1 Introduction

The COVID-19 pandemic, the Russia-Ukraine conflict, and the rise in food prices have caused over 44 million individuals in 38 countries to experience an emergency level of food insecurity. These factors have also increased the risks to plant production due to the exacerbation of plant diseases [1, 2].

One of the most important legumes in the world is the pea (Pisum sativum L.), which is grown in more than 90 countries and has an estimated annual production of 13.5 million metric tons at a producer price of roughly 200 USD/ton. Pea is most frequently utilized as an alternative source for industrial proteins because of its high yields, availability, and affordable manufacturing. As a result, the food industry has given pea protein’s research and use a lot of attention [3].

Due to its capacity to fix nitrogen, the pea (Pisum sativum L.), an important legume, plays a part in organic farming. There are many diseases that lower the overall yield of peas. Evidence suggests that Pythium spp. might harm peas and reduce the overall yield in the production of grain legumes [4].

Diseases of plants are one of the most pervasive global problems endangering agricultural wealth because they result in significant decreases in agricultural production, lower product quality, and the production of toxins that poison consumers and cause a number of serious diseases in animals and humans who consume infected products [5]. Some plant diseases, particularly those affecting economic agricultural plants, have a deleterious impact on plant development at various stages, as well as crop yield [6, 7]. P. irregulare is a soil-borne pathogen that is a major cause of plant damage each year [8]. Different crops are affected by P. irregulare’s plant diseases, which include damping off and wilting [9].

Among the most detrimental seedling diseases, pea damping-off and root rot caused by soil-borne pathogenic fungus led to severe reductions in either seed quality or yield [10]. There is a commitment to defense against pathogens, but the overuse of chemical fungicides is ineffective as a long-term treatment and poses a risk to humans [11]. Fungal diseases have an impact on all of plants' essential physiological and metabolic processes [12]. Reactive oxygen species (ROS) including hydrogen peroxide and hydroxyl radicals, which have been proven to be extremely detrimental to plants, are oxidative stress markers [13]. The best alternative was the inclination to adopt a more effective biological management strategy for wilt disease, which was also cheaper and more environmentally friendly. Chemical control of damping off disease is difficult and dangerous; therefore, it was the best option [14]. Climate change has intensified recently, which has accelerated the spread of plant infections [15]. Researchers’ focus is now on discovering environmentally acceptable options to synthetic pesticides that might be used to reduce the development of plant diseases [16]. Induced resistance is that resistance that is activated by biological factors, which leads to improving the physiological immunity of the plant, as well as inducing the formation and accumulation of natural and chemical barriers to pathogens [17]. As a consequence of improving plant systemic immunity, disease protection, and production, the application of nanobiotechnology in combating pathogens and increasing physiological plant immunity has produced significant and effective outcomes [18,19,20]. Intact biological particles may now be created using biological techniques for nanoparticle and nanocrystal production [21]. The biological strategy is a more viable green option that is energy-efficient and environmentally beneficial [22]. Streptomyces species are being studied for their potential as a biological control for pathogenic fungus and bacteria that affect plants, in addition to their use in the manufacture of antibiotics. By inhibiting their spore germination, Streptomyces sp. strain CACIS-1.5CA, for example, has demonstrated strong bioactivity against a number of plant pathogenic fungi [23].

The biologically produced nanoparticles boost enzyme and metal salt activity and have better catalytic reactivity and specific surface area [24]. Nanofertilizers are unconventional derivatives that are more effective than conventional fertilizers at reducing soil and air pollution. The technology of manufacturing nanofertilizers has become a vital requirement in modern agricultural practices, due to its role in improving plant production, playing the role of therapeutic nutrients, and preserving the ecosystem from dangerous pollutants [25]. The production of nano fertilizers is considered the most important alternative to traditional fertilizers and pesticides, due to their potential role in improving crop production, reducing the use of chemical fertilizers and pesticides, and mitigating their harmful effects on soil and plants [26]. In addition, synthetic nanofertilizers are created from conventional fertilizers using physical, chemical, or biological means that lead to superior results to conventional fertilizers in terms of expansion rates and nutritional values [27]. A rich source of natural compounds like carbohydrates, phenols, flavonoids, tannins, and alkaloids, fungal extracts are used in the biosynthesis of nanomaterials [28], which are also thought to be safe reducing and stabilizing agents and capable of maintaining aseptic conditions throughout the process [29].

Zinc (Zn) is an imperative element in plant growth, and it is classified in terms of nutrition as one of the micronutrients needed by plants, as it has avital role in composition of enzymes that synthesize plant auxin [30, 31]. Additionally, zinc is crucial for the oxidation of carbohydrates in plants and plays a significant part in the production of chlorophyll and photosynthesis [32, 33]. Notably, several research have demonstrated the significance of nanozinc in evoking plant defense against fungus disease [34, 35].

Another essential micronutrient is boron (B), which is primarily used to maintain the structural integrity of plant cell walls [36]. It has an effective role in protein formation, nitrogen metabolism and cell division, cell membrane integrity, formation of cell wall, nucleic acid, and antioxidant system also [37, 38].

This study’s major objective is to biosynthesize bimetallic ZnO-B2O3 NPs by actinomycetes S. gancidicus and compare their effectiveness against P. irregulare (which causes damping off disease in pea plant) to monometallic ZnO NPs and B2O3 NPs. This study’s main objective was to explain how the biosynthesis of a bimetallic ZnO-B2O3 NP, ZnO NPs, and B2O3 NPs decreased P. irregulare’s detrimental effects on pea plant while also enhancing their physiological immune responses.

2 Materials and methods

2.1 Reagents and chemicals

Media components obtained from Difco and Oxoid. Chemicals used, including isopropanol (CH3)2CHOH), boric acid (H3BO3), zinc nitrate hexahydrate (Zn (NO3)2·6H2O), and reagents, were considered standard materials (Sigma-Aldrich).

2.2 Collection and enrichment of soil samples

In February 2021, soil samples were taken from the rhizosphere of plants grown in the Damietta governorate that included wheat (Triticum aestivum), onions (Allium cepa), clover (Trifolium), potatoes (Solanum tuberosum), and garlic (Allium sativum). The samples were taken in sterile polythene sampling bags with the appropriate labels. The samples were physically treated (heated at 55 °C for 60 min) in accordance with a recent standard [39] and chemical treatment; CaCO3: soil (1:10 w/w) [40], for the improvement the growth of actinobacteria.

2.3 Isolation, purification and preservation of actinomycetes

In accordance with accepted publications [41, 42], the direct inoculation approach was adopted, in which soil samples (2–3 mg) were immediately distributed over the surface of starch-nitrate agar petri dishes. The incubation period was 5–25 days at 30 °C. The growing colonies were selected and picked up after incubation based on physical traits such distinctive color and colony form on starch-nitrate agar medium. The process was repeated several times till complete purification as indicated by microscopic examinations to assure the absence of any contamination. The isolated actinomycetes were stored on slant culture and maintained at 4 °C for less than 1 month or in 50% glycerol broth at − 4 °C.

2.4 Morphological and culture characteristics of actinomycetes

Morphological and cultural characters of the isolated actinomycetes were studied by inoculating the selected isolates into sterile International Streptomyces Project (ISP) media. The media were sterilized and poured into sterile Petri dishes. After solidification, the culture of the selected isolates was streaked on the medium surface aseptically and incubated at 30 °C for 7 days. Morphological characters such as the color of areal and substrate hyphae and presence of diffusible pigment were observed [43]. For the purpose of investigating the culture characteristics, the ISCC-NBS color name charts illustrated centroid color detection of aerial and substrate mycelia and diffusible pigment was used.

2.5 Screening of actinomycetes isolates for biosynthesis of different NPs

According to Kalaba et al. [44], each isolate was cultivated into a flask (100 mL) contained 50 mL of sterile starch nitrate broth, and the flask was shaken at 150 rpm for 7–10 days at 30 °C. The culture was then filtered to produce CFF utilizing Whatman No. 1 paper filters. For the biosynthesis of bimetallic ZnO-B2O3 NPs, ZnO NPs, and B2O3 NPs, all actinomycete filtrates were utilized. In order to identify the most effective actinomycetes that have a great potential to biosynthesize nanoparticles, all produced solutions were evaluated for the biosynthesis of various NPs using UV–Vis. spectrophotometer.

2.6 Extraction of S. gancidicus metabolites

Extraction performed by using two solvents (ethyl acetate and chloroform). According to Ahmed [45], the resulting culture filtrate was combined in a 1:1 ratio with ethyl acetate and collected in the top organic layer. Additionally, using a rotary evaporator, the extraction procedure was repeated three to four times, collected, and then condensed at 45 °C.

In the end, ethyl acetate extracts were gathered and kept in a room temperature storage facility. In order to evaluate the pot experiment [46], synthesize a chloroform extract, and compare the two extracts’ in vitro antifungal efficacy against P. irregulare.

2.7 Identification of the most potent actinomycetes

Both morphological and genetic analysis were used to pinpoint the strongest actinomycete isolate. Using a transmission electron microscope, morphological identification was carried out by studying the morphological features and macroscopic characteristics. Using eubacterial universal primers, the 16S rRNA gene was genetically replicated by polymerase chain reaction (PCR). R1492 with the sequence 5′-TACGGGYTACCTTGTTACGACTT-3′ with the sequence 5′-AGAGTTTGATCMTGGCTCAG-3′. The PCR mixture contained 2.5 units of Taq polymerase, 200 M dNTPs, 30 pmol of each primer, 10 µg of chromosomal DNA, and 50 µL of polymerase buffer. In 30 cycles, the PCR was run at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min. PCR purification kit (Qiagen, Germany) was used to clean the PCR product once it had been completed. According to the technique employed by [47]. DNA sequences were acquired employing an ABI PRISM 3700 DNA sequencer and ABI PRISM Big Dye Terminator Cycle Sequencing.

The BLAST tool, which is accessible on the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov), was used to examine sequence data in the GenBank database. To determine the DNA similarities, unidentified genomes were matched to every sequence in the database [48].

2.8 Preparation of ZnO NPs,B2O3NPs, and bimetallic ZnO-B2O3NPs

The specific quantity of salts was utilized for ZnO-B2O3 NPs biosynthesis in order to achieve bimetallic biosynthesis. In further specifics, 10 mL of (2.0 mM) Zn (NO3)2·6H2O and 10 mL of (2.0 mM) boric acid were combined and stirred at room temperature for approximately 30 min. Following that, 80 mL of the produced actinomycetes filtrate was added to them. The solution’s pH was checked after creating the combination, and it was discovered to be 7.2. In order to create the most successful synthesis of ZnO-B2O3 NPs, the reaction conditions were created as the temperature for incubation was set at 30 °C and reaction time around 24 h with stirring (500 rpm) in a shaking incubator [49]. The change in color was observed and fixed as deep off-white after the entire incubation, which explained the bio-production of ZnO-B2O3 NPs. To extract loosely attached actinomycetes biomolecules, the produced ZnO-B2O3 NPs were washed multiple times with distilled water before being cleared by centrifuge at 5000 rpm for around 20 min. The salt solutions were combined individually alongside the actinomycetes supernatant and incubated using the same techniques as when the process of biosynthesis of ZnO NPs and B2O3 NPs was carried out.

2.9 Characterization of ZnO NPs,B2O3NPs, and bimetallic ZnO-B2O3NPs

First, the optical characteristics of the synthesized ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs were examined using a UV–Vis. spectrometer (JASCO V-560) at various wavelengths between 190 and 900 nm. Before taking any measurements, the instrument should be auto-zeroed with a sample lacking the known metal salt. Dynamic light scattering (DLS-PSS-NICOMP 380, USA) was used to determine how the generated ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NP diffuse on an average particle size basis. Additionally, HR-TEM (HR-TEM, JEM2100, Jeol, Japan) was used to determine the average and precise sizes of the produced nanoparticles as well as the observed forms of the created ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs. After performing XRD (XRD-6000, Shimadzu Scientific Instruments, Japan), the crystallinity and crystal size determination were examined. SEM (SEM, ZEISS, EVO-MA10, Germany) was used to analyze the surface morphology and boundary size of the produced ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs.

2.10 Control of P. irregulare by ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs

2.10.1 P. irregulare inoculum, and culture conditions

The P. irregulare pathogen, with accession numbers RCMP 026F001 (1), EMCC 233, and DSM 62956, was obtained from the Regional Center for Mycology and Biotechnology (RCMB) of Al-Azhar University. The pathogen was cultured on Sabouraud dextrose agar (SDA) media and maintained at 4 °C for up to three months after being incubated at 28 °C for up to seven to fourteen days in a state condition. The ready state of the pathogenic fungal inoculum was reported by the following reference [50].

2.10.2 In vitro assessment of antifungal activity and determination of minimum inhibitory concentration (MIC)

Antifungal activity of biosynthesized ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs and ethyl acetate extract of S. gancidicus was performed using an agar well diffusion method used by Shubharani et al. [51] with minor modifications.

On SDA plates, the fungus suspensions were seeded. Then, a sterile micropipette tip was used to create wells 6 mm in diameter. Using a micropipette, the wells were filled with 90 µL of biosynthesized ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs with concentration 1000 µg/mL for all, 1 mg/mL of chloroform extract of S. gancidicus and ethyl acetate extract of S. gancidicus. Negative control (sterile water) and positive control (difenoconazole 25% as antifungal) were placed in the plate.

For seven days, the culture plate was left to incubate at 28 °C, and the areas of inhibition were monitored and quantified. Three duplicates of the experiment were run. MIC was also achieved, where different concentrations of bio-synthesized ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs (100, 10, 1, 0.1, 0.01, and 0.001 µg/mL) and ethyl acetate extract of S. gancidicus with concentration (4, 2, 1, 0.5, and 0.25 mg/mL) were placed in wells.

2.10.3 Ultrastructure study of control and treated P. irregulare

After centrifuging cultures that had been grown for seven days and rinsing control and treated P. irregulare using distilled water, following 3% glutaraldehyde fixation, samples were washed in phosphate buffer before being post-fixed for 5 min at room temperature in potassium permanganate solution. Samples were dehydrated for 30 min in 100% ethanol after 15 min in each ethanol dilution, which ranged from 10 to 90%. Samples were eventually submerged in pure resin after a graded sequence of infusions of epoxy resin and acetone. Very tiny fragments were collected on copper grids. Sections were then given a second coat of stain using lead citrate and uranyl acetate [52], at RCMB, Al-Azhar University, a JEOL-JEM 1010 transmission electron microscope was employed.

2.11 In vivo experiment

2.11.1 Pot experiment design

For the present investigation, seeds of (Pisum sativum) master B were purchased from Legume Crops Research department, Field Crop Research Institute, Agricultural Research Centre. Seeds of Pisum sativum plants were soaked for 2 h in a suspension containing 2 mL/one gram of seeds.

Plastic pots were used to germinate seeds of Pisum sativum, for the plastic pots used for germination, clean sand and clay were combined in a 1:3 ratio. The pots were stored in the greenhouses at an average temperature of 22 °C during the day and 18 °C at midnight, with a humidity level of 70–85%, and 28 seeds per treatment were typically used. After 40 days, post-emergence damping-off and survivability were assessed. The seedling peas were moved to plastic pots after germination as In the Research Garden of the Faculty of Science, Al-Azhar University, Cairo, Egypt, one seedling/pot (30 cm in diameter) having a combination of sand and clay (1:3 w/w) with an overall weight of 7 kg was sown.

For each treatment, 8 replicas of the pots were dispersed. A 100-pressure sprayer was then used to provide treatment to the plants on three separate occasions (50 mL per plant every week), before and after blooming.

The following is how the treatments were set up: T1 represents the healthy control, T2 the infected control, T3 the healthy and treated with ZnO NPs, T4 the infected and treated with ZnO NPs, T5 the healthy and treated with B2O3 NPs, T6 the infected and treated with B2O3 NPs, T7 the healthy and treated with ZnO-B2O3 NPs, T8 the infected and treated with ZnO-B2O3 NPs, T9 the healthy and treated with S. gancidicus, T10: infected and treated with S. gancidicus, and T11: infected and treated with difenoconazole 25%.

To assess plant resistance, track disease symptoms, and collect samples for biochemical analysis from plant samples 40 days after planting, an artificial infection with a pathogen (P. irregulare) 109 CFU/mL culture was made.

When the plant achieves the age of 40 days, morphological and biochemical markers for resistance analysis as well as development and severity were noted for plant resistance evaluation.

In accordance with [53], disease severity and protection ratio were calculated using five score classes: 0 (no symptoms), 1 (water-soaked tissue), 2 (discolored and collapsed tissue), 3 (stunted and poor standing seedlings), and 4 (root rot and seedling rot at soil line then damping off). Disease symptoms were noted 40 days after germination.

The following formula was used to determine PDI:

$$\mathrm{PDI}=\left({1\mathrm{n}}_{1}+{2\mathrm{n}}_{\mathrm{n}}+{3\mathrm{n}}_{3}+{4\mathrm{n}}_{4}\right)\times 100/{4}_{{\mathrm{n}}_{\mathrm{t}}}$$

where Nt is the overall number of plants and n1 through n4 represent the total amount of plants in each class.

Percentage of protection was determined using:

$$\mathrm{Protection\%}=\mathrm{A}-\mathrm{B}/\mathrm{A}\times 100\mathrm{\%}$$

where A is the PDI in the infected control plants and B is the PDI in the treated infected plants.

As stated by Lowry et al. [54], total soluble proteins were evaluated. A total of 5 mL of 2% phenol and 10 mL of deionized water were used to extract the dried shoots. One mL of this extract was combined with 5 mL of alkaline reagent (50 mL of 2% Na2CO3 prepared in 0.1 N NaOH and 1 mL of 0.5% CuSO4 prepared in 1% potassium sodium tartrate) and 0.5 mL of Folin’s reagent (diluted by 1:3 v/v). After 30 min, a color change could be seen at a wavelength of 750 nm.

The amount of phenolics was determined using an earlier method [55, 56]. One g of dried shoots was extracted in 5–10 mL of 80% ethanol for at least 24 h. After dehydrating the alcohol, the remaining residue was extracted three times using 5–10 mL of 80% ethanol each time. The purified extract was then filled to a capacity of 50 mL with 80% ethanol and then, 0.5 mL of the extract was mixed well with 0.5 mL of Folin’s reagent and shaken for 3 min. Thereafter, 3 mL of purified water and 1 mL of saturated sodium carbonate solution were added and thoroughly mixed. The blue color was detected at 725 nm after 1 h.

The quantity of MDA in fresh Pea leaves was quantified using the procedure outlined by Badawy et al. [57]. Fresh leaf samples (0.5 g) were extracted with 5% TCA and centrifuged for 10 min at 4000 × g. 2 mL of the extract was combined with 2 mL of 0.6% thiobarbituric acid (TBA) solution and placed in a water bath for 10 min. The generated color’s absorbance was measured at 532, 600, and 450 nm after cooling. The following equation was used to calculate the MDA content: 6.45 × (A532 − A600) − 0.56 × A450. The Mukherijee technique [58] was utilized to calculate H2O2 content. Fresh pea leaves (0.5 g) were added to 4 mL cold acetone; then 3 mL of the extract was mixed with 1 mL of 0.1% titanium dioxide in 20 percent (v:v) H2SO4; then the mixture was centrifuged at 6000 × g for 15 min. The yellow color generated at 415 nm was detected. PPO and POD enzyme function was evaluated in this study to provide an unambiguous sign of immunity-related enzymes. The adopted method of Srivastava [59] was applied to determine POD activity. The activity of PPO was measured by the method of Matta [60].

2.12 Statistical analysis

One-way analysis of variance (ANOVA) was used to evaluate the pilot data. By utilizing CoStat (CoHort, Monterey, CA, USA) and the least significant difference (LSD) test, statistically significant differences across treatments were shown to exist at p 0.05. The results are displayed as mean standard errors (n = 3) [61, 62].

3 Results and discussion

3.1 Isolation and identification of actinomycetes

Twenty-nine actinomycetes isolates coded from (A1–A29) were isolated from rhizosphere of different soils from Damietta Governorate, Egypt. On starch nitrate agar medium, about 41.3% of isolates give gray color, 31% white, 6.8% orange, 10.3% pink, and 10.43% red as shown in Table 1.

Table 1 Some morphological characters of actinomycetes isolated from different rhizospheric soils of Damietta Governorate, and growing on starch nitrate agar medium
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All isolates screened according to their potency to biosynthesis the formed NPs, and the results showed that A9 isolate was the supreme producer. Depending on the morphological characteristics of the isolate A9 when growing on starch nitrate agar medium showing rough spore surface with 265 medium gray aerial mycelium and 263 white substrate mycelium and the optimal growth of it was studied using seven different ISP culture media.

The isolate grew well on ISP1, ISP3, ISP4, and ISP6, while the growth was moderate on ISP-5 and weak growth on ISP7 and no growth on ISP2 as shown in Table 2.

Table 2 The culture characteristic of S. gancidiucus OR229936 on different ISP media and the color of organism under investigation was consulted using the ISCC-NBS
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Genetically, this isolate showed similarity (92%) with S. gancidicus strain NRRL B-1872 16S ribosomal RNA, partial sequence (GenBank accession number OR229936) as showed in Fig. 1.

Fig. 1
figure 1

A Photograph of isolate A9 growing on starch nitrate agar medium showing rough spore surface with 265; moderate gray aerial mycelium and 263 white substrate mycelium, B transmission electron micrograph of isolate A9 growing on starch nitrate agar media showing spiral spore chain and spiny spore surface, and C phylogenetic tree of S. gancidicus with accession number (OR229936)

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3.2 Biogenic synthesis of ZnO NPs, B2O3NPs, and bimetallic ZnO-B2O3NPs

In this study, actinomycetes were used to produce bimetallic ZnO-B2O3 NPs. At first, these NPs appeared to be a faint white color, but as they were created, the color changed to a deep off-white. This color served as a reducer or capping agent that reduced zinc nitrate and boric acid into ZnO-B2O3 NPs and maintain them in colloidal appearance. The produced extract’s bioactive metabolites function as a capping agent to stop nanoparticle agglomeration and change their biological activity [63, 64].

According to Kumaravel et al. [65], the extracted compounds from Metarhizium anisopliae possess a greater concentration and aid in the reduction of zinc and copper ions to produce bimetallic ZnO-CuO NPs by bio-nano formulation. The primary active metabolites in the extract are in charge of NPs' bio-reduction [66].

According to Šebesta et al. [67], the filtrate contains enzymes with the capacity to nanometrically convert metallic ions to elements metal (Mo). Biomolecules also may combine with metal ions to create intricate electron transport pathways during the production of metals, in which NADPH/NADH transform into NADP+/NAD+ [68, 69]. Actinomycetes’ synthesis of NPs is a simple and straightforward technique because of the simplicity of the biomass treatments and downstream processing, as well as their higher productivity than bacteria [70].

3.3 Characterization of ZnO NPs, B2O3NPs, and bimetallic ZnO-B2O3NPs

As the reducing and capping agent, the produced filtrate’s ability to synthesis ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs was assessed. When bimetallic ZnO-B2O3 NPs were created, the prepared solution’s hue changed from a faint white to a deep off-white. The resulting off-white color, which was due to the stimulation of the SPR of natural bimetallic ZnO-B2O3 NPs, provided a reliable spectroscopic signal of their existence [71].

The experimental peak was visible in the spectra due to the O. D. (1.18; diluted three times) (Fig. 2). The created ZnO-B2O3 NPs were small, visible at 345 nm, and matched our most recent publication, according to the UV–Vis. examinations [72], owing to variations in the synthetic routes, with little alteration. The intensity of the off-white hue correlated with the ability to create ZnO-B2O3 NPs [73, 74]. As shown in Fig. 2, the (O. D.) for B2O3 NPs was found to be 0.76 (dilution three times) at a particular wavelength of 275 nm, which is consistent with the results of the published research [75].

Fig. 2
figure 2

UV–Vis. spectrum of the action-synthesized ZnO NPs, B2O3NPs, and bimetallic ZnO-B2O3 NPs (diluted three times)

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Similar results were obtained for ZnO NPs, where the (O. D.) was discovered to be 0.86 (diluted three times) at a particular wavelength of 365 nm. SPR is greatly influenced by the durability, dimensions, morphology of the surfaces, construction, and dielectric characteristics of any produced nanoparticles [76, 77].

Our blend of ZnO-B2O3 NPs was discovered to be poly-dispersed, ranged in dimensions, and mostly had spherical particles as their dominating shape after contrasting with articles in the field on intermediary particle size and form. It is possible that labor led to the development of several forms [78]. Similar shapes could have been observed as an outcome of the extraction-based synthesis procedure, which is why the identical shape had been discovered, even though all of the freshly created NPs were sphere-shaped. Because only the most practicable reducing and capping agents were used in our work, mono-displayed NPs are a stable form.

The hydrodynamic diameter, particle dimension dispersion, and the polydispersity index (PDI) of produced ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs were all examined using DLS. To ascertain the typical size of these NPs, the acquired data were contrasted with the HRTEM research [79]. The HR-TEM image, as seen in Fig. 3a, revealed the spheroidal shapes of significantly mono-dispersed ZnO NPs with typical diameters that ranged from 33.5 to 48.5 nm and an estimated mean diameter of 40.31 ± 1.0 nm. The biosynthesized B2O3 NPs, which were round in shape and had an average diameter of 55.85 ± 2.5 nm and a size range of 42.10 nm to 92.89 nm (Fig. 3b), were seen to have a similar situation. Last but not least, the synthesized bimetallic ZnO-B2O3 NPs’ HR-TEM images in Figs. 3c and 3d showed that the particles were perfectly spherical, with diameters ranging from 15.98 to 86.58 nm and an overall diameter of 59.14 ± 1.4 nm. The created actinomycetes filtrate, which was abundant in protein and amino acid sequences, had to be reduced, stabilized, and capped using the given mono-dispersed NPs [80].

Fig. 3
figure 3

HR-TEM images of the actino-synthesized ZnO NPs (a), B2O3 NPs (b), and bimetallic ZnO-B2O3 NPs at different magnifications (c, d)

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The HRTEM result demonstrates that there was just one grade system since the line width was same throughout. It proved that the boron was evenly dispersed through the zinc matrix, resulting in a special alloy. The actinomycetes filtrate’s generated radical-multi-position may result in a simultaneous drop in Zn and B, as seen and similarly described in the published research [81].

Our mixture of ZnO-B2O3 NPs were found to be mono-dispersed, variable in size, and mostly had circular particles as their dominant shape after contrasting with articles in the field on intermediary particle size and form. It is possible that the work did not evolve many different forms [78]. Although all of the newly produced NPs were sphere- or orbicular-shaped, different morphologies may have been seen as a result of the extraction-based synthetic process, which explains why the anisotropic type had been detected. A stable form is mono displayed since only the most practical reducers and caps (actinomycetes filtrate) were utilized in our experiment.

The typical particle size distribution of the biosynthesized ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs generated by the actinomycetes filtrate was determined by the DLS technique to be 66.52 nm, 77.45 nm, and 76.85 nm, respectively (Fig. 4).

Fig. 4
figure 4

DLS analysis of the actino-synthesized ZnO NPs (a), B2O3 NPs (b), and bimetallic ZnO-B2O3 NPs (c)

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When the polydispersity index (PDI) results are less than 0.05, samples are said to be monodisperse by international standards organizations (ISOs). The goal of PDI results larger than 0.7, however, is to create particles with a polydispersity distribution [82]. Our research revealed that the PDI values for ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs were, respectively, 0.041, 0.040, and 0.038. The values at hand indicated that the biosynthesized NPs were within a tolerable range.

The findings showed that the estimated sizes of the particles identified by HRTEM imaging were smaller than the mean and prevalent sizes indicated by DLS analysis. The causes for the considerable sizes of the biosynthesized NPs, according to the following reference, include the hydrodynamic radius inside the produced ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs as well as the water layers around them [83].

The surface properties and surface shape of the produced bio-synthesized NPs were investigated using the SEM method. The created actinomycetes filtrate included the equivalent bright spherical particles, and the SEM picture of ZnO NPs produced by the generated filtrate showed variable border sizes (Fig. 5a). The biosynthesized B2O3 NPs, which are shown in Fig. 5b as isolated, rounded, bright particles on the actinomycetes filtrate, also showed the similar pattern.

Fig. 5
figure 5

SEM images of the actino-synthesized ZnO NPs (a), B2O3 NPs (b), and bimetallic ZnO-B2O3 NPs at different magnifications (c, d)

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A consistent ZnO-B2O3 NP surface with a clear appearance can be seen in the combined ZnO-B2O3 NPs and filtrate (SEM findings) (Fig. 5c). It was found that the produced fungal filtrate effectively separated the ZnO-B2O3 NPs as spherical aggregates merged with each other throughout the filtrate's surface (Fig. 5d).

When compared to other research on the subject of morphological form, the produced ZnO-B2O3 NPs (in this work) were evenly dispersed with restricted size and the perfect spherical formation. Citrate reduction was used by Mohsin et al. [84] to create bimetallic Ag and Au core–shell NPs at varied pH and temperature levels. Since the authorized morphological form and border size recommended that they keep the size range from 50 to 65 nm and present as round particles, temperatures and pH levels perform a key role in the synthetic procedure.

Figure 6 displays the XRD analysis for the created bimetallic ZnO-B2O3 NPs. The synthesized bimetallic ZnO-B2O3 NPs and the starting material (actinomycetes filtrate) are both represented by the manufactured NPs in their respective amorphous and crystal configurations. XRD results were evaluated of the generated ZnO NPs and B2O3 NPs as distinct spectra in so that the unique lines of the ZnO-B2O3 NPs was directly discovered (Fig. 6). That 2Ɵ relates to the prepared filtrate in the range of 5° to 25° should be made clear [81, 85]. The generated bimetallic ZnO-B2O3 NPs, and the related XRD data are shown in Fig. 6, which accentuates the ZnO NPs’ diffraction peaks. The peaks at 2Ɵ = 27.47°, 31.14°, 45.90°, 56.12°, 67.33°, and 75.45° are among them. These peaks, which are completed by the JCPDS standard card number 361451, correspond to Bragg’s reflections (002), (101), (102), (110), (103), and (201) [86]. Along with the standard card JCPDS number 300019, they additionally include the B2O3 NP diffraction peaks at 2Ɵ = 15.90°, 28.12°, 31.23°, and 41.22° [87].

Fig. 6
figure 6

XRD analysis of ZnO NPs, B2O3 NPs, and bimetallic B2O3-ZnO NPs

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The produced ZnO-B2O3 NPs were crystallized and possessed a face-centered cubic (fcc) crystal structure, according to the available XRD data (Fig. 6). According to the XRD data, the produced bimetallic NPs were extremely crystalline and associated with amorphous actino-filtrate, which increased their movement in the solution to enhance biomedical use [88].

Finally, the intermediate crystallite sizes of bimetallic ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs were determined using the equation of Williamson-Hall (W H; Eq. 5) and were found to be 30.94, 31.09, and 38.99 nm, respectively [89, 90],

$$\beta \mathrm{cos}\theta =\frac{k\lambda }{{D}_{W-H}}+4\varepsilon \mathrm{sin}\theta$$

3.4 Antifungal activity of ZnO NPs, B2O3NPs, and ZnO-B2O3NPs and extract of S. gancidicus against P. irregulare

In the present investigation, all actino-synthesized ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs at 1000 µg/mL besides to 1 mg/mL from each ethyl acetate extract and chloroform extract of S. gancidicus demonstrated positive antifungal efficacy against P. irregulare as qualitative determination with ZOI as 22, 16, 33, 17, and 9 mm, respectively, in comparison with 25% difenoconazole as a positive control with ZOI as 12 mm as shown in Fig. S1.

Additionally, various actino-generated NP doses (100, 10, 1, 0.1, 0.01, and 0.001 µg/mL) were assessed to find MIC. According to our findings, ZnO-B2O3 NPs demonstrated increasing antifungal efficacy against P. irregulare, with an inhibition zone of 33 ± 0.96 mm at a dosage of 1000 µg/mL and a MIC of 0.01 µg/mL.

In addition, as shown in Fig. 7, both ZnO NPs and B2O3 NPs showed antifungal activity, but to a lesser extent than ZnO-B2O3 NPs, which showed inhibition zones at concentrations of 1000 µg/mL of 22 ± 0.87 and 16 ± 0.99 mm, respectively, and gave MICs for both at 1 µg/mL. This proves that bimetallic NPs have better antifungal activity than mono-metal NP.

Fig. 7
figure 7figure 7

MIC of the actino-synthesized bimetallic ZnO-B2O3 NPs, ZnO NPs, B2O3 NPs and ethyle acetate extract (EAE) of S. gancidicus against pathogenic P. irregulare using agar well diffusion method

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Bimetallic NPs have greater antibacterial action than monometallic NPs, according to earlier studies [91,92,93]. Due to an extra degree of flexibility, bimetallic NPs produced using biological means possess greater possibility than metallic NPs produced independently using biological methods [94].

Additionally, the efficacy may be attributable to the NPs’ rough exterior surface, which damages cell walls and allows NPs to penetrate plasma membranes [95]. A variety of concentrations of ethyl acetate extract (0.25, 0.5, 1, 2, and 4 mg/mL) were investigated to discover MIC as shown in Fig. 7 because ethyl acetate extract had stronger anti-pythium activity compared to chloroform extract.

Results showed that the MIC was 0.5 mg/mL having a ZOI of around 10 mm; hence, it was employed for marijuana research. Based on the most recent publications [96, 97], the generation of antibiotics and hydrolyzing enzymes through antibiosis is the primary biocontrol methods used by Streptomyces species. They produce extracellular hydrating and destroying enzymes such cellulases, amylases, chitinases, and lipases as part of their action against phytopathogens.

3.5 Ultrastructure investigation of control and treated P. irregulare

Examination of ultrathin sections of P. irregulare treated with ZnO NPs, B2O3 NPs and bimetallic ZnO-B2O3 NPs revealed the occurrence of fungal cell at different degree of disorganization, ranging from wall loosening to vacuolation and protoplasm disintegration.

It was juxtaposed to the control P. irregulare and revealed that the protoplast was delimited by a thin cell wall and contained a dense, polyribosome-enriched cytoplasm in which numerous organelles were present, such as mitochondria, strands of endoplasmic reticulum, nuclei, and lipid bodies as shown in Fig. 8a, which is consistent with a recent paper [98].

Fig. 8
figure 8

Ultrastructure of the control non-treated P. irregulare (A), the cells of P. irregulare treated with ZnO NPs (B), treated with B2O3 NPs (C), and treated with bimetallic ZnO-B2O3NPs (D)

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When P. irregulare was treated with ZnO NPs, the cell walls and plasma membrane were largely destroyed, the nucleus had a tiny, damaged appearance, and the chromatin components were dispersed throughout the cytoplasm as numerous darkly colored masses (Fig. 8b). Historically, fungicides containing zinc have been used with great success, such as ethylene thiocarbamate zinc [99].

The nucleus and succulent vacuoles vanished from the P. irregulare treated with B2O3 NPs, and all cell contents showed aberrant appearances (Fig. 8c). Furthermore, ZnO-B2O3 NPs treatment of P. irregulare resulted in the unambiguous destruction of all cell contents, the collapse of the cell wall, and a breakdown of the plasma membrane. Additionally, the nucleus had a tiny size, distorted form, and many dark stained entities dispersed throughout the cytoplasm (Fig. 8d). According to the following reference [100], the consequences of the ultrastructural alterations in pythium cells might have detrimental effects on the cell wall, nucleus, and mitochondria and might be partly to blame for the decline in respiration and essential activities.

3.6 Disease index and protection

Data existing in Table 3 explained that all applied treatments (ZnO NPs, B2O3 NPs, ZnO-B2O3 NPs, S. gancidicus extract, and difenoconazole 25%) significantly diminished percentage of disease severity caused by P. irregulare compared to the non-treated infected plants (control), as revealed in Table 3, and the infection occurrence reached 85%, resulted from P. irregulare infection which agree with the recent work [101,102,103]. Figure 9 explains the symptoms grades from 0 to 4 and clarifies the effects of P. irregulare on pea plant.

Table 3 Effect of ZnO NPs, B2O3 NPs, bimetallic ZnO-B2O3 NPs, EAE of S. gancidius, and difenoconazole 25% on disease severity % and protection % of infected pea plants with P. irregulare
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Fig. 9
figure 9

Symptom grades from 0 to 4 clarify effects of P. irregular on pea plants, where 0: no symptoms, 1: water-soaked tissue, 2: discolored and collapsed tissue, 3: stunted and poor standing seedlings, and 4: root rot and seedling rot at soil line then damping off

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As opposed to that, the synthesized bimetallic ZnO-B2O3 NPs was the best treatment in decreasing PDI by 7.5% and raising the protection by 91.1%, then followed by treatment with S. gancidicus, ZnO NPs, B2O3 NPs, and difenoconazole, where the PDI was recorded as 10%, 15%, 20%, and 22.5%, respectively and protection by 88.3%, 82.3%, 76.5%, and 73.5%, respectively. Figure 10 shows the effect of ZnO NPs, B2O3 NPs, ZnO-B2O3 NPs, EAE of S. gancidicus, and difenoconazole 25% on infected pea plants with P. irregulare in comparison to healthy plant.

Fig. 10
figure 10

Effect of ZnO NPs, B2O3 NPs, bimetallic ZnO-B2O3 NPs, EAE of S. gancidius and difenoconazole 25% on infected pea plants with P. irregulare in comparison to healthy plant where a control healthy, b control infected, c infected and treated with bimetallic ZnO-B2O3 NPs, d infected and treated with S. gancidicus, e infected and treated with ZnO NPs, f infected and treated with B2O3 NPs, and g infected and treated with difenoconazole 25%

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Lessening the severity of the infection is the most potent confirmation for defense occurrence in the plant. Nanofertilizers are considered a magical treatment to stimulate plants to improve growth, increase antioxidants, and resist biological stresses, especially fungal plant diseases, which is reflected in increased productivity [26, 104,105,106].

3.7 Total soluble protein determination

The results in Fig. 11 showed that the contents of the total soluble protein in pea plants highly significantly increased as a result of P. irregulare infection by 40.76% in comparison with healthy control. These results are supported by many previous studies [107,108,109].

Fig. 11
figure 11

Effect of ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs on the total soluble protein content of infected pea plants with P. irregulare, where T1: healthy control, T2: infected control, T3: healthy + ZnO NPs, T4: infected and treated with ZnO NPs, T5: healthy + B2O3 NPs, T6: infected and treated with B2O3 NPs, T7: healthy + ZnO-B2O3 NPs, T8: infected and treated with ZnO-B2O3 NPs, T9: healthy + S. gancidius filtrate, T10: infected and treated with S. gancidius, and T11: infected and treated with difenoconazole. Data represent mean ± SD (n = 3); letters revered to significance in statistical analysis

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On the other hand, it was found that application of ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs, on healthy and infected plants, caused significant increase in total soluble protein when being compared with non-treated plants control. Concerning the effect ZnO NPs, B2O3 NPs, ZnO-B2O3 NPs, S. gancidicus, and difenoconazole, on the infected pea plants with P. irregulare, it was found that ZnO-B2O3 NPs show considerable increase in total soluble protein by 69.77%, followed by S. gancidicus filtrate by 55.85%, B2O3 NPs by 24.68%, ZnO NPs by 20.16%, and came next difenoconazole by 18.33%, respectively, when being compared with untreated infected control (Fig. 11). These results are in harmony with the following references [110,111,112].

The micronutrient zinc is an essential part of the two glycodehydrogenases, which are required for protein synthesis, and glycine dipeptidases, which are required for glycolysis in the last stages of respiration, according to research by [113].

Many of researches showed the valuable effect of boron on plant growth; Emara and Abd El-All [114] found that nanoboron treatment increased the values of plant growth and photosynthetic pigments. The vital role of boron on ornamental plants was attributed to its essential roles in translocation of sugars as well as enhancing the synthesis of proteins, cell division, and root development. There is beneficial effect of boron on preventing the abortion of flowers and the conversion of starch to soluble sugars [115, 116].

The results of this study, together with previous studies, explain the importance of zinc and boron in nano form to enhance plant immune responses and to form proteins that perform a vital part in pathogen resistance. Additionally, our findings support the idea that actinomycetes increase the protein content of both healthy and diseased plants. S. gancidicus is extensively distributed in a variety of habitats, including soil settings, where it participates in the breakdown of decaying organisms, the fixation of nitrogen (actinomycetes perform around 15% of the nitrogen fixation), and the solubilization of phosphates [117, 118].

3.8 Phenol contents

Juxtaposed with healthy control plants, pea plant contaminated with P. irregulare exhibited substantial increases in total phenol levels by 38.88%. This conclusion is corroborated by a number of other studies [107, 119,120,121,122]. Additionally, as compared to untreated plants (control), ZnO NPs, B2O3 NPs, and ZnO-B2O3 NP treatment generated a substantial rise in total phenol on both healthy and afflicted plants.

Regarding the effect of ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs, S. gancidicus, and difenoconazole on the infected pea plants with P. irregulare, it was found that ZnO NPs show considerable increase in total phenol by 204%, followed by bimetallic ZnO-B2O3 NPs by 196%, S. gancidicus filtrate by 144%, difenoconazole by 112%, and came B2O3 NPs by 104% respectively, when being compared with untreated infected control as shown in Fig. 12.

Fig. 12
figure 12

Effect of ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs on the total phenol content of infected pea plants with P. irregulare, where T1: healthy control, T2: infected control, T3: healthy + ZnO NPs, T4: infected and treated with ZnO NPs, T5: healthy + B2O3 NPs, T6: infected and treated with B2O3 NPs, T7: healthy + ZnO-B2O3 NPs, T8: infected and treated with ZnO-B2O3 NPs, T9: healthy + S. gancidius filtrate, T10: infected and treated with S. gancidius, and T11: infected and treated with difenoconazole. Data represent mean ± SD (n = 3); letters revered to significance in statistical analysis

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Phenols have avital impact on maintaining antioxidant levels and scavenging ROS from cells in pea plants [123]. The phenolics accumulation in pea plant considered as an adaptive strategy against damping off disease. Other studies confirmed that ZnO NPs induces the formation of phenols and strengthens cell walls, thus reducing the severity of disease [124, 125]. However, B2O3 NPs works to increase phenols and substances responsible for plant resistance [126, 127]. It has been proposed that B2O3 NPs probably improve the phenol and antioxidant content of distress pea plants and decrease oxidative stress indicators, protecting plant health [127].

3.9 Oxidative stress

Results in Fig. 13a, b showed that P. irregulare cause increased contents of MDA and H2O2 by 46.21%, and 122%, respectively comparing to non-infected pea plants. These results are supported by many previous findings [128, 129].

Fig. 13
figure 13

Effect of ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs on MDA (a) and H2O2 (b) levels of infected pea plants with P. irregulare, where T1: healthy control, T2: infected control, T3: healthy + ZnO NPs, T4: infected and treated with ZnO NPs, T5: healthy + B2O3 NPs, T6: infected and treated with B2O3 NPs, T7: healthy + ZnO-B2O3 NPs, T8: infected and treated with ZnO-B2O3 NPs, T9: healthy + S. gancidius filtrate, T10: infected and treated with S. gancidius, and T11: infected and treated with difenoconazole. Data represent mean ± SD (n = 3); letters revered to significance in statistical analysis

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The infected pea plants’ levels of MDA were declined in response to S. gancidicus filtrate, ZnO NPs, B2O3 NPs, ZnO-B2O3 NPs, and difenoconazole by 41.68%, 36.51%, 26.15, 26.15, and 15.25%, respectively as shown in Fig. 13a.

While contents of H2O2 in infected pea plant were decreased by 50%, 45%, 40%, 37.5%, and 22.5% at ZnO-B2O3 NPs, S. gancidicus filtrate, ZnO NPs, difenoconazole and B2O3 NPs comparing to P. irregulare infected pea plants as shown in Fig. 13b. By boosting antioxidant molecules that scavenge ROS and protect cellular membranes against oxidative stress, the application of ZnO-B2O3 NPs reduced the generation of MDA and H2O2 [130, 131].

One of the most important signs of resistance to stress is avoidance and reduction of oxidative stress and capture of free radicals [132]. The findings of this investigation are consistent with other studies that showed a reduction in MDA and H2O2 levels following treatment with ZnO NPs and bimetallic ZnO-B2O3 NPs [133,134,135].

3.10 Oxidative enzyme activities

Results in Fig. 14a, b showed that P. irregulare cause increased the activity of POD and PPO enzymes by 36.36% and 23.23%, respectively comparing to non-infected pea plants. These results are supported by many previous findings [136, 137].

Fig. 14
figure 14

Effect of ZnO NPs, B2O3 NPs, and ZnO-B2O3 NPs on on POD (a), and PPO (b) activites of infected pea plants with P. irregulare where, T1: healthy control, T2: infected control, T3: healthy + ZnO NPs, T4: infected and treated with ZnO NPs, T5: healthy + B2O3 NPs, T6: infected and treated with B2O3 NPs, T7: healthy + ZnO-B2O3 NPs, T8: infected and treated with ZnO-B2O3 NPs, T9: healthy + S. gancidius filtrate, T10: infected and treated with S. gancidius, and T11: infected and treated with difenoconazole. Data represent mean ± SD (n = 3); letters revered to significance in statistical analysis

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The activities of POD in infected pea plants were increased in response to S. gancidicus filtrate, difenoconazole, B2O3 NPs, bimetallic ZnO-B2O3 NPs, and ZnO NPs by 89.09%, 87.27%, 34.54%, 21.81%, and 10.90%, respectively as shown in Fig. 14a.

While the activities of PPO in infected pea plants were raised by 92.50%, 92.20%, 85.90%, 79.01%, and 65.66% at ZnO-B2O3 NPs, ZnO NPs, B2O3 NPs, S. gancidicus filtrate, and difenoconazole and comparing to P. irregulare infected pea plants as shown in Fig. 14b.

Enhancing the efficiency of antioxidant enzymes is crucial for improving plant physiological resistance and preventing infection-related cell oxidation [138, 139]. The accumulation of both antioxidants and phenolic compounds are observed [140]. H2O2 and MDA levels in the plants are significantly reduced after zinc treatment [141].

4 Conclusion

In this work, S. gancidicus (OR229936) was effectively used to actino-synthesize bimetallic ZnO-B2O3 NPs as well as monometallic ZnO NPs and B2O3 NPs utilizing an economical and environmentally friendly technique. In our study, the actinomycete generation of bimetallic ZnO-B2O3 NPs was detected by the emergence of a faint off-white hue in the solution, which functioned as a stabilizing and reducing agent. With typical diameters varying from 33.5 nm to 48.5 nm and an estimated mean diameter of 40.31 ± 1.0 nm, the ZnO NPs in the HR-TEM image had spheroidal morphologies. The biosynthesized B2O3 NPs, that were round in shape and varied in size from 42.10 nm to 92.89 nm with an average diameter of 55.85 ± 2.5 nm, were seen to have a similar situation. The synthesized bimetallic ZnO-B2O3 NPs were pure-spherical, with diameters ranging between 15.98 nm and 86.58 nm and a mean diameter of 59.14 ± 1.4 nm, as seen by the HR-TEM images. The produced bimetallic ZnO-B2O3 NPs shown encouraging antifungal efficacy against P. irregulare, a plant pathogen that causes damping off. The outcomes shown that actino-synthesized ZnO-B2O3 NPs might increase plant biochemical resistance while also inhibiting P. irregulare’s damaging impacts on pea plant. Significantly, the synthesized bimetallic ZnO-B2O3 NPs was the best treatment in decreasing PDI by 7.5% and raising the protection by 91.1%, then followed by treatment with S. gancidicus, ZnO-NPs, B2O3 NPs and difenoconazole, where the PDI was recorded as 10%, 15%, 20%, and 22.5%, respectively, and protection by 88.3%, 82.3%, 76.5%, and 73.5%, respectively. Concerning the effect of ZnO NPs, B2O3 NPs, ZnO-B2O3 NPs, S. gancidicus, and difenoconazole, on the infected pea plants with P. irregulare, it was found that ZnO-B2O3 NPs show considerable increase in total soluble protein by 69.77%, followed by S. gancidicus filtrate by 55.85%, B2O3 NPs by 24.68%, ZnO NPs by 20.16%, and came next difenoconazole by 18.33%, respectively, when being compared with untreated infected control. The in vivo findings demonstrated that biological agents and chemical pesticide alternatives, such as ZnO NPs, B2O3 NPs, and bimetallic ZnO-B2O3 NPs, may be used on pea plant infected with P. irregular, which are encouraging for the agricultural sector. Our study is limited in that further research must be done to determine the toxicity level and the amount of synthesized NPs that accumulate in the pea plant before we can be convinced that they are safe for use. Along with using our synthesized NPs in other tested plants, which will be taken into account in our future study.