M. haplocalyx dry leaf powder (∼5 g) was added to a blue cap bottle containing de-ionized water (100 mL), which was then placed in a microwave at medium-high heat for 5 min. The leaf extract was then obtained by filtration through a Millipore filter (φ = 0.22 μm). For biosynthesis of nano silver, filtrate (10 mL) was mixed with deionized water and the concentration of AgNO₃ was set as 1 mmol.L⁻¹. The mixture was heated in a microwave for 2 min (Gao et al., 2017).

A UV-vis spectrophotometer (TU-1950, PERSEE, China) was used to establish the biosynthesis of nano silver. After heating for 2 min, 2 mL of solution was scanned over a wavelength range of 350–550 nm and the diluted filtrate was used as a blank. Sample was dripped on a copper grid until totally dried. TEM (JEM-2100F, JEOL, Japan) was utilized to determine the morphology and particle size of the biosynthesized nano silver. About 200 particles were selected randomly, and the size distribution was analyzed using ImageJ software. The sample was suspended in ethanol and added to substrate for SEM (S4800, Hitachi, Japan) analysis to determine the size of the nano silver agglomerates. The attached EDX system was used to verify the presence of elemental Ag. Nanoparticles on a mica surface were subjected to AFM (BioScope) in non-contact mode and the initial position of the nanoparticle was observed. FTIR was used to identify functional groups on the surface of the biogenic nano silver. Dried nano silver (∼0.5 g) was mixed with KBr and pressed into a thin pellet. A FTIR spectrum of nano silver was obtained in the range of 4,000–500 cm⁻¹. A zeta potentiometer was used to determine surface charge.

Nano silver and PDA medium were mixed at a ratio of 1:9 (v/v) and the concentrations of nanoparticles were 10, 20, 50, 100, and 200 μg.mL−1. PDA medium with sterile water was the control. A fungus disk (φ = 8 mm) was inoculated in the center of the PDA plate and then incubated at 28 °C for 5–7 days. Each treatment or control was repeated 3 times. The inhibition rate was calculated using the following equation:

I n h i b i t i o n   r a t e   % = * 100 %

A spore suspension of C. lunata was prepared in sterile water and its concentration was adjusted to 106 mL−1 using a blood counting chamber. Subsequently, spore suspension (100 μL) was dripped and smeared evenly on solid PDA plates. Several wells were drilled with a hole puncher and samples (30 μL) of different concentrations (10, 20, 50, 100 and 200 μg.mL−1) of nano silver were dropped into the wells. The control well con-tained sterile water (30 μL). The inhibition zone diameter was measured after incubation at 28 °C for 2–3 days.

Maize leaves (Anke985, AK985) were washed several times under running water and then dipped in 70% ethyl alcohol and 2% aqueous sodium hypochlorite solution for 1 min and 30 s, respectively. The sterilized leaves were then soaked in sterile water for 1 min to remove solution residue. Subsequently, the air-dried leaves were placed on solid PDA plates. A mixed solution (20 μL) containing different concentrations of nano silver and spore suspension was dripped onto the plates. Spore suspension (∼20 μL) was added to the leaves as control. All plates were replicated 3 times and incubated at 25 °C for 72 h to observe infection in detached leaves under the microscope.

Seeds of AK985 were sterilized by soaking in 5% aqueous sodium hypochlorite solution for 10 min and then rinsed three times with sterile water. The seeds were then placed in a germinating box with double sterilized gauze. Seven-day-old seedlings were transplanted into a pot containing 400 g of substrate and soil (3:1, w/w). Plants were grown in a climate chamber with a cycle of 14 h light at 25 °C and 10 h dark at 22 °C. Hoagland nutrient solution (Phygene Biotechnology Co. Ltd.) was applied once a week as fertilizer. After growing for 30 days, three treatments were sprayed onto the maize leaves as follows: 1) 5 mL/plant of sterile water, 2) 5 mL/plant of spore suspension (106 mL−1) containing 1% Tween-20, 3) 5 mL/plant of spore suspension (106 mL−1) containing 1% Tween-20 and 200 μg mL−1 nano silver. All treated plants were grown in the climate chamber described above, keeping the humidity above 80%. The infection on maize leaves was observed after incubation for 120 h.

The morphology of nano silver is not exactly the same when synthesized using different living organisms or even the same one. As shown in Figure 2, most of the synthesized particles were near spherical, similar to previous reports (Khatami et al., 2017; Helmlinger et al., 2016; Mollahosseini et al., 2012). Some other shapes were also present, including triangular, rectangular and irregularly shaped. Helmlinger et al. reported that multiple morphologies of silver nanoparticles, including spheres, platelets, cubes, and rods, were prepared and their antibacterial activities were also measured (Hussain et al., 2019). Figure 2B shows that the sizes of synthesized particles were in the range of 6–48 nm and the average particle size was about 23 nm. The prominent antimicrobial activity of nanomaterials is closely related to their superfine size. It has been reported that the smaller the particle size, the stronger the antimicrobial activity (Hussain et al., 2019; Franzolin et al., 2022).

The morphology of synthesized nano silver was also evaluated by SEM. The particles were almost spherical, either as individuals or gathered into clusters (Figure 3A). The elemental composition was determined using an EDX detector attached to the scanning electron microscope. Figure 3B shows a strong peak for elemental silver (Ag) at around 3 keV. The presence of other elements, including carbon C), oxygen O), chlorine (Cl) and unknown, might be due to components of plant extract. It has been reported that polyphenols and flavonoids present in plant extract are likely to reduce silver ion, and some inherent secondary metabolites, such as alkaloids and saponins, might contribute to the stability of nanoparticles (Binsalah et al., 2022).

The particular morphological features of the green synthesized nano silver were determined by AFM. The particles deposited on the substrate with uniform distribution (Figure 4A). The 3D topographic image is shown in Figure 4B.

The FTIR spectrum of synthesized nano silver was recorded, and the bioactive compounds that might be related to biosynthesis and stability of nano silver were investigated. The FTIR spectrum of biosynthesized nano silver contained bands at 2,996.67, 1987.75, 1523.54, 1234.72, 1101.06, 954.07, and 845.56 cm⁻¹ (Figure 5). The band observed at 2,996.67 cm⁻¹ indicates a C–H (alkane) group. The band at 1987.75 cm⁻¹ represents C=C (alkene) stretching. The peak seen at 1523.54 cm⁻¹ indicates a C–H (alkane) group. The bands at 1234.72 and 1101.06 cm⁻¹ are attributed to C–O (alcohol/ether) and C–N (amine) stretching, respectively. The bank appeared at 954.07 and 845.56 cm⁻¹ indicate a O-H and C-Cl group (Paul and Yadav, 2016; Toro et al., 2021; Al-Otibi et al., 2023; Khatami et al., 2017). These characteristic bands appearing on the surface of nano silver are due to the presence of proteins, flavonoids, phenolic compounds or other substances that contribute to capping and stabilization of nano silver (Liaqat et al., 2022).

To ascertain the surface charge of nano silver biosynthesized by M. haplocalyx leaf extract, the zeta potential was determined. Figure 6 shows that the zeta potential value was −19.7 mv, which indicates that the synthesized nano silver was relatively stable. Compared with physical and chemical approaches, plant- or microorganism-mediated synthesis of nano silver does not require extra capping or stability agents, since the presence of bioactive compounds prevents particle aggregation (Sathiyabama and Manikandan, 2018; Khodadadi et al., 2021).

Amycelial growth rate method was used to determine the effects of nano silver at different concentrations against C. lunata. As shown in Figure 7, growth of C. lunata was inhibited in a concentration-dependent manner by nano silver. On the control plate, the colony covered the Petri dish (φ = 9 cm). The colony area decreased gradually as the concentration of nano silver was increased. At a concentration of 200 μg mL⁻¹, the colony diameter was 4.30 cm, and the inhibition rate reached 57.3%.

An obvious inhibition zone will appear around the agar wells if there is antimicrobial activity. The zone diameter is positively correlated with inhibition (Figure 8). Table 3 shows that synthesized nano silver at high concentrations (≥50 μg mL⁻¹) exhibited antifungal activity against C. lunata. There appeared no inhibition zone at low concentrations (10 and 20 μg mL⁻¹), however, when the concentration increased to 200 μg mL⁻¹, the diameter reached 12.50 ± 1.18 mm. There is a positive relationship between nanoparticle diffusion in agar and inhibition zone diameter. Nano silver can diffuse more into agar well and obtain more chance to interact with C. lunata at a higher concentration, however the diffusion of lower concentration nano silver is limited. In addition to the agar well diffusion method, the filter paper diffusion approach has also been used to determine the inhibition zone diameter. The latter method is influenced by parameters such as volume of addition, type of nanoparticle and species of pathogen (Hasson et al., 2019; Huang et al., 2020; Al-Otibi et al., 2023).

An in vitro inoculation assay was carried out to estimate the antifungal effect of nano silver against C. lunata on detached maize leaf (Huang et al., 2020). At the correct temperature, high humidity is essential for conidia germination and disease spot extension. For maize leaf inoculated with conidia suspension alone, there was obvious necrosis near the inoculation point and a large part of the leaf turned yellow as pathogen infection progressed (Figure 9A). However, maize leaf inoculated with conidia suspension and nano silver (200 μg mL⁻¹) suffered less pathogen damage and the leaf looked much healthier than the control (Figure 9B). The microscopic image of the control showed that most of the conidia germinated and the mycelia filled the whole leaf tissue (Figure 9C), while almost all of the conidia failed to germinate in the presence of nano silver and few mycelia appeared in the leaf tissue (Figure 9D). Conidia germination is the initial and indispensable step for pathogen invasion, so it is desirable to reduce the germination rate of conidia (Huang et al., 2020; Khleifat et al., 2022). Besides, the growth of germ tubes was significantly suppressed, and the integrity and permeability of cell membranes was also disrupted by silver nanoparticles (Jian et al., 2022). In the in vitro inoculation experiment, 200 μg mL⁻¹ nano silver synthesized by M. haplocalyx leaf extract prevented C. Lunata infection of detached maize leaves.

An in vivo inoculation assay was conducted to evaluate the antifungal activity of nano silver against C. lunata on living maize leaf. Figure 10A shows that numerous sub-round lesions appeared on leaves inoculated with conidia suspension alone. However, leaves inoculated with a mixture of conidia suspension and 200 μg mL⁻¹ nano silver suffered few lesions (Figure 10B). Several factors, such as pathogen, environment, host plant and human activity, influence plant disease epidemics. The result confirmed that 200 μg mL⁻¹ nano silver could greatly reduce the incidence of disease. In the case of foliar inoculation, it can definitely inhibit extend of disease spots, but where did the nanparticles go or transport is an interesting task for further research (Pérez-de-Luque, 2017), and which will evaluate its safety to plants.