Zinc nitrate hexahydrate, sodium borohydride, and sodium hydroxide were obtained from Merck (KGaA, Darmstadt, Germany). Switch 62.5WG Fungicide was obtained from Syngenta USA (Greensboro, NC, USA).

The ZnO-NPs were prepared following the Wet-chemical method as reported by Arora et al. (2014). In particular, zinc nitrate and sodium hydroxide have been used as precursors, whereas sodium borohydride has been used as a stabilizing agent. Following the reaction and the formation of zinc hydroxide, calcinations should be performed to obtain the required size. Briefly, 2.79 g of Zn(NO₃)₂·6H₂O was weighed and dissolved in 100 ml of distilled, deionized water, which was then agitated vigorously for 1 h. Then 0.48 g NaOH dissolved in 60 ml distilled deionized water was prepared, and then 1 ml NaBH₄ (1%) was added dropwise after zinc nitrate dissolution, and stirred overnight for 12 h to obtain a low nano size. The precipitate was then allowed to settle before being filtered under suction and washed several times with distilled, deionized water (pump). The precipitate was then dried in an oven at 80°C. Zn(OH)₂ was formed and then converted to ZnO. The muffle calcination had been performed at 500°C to obtain the smaller size.

The green synthesis of ZnO-NPs has been carried out using M. oleifera as follows. Ten grams of M. oleifera were obtained from the greenhouse of the Desert Research Center (Cairo, Egypt), washed with distilled water, and bot leaves surfaces were sterilized using alcohol by gentle rubbing. These leaves were heated for 40 min in 100 ml of distilled water at 50°C. The extract was filtered with Whatman 41 (Merck, KGaA). The filtrate was stored in a cool and dry place.

Zinc oxide nanoparticles were prepared by mixing 10 ml of M. oleifera leaf extract with 90 ml of a 0.05 mM zinc nitrate hexahydrate [Zn(NO₃)₂·6H₂O] solution. The mixture was heated on a hot plate at 80°C for 30 min with continuous stirring. The presence of white-colored particles indicated the formation of nanoparticles. The extract was centrifuged and the pellets were collected, dried, and calcinated for 2 h at 300°C. White-colored nanoparticles were obtained as per the protocol as follows (El-Saber, 2021; Pal et al., 2018; Zambri et al., 2019).

The shape and size of ZnO-NPs were practically obtained using high-resolution transmission electron microscopy (TEM; HRTEM, JEOL JEM-2100, JEOL, Tokyo, Japan). Specimens for TEM measurements were prepared by placing a drop of colloidal solution on a 400-mesh carbon-coated copper grid and evaporating the solvent in the air at room temperature. The programme (ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA) was used to calculate the average size of nanoparticles. The crystalline structure of the ZnO-NPs was confirmed via X-ray diffractometer (XRD; X‘Pert Pro, PanAlytical, Almelo, The Netherlands). Malvern zeta sizer nano was used to measure particle size and zeta potential. The sample in solution form was placed in a cuvette and mitigated to become transparent until the error in the reading was reduced, then was placed inside the device, and the device start required the diameter of the particles and determination of the extent of homogeneity through the polydispersity index (PDI; dispersed index), which ranges from 0 to 1, and when approached zero increased homogeneity.

Samples with visible disease symptoms were removed by a sterilized knife in such a manner as to contain the lesion edges too. These were then immersed in HgCl₂ (1:10,000) for 2-3 min for surface sterilization and washed thoroughly in sterile distilled water until all the HgCl₂ was removed. The surface-sterilized tissues were further cut into smaller pieces using a sterile blade and placed on potato dextrose agar (PDA) medium using disinfected forceps. Plates were incubated at 28°C for 7 days. The isolated fungi were identified by molecular methods based on polymerase chain reaction (PCR) (Camele et al., 2005) at Laboratory of Mycology, School of Agricultural, Forestry, Food and Environmental Sciences (SAFE), University of Basilicata (Potenza, Italy). The genomic DNA (gDNA) were extracted from each pure fungal isolate using DNeasy Plant Mini Kit (Qiagen, Heidelberg, Germany). The gDNAs were amplified using the universal primer pair ITS4/ITS5 of rDNA (White et al., 1990). The obtained amplicons were directly sequenced and the obtained sequences were compared with those available in the GenBank nucleotide archive using Basic Local Alignment Search Tool software BLAST (Bethesda, MD, USA) (Altschul et al., 1997). The purified isolates were maintained on PDA slants for further studies (Talibi et al., 2014).

The poisoned food in vitro technique described by Das et al. (2010) was employed to evaluate the antifungal activity of the tested materials (moringa leaves aqueous extract, ZnO-NPs, and green zinc nanoparticles) against A. citri at different concentrations (0, 500, 1,000, and 2,000 μg/ml) by incorporating appropriate quantities of the treatments with an autoclaved PDA at 45°C, mixing very well, then plating in sterile Petri dishes and allowing to solidify (Elshafie et al., 2020a, 2020b, 2021). With the assistance of a sterile cork borer, a 5 mm mycelial disc of the old culture of A. citri was taken and aseptically placed in the center of the treated plates. The inoculated plates were incubated at 28°C for 7 days, or until the control plate grew to 9 cm. The inhibition percentage was measured with the help of the mean colony diameter and computed according to the following formula (O’Rourke et al., 2009).

Where, C = Average mycelial growth in control petri plates, and T = Average mycelial growth in treated Petri plates.

Scanning electron microscopy (SEM) of A. citri treated with studied antifungal agents at 2,000 μg/ml compared to control (without treatment) for 4 h at room temperature was estimated according to Sitohy et al. (2013) as described by Abbas et al. (2020).

An in situ experiment has been carried out using navel orange fruit obtained from the local market. Harvested orange fruits were immediately transported to the Plant Pathology Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt some fruits were selected based on uniformity of size and color, then were disinfected using 70% ethanol for 1 min, 1% sodium hypochlorite for 1 min, and washed three times with sterilized distilled water, then drained, rinsed with fresh air, and air dried at room temperature (25°C) before being treated on the same day. A fresh PDA culture of A. citri, was prepared and used for artificial infection of navel orange fruits (six groups of six fruits each, in three replicates). Twenty microliters (2,000 μg/ml) of each tested material (moringa leaves aqueous extract, ZnO-NPs, and green zinc nanoparticles) were injected into the central axis of a sterilized fruit using a micropipette. Switch fungicide was similarly applied using the recommended dose as positive control. The negative control was treated likewise, except that the fruits were neither infected with the fungus nor treated with nanoparticles. After that, 100 μl of spore suspension containing 10⁶ spores/ml was injected directly into the central axis of a sterilized fruit following the method described by Katoh et al. (2006). The inoculated fruits were then incubated in a sealed, sterilized plastic box at 24°C for 3 weeks, and the fruits were sliced in half for observation of symptom development. Disease severity was determined according to the area of the lesion on each fruit using the following formula:

The PCR amplification of the isolated fungi produced amplicons with molecular weights of approximately 600 bp. No amplification was observed in the case of the negative control. The obtained amplicons were directly sequenced (BMR Genomics, Padova, Italy) and the obtained sequences were compared with those available in the GenBank nucleotide archive using the Basic Local Alignment Search Tool software (BLAST) (Altschul et al., 1997). The sequences analysis for the isolated fungi showed high similarity with the sequences already presented in the GenBank database >99% for A. citri.

The chemical characterization of ZnO-NPs showed a well-dispersed, nearly spherical shape with a particle size of approximately 80 ± 5 nm as shown in the TEM image (Fig. 1A) and for extra-confirmatory identification by dynamic light scattering, which was performed to evaluate the nano-dispersity and particle size determination, an average particle size of 80 ± 5 nm with 0.552 PDI indicated a narrow size range (Fig. 1B). Stability and surface charge were estimated by the zeta potential exhibited by the −11.5 mV moderately stable NPs (Fig. 1C). For phase analysis and chemical structural identification, the XRD technique was conducted, where the XRD pattern of ZnO-NPs showed peaks at 2 = 31.69, 34.39, 36.21, 47.51, 56.47, 62.97, and 68.13, card number 01-078-2585 hexagonal (Fig. 1D).

Different characterization techniques including TEM, XRD, zeta potential, and zeta size were used to ascertain the formation of ZnO-NPs green synthesized by moringa leaf. The shape and the size of the biosynthesized ZnO-NPs were studied and investigated by using TEM (Fig. 2A). The average size and the stability of the nanoparticles were confirmed by the zeta size (Fig. 2B) and the zeta potential analysis (Fig. 2C). The obtained results showed that the mean diameter of ZnO-NPs was about 65 nm, and the zeta potential was −13.7 mv. Finally, The XRD pattern of the ZnO-NPs powder showed various diffractions peaks at 2 theta degree 31.24, 34.11, 35.71, 47.77, 56.22, 62.72, 66.34, 67.78, 72.84, and 76.79. As matched with the crystallographic database JCPDS 36-1451, these peaks were assigned to (100), (002), (101), (102), (110), (103), (200), (112), (004), and (202) planes of zinc, respectively (Fig. 2D).

Figs. 3 and 4 illustrate the in vitro antifungal activity of M. oleifera leaf aqueous extract and ZnO-NPs prepared either chemically or green-synthesis against A. citri mycelium growth. All treatments showed a promising effect on the linear growth of A. citri in a dose-dependent manner compared to control. A linear growth percentage reduction of A. citri at a concentration was 2,000 μg/ml, which was the most effective, being 3.5, 4.36, and 5.3 cm for greenly synthesized ZnO-NPs, chemically-synthesized ZnO-NPs, and moringa aqueous extract, respectively. Furthermore, data showed that the green-synthesized ZnO-NPs at 2,000 μg/ml was the most effective dose decreasing the linear growth of A. citri, where it showed 61.1%, followed by chemical NPs (51.55%), and finally moringa aqueous leaf extract (41.11%).

SEM was used to examine the morphological features of A. citri mycelia and cells treated with green-synthesized ZnO-NPs at 2,000 μg/ml to investigate the possible antifungal mechanism of these NPs against A. citri. The observation explicated that the mycelia and conidia of A. citri grown on PDA were smooth, homogeneous mycelial morphology. In contrast, the mycelial and conidial morphology was conspicuously changed when exposed to green-synthesized ZnO-NPs at 2,000 μg/ml (Fig. 5).

Table 1 and Fig. 6 showed the effect of moringa leaf aqueous extract and new green and chemically-synthesized ZnO-NPs on the severity of black rot disease on navel orange. Data presented in Table 1 indicates that switch fungicide completely inhibited black rot disease severity. Furthermore, data showed that green ZnO-NPs at 2,000 μg/ml was the most effective in reducing black rot disease severity (93.08%), followed by chemical ZnO-NPs (90.77%), whereas moringa aqueous extract exhibited only 76.93% effectiveness.