Green synthesis and evaluation of silver nanoparticles for antimicrobial and biochemical profiling in Kinnow (Citrus reticulata L.) to enhance fruit quality and productivity under biotic stress

Abstract

Green synthesis of silver nanoparticles (AgNPs) by utilising plant extract is an emerging class of nanotechnology. It revolutionizes all the field of biological sciences by synthesizing chemical free AgNPs. In the present study, AgNPs were synthesised by utilising Moringa oleifera leaves as the main reducing and stabilising agent and characterised through UV–visible spectroscopy, zeta analyser, X‐ray diffraction spectroscopy (XRD), energy dispersive X‐ray (EDX), and scanning electron microscopy (SEM). The different concentrations of biosynthesised AgNPs (10, 20, 30, and 40 ppm) were exogenously applied on the already infected plants (canker) of Citrus reticulata at different day intervals. The AgNPs at a concentration of 30 ppm was found to be most suitable concentration for creating the resistance against canker disease in Citrus reticulata. The enzymatic activities were also explored and it was found that 30 ppm concentration of biosynthesised AgNPs significantly reduced the biotic stress. Fruit quality and productivity parameters were also assessed and it was found that fruit quality and productivity were significant in response to 30 ppm concentration of biosynthesised AgNPs. The present work highlights the potent role of biosynthesised AgNPs, which can be used as biological control of citrus diseases and ultimately improving the quality and productivity of Citrus.

1 Introduction

Citrus is one of the most horticultural and economically important tree fruit crops in the world [1, 2]. Citrus belongs to family rutaceae and comprises 18% of the total fruit production [3]. Among the citrus cultivar, Citrus reticulata (Kinnow) is the most extensively growing citrus fruit crop in Pakistan [4]. About 95% of the total Kinnow mandarin is produced in Pakistan [4]. The annual production of Kinnow mandarin in Pakistan is less when compared with the industrialised countries of the world [5]. The low yield is due to an attack of a diverse range of pathogens, i.e. viruses, bacteria, and fungi [6]. From the development of Kinnow fruit to feeding and various other purposes, Kinnow fruit is affected by a wide variety of pathogens during the long‐term storage [7].

Among the various citrus diseases, Asiatic citrus canker (ACC) whose causal agent is Xanthomonas axonopodis pv. citri is the prevailing disease in the citrus crop and it is a pathogen of major concern to the citrus industry in the subtropical regions of the world [8]. This disease is characterised by conspicuous necrotic lesions surrounded by yellow halo that develops on leaves, twigs, and fruits [9]. Different methods can be utilised for protecting the plant cells infected from X. axonopodis. Different bactericides are currently utilised to control ACC [10]. Unnecessary use of pesticides triggered the contamination in the environment; therefore, scientists are searching different protection measures against various types of pathogens. Nanotechnology offers a great promise in this regard.

Nanotechnology is renowned the science of recent century and its applications have been extended in different fields of physics, biology, and chemistry [11]. Green synthesis of nanoparticles (NPs) is an expending area due to their applications in different fields such as functional biology, biosensor, and antimicrobial [12]. Green synthesis of NPs by utilising plant extract is the most commonly used method having distinctive advantages that plants are widely distributed, less bio hazardous, easily assessable, and act as a source of active constituents [13, 14]. Among the NPs, silver nanoparticles (AgNPs) represent one of the most comprehensively studied nanomaterials which fascinate researchers due to their broad‐spectrum antimicrobial efficacy [15].

The production of reactive oxygen species (ROS) under stress conditions can interact with biological molecules and prevent the growth of plants. In such unfavourable conditions, plant cells use endogenous enzymes, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), that neutralise the damaging effects of ROS [16]. Antioxidative enzymes are key contributors in mericlones development and the alteration of oxygen radical to H₂ O₂ [17, 18]. Most of the recent studies are focused on the role of NPs in plant growth, various physiological, and biochemical attributes and fruit quality and productivity parameters [19, 20, 21].

So far, few studies have been reported regarding the exogenous application of NPs in different plant species. The role of green synthesised AgNPs in ameliorating the disease intensity, biochemical profiling, and various quality and productivity parameters in Kinnow mandarin is still to be explored. The present study has been planned to check the effect of M. oleifera leaves extract‐mediated AgNPs in directing the disease intensity against canker, biochemical profiling, fruit quality, and productivity in Kinnow mandarin. According to the research, this appears to be the first study that utilises M. oleifera leaves extract‐mediated AgNPs in directing the infection index value and biochemical profiling to enhance the fruit quality and productivity of Kinnow under biotic stress.

2 Materials and methods

2.1 Green synthesis of AgNPs

The green synthesis of AgNPs was carried out by following the protocol described by Hussain et al. [21] with slight alterations. The most commonly used salt for the synthesis of AgNPs is the silver nitrate (AgNO₃). The solution of AgNO₃ (5 mM) was prepared by dissolving the 0.85 g AgNO₃ in 1 l distilled water. The M. oleifera leaves extract were used for the reduction in silver salt to AgNPs. The extract of M. oleifera was gradually added in silver nitrate solution (SNS) with continuous boiling at 100°C for 10 min. The solution was then centrifuged at 14,000 rpm for 10 min. The pellet was collected and centrifuged again at 14,000 rpm for 10 min. This process was repeated thrice to remove remains of unreacted plant extract and silver nitrate. The pellet was dried by utilising SpeedVac concentrator. The biologically synthesised AgNPs were used for assessing disease incidence against canker, biochemical profiling, fruit quality, and productivity parameters of C. reticulata under biotic stress.

2.2 Characterisation of AgNPs

2.2.1 UV–Visible spectroscopy

The reduction in AgNO₃ to AgNPs was observed by recording the UV–Visible spectrum at the International Islamic University, Islamabad. The synthesised AgNPs were added to sterilised water and then subjected to ultra‐sonication for 5–10 min. UV–Visible spectrum analysis was done by recording the UV–Visible spectrum from 300 to 700 nm.

2.2.2 Zeta analyzer

The size range of AgNPs was determined through a zeta analyzer from the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad. The synthesised NPs were ultrasonicated for 15 min, and then the size range was determined through zeta potential.

2.2.3 X‐ray diffraction spectroscopy

The crystalline nature of the synthesised AgNPs was determined by using X‐ray diffraction (XRD) at the National Centre for Physics (NCP), Islamabad. For the diffraction pattern analysis, powdered samples were used on a Shimadzu XRD‐6000 in the range of 5°–50° at a 2θ angle. The average size of the synthesised AgNPs was calculated by using the Debye–Scherrer equation

$$D=k\lambda /\beta \cos \theta$$

where k is the shape factor, λ the wavelength of the X‐ray, β the full width in radians at half maximum, and θ the Bragg's angle.

2.2.4 Energy‐dispersive X‐ray spectroscopy

The elemental analysis of the green synthesised AgNPs was also done at the IST. The energy‐dispersive X‐ray (EDX) detector was utilised for the elemental analysis of the green synthesised AgNPs by dropping the synthesised NPs on carbon film.

2.2.5 Scanning electron microscopy

The structural analysis of the synthesised AgNPs was examined by scanning electron microscopy (SEM) using a SIGMA model operated at 5 kV, magnification ×10 K, from the Institute of Space and Technology (IST), Islamabad. Then, a film of the sample was prepared on a carbon‐coated copper grid by dropping the suspension of AgNPs in water on the grid. The extra solution was removed by utilising the blotting paper, and then the film on the SEM grid was allowed to dry by putting it under a mercury lamp for 5 min. The sample surface images were taken at different magnifications.

2.3 Survey of canker incidence

Survey was conducted in Kinnow mandarin orchids at three different localities (Ratto Kala, Chak No. 6 AML, and Ahli Sheikh Raju) of Bhalwal Tehsil of District Sargodha, Pakistan. Ten Kinnow mandarin plants from each locality were randomly selected and disease intensity was then recorded by utilising the disease rating scale as determined by Horsfall and Heuberger [22].

• 0, free from infection,

• 1, 0–25% leaf area infected,

• 2, 25–50% leaf area infected,

• 3, 50–75% leaf area infected,

• 4, 75–100% leaf area infected.

An infection index was then recorded by the following formula

$$\mathrm{Disease}\mathrm{incidence}=\mathrm{Sum}\mathrm{of}\mathrm{individual}\mathrm{rating}/\mathrm{Total}\mathrm{number}\mathrm{of}\mathrm{plants}\mathrm{assessed}\times 100/4$$

2.4 Evaluation of different concentrations of green synthesised AgNPs against X. axonopodis

Most susceptible 3‐year‐old Kinnow plants of Chak No. 6 AML were sprayed with different concentrations (10, 20, 30, and 40 ppm) of green synthesised AgNPs for five times with an interval of 3 days to record the disease intensity. The disease incidence was recorded at 5 day intervals up to 30 days. A correlation was established between the appropriate concentration of M. oleifera leaves extract‐mediated AgNPs and disease incidence (Table 1).

Table 1.

Layout

Treatments	Disease incidence
T0	control
T1	pathogen
T2	pathogen + 10 ppm AgNPs
T3	pathogen + 20 ppm AgNPs
T4	pathogen + 30 ppm AgNPs
T5	pathogen + 40 ppm AgNPs

2.5 Biochemical parameters

2.5.1 Extraction of leaves

Fresh leaves of Kinnow mandarin were collected and extract of endogenous enzymes was prepared by following the protocol described by Nayyar and Gupta [23] with minor modification. About 100 mg fresh leaves were extracted in 10 ml extraction buffer. The extract was then sonicated three times with a resting period of 30 min. The extract was then centrifuged at 10,000 rpm for 10 min and supernatant was collected and utilised for enzymatic assays.

2.5.2 SOD activity

SOD activity was determined by following the protocol described by Ullah et al. [24] with minor modifications. The reaction mixture (2 ml) was composed of 600 µl enzyme extract, 200 µl of 0.075 mM NBT, 200 µl of 130 mM methionine, 200 µl of 1 mM EDTA, 20 µl of 0.02 mM riboflavin, and 780 µl phosphate buffer. The blank was prepared in the same way except the phosphate buffer was added instead of enzyme extract. The blank and reaction mixture were placed for 7 min under fluorescent light and absorbance was recorded at 560 nm by using spectrophotometer. The Lambert–Beer law was then applied to calculate the SOD activity

$$A=\epsilon LC$$

where $\epsilon$ is the extinction coefficient, L the length of wall, and C the concentration of enzymes.

2.5.3 POD activity

POD activity was determined by following the protocol described by Lagrimini [25] with minor modifications. The reaction mixture (2 ml) was composed of 200 µl enzyme extract, 200 µl of 27.5 mM hydrogen peroxide (H₂ O₂), 200 µl of 100 mM Guaiacol, 400 µl phosphate buffer, and 1 ml distilled water. The blank was prepared in the same way except the phosphate buffer was added instead of enzyme extract. The blank and reaction mixture were placed for 7 min under fluorescent light and absorbance was recorded at 470 nm by utilising a spectrophotometer.

2.5.4 CAT activity

CAT activity was determined by following the protocol described by Aebi [26] with minor modifications. The reaction mixture (2 ml) was composed of 500 µl enzyme extract, 200 µl of 27.5 mM hydrogen peroxide (H₂ O₂), 400 µl phosphate buffer, and 900 µl distilled water. The blank was prepared in the same way except the phosphate buffer was added instead of enzyme extract. The blank and reaction mixture were placed for 7 min under fluorescent light and absorbance was recorded at 240 nm by using a spectrophotometer.

2.6 Fruit quality parameters

2.6.1 Determination of juice contents

The juice contents were weighed and recorded in grams [27]. The % juice contents were calculated by using the following formula

$$\text{\%}\mathrm{juice}\mathrm{contents}=\mathrm{juice}\mathrm{weight}÷\mathrm{fruit}\mathrm{weight}\times 100$$

2.6.2 Determination of pH

The juice pH for each sample was determined using calibrated pH meter.

2.6.3 Vitamin C content

Ascorbic acid (AA) content was determined by following the protocol described by Hans [28]. About 5 g pulp was grinded in 5 ml of 1% HCl by utilising mortar and pestle and mixture was then centrifuged at 10,000 rpm for 10 min. Absorbance was recorded at 243 nm by using a spectrophotometer.

2.7 Fruit productivity parameters

The fruit productivity parameters are:

1. fruit weight,

2. yield/plant,

3. equatorial diameter,

4. polar diameter (PD),

5. peel thickness (PT).

2.8 Statistical analysis

The experiment consisted of three replicates per treatment and each experiment was repeated twice. These results were interpreted as mean standard deviation. Randomised complete block design was applied for the statistical analysis of the experiment data utilising statistical package for social sciences (SPSS 16.1).

3 Results and discussion

3.1 Synthesis and characterisation of AgNPs

The biosynthesis of AgNPs was carried out by utilising M. oleifera leaves as main reducing and stabilising agent. The aqueous silver ions were reduced to AgNPs when the prepared plant extract of M. oleifera was added. Plant extracts may contain certain concentrations of active ingredients, which are intended to play a key role in the synthesis of NPs [29]. Hussain et al. [30] reported that functional groups of plant flavonoids are actually involved in the reduction and capping of AgNPs (Fig. 1).

Fig. 1.

Flowchart for the synthesis of AgNPs

The M. oleifera leaves extract‐mediated AgNPs were characterised through UV–Visible spectroscopy, zeta analyser, EDX, XRD, SEM, and atomic force microscopy. UV–Visible spectroscopy is the most commonly used technique for examining the reduction and capping of AgNPs. The combination of different techniques is usually required because a single technique is unable to study all the characters of the synthesised AgNPs. Different characterisation peaks usually in the range of 410–480 nm are obvious for AgNPs synthesis [31, 32]. However, different wavelengths may attribute different sizes, shapes, and nature of the synthesised AgNPs [33] (Fig. 2).

Fig. 2.

UV–Visible spectrum of the green synthesised AgNPs

The synthesised AgNPs were characterised through zeta potential for determining the size range. Fig. 3 clearly elucidates that M. oleifera leaves extract‐mediated AgNPs ranged from 8 to 28 nm. The presence of metallic silver ions was confirmed by the EDX detector (Fig. 4). EDX spectrum elucidated strong absorption peak of metallic silver ions in the range 2.5–3.5 keV, while silver nanocrystal showed absorption peaks in the range of 2.5–4 keV. Our findings are in line with some other researchers who reported the absorption peaks of the silver ions presence by utilising an EDX detector [34, 35].

Fig. 3.

Size distribution analysis of the green synthesised AgNPs

Fig. 4.

EDX spectrum of the green synthesised AgNPs

The crystalline nature of the M. oleifera leaves extract‐mediated AgNPs was confirmed by XRD (Fig. 5). The average size of the synthesised AgNPs was calculated to be ∼ 21.64 nm. Our findings are in line with Mie et al. [35] who reported similar nature of green synthesised ANPs. Some non‐crystalline peaks were also observed at 35.63°, 38.23°, and 47.25° which are also apparent in many other works [34, 36]. These diffraction peaks might be due to the presence of active constituents which are involved in capping of AgNPs. The morphology of the green synthesised AgNPs was observed by SEM (Fig. 6). The SEM elucidated that green synthesised AgNPs were rectangular segments fused together in shape.

Fig. 5.

XRD diffractogram of the green synthesised AgNPs

Fig. 6.

SEM micrograph of the green synthesised AgNPs

3.2 Citrus canker incidence in Tehsil Bhalwal

Survey was conducted regarding the canker disease incidence in three different localities of Tehsil Bhalwal, District Sargodha (Table 2). Kinnow orchids situated at Tehsil Bhalwal revealed 6.5, 8.0, and 7.5% disease incidence of ten randomly selected plants of Ratto Kala, Chak No. 6 AML, and Ahli Sheikh Raju, respectively. The Kinnow plants situated at Chak No. 6 AML were more susceptible to ACC when compared with the other two localities of Tehsil Bhalwal, District Sargodha. The major factor which is involved in the enhanced susceptibility of citrus in Chak No. 6 AML might be due to edaphic and climatic factors.

Table 2.

Incidence of citrus canker in three different localities of Tehsil Bhalwal, Punjab, Pakistan

Sr. No.	Location	Per cent disease of 10 randomly selected plants
	Tehsil Bhalwal
01	Ratto Kala	6.5
02	Chak No. 6 AML	8.0
03	Ahli Sheikh Raju	7.5

3.3 Field evaluation of various concentrations of green synthesised AgNPs against Xanthomonas axonopodis at Chak No. 6 AML (Jhlaran Warraicha)

Disease intensity was recorded against X. axonopodis pv. citri in response to various concentrations of green synthesised AgNPs at different time intervals: 5, 10, 15, 20, 25, and 30. The effect of the green synthesised AgNPs on the occurrence of canker disease varied greatly depending upon the number of days passed after applying different concentrations of the green synthesised AgNPs. The experiment was conducted for 30 days and the first data was collected after 5 days (Table 3). It was observed that none of the concentration of the green synthesised AgNPs completely inhibited the symptoms of canker disease at different day intervals; however, the intensity of disease mainly depends upon the concentration of the exogenously applied AgNPs.

Table 3.

Evaluation of disease incidence at various concentrations of AgNPs against different day intervals

Tr.	Disease incidence at different day intervals
	05	10	15	20	25	30
T0	0.00	0.00	0.00	0.00	0.00	0.00
T1	3.13 ± 0.15	4.27 ± 0.21	4.76 ± 0.23	5.83 ± 0.29	7.50 ± 0.37	8.00 ± 1.37
T2	7.00 ± 0.35	4.51 ± 0.22	3.11 ± 0.15	2.03 ± 0.10	2.00 ± 0.10	2.81 ± 0.57
T3	4.15 ± 0.20	3.74 ± 0.18	3.72 ± 0.36	2.57 ± 0.12	1.79 ± 0.08	3.38 ± 0.51
T4	2.47 ± 0.12	2.03 ± 0.10	1.87 ± 0.09	1.56 ± 0.07	1.08 ± 0.05	1.87 ± 0.42
T5	4.79 ± 0.23	4.51 ± 0.25	4.01 ± 0.20	3.79 ± 0.18	3.67 ± 0.18	4.92 ± 0.34

The incidence of canker disease intensity was progressively decreased with the passage of time in all the treatments of green synthesised AgNPs up to 25 days. The Kinnow plants which were not treated with the green synthesised AgNPs under biotic stress revealed highest infection index values at different day intervals. The minimum disease incidence was recorded in the plants which were exogenously sprayed with a 30 ppm concentration of the green synthesised AgNPs. The infection index values were significantly reduced after 25 days (2.00, 1.79, 1.08, and 3.67) in response to 10, 20, 30, and 40 ppm concentration of M. oleifera leaves extract‐mediated AgNPs when compared with T1 treatment. Disease intensity was increased after 25 days in all the applied treatments.

We found that AgNPs at the concentration of 30 ppm reduced the disease intensity against canker at different day intervals in comparison to the other concentrations of AgNPs. The present findings are in line with Hussain et al. [37] who reported similar results in an attempt to find the effect of green synthesised AgNPs on brown spot disease in Kinnow mandarin. Our findings are also in strong agreement with Sahi et al. [38] who recorded the incidence of citrus canker disease caused by X. axonopodis pv. citri on Kinnow mandarin at different localities. Under biotic stress, ROS are produced via the enhanced production of NADPH oxidase, amine oxidases, and peroxidases in the cell [39].

3.4 Antioxidative enzymes

In the present study, the major antioxidative enzymes (SOD, POD, and CAT) of Kinnow leaves were studied in response to different concentrations of green synthesised AgNPs under biotic stress. The enhanced production of SOD, POD, and CAT was found when Kinnow plants were under biotic stress. The application of AgNPs significantly reduced the production of antioxidative enzymes by reducing the stress (Figs. 7 and 8). It is evident from Fig. 7 that the SOD (0.51 nM/min/mg FW) and POD (0.27 nM/min/mg FW) activities were significant in Kinnow under biotic stress. The findings of Hussain et al. [21, 30] also affirmed our results in an attempt to find the effect of pathogen and application of AgNPs in C. reticulata. POD and SOD show dependency and linear correlation with CAT. CAT activity was also significant (0.16 nM/min/mg FW) in Kinnow plants which were susceptible to X. axonopodis and exogenous application of AgNPs was not done. The major cause of the enhanced production of endogenous enzymes could be the stress. Various researchers reported the enhanced production of antioxidative enzymes in different plants [17, 40]. Both biotic and abiotic stresses result in the enhanced production of ROS which ultimately damage the cells [41]. Different enzymes are produced to neutralise the effect of ROS [42]. These antioxidative enzymes such as CAT, POD, SOD, and APX form a complex system of endogenous enzymes which sift both toxic‐free radicals and other non‐radical oxygen species [41]. The application of AgNPs significantly reduced the stress and ultimately reduced the production of endogenous enzymes. The present work is in agreement with Iqbal et al. [43, 44] who reported that AgNPs significantly reduced the stress.

Fig. 7.

SOD and POD activities of C. reticulata in response to different treatments

Fig. 8.

CAT activity of C. reticulata in response to different treatments

3.5 Fruit quality parameters

The juice content and pH of Kinnow fruits were assessed in response to different concentrations of the green synthesised AgNPs under biotic stress (Fig. 9). X. axonopodis significantly affect the juice yield of Kinnow mandarin. The juice content was significantly reduced when Kinnow plants were not treated with synthesised AgNPs under biotic stress. The maximum juice content (45.2%) was observed when Kinnow plants were sprayed with 30 ppm concentration of the green synthesised AgNPs under biotic stress. The present findings are in agreement with the previous reports [45]. The statistical analysis of Kinnow juice pH elucidated non‐significant difference among the different treatments of the synthesised AgNPs under biotic stress. The exogenous applications of various concentrations of green synthesised AgNPs under biotic stress did not affect the Kinnow juice pH which ranged from 3.4 to 3.7. Similar results were obtained by Najar et al. [46] in an attempt to find the effect of pathogen infection on pH of different citrus cultivars.

Fig. 9.

Juice yield and pH of C. reticulata in response to different treatments

The AA content was also assessed in Kinnow fruits in response to different concentrations of the green synthesised AgNPs under biotic stress. It is clear from Fig. 10 that AA content was significantly reduced in Kinnow plants under biotic stress. The maximum AA content was observed under control treatment (T0) followed by T4, T5, T3, T2, and T1, respectively. The vitamin C content was also investigated by other researchers for comparative analysis in the fruits of different citrus cultivars [47, 48].

Fig. 10.

AA content of C. reticulata in response to different treatments

3.6 Fruit productivity parameters

Fruit weight and yield per plant of Kinnow were explored in response to different concentrations of green synthesised AgNPs under biotic stress (Fig. 11). The statistical analysis of fruit weight and yield per plant elucidated the significant increase in response to 30 ppm concentration of the synthesised AgNPs when compared with the other treatments. The plants which were not sprayed with AgNPs under biotic stress showed significantly less fruit production per plant (97.1 kg) as well reduced fruit size (178.2 g). The present findings are in line with some other researchers who reported the effect of stress on yield in different commercial citrus orchids [49, 50]. A similar pattern was observed by Velez et al. [51] who reported that an increase in fruit weight resulted in the enhanced production of yield per tree.

Fig. 11.

Fruit weight and yield per plant of C. reticulata in response to different treatments

Some other productivity parameters such as equatorial diameter (ED), (PD), and (PT) were also explored in response to different concentrations of green synthesised AgNPs under biotic stress (Fig. 12). The significant results were obtained for ED (71 mm), PD (57 mm), and PT (5.1 mm) in C. reticulata plants when supplemented with a 30 ppm concentration of the synthesised AgNPs under biotic stress. The ED, PD, and PT were significantly reduced in plants which were susceptible to X. axonopodis and exogenous application of the synthesised AgNPs was not done. A similar pattern was reported by Tejero et al. [50] with a fall in PD of fruit and slight rise in ED of fruit.

Fig. 12.

ED, PT, and PT of C. reticulata in response to different treatments

4 Conclusion

In the present study, a novel protocol has been developed for safe, rapid, easy, and cost‐effective biosynthesis of the green synthesised AgNPs by utilising M. oleifera leaves as the main reducing and capping agent. The synthesised AgNPs have the potential in developing the resistance against X. axonopodis pv. citri and alters the biochemical profiling in C. reticulata. The fruit quality and productivity parameters were significantly enhanced in response to green synthesised AgNPs. The present findings pave the way for the more comprehensive further study about the ecotoxicity of NPs and changes at molecular level by the application of NPs under biotic stress in future.