Using of Biosynthesized Zinc Oxide Nanoparticle and Vinegar as a Substitute of Insecticides on Vicia faba Plant

doi:10.21203/rs.3.rs-3455855/v1

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Research Article

Using of Biosynthesized Zinc Oxide Nanoparticle and Vinegar as a Substitute of Insecticides on Vicia faba Plant

https://doi.org/10.21203/rs.3.rs-3455855/v1

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The current research study includes a process of green safety method for synthesizing zinc oxide nanoparticles (ZnO NPs) from Musa paradisiaca L. leaf plant extract as an efficient chelating and capping agent for ZnO NPs from Zinc acetate dihydrate salt, and use with vinegar as a substitute of Carbaryl insecticide on Vicia faba plant. UV-Vis and FTIR studies of Musa paradisiaca L. leaf extract and ZnO NPs demonstrated that this plant is an excellent candidate for the green synthesis of ZnO NPs. From using various characterization techniques of XRD, FTIR, UV-Vis. spectroscopy and TEM, the structure, morphology, chemical composition and crystallinity of ZnO NPs were investigated. The XRD pattern spectrum and TEM, and FTIR confirmed the pure crystallinity and the spherical-shape of the hexagonal wurtzite ZnO NP structure with an average size 47 nm and with the measurement results of ZnO NPs band gap was around 4.52 eV. Seven treatments each with three replications were sprayed by the leaves including: 100 ppm ZnO NPs, 200 ppm ZnO NPs, 1% vinegar, 10% vinegar, recommended dose (1X) of Carbaryl, double dose (2X) of Carbaryl and a control (without any treatment). Following Carbaryl (2X) spraying, 100 ppm of ZnO NPs has shown the greatest effects on death rate and corrected mortality rate (Mc %) of Aphis fabae by values of 87.5% and 5.15% respectively. Moreover, 200 ppm ZnO NPs increased soil TDS and EC to 270.65 ± 0.029 ppm and 189.49 ± 0.003 µS.cm^− 1 respectively. Thus, it can be concluded that the biosynthesized ZnO NPs and vinegar are good and natural substitutes of chemical pesticides.

Biosynthesized ZnO nanoparticles

Characterizations

Vinegar

Insecticide

Vicia faba L.

Soil Pollution

In recent decades, nanotechnology has played a crucial role, particularly in enhancing the quality of life [1]. The functionality of micro-particles is strongly influenced by their properties, including size, shape, and wettability [2]. Nanomaterials (NMs) are generally defined as materials with at least one dimension within the range of 1-100 nanometers, with the diameter falling typically within the same range. Various methods have been employed for the synthesis of ZnO nanoparticles (NPs) [3]. In response to growing environmental concerns, a significant shift in research focus has taken place. Many researchers now prioritize eco-friendly approaches to sustain our planet [4]. As a result, there is an increasing demand to develop environmentally friendly methods for nanoparticle production that do not depend on harmful ingredients. Several techniques have been employed for nanoparticle preparation, including chemical, physical, and biological methods. Among these approaches, microorganisms, enzymes, and plant extracts have been proposed as alternatives to traditional chemical methods [5]. Biological synthesis by plant species has proven to be an effective method for large-scale production of metal or metal oxide nanoparticles, all without the use of toxic or hazardous chemicals [6]. Recently, various plant parts, such as leaves [7], peels [8], bark [9], flowers [10], seeds [11], and tubers [12], have been employed in the process to extract and synthesize ZnO nanoparticles. It have been investigated as potential insecticides for controlling pest populations in agriculture and other settings [13]. While the use of ZnO NPs for insect control is still in the early stages of research, there is evidence to suggest that they could be effective in controlling insect populations in some situations [14]. However, it is important to note that the use of ZnO NPs for insect control raises concerns about their potential impact on non-target organisms and the environment [15]. Various studies have demonstrated that ZnO nanoparticles can exert a range of effects on different insect species, encompassing toxicological, behavioral, and physiological impacts. These findings hold significant implications for the application of ZnO in agriculture and pest control [16–19]. Moreover, wood vinegar, has excellent biocidal properties and its application would avoid the use of chemical pesticides that have serious effects on human health and environment [20]. It has been widely used as insect repellants from plants and households in different areas of the world [21].

In the world, the fourth great important crop is faba bean (Vicia faba L.) or field bean [22]. that has a high nutritive value and considered as the most important grain legumes for food and feed of human and animal [23, 24]. Faba bean is an excellent nitrogen fixer and contributor to sustainable agriculture through improvement of soil fertility and mimic the key role in crop rotation [25]. Nowadays, devastating diseases, reduced soil fertility, yield instability as well as insufficient, and absence of effective and compatible Rhizobium strains in the soil always have declined the cultivation and production of faba bean [26]. Nitrogen fixation (N₂-fixation) process is very sensitive to the availability of N and P nutrients [27]. Therefore, this study aimed; first the biosynthesis of ZnO NPs from Musa paradisiaca L. leaf plant extract and their characterizations, and second, to show and probe the effects of this biosynthesized ZnO NPs as well as wood vinegar on the yield and the productivity of faba bean, as well as their effects on soil microbial population when they applied as a substitute of chemical insecticides such as carbaryl. Since, there is minimal scientific evidence on the insect repellent efficacy of wood vinegar because it has not been widely studied or published. The plant growth parameters like plant height, leaves number and branches, chlorophyll at leaf and seed germination were investigated in this work beside microbial population including total bacteria, total fungi and total actinomycetes.

2.1 Materials

Musa paradisiaca L. leaves were collected in Erbil City. The city of Erbil, located at Latitude 36°12’ Longitude 44° 04’, is the capital of Iraqi Kurdistan Region. The plant material was identified by Prof. Dr. Abdullah Sh. Sardar, College of Education Salahaddin University-Erbil, and the voucher specimen was deposited at (ESUH) with ID 9028. Distilled water, deionized water, Zinc acetate dihydrate [Zn (CH₃CO₂)₂·2H₂O] and sodium hydroxide (NaOH) were purchased from sigma-Aldrich. Vinegar and the insecticide (Carbaryl) on Vicia faba plant has used for the purpose of application.

2.1.1 Preparation of plant extract

The Fig. 1 (A-E) showing the plant extraction process as photographically. It can be seen that Musa paradisiaca L. extract was prepared from 100 gm thoroughly washed fresh Musa paradisiaca L. leaves with distilled water (Fig. 1A). They cutting to small pieces (Fig. 1B), then 20 grams of chopped Musa paradisiaca L. leaves, made into a paste, using mortar when mixed with 5 ml of distilled water (Fig. 1C). After that, 6 gm of the paste is heated and stirred in 100 ml of distilled water for an hour at 70°C (Fig. 1D), filtered with Whatmann No 1 filter paper and the resulting extract was cooled down at room temperature (Fig. 1E) and used for synthesis of metal oxide (ZnO NPs).

2.1.2 Green synthesis of Zinc Oxide NPs

ZnO NPs were synthesized using the green synthesis method according to the steps shown in Fig. 2 (A-F). 30 ml aqueous of plant extract was mixed with 3 gm of Zinc acetate dehydrate and boiled over magnetic stirrer with hot plate at temperature 90 ℃ and stirrer with 400 rpm, then drops of NaOH solution with 1M added until the solution color changed from dark yellow to yellowish-white color. After completion of reaction, 50 ml of deionized water was added to the paste and settled for 24 hours in order to precipitate the NPs, then dried in oven (at 90 ℃) to remain only the NPs. The paste was transferred into a crucible, which was calcinated at 400 ℃ for 2 in order to obtain pure ZnO NPs, and to remove the basic solution, the content was washed thoroughly and repeatedly with distilled water. Finally, the resulting sample was kept in a dry area for further characterization.

2.1.3 Preparation of solutions

2.1.3.1 Wood vinegar

Wood vinegar obtained from the local markets of Erbil City. Two concentrations of commercial vinegar were prepared 1% and 10%.

2.1.3.2 Preparation of Carbaryl insecticide

Carbaryl insecticide (85% WP) (Table 1 and Fig. 3) was prepared at two different doses Commercial recommended dose (1X) and two-fold dose (2X) which was prepared by adding a double amount of the insecticide [28].

Table 1

Description of Carbaryl insecticide.
ISO Common name	Carbaryl (E-ISO, (m) F-ISO, ANSI, BAN, BSI, ESA, JMAF)
Chemical name	IUPAC: 1-naphthyl methylcarbamate; CA: 1-naphthalenyl-methylcarbamate
Trade name	Adios, Bugmaster, Carbamec, Carbamine, Crunch, Denapon, Dicarbam, Hexavin, Karbaspray, Nac, Rayvon, Septene, Sevin, Tercyl, Torndao, Thinsec, Tricarnam, and Union Carbide 7744
Chemical class	Carbamate
Empirical formula	C₁₂H₁₁NO₂
Relative molecular mass	201.2 g.mol^-1
Formulation	Bait, dusts, wettable powders, granules, dispersions, and suspensions
Usage class	Insecticide
Mode of action	Disrupts the normal functioning of the insect nervous system and may cause toxicity by contact or ingestion
CAS Registry number	63-25-2
CIPAC number	26
EINECS/ELINCS number	200-555-0
Identity tests	HPLC retention time, ¹H-NMR
Recommended dose	300–400 g.200 L^-1 water
Half-life in soil	7–14 days in sandy loam soil and 14 to 28 days in clay loam soil
Half-life in vegetation	˂2 weeks
LD50	The oral LD50 (250–850 mg.kg^-1 in rats), (100–650 mg.kg^-1 in mice). The inhalation LC50 in rats is ˃200 mg/L. Dermal LD50 is ˃2000 mg/kg.
WHO toxicity classification	II = Moderately hazardous
Signal Word	DANGER - POISON, WARNING, or CAUTION depending on the product formulation

Source: [28–31].

2.2 Methods

2.2.1 Soil sample selection

In September 2022, a soil with no prior history of pesticide use was collected from Aski-Kalak in Erbil province (Table 2). According to [32], samples were taken from the upper soil layer (0–30 cm depth) at a random pattern around each area.

Table 2

Description of the studied soil.
Location			Aski-Kalak
1.	Coordination of locations	North	36˚15' 22.9"
1.	Coordination of locations	East	43˚38' 20.6"
2.	Particle size distribution (g.kg^− 1)	Clay	52.10
		Silt	251.10
		Sand	696.80
		Texture class	Sandy Loam
3.	Organic matter (g.kg^− 1)		12.5
4.	pH		7.79
5.	Electrical conductivity (µS.cm^− 1)		695

Information were taken from [33].

2.2.2 Pot experiment layout and plant cultivation

A completely randomized design with three replications (Table 3) was performed including seven treatments [ZnO NPs 100 ppm, ZnO NPs 200 ppm, Vinegar 1%, Vinegar 10%, Carbaryl 1X, Carbaryl 2X and control (no treatment)]. For this purpose, 21 pre-labeled and similar plastic pots with average diameter 27 cm and height 25 cm were prepared and each filled with 5 kg soil. The pots were supplied with below containers to collect and return the leached irrigation water. The pots were irrigated daily by tap water at 60% of field capacity. This experiment last for four months.

During the autumn growth season of 2022 and under controlled environmental conditions in the glasshouse/ College of Science/ Salahaddin University, at 25/14 ℃ and natural light [34–36], pots were planted with seeds of the Spanish variety (Lashbona) of (Vicia faba L.) that obtained from Erbil Alwa for agricultural materials. For each pot, 4 faba bean seeds were sown in about 2–5 cm deep, and spaced 3–6 cm apart. Seeds were then thinned to one plant.

After that, each triplicate was sprayed separately with above concentrations of ZnO NPs, wood vinegar and Carbaryl insecticide and left. The following vegetative growth measurements were recorded two weeks before the end of the experiment on February 15, 2022; these were plant length (cm), number of leaves per plant, number of branches per plant and Chlorophyll at leaf.

2.2.3 Laboratory analysis

In the laboratory, approximately 200 g of soil samples were collected in pre-labeled glass bottles from each pot, and presented for assessment of microbiological and soil chemical properties. Laboratory works were performed in Biology and Environmental Science and Health Departments.

2.2.3.1 Vegetative growth characteristics

The plant growth characteristics included in this study were plant fresh and dry weights, plant height, number of leaves and branches as well as chlorophyll at leaf.

2.2.3.2 Seed germination and difference from control (DFC %)

The percentage of seed germination was measured 10 days after seed planting. Inside separate petri plates containing absorbed filter papers with ZnO NPs, wood vinegar and Carbaryl insecticide as well as distilled water as control, 100 seeds were placed on the filter papers. For each of 10 days, the filter papers have kept moist and the number of germinated seeds were recorded. Seed germination was estimated according to the equation given by [37].

$\text{\%} \text{S}\text{e}\text{e}\text{d} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n}= \frac{\text{s}\text{e}\text{e}\text{d}\text{s} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{e}\text{d} \text{i}\text{n} \text{p}\text{e}\text{s}\text{t}\text{i}\text{c}\text{i}\text{d}\text{e} \text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}{\text{s}\text{e}\text{e}\text{d}\text{s} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{e}\text{d} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}$ × 100 …………… (1)

The percentage of difference from control (% DFC) for germination was calculated from the formula of Mhatre and Chaphekar (1982) given by [38]:

$\text{\%} \text{D}\text{F}\text{C}= \frac{\text{\%} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{\%} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} P. farcta \text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\text{s} }{\text{\%} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}$ × 100 …………...…. (2)

2.2.3.3 Microbiological analysis

Aseptically collected soil samples were serially diluted from 10^− 1-10^− 7 for each treatment according to [39]. Standard plate technique was used for counting the whole soil bacteria, fungi and actinomycetes using nutrient agar, Sabouraud dextrose agar contained 0.3 mg penicillin and water agar respectively. Media were prepared according to [40]. Total number of bacteria expressed as number of bacteria ×10⁶ cfu.g^− 1 dry soil, while fungi and actinomycetes as total fungi and actinomycetes ×10⁵ cfu.g^− 1 dry soil.

2.2.3.4 Toxicity tests on black aphid (Aphis fabae)

The use of an analytic technique to estimate the risks of insect death in the presence or absence of multiple causes is called mortality, therefore, potentially has far-reaching implications for population ecology [41].

After 96 hours of aphid infestation, pots were sprayed with zinc oxide nanoparticles, vinegar and Carbaryl insecticide, and then broad bean plants of the same size were chosen and were placed on benches in the greenhouse. Effects of aphid (Aphis fabae) feeding were estimated at initial infestation levels of 10 aphids per plant. Treatments were arranged in a completely randomized design (CRD) as [ZnO NPs 100 ppm, ZnO NPs 200 ppm, Vinegar 1%, Vinegar 10%, Carbaryl 1X, Carbaryl 2X and control (no treatment)] and each treatment was replicated three times. Screen cloth mesh was mounted over each treatment to prevent aphid contamination between treatments.

2.2.3.4.1 Calculation of death rate

Death rate was calculated according to the below formula [42].

$$\text{D}\text{e}\text{a}\text{t}\text{h} \text{r}\text{a}\text{t}\text{e}=\frac{EquationNumber of death }{total EquationNumber} \times 100$$

…………………………

2.2.3.4.2 Corrected mortality rate

The calculation of mortality rate was corrected for control mortality according to Abbott’s formula given by [42].

$$\text{M}\text{c}=\frac{Mo - Me}{100-Me} \times 100$$

……………………………….

Mo = Mortality rate of treated adults (%)

Me = Mortality rate of control (%)

Mc = Corrected Mortality rate (%).

2.2.3.5 Chemical analysis

Soil pH and EC were estimated in 1:1 soil solution according to [43]. Soil organic matter was estimated according to Walkley-Black method as described by [43].

2.2.3.6 Statistical analysis

Statistical analysis of the treatments was conducted using Microsoft Office Excel 2019 and expressed as mean ± standard error values. Each of the experimental values was compared to control. The results were analyzed by one-way ANOVA and Duncan’s test at the level of significant 0.05 [44] with used Statistical Package for Social Sciences (SPSS) Version 26.

3.1 ZnO nanoparticles characterization

3.1.1 UV-Vis spectra analysis:

Zinc oxide nanoparticles (ZnO NPs) synthesized using Musa paradisiaca L. plant extract was studied. The formation of ZnO NPs was confirmed by UV-vis spectrophotometer. UV–vis absorption spectrum of synthesized zinc oxide nanoparticles was recorded for the sample in the range of 200–800 nm. The strong absorption was make out 274 nm as shown in (Fig. 4) corresponding to band gap energy was 4.52ev, by the using of the calculation based on the formula E_g=1240/λ which is in small variance with results obtained by [45] and previously reported values [46] could be due to disagreeing in average crystal size of the NPs.

3.1.2 FTIR analysis

In order to determine the functional groups in ZnO NPs synthesized from Musa paradisiaca L. leaves aqueous extract the FTIR spectroscopy technique instruments were

used. (Fig. 5) shows the fingerprint region of green synthesized Zinc Oxide nanoparticles appears in the band width range of 604.37–400 cm^− 1. The peak around 2924 cm^− 1, 2852 cm^− 1 corresponds to C-H stretching. The peak at 1440.83 refers to C = C stretching vibrations of the alkene group. Due to the water adsorption of the surface of synthesized Zinc Oxide nanoparticles, the OH stretching bond vibration has appeared as shown as a broad peak around 3435.22 cm^− 1.

3.1.3 XRD analysis

The X-ray diffraction (XRD) pattern of green synthesized ZnO NPs has been illustrated in (Fig. 6). Synthesized ZnO NPs patterns were exhibited three predominant crystalline peaks 2θ value at 31.9, 34.55 and 36.4° which indexed to the main three planes 100, 002, and 101, respectively, and eight low peaks at 47.8, 56.85, 63.15, 66.5, 68.1, 69.2, 72.7 and 77.15° attributed to (102), (110), (103), (200), (112), (201), (004) and (202) planes, respectively. These crystalline peaks confirm the existence of pure formation of the hexagonal wurtzite phase. The detected dominant diffraction peak on the (101) plane and crystal dimension can be calculated by using the famous Shearers equation:

$$\varvec{D}= \frac{0.94 \varvec{\lambda }}{\varvec{\beta } \varvec{c}\varvec{o}\varvec{s} \varvec{\theta }}$$

………………………..

Where, D is the crystalline dimension, $\varvec{\lambda }$ is the X-ray wavelength, equal to 1.5406 A°, $\varvec{\beta }$ is the full width of the peak. The calculated average crystal size of ZnO NPs was 49 nm as our results agree with those of [47, 48] for different plant-mediated ZnO NP synthesis.

3.1.4 TEM analysis

Transmission electron microscopy of the synthesized of zinc oxide nanoparticles illustrated in (Fig. 7) at 50 nm scale It was observed that the ZnO NPs have spherical shape with an average size about 47 nm.

3.2 Vegetative growth characteristics

Effects of nanoparticles can be either positive or negative depending upon the plant species and type of nanoparticles used as well as its concentration [21]. By the present study, 100 ppm of ZnO NPs has shown the shortest (35 cm) plant height (Table 3), indicating that ZnO NPs and vinegar exerted non-significant negative effect on plant height. While, double dose of Carbaryl insecticide has shown the tallest plant height 47.5 cm.

However, the greatest numbers of leaves were 46 observed in pots sprayed with double dose of Carbaryl insecticide, while, the least leaf number was 17 which detected in control indicating that all treatments increased leaf number significantly when compared to control.

Zinc oxide nanoparticles (100 ppm) showed significant increase in numbers of branches (35) and the same results were observed by [35] in Mosul of Iraq when they treated broad bean with 100 and 200 ppm zinc oxide nanoparticles. Whereas, 1% vinegar gave little branch numbers (17) when compared with control.

Vinegar 10% and ZnO NPs (100 ppm) showed significant increase in chlorophyll at leaf, so the greatest chlorophyll at leaf was 49.48 in pots sprayed with 10% vinegar followed by 46.23 chlorophyll at leaf in pots sprayed with ZnO NPs (100 ppm), while double dose of Carbaryl insecticide suppressed chlorophyll synthesis to 39.57 chlorophyll at leaf. The mechanism of action of vinegar is the lack of metabolic degradation in plants or residual activity in the soil made it a very popular herbicide choice [49]. In a related study of [20] observed highest rate of chlorophyll in maize plant when sprayed the leaves with nano-iron. Moreover, [50] found that showed that, faba bean plots treated with bioorganic fertilizers combined with nanoparticles gave statically maximum effective number of tillers, number of pods per plant, hundred seeds weight, grain yield, biomass yield and harvest index than over other treatments. Furthermore, [51] found that foliar spraying of citric acid caused a significant increase in plant height, branch number, leaf number, leaf area per plant, pods number, green pod yield per plant, green pod productivity, fresh seed yield per plant and productivity, pod length, weight of 100 seeds, percentage of dry matter on seeds and total soluble solids compared with control treatment.

Table 3

Average growth parameters of *Vicia faba* two weeks after spraying with vinegar, ZnO NPs and Carbaryl (Mean ± S.E.).
Treatments		Plant height (cm)	No. leaves	No. branches	Chlorophyll at leaf
ZnO NPs	100 ppm	35.0 ± 1.732^a	38 ± 6.640^a	35 ± 5.485^a	46.23 ± 0.652^ab
ZnO NPs	200 ppm	37.5 ± 3.175^a	45 ± 4.041^a	24 ± 1.732^b	41.98 ± 1.316^cd
Vinegar	1%	38.0 ± 0.577^a	39 ± 7.506^a	17 ± 2.887^b	43.88 ± 1.224^bc
Vinegar	10%	36.5 ± 0.866^a	37 ± 0.000^a	23 ± 0.866^b	49.48 ± 1.068^a
Carbaryl insecticide	R. dose	40.0 ± 2.309^a	30 ± 1.155^b	20 ± 1.732^b	41.15 ± 0.318^cd
Carbaryl insecticide	D. dose	47.5 ± 3.175^a	46 ± 6.928^a	26 ± 3.753^b	39.57 ± 0.713^d
Control (without treatment)		42.0 ± 5.196^a	17 ± 0.577^b	23 ± 0.577^b	45.67 ± 1.637^b
Maximum		47.5 ± 3.175	46 ± 6.928	35 ± 5.485	49.48 ± 1.068
Minimum		35.0 ± 1.732	17 ± 0.577	17 ± 2.887	39.57 ± 0.713
Different letters indicate significant differences among the treatments

3.3 Seed germination and difference from control (DFC %)

Maximum seed germination of 80% was observed in treated pots with both doses of ZnO NPs (Fig. 8) with no difference from control (Fig. 9), whereas minimum germination was 55% that observed in pots treated with double dose of Carbaryl insecticide when compared with control as illustrated in (Fig. 8) with 31.25% difference from control (Fig. 9). In a relative study conducted by [52] they found that the seed germination percentage and sprout growth rates were significantly higher in the plants treated with 500 mg.L^− 1 of ZnO NPs (100% ± 0.00), compared to the control plants (93.33% ± 1.66).

3.4 Microbiological analysis

By this study, 200 ppm of ZnO NPs have enhanced bacterial growth to 18.82×10⁶ cfu.g^− 1 dry soil with compared with control; however, recommended dose of Carbaryl has reduced bacterial count to 0.99×10⁶ cfu.g^− 1 dry soil when compared with control (Table 4). Furthermore, 100 ppm of ZnO NPs have increased both fungal and actinomycetes population to 12.29 and 7.59×10⁵ cfu.g^− 1 dry soil respectively in comparing with control which showed the lowest fungal 2.27×10⁵ cfu.g^− 1 dry soil and actinomycetes 2.61×10⁵ cfu.g^− 1 dry soil population. There is no sufficient literatures about the effects of ZnO NPs on soil microorganisms, however, some of them indicated that ZnO NPs reduced microbial biomass, diversity and enzyme activities and there are no reports on the influence of ZnO NPs on ammonification yet. The adverse effect of ZnO NPs on soil microorganisms in neutral and acid soils is worthy mentioned [53]. Moreover, the effects of spraying vinegar on faba bean showed no significant effects on soil microorganisms as the same as obtained by [54] in Turkey, hence, the statement of using different wood vinegar doses to protect plant and/or crop in wheat cultivation system without negative effects on microbial communities in soil may be true, especially in terms of 3% which showed a positive effect on soil bacterial number and Beta Glucosidase enzyme activity.

Table 4

Total bacteria, fungal and actinomycetes population after two weeks of spraying pots with vinegar, ZnO NPs and Carbaryl (Mean ± S.E.).
Treatments		Total bacteria (×10⁶ cfu.g^− 1 dry soil)	Total fungi (×10⁵ cfu.g^− 1 dry soil)	Total actinomycetes (×10⁵ cfu.g^− 1 dry soil)
ZnO NPs	100 ppm	3.86 ± 0.001^b	12.29 ± 0.001^a	7.59 ± 0.001^a
ZnO NPs	200 ppm	18.82 ± 0.001^a	6.07 ± 0.067^b	7.29 ± 0.001^b
Vinegar	1%	1.52 ± 0.001^e	4.27 ± 0.001^f	5.12 ± 0.001^d
Vinegar	10%	1.84 ± 0.001^c	4.94 ± 0.001^d	4.37 ± 0.001^f
Carbaryl insecticide	R. dose	0.99 ± 0.001^g	5.68 ± 0.003^c	4.57 ± 0.003^e
Carbaryl insecticide	D. dose	1.18 ± 0.001^f	4.66 ± 0.001^e	5.80 ± 0.001^c
Control (without treatment)		1.55 ± 0.001^d	2.27 ± 0.001^g	2.61 ± 0.001^g
Maximum		18.82 ± 0.001	12.29 ± 0.001	7.59 ± 0.001
Minimum		0.99 ± 0.001	2.27 ± 0.001	2.61 ± 0.001
Different letters indicate significant differences among the treatments

3.5 Mortality analysis of black aphid (Aphis fabae)

Generally, spraying of faba bean plants with ZnO NPs and vinegar provoked a highly significant effects on mortality of Aphis fabae aphids and showed great insecticidal activities, so this study is the first performance with regard to insecticidal activities of nanoparticles and vinegar in Kurdistan Region. Following Carbaryl (2X) spraying, 100 ppm of ZnO NPs has shown the greatest effects on death rate and corrected mortality rate (Mc %) of Aphis fabae by values of 87.5% and 5.15% respectively (Fig. 10 and Fig. 11). So, these values are greater than the death rate (69.23%) and corrected mortality rate (4.12%) recorded by the recommended dose of Carbaryl. This situation may refer to the dissolution of ZnO NPs to ionic zinc and particle-induced generation of reactive oxygen species (ROS) which represent the primary modes of action for ZnO NPs toxicity on Aphis fabae, and photo-induced toxicity associated with its photocatalytic property may be another important mechanism of toxicity under environmentally relevant UV radiation inside the greenhouse [55]. Moreover, 10% vinegar showed good death rate (62.5%) and corrected mortality rate (3.09%) in comparing with control. Wood vinegar is a natural product containing many types of organic acids and phenols, and applied in agriculture field to combat disease, pest control, improve growth and fruit quality, seed germination accelerator as well as herbicide [56].

3.6 Chemical analysis

Double dose of Carbaryl insecticide has showed the highest pH of 8.23 ± 0.003 in comparing with control (Table 5). Soil pH is probably the most informative single measurement that can be made to determine soil characteristics because it is closely correlated to many other soil properties like the presence of essential nutrients, pesticides and potentially toxic trace elements [39]. The greatest TDS and EC values were observed in treated pots sprayed with 200 ppm of zinc oxide nanoparticles which were 270.65 ± 0.029 ppm and 189.49 ± 0.003 µS.cm^− 1 respectively. It is obvious that the application of dissolved inorganic salts to soils, refers to the soluble plus readily dissolvable salts in the soil, or more usually in an aqueous extract of a soil sample. Soil salinity refers to total concentration of such soluble salts [39]. Moreover, the highest organic matter (0.44%) was observed in sprayed pots with 100 ppm of zinc oxide nanoparticle.

Table 5

Chemical properties of the soil samples after two weeks of spraying pots with vinegar, ZnO NPs and Carbaryl (Mean ± S.E.).
Treatments		pH	TDS (ppm)	EC µS.cm^− 1 25 C°	OM %
ZnO NP	100 ppm	8.21 ± 0.003^b	249.45 ± 0.029^c	174.59 ± 0.003^c	0.44 ± 0.000^g
ZnO NP	200 ppm	8.21 ± 0.003^b	270.65 ± 0.029^a	189.49 ± 0.003^a	0.87 ± 0.000^b
Vinegar	1%	8.22 ± 0.003^ab	251.35 ± 0.029^b	175.91 ± 0.003^b	0.49 ± 0.000^e
Vinegar	10%	8.21 ± 0.003 ^b	222.15 ± 0.029^f	155.47 ± 0.003^f	0.59 ± 0.000^c
Carbaryl insecticide	R. dose	8.21 ± 0.003^b	210.35 ± 0.029^g	147.29 ± 0.003^g	0.53 ± 0.000^d
Carbaryl insecticide	D. dose	8.23 ± 0.003^a	237.45 ± 0.029^e	166.24 ± 0.003^e	1.24 ± 0.000^a
Control (without treatment)		8.21 ± 0.007^b	244.35 ± 0.029^d	171.02 ± 0.006^d	0.47 ± 0.000^f
Maximum		8.23 ± 0.003	270.65 ± 0.029	189.49 ± 0.003	1.24 ± 0.000
Minimum		8.21	210.35 ± 0.029	147.29 ± 0.003	0.44 ± 0.000
Different letters indicate significant differences among the treatments

A green safety method for synthesis of zinc oxide nanoparticles from Musa paradisiaca L. leaves has been used successfully and they were annealed using UV-Visible, FTIR, XRD and TEM techniques with particle size of ~ 47 nm comparable with the Scherrer’s results. Plant protection strategies are governed by directives and rules pertaining to the sustainable use of pesticides. These policies aim to reduce the use of synthetic chemicals by substituting them with natural products ZnO nanoparticles, which can be used as pesticides and biocides. Given that a decreased reliance on fossil fuel is a general objective in the global food and feed production sector as well as exert a lower danger to the environment, it is envisaged that many megatrends of global policies would enhance the use of plant-based products.

Conflicts of Interest:

The authors declare no conflict of interest.

Funding:

This research received no external funding.

Author Contributions

A.S.K: writing- original draft preparation and corresponding author. Investigation, N. S. Kh., S. A. S., H. R. Q., A.S.K.: data curation and supervision, N. S. Kh., A.S.K.: All authors have read and approved the published version of the manuscript.

Acknowledgments:

Authors are grateful to the Salahaddin University-Erbil, Iraq, for support the researchers.

Data Availability Statement:

All available in this paper.

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Abbreviations

ZnO Zinc Oxide

NPs Nanoparticles

UV-Vis Ultraviolet visible

FTIR Fourier transform infrared

XRD X-Ray diffraction

TEM Transmission Electron Microscope

eV Electron volt

ppm part per million

1X One-fold (single dose)

2X Two two-fold (double dose)

Mc % Corrected mortality rate percent

TDS total dissolved solids

EC electrical conductivity

NT Nanotechnology

Zn (CH₃CO₂)₂·2H₂O Zinc acetate dihydrate

NaOH Sodium hydroxide

gm gram

No 1 Number one

ml milliliter

℃ Degree Celsius

WP wettable powder

Kg Kilogram

Cm Centimeter

pH Potential of Hydrogen

DFC Difference from control

CRD completely randomized design

Mo Mortality rate of treated adults (%)

Me Mortality rate of control (%)

Mc Corrected Mortality rate (%).

ANOVA Analysis of variance

SPSS Statistical Package for Social Sciences

E_g Band gap Energy

C = C functional group (alkene)

C-H Carbon-Hydrogen bond

D Crystalline dimension

$\varvec{\lambda }$ X-ray wavelength

A° Angstrom degree

$\varvec{\beta }$ full width of the peak

ROS Reactive oxygen species

No competing interests reported.

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Using of Biosynthesized Zinc Oxide Nanoparticle and Vinegar as a Substitute of Insecticides on Vicia faba Plant

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Abstract

1 Introduction

2 Materials and methods

2.1 Materials

2.1.1 Preparation of plant extract

2.1.2 Green synthesis of Zinc Oxide NPs

2.1.3 Preparation of solutions

2.1.3.1 Wood vinegar

2.1.3.2 Preparation of Carbaryl insecticide

2.2 Methods

2.2.1 Soil sample selection

2.2.2 Pot experiment layout and plant cultivation

2.2.3 Laboratory analysis

2.2.3.1 Vegetative growth characteristics

2.2.3.2 Seed germination and difference from control (DFC %)

2.2.3.3 Microbiological analysis

2.2.3.4 Toxicity tests on black aphid (Aphis fabae)

2.2.3.4.1 Calculation of death rate

2.2.3.4.2 Corrected mortality rate

2.2.3.5 Chemical analysis

2.2.3.6 Statistical analysis

3 Results and discussion

3.1 ZnO nanoparticles characterization

3.1.1 UV-Vis spectra analysis:

3.1.2 FTIR analysis

3.1.3 XRD analysis

3.1.4 TEM analysis

3.2 Vegetative growth characteristics

3.3 Seed germination and difference from control (DFC %)

3.4 Microbiological analysis

3.5 Mortality analysis of black aphid (Aphis fabae)

3.6 Chemical analysis

4 Conclusion and recommendations

Declarations

Conflicts of Interest:

Funding:

Author Contributions

Acknowledgments:

Data Availability Statement:

References

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