Using Biosynthesized Zinc Oxide Nanoparticles as a Pesticide to Alleviate the Toxicity on Banana Infested with Parasitic-Nematode

We appraised the use of zinc oxide nanoparticles, (ZnO-NPs) and zinc oxide bulk (ZnO-bulk) or zinc acetate, as a natural nematocide, alone or in combination with oxamyl in vitro and in vivo trials in order to improve systems for root-knot nematode (RKNs) control in banana plants. Especially, ZnO-NPs were biosynthesized from the alga, Ulva fasciata. In general, all applications of ZnO-NPs were more effective to control RKNs than ZnO-bulk as well oxamyl alone (chemical control). In in vitro conditions, ZnO-NPs with oxamyl showed 98.91% second stage juveniles2 (J2s) mortality of Meloidogyne incognita after 72 h, while 72.86% mortality was observed at the same NPs treatment without oxamyl at the same exposure time. The same treatment was the most significantly effective in diminution of J2s community (82.77%) in soil and galls number (81.87%) in roots under in vivo conditions. In contrast, the highest weight and height of the shoot was observed in Zn-bulk treatment in combination with oxamyl as well oxamyl only (nematocides check). Scanning electron microscopy (SEM) reports displayed the distributions and accumulations of ZnO-NPs on the nematode (J2s) body under direct exposure, which might be the reason of NP-mediated toxicity and disruption for M. incognita.


Introduction
Nanoparticles (NPs) are widely employed in the environment due to their unique optical, electronic and magnetic characteristics [1][2][3]. Among the NPs, Zinc oxide nanoparticles (ZnO-NPs) are used in several industries, medicine and agriculture [4,5], due to their unique characteristics which differ from their particular bulk counterparts of the same synthesis [6,7], such as morphology, size and distribution [8]. The NPs are also used in the gene delivery [9] or as a synthetic drug [10][11][12]. The use of NPs in agriculture has become increasingly popular over the few past decades [13,14]. Furthermore, the optical characters of ZnO-NPs can be controlled by producing these particles in combination with suitable elements [15]. The NPs can be synthesized through several ways and the bio-synthesis of NPs using marine algae has been under manipulated [16]. Bio-synthesis of NPs using marine algae has become of great interest [17]. Whereas, microalgae and macro-algae are being utilized as bio-factories for the production of NPs. Significantly, little information are available regarding the biosynthesis of NPs using microalgae, which are being utilized as a bio-factory for the production of NPs [18]. However, the microalgae have a renewable origin; it could be effectually studied in the biosynthesis of nanoparticles [19]. In general, ZnO-NPs have also unique toxic properties which might be ascribed to the production of reactive oxygen species (ROS) in living cells [20,21]. ROS formation can occur in protein, membrane and DNA of host cells, and it may cause damage in biological systems [3,22,23].
The plant-parasitic nematodes (PPNs) are among the major factors which affect the growth and development of several crops [24,25]. Owing to the intense cultivation of crops, the pests increase at an alarming rate leading to crops yield reduction. Thus, there is an urgent need to manage the pests to boost crops growth [26]. A chemical nematocide is an artificial compound used to inhibit PPNs. The use of these chemical nematocides to manage PPNs can result in extreme hazards in the environment and also cause mammalian health risks [27,28]. Thus, more environment friendly techniques are required to control pests in an appropriate way. Accordingly, the use of bio-NPs for the purpose of controlling plant pathogens is a novel method [13] that may be effective in the future through the progress of nanotechnology applications [29][30][31].
Memorable, ZnO-NPs may affect (i) the creatures causing physical damage a result of direct interaction, (ii) the zinc ions dissolution and (iii) the ROS production which represent the first mode of action for nano-ZnO toxicity in all species tested [32], and photo-induced toxicity associated with its photo-catalytic property may be another vital mechanism of NPs toxicity under ecologically relevant UV radiation [33]. The other mechanism of ZnO-NPs toxicity in eukaryotic living cells with pinocytosis, is through allowing ZnO nanoparticles to pass through the host cells and unite with the cell organelles, leading to the cells being damage [34]. However, as dissolved free Zinc from nano-ZnO is a crucial contributor to toxicity, and it is necessary to appraisal the dissolution rate [35]. The dissolution rate of Zn oxide under present experimental condition (pH ¼ 6.8) was evaluated as < 15%, mostly lower than the 100% free Zinc ion in the ZnCl2 solution [36].
It was hypothesized that ZnO-NPs-as a biosynthesis nematicides-ould be a novel and safe technology to constrain the usage of chemical hazards in the environment like artificial oxamyl. Besides this, ZnO-NPs may help in depressing RKNs, M. incognita and may cause beneficial effects on banana health. The present research has demonstrated the toxicity of ZnO-NPs and their particular bulk (Zn acetate or ZnO-bulk) treatments alone or in combination with oxamyl against RKNs, M. incognita in potted banana plantlets.

Biosynthesis of ZnO-NPs by Ulva Fasciata and their Characterization
Ten mL of U. fasciata extracts (1 g of dry alga in 100 mL distilled water, 1 h. boiling and then filtered) were added to 40 mL of distilled water containing 0.02 M Zn acetate dehydrates by constant stirring. After 10 min of stirring, 2.0 M NaOH was added and then stirred for 2 h The pale white precipitation was obtained and then filtrated and washed two times with distilled water and one time by absolute ethanol and then dried in electric oven at 60 °C overnight [37]. Chemical structure of the green synthesis of ZnO-NPs were determined by Energy Dispersive X-ray spectroscopy (EDX). The morphology and particle size of NPs was determined by Transmission Electron Microscope (TEM).

Nematode Inoculation
Eggs of the root-knot nematode (RKN), Meloidogyne incognita, were extracted from tomato (Lycopersicon esculentum cv. Castle Rock) roots infested with the RKN using 0.5% sodium hypochlorite (NaOCl) solution [38]. Juveniles2 (J2s) were stored daily from the eggs at 15 °C. The J2s used in the trials were less than 5 days old.

The In Vitro Treatment
A volume of 1 mL active RKNs suspension containing about 100 J2s of M. incognita was placed in sterilized Petri-dishes (9 cm diameter) and mixed with constant volume of each treatment to prepare (ZnO-NPs and ZnO-bulk in concentration 0.5 mg/mL); (ZnO-NPs and ZnO-bulk in concentration 0.25 mg/mL mixed with 12.5 µl oxamyl and complete to 4 mL by distilled water, replicated five times of the previous treatments. Also, five inoculated petri-dishes were treated with 25 µl of oxamyl only (nematocides check). The five remaining injected petri-dishes served as nematode only without treatment (nematode check). Distilled water was used as a check. All applications were incubated at 27 °C. The number of active and inactive J2s was counted after 24, 1 3 48 and 72 h Abbott formula [39] was used to determine the percentage of mortality in comparison with control whereas X = ME-MC / 100-MC × 100*. *(ME: The percentage of mortality in each treatment; MC: The percentages of mortality in check; X: The percentage of mortality in comparison with check).

The Physical and Chemical Properties of the Used Soil
The structure of the artificial soil was prepared in laboratory conditions (containing 4.5 kg/pot) of sterilized soil mixture (clay/sand 1:2) with pH 7.26. It included sand (25.33%), silt (35.30%), and clay (39.37%). The other physical and chemical characteristics of the soil texture are recorded in Table 1. Micro and macroelements were measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES, Thermo Scientific).

The In Vivo Treatment
The banana plantlets cv. Grande-Naine (2 months-old) were received from the Tissue Culture Lab. of the Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat University, Egypt. Plantlets were placed in pots (30 cm diameter) already filled with 4.5 kg of artificially sterilized soil (1:2; clay: sand ratio) with a final pH of 7.0. The temperature of the soil was maintained at 28 ± 2 °C during the whole experimental period. 30 pots were inoculated with 2000 active J2s per pot at the time of seedling transplantation. Five days later, 10 pots were injected with 2 g per pot of each previously treatment (replicated five times) after preparation, with ZnO-NPs and ZnO-bulk. In addition, the same numbers were inserted with 1 g per pot of the previous treatments followed by 0.5 mL oxamyl. Five numbers of infested pots were treated with 1 mL oxamyl per pot (nematocides check). The remaining five inoculated pots were used as untreated nematode control (nematode check). Moreover, five pots were determined as untreated and non-inoculated control (check free). All tested applications were applied to the soil in 3 holes around the plant and followed by the application of 50 mL of water. All the pots were placed in a completely randomized design under greenhouse conditions. Plants were harvested after two months of inoculation. The roots were washed carefully with tap water to remove the soil particles and stained with artificial "Phloxine B" solution (3.5 g in 750 mL distilled water + 250 mL acetic acid 5%) for 5 min to facilitate the counting of all nematode stages in the root. Nematode variables were observed per root system including galls, females and females with egg-masses. The second stage J2s per 250 g soil was extracted and counted according to Cobb's sieving and decanting method, using sieves (20 meshes, 60 meshes and 325 meshes). The banana growth variables such as shoot length, shoot weight, rhizome length and weight and corm weight were measured and recorded.

Scanning Electron Microscope (SEM) Analysis of Juveniles2
The juveniles2 (J2s) of M. incognita treated with ZnO-NPs within 24 h obtained from the laboratory assays study were fixed in a 2% glutaraldehyde with 2% paraformaldehyde solution in a (0.  Finally, the specimens were placed and covered with gold-palladium membranes and were noticed in a Jeol JSM-6510 L.V SEM. The microscope was functioned at 30 kV at electron microscope (EM) Unit, Mansoura Univ., Egypt.

Statistical Analysis
All data were subjected to analysis of variance (ANOVA). Significance of the variable mean differences was prescribed using Duncan's multiple range tests (p ≤ 0.05). All data analyses were conducted using SPSS 16 software [40].

ZnO Nanoparticles Characterization
The results obtained from the Transmission Electron Microscopy (TEM) image demonstrated that biosynthesis of ZnO-NPs had a nanorod shape (Fig. 1). The major composition of the biosynthesized nanoparticles contains carbon, oxygen and zinc as depicted from the energy dispersive X-ray spectrophotometry analysis (Fig. 2).

Evaluation of the In Vitro Trial
Influence of ZnO-NPs and their bulk alone or in combination with oxamyl on mortality of second stage J2s in in vitro experiment, was observed and the results are reported in Fig. 3. Apparently, the percentage of nematode mortality was increased with the increase of exposure time (minimum at 24 h and maximum at 72 h). In general, ZnO-NPs with oxamyl showed 98.91% J2s mortality at 72 h, while the same treatment without oxamyl recorded 72.86% mortality at the same exposure time. At 24 h exposure, green ZnO-NPs with oxamyl provided the greatest nematode mortality (90.51%) while nematode control gave a minimal of 2.74%. Similarly, oxamyl alone as a chemical control showed 91.76% and 100% nematode mortality at 24 h and at 72 h, respectively. Results in Fig. 3 indicated that the significant effect occurs in in vitro trial of ZnO-NPs compared to zinc bulk after 24, 48 and 72 h However, significant effect was obtained when each of ZnO-NPs and ZnO-bulk is combined with oxamyl after 72 h.

Nematocidal Evaluation
The influence of the current applications on the RKN, M. incognita contaminating banana plantlets caused a marked depression of the nematode community in in vivo experiment ( Table 2). All the tested materials were compared with oxamyl only (nematocides check) and the results indicated a significant inhibition in number of galls, females and females with egg-masses per rhizome system; and number of J2s in soil (Table 1). Notable, the treatment of ZnO-NPs with oxamyl was significantly effective in reducing J2s community (82.77%) in soil and galls number (81.87%) in roots compared to ZnO bulk with oxamyl. However, ZnO-bulk with oxamyl reduced the number of females (82.09%) and females with egg-masses (79.53%) in roots (Fig. 4).

Plant Health Measurements
Growth criteria of banana plants, such as length and weight of shoots and rhizomes, and weight of corms, are documented in Table 3. All the applications exhibited enhancement in plant health. On the contrary, no notable increase  in corm weight was established. The highest outcome for weight and height shoot was stipulated by bulk-ZnO with oxamyl than nematode check ( Table 3). Figure 5 displays the SEM pictures, the in vitro investigations have been developed with a relatively high disruption of the nematode cuticle, in addition ZnO-NPs distributions on the body of treated nematode comparing nematode control. These finding demonstrated that NPs had obviously disrupted in the nematode body after treatment in vitro after one day (Fig. 5c and d). The nano-ZnO are clearly pictured as spherical nano-organized particles on the nematode body ( Fig. 5e and f).

ZnO Nanoparticles Characterization
The results obtained from the Transmission Electron Microscopy (TEM) image demonstrated that biosynthesis of ZnO-NPs had a nanorod shape (Fig. 1). A nanorode shape of green ZnO-NPs that synthesis by using green alga like, Chlorella vulgaris [41], Chlamydomonas reinhardtii [42] and Santalum album [43]. The major composition of the   biosynthesized nanoparticles contains carbon, oxygen and zinc as depicted from the energy dispersive X-ray spectrophotometry analysis (Fig. 2). Whereas, the weight of Zn and oxygen was over 50% of the total constitute [44].

Effect of the ZnO-NPs on Nematode Community in Petri-Plates
In the current study, nano-ZnO can produce toxic effects in the RKN under different exposure times in in vitro conditions. Using different ZnO-NPs applications gave high toxicity compared to ZnO-bulk for M. incognita through 72 h Toxicity assay of synthesized nano-ZnO were produced using the free-living nematode, Caenorhabdities elegans, which has been applied commonly in ecotoxicological researches [6,45,46]. Generally, ZnO-NPs in combination with oxamyl presented 98.91% nematodes J2s mortality at 72 h, while the same treatment without oxamyl recorded 72.86% mortality at the same exposure time. In vitro assays also showed the nematocidal action of bio-synthesized nanoparticles that may provide an alternative method to avoid high-risk of artificial nematocides [13,47].

Effect of Nano-ZnO and Bulk-ZnO on Nematode Community in Soil and Roots
Results established that NPs successfully controlled the RKNs counts in soil samples and plant-roots by using ZnO-NPs and its bulk, alone or in combination with oxamyl which presented nematocidal action to a variety of PPNs that may extremely harm the banana plants [48]. Evaluation of ZnO-NPs trial in in vivo and bio-synthesized NPs by algae confirmed its beneficial role for RKN control and nematode community reduction in soil and roots. Currently, there were several studies that examined the toxicity of nano-ZnO towards various pests, especially nematodes [49][50][51]. The field assessment revealed its benefits for modifying injury caused by RKN in plants [52][53][54]. Overall, all treatments of ZnO-NPs compared to bulk-ZnO as well oxamyl alone (chemical check) were more effective to RKN control due to their defensive power. ZnO-NPs also revealed superior high toxicity than its bulk due to the smaller particles of ZnO-NPs having a larger surface area/ unit mass [55]. The toxic effects of nano-ZnO were examined in the case of several animals utilized in bio-monitoring, for instance nematode, C. elegans [6]. In general, the treatment of ZnO-NPs with oxamyl was the most effective in the reduction of M. incognita community J2s (82.77%) in soil and count of galls (81.87%) on roots.

Effect of the ZnO-NPs and its Bulk on Banana Growth
All applications of the tested treatments as well oxamyl caused relatively an incredible increase in banana growth than nematode check. In addition, the highest outcome of the weight and height of the banana shoot was achieved by bulk-ZnO with oxamyl than nematode check. The Zn-bulk enhanced the vegetative growth of plants [56], for this reason it has been mainly applied in lettuce plant that obtained the maximum weight [57]. Zinc ion is the main component of several proteins and it is playing a crucial role in organizing domain fixation of plant protein through association with the plant DNA [58]. Consequently, Zn deficiency can affect the plant growth [59] and creation of the plant proteins [60,61].

Effect of the ZnO-NPs Distribution on Nematode Body and Toxicity Mechanism
In the published research, a novel management strategy was discovered for controlling PPNs based on biosynthesis of ZnO-NPs by microalga [18,62]. The successful applications of bio-ZnO NPs discovered from laboratory experiments were utilized to a continuous banana-potting to study its effects on nematodes control and the RKNs community both in soil and roots. Many potential RKNs depression approaches have revealed exceptional effects in vitro and in vivo experiments; notwithstanding, these methods are costly because they used artificial ZnO-NPs to mainly inhibit pests [44]. Therefore, there is now excessive stimulation to create bio-nanopesticides, which are less injurious to the environment and costs little compared to synthesis formulations [13]. Generally, The SEM alterations observed in the laboratory experiments demonstrated the occurrence of relations between both the ZnO-NPs and some J2s structures, especially the nematode cuticle, which has a crucial role in parasitic-nematode functions. These laboratory investigations have been developed with a relatively high disruption of the nematode body and occurred of nano-ZnO particles after one day with nematodes. These similarities showed that both nanoparticles and their bulk ZnO, Al 2 O 3 and TiO 2 equals were toxic and inhibited the development of the parasitic-nematode in in vitro trial [63,64]. Furthermore, the observations suggested that one ZnO nanoparticles uptake route is through the cuticle of nematode juveniles, and they may interact with tissue structures, and contribute to the detected increases in germ cell apoptosis [35]. Consequently, as dissolved free Zinc from nano-ZnO is a crucial contributor to nematode toxicity, and it is necessary to appraisal the dissolution rate [36]. The dissolution rate of Zn oxide under present experimental condition (pH ¼ 6.8) was evaluated as < 15%, mostly lower than the 100% free Zinc ion in the ZnCl2 solution [36].
The several mechanisms of nanoparticles against pests are frequently related with their high surface to particle size ratio [65] and with their unique physical and chemical properties [8]. In general, nematocidal effect of nanoparticles is similar on both PPNs and plant pathogenic fungi [66], because its mechanism of action is not specialized according to the type of host cells. Nevertheless, the mode of action is associated with disrupting multiple cellular mechanisms [67] including: (i) cell wall damage due to Zinc oxide-localized interaction, (ii) improved membrane permeability [68], (iii) ATP synthesis and (iv) response to oxidative stress in eukaryotic cells [69], and prokaryotic cells as a results of mitochondria weakness and intracellular outflow [70]. Consequently, (v) inhibition of cell growth and ultimately cell death, take place [71]. Hence, this research used bio-ZnO nanoparticles toxicity and its bulk alone or in combination with oxamyl against plant-nematodes infestation in in vitro and in vivo conditions as a novel method.

Conclusion
This study presented an alternative method for controlling RKNs, M. incognita in banana which might be applied to several crops. We have also described a new way to biosynthesis nematocidal of ZnO-NPs using microgreen alga, U. fasciata. The toxicity of nano-ZnO and its bulk against parasitic-nematodes is mainly determined by the exposure time in in vitro conditions. Also, our results of the in vivo experiment confirmed that all treatments were effectual approaches to reduce the nematode community and to promote banana health. Electron microscopy studies showed that the distributions and accumulations of nano-ZnO on the body of nematode J2s under direct exposure, which aim to assess the influence of NPs to the toxicity for M. incognita.