Evaluation of natural products and chemical compounds to improve the control strategy against cucumber powdery mildew

Powdery mildew, caused by Podosphaera xanthii, is a devastating disease that can wipe out a cucumber crop in optimal weather conditions. Disease control management depends mainly on fungicides to inhibit the pathogen. However, they have fatal side effects on most organisms. This study evaluated the use of natural products as safe alternatives to fungicides for controlling cucumber powdery mildew. The effectiveness and phytotoxicity of the studied products, as well as their effects on leaf anatomy and pollen fertility, were evaluated. Although the fungicide tested (Score 25% EC) was the most effective treatment, it caused the highest phytotoxicity, leaf morphological changes, and pollen sterility. All the treatments used significantly reduced disease severity under greenhouse and field conditions, except for Spirulina, which recorded the lowest efficacy rate. Lemon oil, garlic oil, and Blight stop achieved the desired goal of controlling the disease and improving the plant’s physiological state. Therefore, we recommend using any of them to control cucumber powdery mildew, except for Spirulina, which we recommend as a biofertilizer.


Introduction
Cucurbitaceae is one of the most famous plant families containing various species around the world, including cantaloupe, pumpkin, squash, melon, cucumber and watermelon. Cucumber (Cucumis sativus L.) is one of the most economically important vegetables in Egypt, both for local consumption and exportation. Powdery mildew on cucurbits may be caused by two fungi, Podosphaera xanthii (syn. Sphaerotheca fuliginea) which is common in warmer regions, and Golovinomyces cichoracearum (syn. Erysiphe cichoracearum) which needs cold weather (Lebeda et al., 2011). The conidia of Podosphaera xanthii germinate laterally, while the conidia of Golovinomyces cichoracearum germinate terminally (Boesewinkel, 1980). On cucumbers, powdery mildew caused by Podosphaera xanthii (Castagne) U. Braun & Shishkof is considered the most destructive cucumber foliar disease; it attacks the leaves, petioles, stems, and occasionally fruits in the greenhouse and open field (Bettiol et al., 2008). Podosphaera xanthii is an obligate biotrophic ectoparasite that depends on living host cells for growth, reproduction, spread, and recurrence of infection, which mainly results in a shortened cucumber growing season (Martínez-Cruz et al., 2014). After the initial infection, disease progression can occur rapidly under favourable conditions. The typical symptoms appear after 3-7 days as powdery white spots on the surfaces of the leaves, consisting of fungal hyphae and conidia. Severely infected plants show a decrease in physiological processes and metabolic activities, leading to defoliation and fruit deformation, causing a reduction in the crop quality and negatively affecting productivity (Pérez-García et al., 2009).
Control of powdery mildew depends mainly on the use of fungicides as the most effective method to limit disease severity, and on the cultivation of diseaseresistant varieties (Kiss, 2003). With changing environmental conditions and climate change, the effectiveness of fungicides is no longer the same as before, especially with the emergence of new fungal strains that are more tolerant to the fungicides and more able to bypass the defenses of resistant varieties. Furthermore, the accumulative effects of fungicides pollute the surrounding environment and harm beneficial organisms and humans, thus prompting researchers to find safer alternatives to fungicides that provide the same effectiveness in controlling the disease. Natural substances, such as bioproducts obtained from plants, algae, and microorganisms have been suggested as particularly promising safe alternatives (Masheva et al., 2014).
Our current study aims to evaluate some natural and chemical products for the control of cucumber powdery mildew by foliar spray under greenhouse and field conditions. Plant essential oils are secondary plant metabolites rich in bioactive compounds that are classified into two main groups: the terpene hydrocarbons group consists of monoterpenes (with ten carbon atoms) and sesquiterpenes (with 15 carbon atoms); and the oxygenated compounds group is made up of terpenoids, alcohols, phenols, aldehydes, oxide ethers, ketones, esters, sulphur-containing compounds, and flavonoids (Dhifi et al., 2016;Nazzaro et al., 2017;Raveau et al., 2020). These bioactive compounds work synergistically together; therefore, they are more efficient in commercial application than compounds containing a single bioactive compound (Tian et al., 2012). Essential oils have biological and antifungal activity, are biodegradable, economically efficient, and almost non-toxic (Abdullahi et al., 2020). Because of this, plant essential oils are ideal for plant protection and the management of phytopathogens under greenhouse and field conditions (Lanzotti et al., 2013). In the present study, we used three essential oils: lemon (Citrus limon), garlic (Allium sativum), and ginger (Zingiber officinale) and tested them as control agents against cucumber powdery mildew.
One of the biological products used in this study is Blight stop, which combines two bioagents, Trichoderma harzianum and Bacillus subtilis, which are capable of controlling several phytopathogens and have demonstrated an inhibitory effect on obligate parasites, such as powdery mildew and rust diseases (Hafez et al., 2018). The second biological product is Spirulina, a powder bioproduct of Spirulina platensis. Spirulina is a filamentous blue-green algae and lives in the sea and freshwater. It contains essential amino acids, proteins, vitamins, minerals, fatty acids, pigments (carotenoids, beta-carotene, phycocyanin, and chlorophyll), and phenolic compounds (caffeic, chlorogenic, salicylic, and trans-cinnamic acids). Spirulina platensis can be used as a biofertilizer and biostimulator that directly provides plants with essential elements required for growth (Jufri et al., 2016) and controls some phytopathogens, e.g., Fusarium oxysporum, Aspergillus flavus, and Aspergillus niger (Al-ghanayem, 2018).
The compound Copperal max used in our research is a chemical contact fungicide that contains 10% copper sulphate. Antifungals containing copper are preferable due to their low cost, chemical stability, partial safety for organisms, and effectiveness in controlling phytopathogens. Copper sulphate prevents many diseases, e.g., powdery mildew in sugar beet and cucumber (Mahmoud & Farahat, 2020). The other chemical compound is Score 25% EC, a systemic fungicide containing 25% difenoconazole, a commonly used triazole that treats plant diseases caused by fungi. It inhibits fungal ergosterol biosynthesis by targeting sterol-1-4demethylase (Elansky et al., 2016).

Source of plants and fungal inoculum
Seeds of cucumber susceptible cultivar Beit Alpha were obtained from the Department of Vegetables Production Research, Horticultural Research Institute, Agricultural Research Center, Dokki, Egypt. Podosphaera xanthii was obtained from infected plants collected from cucumber cultivations in the Kafr El-Sheikh governorate, Egypt. The typical symptoms of powdery mildew were confirmed by field inspection and microscopic examination. To confirm that the fungal pathogen is Podosphaera xanthii, spores were dusted from the infected leaf onto a microscope slide and treated with 3% aqueous KOH to check for fibrosin bodies. Spores were dusted on another slide, and then the slide was kept for 24 h in a humid chamber at 25°C to induce germination and determine the position of the germ tube (Miazzi et al., 2011). Leaves infected with Podosphaera xanthii were used as a source of infection under greenhouse conditions.

Pathogenicity test
Cucumber seeds were sown in trays filled with peat moss under greenhouse conditions in the Vegetable Diseases Research Department, Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt. After 4 weeks, three plants were transplanted into each plastic pot (25 cm diameter), filled with autoclaved sand:clay (1:1, v/v). Plants were then inoculated artificially with Podosphaera xanthiii by gently wiping infected cucumber leaves using a brush to drop fresh conidia on the leaves. Plants without inoculation were kept in closed plastic cages and served as controls. All the plants were fertilized with the recommended dose of NPK (1 g/L dissolved in irrigation water) and regularly watered. The temperature was maintained between 22 and 25°C with high relative humidity for 24 h to stimulate powdery mildew infection. Disease severity was calculated after 7 days (Abd-Elsayed et al., 2019).
Disease assessment Disease severity was calculated based on the percentage of leaf area covered with fungal colonies following the scale of Morishita et al. (2003) as follows: 0 = no visual symptoms, 1 = more than 0% up to 5%, 2 = more than 5% up to 25%, 3 = more than 25% up to 50%, and 4 = more than 50% up to 100% of leaf area covered by fungal growth. The percentage of disease severity was assessed 7 days after inoculation following the formula from Descalzo et al. (1990): Disease severity (%) = [Σ (n × v) / N × K] × 100. Where n is the number of infected leaves in each category; v is the numerical value of each category; N is the total number of infected leaves; K is the highest degree of infection in the scale.
The effect of treatments on Podosphaera xanthii disease severity The effect of the treatments -lemon oil, garlic oil, ginger oil, Spirulina, Blight stop, Copperal max, and Score 25% EC (Table 1)on disease severity was evaluated in the greenhouse of the Vegetable Diseases Research Department and in the open field at Kafr El-Sheikh governorate, Egypt. The design of the experiments was a complete randomized block design.
Greenhouse experiment Three week old cucumber seedlings were transplanted into 25 cm pots (3 seedlings per pot, 5 pots were used for each treatment). The seedlings were left adjacent to plants infected with powdery mildew as an inoculum source, in which each infected pot faced five un-inoculated pots. The plants were fertilized as recommended and regularly watered.
Open field experiment The field experiment was carried out in Qallin, in the Kafr El-Sheikh governorate. The field was chosen based on the epidemic history of powdery mildew in cucumber cultivations surveyed over the previous years. The experimental plot area was 42 m 2 containing six rows, each of 1 m width × 7 m long. One seedling was transplanted in each hill with 50 cm between hills. Three plots were used for each treatment. All agricultural practices were carried out in accordance with the recommendations of the Egyptian Ministry of Agriculture.
The first treatment spray was applied for all treatments before powdery mildew infection on the cucumber seedlings. All plants were subjected to the foliar spray of the treatments separately using a sprayer pump until the foliage was covered entirely. Plants sprayed with sterile distilled water served as control. All treatments were applied three times with 10 days intervals between each spray at the concentrations recommended by the manufacturer or literature (Table 1) (Hamza et al., 2015). The oils were emulsified with 0.05% Tween 20 individually (Terzi et al., 2007). The disease severity was assessed 7 days after the last treatment, according to Morishita et al. (2003), and the efficacy of the treatments was calculated according to the following formula: Treatment efficacy (%) = [(Control-treatment) / Control] × 100. The toxic effect of treatments on healthy cucumber plants Phytotoxicity was evaluated for all treatments to determine their toxic effects on healthy cucumber plants.
Treatments were sprayed three times every 10 days individually at the recommended concentrations (Table 1) on cucumber seedlings at 3 weeks of age; control plants were sprayed with water only. Phytotoxicity was measured 3 days after the third spray. The symptoms of phytotoxicity are stunting, distortion, and edge burning on the leaf surface. Phytotoxicity was assessed by placing the treated leaves facing a light source to look for damage and rate them on a 0 to 3 scale where 0 corresponds to no damage, 1 to slight damage (spots covering <20% of the leaf surface), 2 to moderate damage (spots covering >20% and < 50% of the leaf surface), and 3 for heavy damage (spots covering >50% of the leaf surface) according to Sharma, 2020 with slight modification.

Anatomical differences of infected cucumber leaves after applying treatments
Three infected cucumber leaves were collected from each treatment 3 days after the third spray. Five sections were prepared from each leaf, and the clearest section was measured. Plants sprayed with water served as the control; infected plants without treatments were considered negative controls. Epidermal peels of leaves were cleared in warm lactic acid and were examined microscopically to study the cell walls surface and fungal structure. Moreover, leaf samples were fixed in formol acetic alcohol fixative (90 mL ethanol 50%, 5 mL glacial acetic acid, 5 mL formalin) for at least 48 hours (Yeung et al., 2015). Samples of half a centimetre length were taken from the leaf blade near the central veins between the second and third lateral veins. Samples were dehydrated in successive series of solutions containing increasing concentrations of ethanol, ranging from 50% to 100%. The samples were embedded in paraffin wax using xylol as a solvent. Sections were cut to a thickness of 15 μm using a rotary microtome and mounted on slides using egg albumin as an adhesive. Wax was dissolved in xylol, and slides were passed through a descending series of ethanol solutions ranging from 100% to 50%. The sections were stained with double-staining (safranin T and light green SF) and permanently preserved using Canada balsam (Ruzin, 1999). All photomicrographs were taken using a Zeiss microscope with a Scmos Digital Camera.

Pollen grain examination
Flower buds were randomly collected from cucumber plants for each treatment 3 days after the last spray to examine pollen grain viability. Unopened flower buds of appropriate sizes were fixed in freshly prepared Carnoy's fixative (alcohol: chloroform: glacial acetic acid in a volume ratio of 6:3:1). Anthers were stained with a 1% acetocarmine staining solution (Crespel et al., 2006). Specimens were examined using an Olympus CX31 Binocular Microscope, and micrographs were taken using XCAM1080P HDMI ToupCam camera, according to Kumar and Singhal (2012). A total of 1000 pollens from each treatment were used to estimate the percentages of fertile and sterile pollens. Stained pollens were considered as apparently fertile while shrivelled and unstained pollen grains were listed as sterile; moreover, the number of burst pollen were taken into account. The fertile pollens were categorized based on size into two types: normal reduced (n) and unreduced (2n), where (2n) is 1.5-times larger than (n) (Xue et al., 2011).

Statistical analysis
Experiments were repeated three times for three independent biological replicates. The data was statistically analyzed by ANOVA using CoStat software. Values reported are the means of all measurements, and mean comparisons were made by Tukey's HSD tests at P ≤ 0.05 (Nanda et al., 2021).

Morphological characteristics of the pathogen
Microscopic examination of the infected exfoliated cucumber leaf revealed that the mycelium is septate and grows externally on the leaf surface. The conidiophore is un-branched and formed from foot cell followed by three short cells bearing conidia. The shape of the conidia is ovoid with lateral germination, and fibrosin bodies are visible as rod-shaped structures after using KOH. The tested fungus caused the typical powdery growth on the artificially infected leaves. There were no fruiting bodies in either the field or the greenhouse. These characters confirm that the pathogen under study is Podosphaera xanthii (Castagne) U. Braun & Shishkoff.
Pathogenicity assessment of P. xanthii

Artificial inoculation of cucumber plants by
Podosphaera xanthii showed typical powdery mildew symptoms with a disease severity of 85%, demonstrating the virulence of the pathogen.
Effect of treatments on powdery mildew disease severity In both the greenhouse and open field, all treatments except Spirulina had a significant effect in reducing the disease severity compared to the untreated infected plants, which showed a disease severity of 80.8% in the greenhouse and 65% in the open field. Treatment with Score 25% EC resulted in the highest decrease in the powdery mildew with a disease severity of 8.3% in the greenhouse and 4.1% in the open field, followed by lemon oil, garlic oil, and copperal max . Spirulina had the lowest reduction in both greenhouse and open field with disease severity values of 77.5% and 61.6%, respectively (Fig. 1a).
The efficacy of all treatments in both the greenhouse and the open field was almost the same. Score 25% EC had the highest efficacy in the greenhouse and open field, being 89.7% and 93.5%, respectively, followed by lemon oil and garlic oil. In contrast, Spirulina had the lowest efficacy, 4.1% in the greenhouse and 5.03% in the open field (Fig. 1b).

Phytotoxicity assessment after treatments application
The phytotoxicity effect of the tested treatments on cucumber plants was evaluated. All treatments showed weak toxic effect. Spirulina resulted in the lowest rate of phytotoxicity followed by Blight stop, lemon oil, garlic oil, Copperal max, and ginger oil. Score 25% EC showed moderate toxicity compared with the control (Fig. 2, Table 2). In the leaf anatomy study, all treatments had no significant effects on the cuticle thickness compared with the healthy control (1 μm) except for lemon oil (1.4 μm) and Spirulina (1.3 μm), which both showed a slight increase in the cuticle thickness. Treatment with garlic oil resulted in the highest increase in the thickness of the upper and lower epidermis, while the negative control with pathogen showed the highest decrease in upper and lower epidermal thicknesses compared with the healthy control (Fig. 3, Table 3).
The palisade tissue thickness was highest in two treatments, garlic oil (147.3 μm) and lemon oil (128.0 μm); the lemon oil treatment increased the size of the palisade cells, while the garlic oil treatment increased the number of palisade cells that appeared arranged in two rows. The lowest palisade cell thickness was in the negative control with pathogen (48.4 μm) followed by Score fungicide treatment (55.5 μm), compared to the control without pathogen (72.0 μm) (Fig. 3, Table 3).
Regarding spongy tissue thickness, the lemon and garlic oils had the highest readings of 115.2 μm and 109.5 μm, respectively, compared to controls without the pathogen (93.0 μm), whereas ginger oil, Blight stop, Eur J Plant Pathol (2023)  and Spirulina had almost the same values. The negative control treatment with the pathogen and treatment with the Score fungicide had the lowest spongy thickness of 51.1 μm and 64.8 μm, respectively. The ratios of palisade tissue to spongy tissue were less than one for all treatments, implying that spongy tissue is greater than the palisade tissue except for the treatment with lemon and garlic oils (Fig. 3, Table 3). The midrib of cucumber leaves contains two vascular bundles; the number of xylem vessels in the central vascular bundle was elevated in plants treated with lemon oil, garlic oil, and Spirulina compared to control plants without the pathogen. On the contrary, in Score fungicide-treated leaves, the bundle showed a decrease in xylem vessels number as well as deformation, whereas in leaves with the pathogen applied, the vascular bundle only showed a decrease in xylem vessels number.
The upper vascular bundle between the palisade and spongy tissues disappeared in fungicide-treated leaves and in the negative control with the pathogen, while it increased in size and cell number in treatments with lemon oil, garlic oil, and Copperal max compared to the control without pathogen. The highest total number of cells in the central and upper vascular bundles were present in the lemon oil treatment (52 cells), while the lowest total cells were observed in treatments with the Score fungicide (4 cells) compared to the control without pathogen (24 cells) (Fig. 3, Table 3).
The highest midrib thickness was recorded in treatments with lemon oil and Spirulina with 803 μm and 648.4 μm, respectively, while the lowest thicknesses were recorded in the negative control and Score fungicide treatments with 402.4 μm and 404.7 μm, respectively, compared to the control without pathogen with 449.5 μm. Treatment with garlic oil resulted in the highest leaf blade thickness at 272 μm, followed by lemon oil and Spirulina, while the lowest thicknesses were observed in the negative control with the pathogen and treatment with ginger oil, with 145.6 μm and 155.7 μm, respectively, compared to the control without pathogen at 190.5 μm (Fig. 3, Table 3). Table 4 shows the effect of each treatment on the viability of cucumber pollen grains. The treatments resulted in various types of cucumber pollen grains, including unstained (Figs. 4d and 6d), wrinkled (Fig. 4c), bigsized 2n pollen (Fig. 4e), burst (Fig. 6c-e), and fertile cells (Figs. 4a, b and 6a, b). In comparison to the control without the pathogen, which had a fertility rate of 98.8%, sterility of 1.2%, and bursting of 0.0%, treatments with lemon oil and garlic oil had the highest fertility rates of 88.1% and 85.8%, respectively, followed by Copperal max 83.9%, Spirulina 81.5%, Blight stop 81.3%, and ginger oil 80.7%. There was no significant difference between Spirulina and Blight stop in either the fertility or sterility rates. The lowest fertility rates were observed upon treatment with Score fungicide 72.7% and in the negative control with pathogen 61.4%. The highest percentage of sterile and bursting pollen grain was observed upon treatment with the Score fungicide, 23.40% and 3.9%, respectively, while the lowest percentage of sterile pollen was recorded in the lemon oil treatment, 10.5%, and the lowest percentage of bursting was recorded from the Spirulina treatment, 0.0%. Our data showed that some pollen grains were larger than the standard size, which were referred to as unreduced (2n) (Fig. 4e). The highest 2n pollen percentage was recorded in the Score fungicide treatment, at 21.1%. The control without pathogen, garlic oil and ginger oil had no 2n pollen.

Discussion
Powdery mildew on cucumber is an obligate parasitic fungus that cannot be grown on artificial media, making laboratory trials difficult (Pérez-García et al., 2009). The use of fungicides to manage powdery mildew is highly effective in minimizing the danger of this disease; nonetheless, it produces cumulative toxic residues that have Ratings are taken on cucumber leaves after 3 days of treatment application; Values followed by the same letter are not significantly different at P ≤ 0.05 an impact on the ecosystem. Because of this, we studied the use of natural products to combat powdery mildew. Natural products are biodegradable, bioactive, non-polluting, have few residues, little phytotoxicity (Brusotti et al., 2014), and contain ingredients with various modes of action. Therefore, they are considered an effective and safe solution against several plant diseases (Pandey et al., 2014).
Anti-fungal mechanism of plant essential oils against Podosphaera xanthii Plant essential oils contain low molecular weight terpenes that can cross and disrupt fungal cell walls by inhibiting β-glucan and chitin synthesis, causing the cell to lose its shape and adhesion (Dhifi et al., 2016;Lagrouh et al., 2017). In addition, terpenes possess hydrophobic and lipophilic properties that allow plant essential oils to penetrate into the lipids of cell membranes and mitochondria, resulting in increased fungal membrane permeability, causing cell content leakage (Friedly et al., 2009) and disrupting mitochondrial functions, resulting in a decrease in ATP production (Dhifi et al., 2016). Moreover, terpenes obstruct the membrane functions by causing a significant reduction in the amount of ergosterol, responsible for maintaining cell function, permeability, fluidity, and integrity (Hussein & Joo, 2018). Additionally, plant essential oils trigger the resistance mechanism in plants by releasing H 2 O 2 , which causes oxidative stress, and the plants respond similarly to when they experience biotic stress (Nazzaro et al., 2017). Our results showed significant antifungal activity of lemon oil against P. xanthii. This may be attributed to the presence of monoterpene (S)-limonene, which suppresses pectin methylesterase. Lemon oil also inhibits the fungus' ability to invade plant cell walls by inhibiting cellulase activity. Marei et al. (2012) reported the antifungal activity of (S)-limonene against F. oxysporum, F. solani, Rhophitulus solani, A. solani, B. cinerea, and P. infestans by the inhibition of cellulase and pectin methylesterase.
The antifungal properties of garlic oil may be related to the presence of sulphur compounds in allicin, alliinase, and allin (Suleiman & Abdallah, 2014). Allicin is rapidly decomposes into sulphur-derived compounds such as diallyl sulphide, sulphur dioxide and allyl propyl disulphide (Daniel et al., 2015). The effect of garlic oil on Fusarium oxysporum, Botrytis cinerea and Phytophthora capsici has been reported by Hayat et al. (2016).
Ginger oil has been shown to reduce the severity of cucumber powdery mildew disease. This may be due to the presence of monotorpene citral, which impede the development and adherence of fungal reproductive structures, protease activity, and phospholipid hydrolysis (Noshirvani et al., 2017) and prevents mycelial development leading to harmful changes that are impossible for pathogens to fix (Tu et al., 2018). The fungicidal effect of ginger oil against Phytophthora colocasiae on taro was reported by Kalhoro et al., 2022. The mode of action of the biological products Powdery mildew infection was controlled using Blight stop, a commercial biological compound. It is a mixture of a filamentous fungus, Trichoderma harzianum, and a Gram-positive bacterium, B. subtilis. Together, they can These variables were transformed according to this equation ffiffiffiffiffiffiffiffiffiffiffiffi X þ 1 p N refers to the total number of counted pollen grain cells, PGs refers to pollen grains, n refers to haploid chromosomes number, and 2n refers to diploid chromosomes number Values are the average of 3 samples. Values in each column followed by the same letter are not significantly different at P ≤ 0.05 Fig. 4 Pollen grain viability: a fertile pollen with an equatorial view, b fertile pollen with a polar view, c wrinkled sterile pollen, d unstained sterile pollen, and e different sizes of heterogeneous pollen grains protect crops by enhancing plant-induced resistance against powdery mildew infection, mainly if applied before infection by triggering the plant defense-related enzymes (peroxidase, polyphenol oxidase) and the accumulation of phenolic compounds (Singh et al., 2018). Trichoderma harzianum competes with Podosphaera xanthii for nutrients and space (Spadaro & Droby, 2016), while B. subtilis produces amphiphilic membrane-active peptide antibiotics, e.g., surfactin, fengycin, and iturin, which directly affect the hyphae and spores of powdery mildew fungus (Gilardi et al., 2008). This explains the significant effect of Blight stop against powdery mildew disease. However, the antifungal effect of Blight Stop was less than the effect of plant essential oils. This may be due to many factors such as the effect of climate change, disease virulence, the diversity of pathogen strains, crop health, and agricultural practices (Nega, 2014), which weaken the efficiency of Blight stop and do not allow comprehensive disease control.
Another biological product used in our study was Spirulina; it was ineffective in suppressing powdery mildew potentially because it contains macro and microelements, salts, and amino acids (Jufri et al., 2016), which may serve as a nutrient medium for the powdery mildew pathogen. Spirulina acts as a fertilizer and enhancer for plant growth by providing the nitrogen and carbon required for plant nutrition (Prasanna et al., 2009). We noticed an increase in leaf surface area, leaf greenness, and improvement in leaf texture and succulence, which creates the appropriate conditions for Podosphaera xanthii to obtain the nutritional requirements with ease; this explains the high disease severity and the minimal phytotoxic effects after using Spirulina. S. platensis acts as a biostimulator that stimulates growth parameters and increases the yield of chili pepper (Jufri et al., 2016). We recommend using Spirulina as a biofertilizer for crops, especially when the pathogen is not present, and not as a biocide.

The effect of chemical compounds on Podosphaera xanthii
We used Copperal max, a chemical contact fungicide, and Score 25% EC, a chemical systemic fungicide, to control powdery mildew infection. Copper sulphate, the active ingredient in Copperal max, is well-known for its antifungal properties, as it destroys DNA, proteins, and lipids; disrupts the cell membrane, enzyme active sites, and other biomolecules; and thus plays an important role in disease prevention in various plant species (Mahmoud & Farahat, 2020). On the other hand, copper acts as a micronutrient responsible for protein and carbohydrate metabolism as well as the synthesis of chlorophyll and other plant pigments; the chloroplasts contain 70% of the total copper inside the plant (Mengel & Kirkby, 2001). These reasons explain the significant effect of Copperal max in reducing the disease severity of powdery mildew and increasing cucumber pollen vitality compared to the negative control with the pathogen. Our finding agrees with Eliwa et al. (2018), who reported that CuSO 4 effectively reduced powdery mildew of sugar beet and delayed the spore germination of Erysiphe betae.
Difenoconazole is the main component of Score 25% EC, and is used widely in agriculture for its diseasecontrol efficacy . It interferes with fungal cell wall sterol synthesis by interrupting the biosynthesis of ergosterol, causing inhibition of fungal growth . However, the accumulation of difenoconazole inside the plant has a toxic effect on plant leaves. It leads to disturbances in plant respiration, photosynthesis, secondary metabolic processes, imbalances of the plant hormones, and changes in the oxidative stress in plants (Liu et al., 2021). This explains the physiological stress, morphological and anatomical changes, toxic effects, and sterility of cucumber plants treated with Score 25% EC.

Effect of treatments on pollen grain viability
When a pathogen infects a host plant, it obtains its nutritional requirements at the expense of the host's cells and tissues, resulting in symptoms and weakening the plant. The damage includes a defect in the division process, resulting in abnormal pollen production, which reduces the fertility of the plant. The amount of fertile pollen determines the crop production rate; therefore, it is necessary to apply treatments against plant diseases without affecting the fertility rate of pollen grains. In the present study, the pathogen lowered fertility to 38.6%. The fertility rate was increased by up to 88% after using treatments on plants in the early phases of infection. If the treatments are not applied in a timely manner, the fertility of pollen grains and fruits will be decreased, which will negatively affect the productivity of the crop.
Although Score 25% EC increased the fertility rate compared to the control with the pathogen it was the least effective treatment in increasing the percentage of fertility. Our results showed that Score 25% EC increased the percentage of sterile pollens (Fig. 4c, d) and bursting (Fig. 6c-e) that had a negative effect on the yield and quality of cucumber fruits; this corroborates the report of Pavlik and Jandurova (2000) that fungicides caused a decrease in pollen germination and deformation. Tort et al. (2005) studied the effects of fungicides on tomatoes infected with Botrytis cinerea and reported changes in pollen anatomical features of tomato plants.
The application of Score 25% EC caused an increase in the 2n pollen grain percentage due to the abnormality in the formation of spindle fibers during meiosis II. The homologous chromosomes can separate unequally, leading to unequal cytoplasmic division and formation of the triad stages (Fig. 5b) (Souza et al., 2012), and this erratic meiotic behaviour causes two different sizes of pollen: small haploid cells (n = x = 7) and big diploid cells (2n = 2x = 14) (Fig. 4e). While in the normal cell division process the chromosomes form dyads in meiosis I (Fig.  5a) and tetrads in meiosis II (Fig. 5c). When a haploid male gamete fertilizes a haploid female gamete, a diploid plant (2n = 2x = 14) results, which is typical behavior. However, when the diploid male gamete (2n = 2x = 14) fertilizes the haploid female gamete (n = x = 7), a triploid plant (3n = 3x = 21) is formed, which is abnormal.
One from of sterility is evidenced by the unstained pollen that may arise due to the contraction of the cytoplasm during the development of the gametophyte in the microspore stage (Fig. 4c) and/or due to the absence of cytoplasm, which can vanish in the early stages of pollen formation (Fig. 5d) (Souza et al., 2012). Another type of abnormality is the presence of burst cells; normally when pollen grains are exposed to water, some pollen cytoplasmic granules (PCG) come out through the pores (Fig. 6a, b), however PCG may be completely released through cracks of the exine if the pollen is brittle or damaged (Motta et al., 2006). Application of the fungicide Score 25% EC induced structural damage of pollen, which increased pollen bursting (Fig. 6c-e).

Conclusion
The effect of plant essential oils, Spirulina, Blight stop, Copperal max, and Score 25% EC treatments on disease severity provided valuable insight into selecting the best approach for powdery mildew management on cucumber. Among the treatments with natural compounds, lemon oil showed the highest efficacy, and Spirulina treatment exhibited the lowest efficacy against fungal progression in terms of resisting disease severity. On the other hand, the chemical product Score 25% EC caused the highest decrease in disease severity.
The phytotoxicity assessment clearly demonstrated that all the natural substances posed trivial toxicity Fig. 5 Meiocytes during meiosis I and II: a normal dyad during meiosis I, b abnormal triad during meiosis II, c normal tetrad during meiosis II, and d tetrad without PCG Fig. 6 Releasing of pollen cytoplasmic granules (PCG) from pollen grains: a, b PCG are expelled from the grain via the pore while the remaining grains stay intact, and c, d, e PCG are released through breaks of the exine from fragile or damaged pollen compared to Score 25% EC. Microscopic examination of the cucumber leaf anatomy in terms of the thickness of the cuticle, epidermis, palisade and spongy tissues, and the integrity of vascular bundles in the midrib revealed that all other treatments had a positive effect on the treated leaves, whereas Score 25% EC had a negative effect by reducing the thickness of the above-mentioned tissues and deforming the vascular bundles. Analysis of the fertility of pollen grains showed that the use of all other substances gave the best fertility rates, while the use of Score 25% EC was associated with the highest rate of sterility and disintegration of pollen grains.
All this experimental data has led us to recommend using any of the other tested substances over the fungicide Score 25% EC for cucumber powdery mildew disease management. In particular, they possess reasonable fungicidal effects with low toxicity; additionally, they may have favorable effects on the host plant and are inexpensive. Therefore, essential oils and natural products can be used as a part of an integrated disease management strategy. They can also be used as part of a curative spraying program in alternation with fungicides in the case of severe powdery mildew infection to reduce the residual effect of fungicides and avoid fungal resistance to fungicides as a result of recurrent spraying. We also suggest using Spirulina as a biofertilizer to improve the morphological characteristics of cucumber.
Acknowledgments The authors thank Prof. Dr. M.F. Attia, professor of Plant Pathology, Department of Plant Pathology, Faculty of Agriculture, Cairo University, for revising the manuscript.
Author contributions EE and MA conceived and designed the experiments. EE, MA, EA and MH performed the experiments. EE and MH carried out the data analysis. EE illustrated and graphical represented. EE and MA discussed the study and wrote the article. All authors read and approved the final manuscript.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Declarations
Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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