03 ± 0.10
1.03 ± 0.11
1.36 ± 0.10
0.86 ± 0.08
(5) E. coli
0.96 ± 0.10
1.06 ± 0.10
1.06 ± 0.08
1.13 ± 0.11
0.86 ± 0.08
(6) Stp. Slv
1.13 ± 0.10
1.16 ± 0.10
1.16 ± 0.03
1.26 ± 0.10
0.86 ± 0.08
Note: zone of inhibition was measured in mm after incubation period of 24 h at 29°C in Agar medium and each value represents the average of three replicates. ± Values are the standard error. Cmm = Clavibacter michiganensis subsp. michiganensis, B. sub = Bacillus subtilis subsp. subtilis, Ag. tf = Agrobacterium tumefaciens subsp. tumefaciens, X. cmp = Xanthomonas campestris subsp. campestris, E. coli = Escherichia coli, Stp. Slv = Streptococcus thermophilus subsp. salivarius, S.E. = standard error, SN = silver nitrate.


## 3.7. Impact of Biogenic AgNPs on Germination of Seeds

As the synthesized AgNPs exhibited excellent inhibitory potential againstCmm,our work could proceed to experiments conducted on plants in a green house. Before this step, it was necessary to investigate in the laboratory possible effects of AgNPs on the germination rate of seeds. Both positive and negative impacts of AgNPs on seed germination have been reported [47, 48]. Previous studies reported that the toxicity of AgNPs could be specific to plant species, concentration, size of NPs, and exposure conditions [47, 49–51]. Figure 8 shows the germination rate measured on tomato seeds in this work. The germination rate was higher than the control (only water), even at the lowest concentration of 0.088 mg/L, suggesting that this concentration promotes seed germination. On the other hand, exposure to AgNO3solution resulted in a reduced germination rate while the fungal extract had no measurable effect on the germination rate. We suppose that the stability of AgNPs is enhanced by the fungal extract of P. oligandrumand that the toxicity is reduced, and this was supported by a previous study [48].Figure 8
A graph presenting the effect ofP. oligandrum assisted AgNPs on the germination of tomato seeds.


## 3.8. Greenhouse Trial

The germinated seeds were transferred to the greenhouse on the 12th day of germination. They were planted in pots and left for the next 6 weeks. On the 7th week of growth, the plants were exposed to AgNPs once after the symptoms appeared. Several plant growth parameters including plant height, fresh, and dry biomass, three shoots per plant and the disease incidence were measured during the experiment and the results are listed in (Table 3). Exposure to AgNPs inhibited the growth of Cmmwhile in the case of the control treatment Cmm appeared with clear symptoms on the leaves, shoots, and stem of the plant as shown in Figure 9. All of the growth parameters under consideration showed normal healthy growth while reduced growth was observed in case of control (AgNO3) treatments (Table 3). Significant increases in the plant growth parameters showed a beneficial effect of biogenic AgNPs against the harmful effects of bacterial pathogen Cmm. A large in-vitro inhibitory effect against Cmmwas observed for all concentrations of AgNPs (2−13 nm) and the application of the same concentrations in in the green house inhibited the growth of Cmm and enhanced plant growth as compared to control treatments. A comparable effect was observed for standard antibiotic and biogenic AgNPs application in the green house where as the control experiment did not reduce in bacterial pathogen Cmm. Importantly, the synthesized AgNPs appeared not to produce any undesirable effects on the tomato plant during the entire experiment. The in-vitro and screen house experiments were successful against BCT and showed similar pattern of the results as observed in seed germination.Table 3
Impact of AgNPs on disease incidence percentage, plant height, fresh biomass, dry biomass, and the shoot per plant grown under greenhouse conditions.
Treatment
Plant height cm
Fresh biomass (g)
Dry biomass (g)
Shoot/plant
Disease incidence (%)
0.088 mg/L
65.2
284.38
70.79
5
28.57
0.176 mg/L
66
308.32
75
5
23.8
0.44 mg/L
66.8
315.28
75.07
6
23.8
Streptomycin
72.8
381.6
95.2
7.6
20
AgNO3
39
105.2
39.5
2.4
100
Water
36.8
159.4
27.88
2.4
100Figure 9
Taken during greenhouse experiment, showing infestation of tomato plants withCmm culture, BCT symptoms, and normal healthy growth of tomato plants.


## 4. Discussion

This study was focused on the antibacterial potential of biologically synthesized AgNPs against BCT caused byCmm. From the disk diffusion method it was determined that the synthesized AgNPs inhibited bacterial growth and deactivated a number of different groups of Gram-positive and Gram-negative bacteria. Results suggest that AgNPs act as a broad spectrum antibacterial agent as there was no significant difference in the bacterial assay for six tested bacterial strains. It was also observed that AgNPs lead to a bactericidal effect rather than bacteriostatic which is in agreement with previous research [52]. This bactericidal potential of AgNPs makes it a potential candidate to eliminate rather than merely inhibit the spread of infection, and many research studies support it [53]. But since the target bacteria was Cmmit was hypothesized that it could be an option to inhibit bacterial canker. So based on the results obtained in the in-vitrotrial, a greenhouse experiment was designed to observe the effect of AgNPs not only on seed germination but also on the mature tomato plant. Seedlings exposed to both concentrations of AgNPs showed full germination after 12 days. Some studies in the past considered a threshold value of AgNPs toxicity of 5 mg/L–1000 mg/L [54–56] whereas here 0.088 mg/L, 0.176 mg/L, and 0.44 mg/L were used which as the lowest possible concentration in this study. We found that the toxicity of AgNPs depends on the concentration used, as this study has not observed any significant toxic effect for root elongation and germination of tomato seedlings. There could be two possible reasons (i) an agglomeration of AgNPs that takes place when they are exposed to the ecosystem and this reduces their toxicity [55] (ii) the ability of the seed coat and endosperm to serve as a filter, passing water and absorbing the metals [56]. Overall the biosynthesized AgNPs did not noticeably affect seed germination. So the germinated seeds were transferred to the green house to evaluate the effect of AgNPs against BCT. To compare the outcome of the trial, standard commercial soil was used and the seedlings were left to grow for six weeks. Very low concentration 0.088 mg/L, 0.176 mg/L, and 0.44 mg/L were used as treatment dose. The synthesized AgNPs suppressed the occurrence of tomato death due to bacterial canker compared to the control experiment under greenhouse conditions. Compared to the control, enhanced plant growth parameters were also assessed in the greenhouse. The obtained results suggested that P. oligandrum mediated AgNPs could be a positive step to treat or prevent the damages caused by Cmm. Next study will be carried out to explore if any possible toxicity exists in the soil system and edible tomatoes due to the application of AgNPs.


## 5. Conclusions

This study has successfully synthesized AgNPs using the aqueous extract of agriculturally beneficial fungiP. oligandrum. The synthesized AgNPs exhibited strong broad spectrum bactericidal potential even at the small concentration of 0.088 mg/L. This study has not observed any negative influence of AgNPs on in-vitro seed germination rate as well on plant physiology during the green house experiment. Results suggested that the biogenic AgNPs can be a promising biocide against the propagation of Cmminfection in tomato plant and a suitable candidate to be used in pharmaceutical products and medical devices against the transmission of drug-resistant pathogens. So the prepared biogenic AgNPs could be an economical, eco-friendly, easy, and extensively useful facility in agricultural applications.



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*Source: 9630785-2020-01-03.xml*