Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive
Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Effectiveness of Ruta graveolens and Syzygium aromaticum Extracts and the Nanoparticles of ZnO against Rhizoctonia solani and Fusarium incarnatum Causing the Damping-off Disease on Eggplants

N
Noor Tariq Abid1,*
R
Rabab Majead Abed1
N
Najm Abdullah Alzubaidy1
1Faculty of Education for Pure Sciences, University of Diyala, Iraq.

ABSTRACT

Background: Rhizoctonia and Fusarium are the most prevalent soil-borne fungi that lead to decline in a number of plants. Rhizoctonia solani is a pathogenic parasite that causes dangerous diseases on various vegetative crops. To evaluate the impact of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO against damping-off disease caused by Rhizoctonia solani and Fusarium incarnatum.

Methods: A pot experiment was conducted in the nursery of the Diyala Agriculture Directorate, Diyala Province, Iraq, in the season 2024-2025 to evaluate the effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO against the disease of damping off caused by Rhizoctonia solani and Fusarium incarnatum.

Result: The treatment of R. graveolens extract 4% + nano zinc oxide recorded the lowest infection incidence and disease severity with F. incarnatum, which reached 11.11%. The same treatment with the absence of pathogenic fungi achieved the highest chlorophyll at 62.30 SPAD followed by the treatment of S. aromaticum extract 4% + nano zinc oxide with Fusarium incarnatum  at 61.37 SPAD. The treatment of S. aromaticum extract 4% + nano zinc oxide with the absence of pathogenic fungi recorded the highest wet and dry vegetative weight, which were 363.8 g and 34.10 g, respectively.

INTRODUCTION

Eggplant is one of the important crops in Iraq, both nutritionally and economically. It is grown in many regions of Iraq. Rhizoctonia and Fusarium are the most prevalent soil-borne fungi that lead to decline in a number of plants. Rhizoctonia solani is a pathogenic parasite that causes dangerous diseases on various vegetative crops. It exists spontaneously as vegetative hyphae and sclerotia due to the absence of conidia (Abdelaziz et al., 2022). One of the most potent and destructive soil-borne diseases is Fusarium wilt, which is caused by the Fusarium fungus. By infecting host roots and colonizing xylem vessels, it causes plant wilting and substantial economic losses (Ahmed et al., 2017). Plant extracts are the best alternatives to the hazardous chemicals, where decreasing their use is crucial to reducing the pollution. Therefore, it is necessary to choose friendly manners to promote plant growth (Al-Dhabaan et al., 2017). Ruta graveolens, a plant belonging to the Rutaceae family, has the ability to produce furanocoumarins, which are phytoalexins play a role in the activity of antifungal and antimicrobial (Alharbi et al., 2023). Clove (Syzygium aromaticum L.) is an important aromatic and medicinal plant belonging to the Myrtaceae family. It is rich in phenolic compounds, primarily eugenol, as well as terpene compounds and volatile esters such as eugenyl acetate, which give clove its unique aromatic and medicinal properties (He et al., 2011).
       
ZnO-NPs nanoparticles possess a high capacity to penetrate fungal cell walls and interact with their internal vital components such as proteins, nucleic acids and enzymes, leading to disruption of their basic vital processes, damage to the cellular structure, breakdown of plasma membrane functions and ultimately fungal cell death. Recent studies have confirmed that ZnO-NPs cause cell wall lysis, induce anatomical changes in the internal structure of fungal cells and reduce reproduction and germination rates, making them an effective agent in halting the spread of pathogenic fungi (Hassan and Yousif, 2013).

One of the most important aspects of using ZnO-NPs in modern agriculture is the biogenic synthesis of these nanoparticles using plant extracts as natural reducing agents and stabilizers. This method is an environmentally safe alternative to traditional chemical methods that may leave toxic residues or contribute to soil and water pollution. Studies have shown that using plant extracts such as Ruta graveolens and Syzygium aromaticum in ZnO-NP synthesis produces stable and effective particles characterized by low toxicity to beneficial organisms and greater sustainability when applied in agricultural environments (He et al., 2011). The aim of this study was to evaluate the impact of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO against damping-off disease caused by Rhizoctonia solani and Fusarium incarnatum.

MATERIALS AND METHODS

A pot experiment was conducted in the nursery of the Diyala Agriculture Directorate, Diyala Province, Iraq, in the season 2024-2025. The study fungi  were acquired from the Directorate of Diyala Agriculture’s Plant Pathology Lab. They were molecularly identified using the PCR technique, with accession numbers OQ357844-OQ357845 for R. solani and OQ357846-OQ357847 for F. incarnatum.
 
Preparation and sterilization of soil
 
Soil from one of the fields in Diyala Governorate was used. It was cleaned and sifted to remove plant and weed residues. The soil was moistened and sterilized with formalin at a concentration of 3% of the commercial solution (37%). It was covered with nylon for 7 days and then ventilated for 7 days to get rid of the formalin vapors (He et al., 2011).
 
Soil analysis
 
The soil was analyzed to identify some of its physical and chemical properties, as shown in Table (1), at the Soil and Water Laboratory-Diyala Agriculture Directorate and the Agricultural Research Department laboratories.

Table 1: Physical and chemical properties of soil.


 
Preparing the fungal inoculum
 
An inoculum of R. solani and F. incarnatum was prepared, each individually, by growing on local millet seeds (Panicum miliaceum L.) after sterilizing the millet seeds inside 250 ml glass flasks using an autoclave (at a temperature of 121°C for 20 minutes) twice consecutively. Then, they were inoculated with 5 discs with a diameter of 0.5 cm, taken from the edges of fungal cultures grown on PDA medium at 7 days old. Then, the flasks were incubated at a temperature of 25±1°C for 10 days, taking care to shake continuously from time to time to ensure aeration and uniform distribution of the inoculum on all seeds (Milesi et al., 2001).
 
Preparation of R. graveolens and S. aromaticum extracts
 
150 millilitres of deionised water (DW) and 350 millilitres of ethanol were mixed with ten grams of powdered R. graveolens and S. aromaticum. After that, the mixture was heated to 70°C for three hours while being stirred magnetically. Aluminium foil needed to be used to seal the glass flask. The extract was then stored in the refrigerator after the precipitation was separated using filter sheets (Whitman No. 1, pore size 25 μm).
 
Biosynthesis of zinc oxide nanoparticles from R. graveolens and S. aromaticum extracts
 
According to Otunola (2022), ZnO NPs were prepared by dissolving 10 g of Zn(NO3)2. 6H2O in 150 ml of D.W. while stirring for 30 minutes. Next, add 45 ml of either R. graveolens or S. aromaticum and stir for 60 minutes. After that, 20 ml of D.W. was mixed with 2.5 g of sodium hydroxide (NaOH) drop by drop. The mixture was then agitated for ten minutes until a milky precipitate formed. To get rid of any contaminants, the precipitate was cleaned four times using D.W. and ethanol before being dried for an hour at 150°C in an oven. Lastly, the white powder was calcined for three hours at 550°C.
 
Preparation of zinc oxide nanoparticles loaded on extracts of R. graveolens and S. aromaticum
 
Ten g of the previously prepared powder for each extract was weighed and dissolved in 100 ml of deionized distilled water; then 100 ml of each extract was added with continuous stirring on a hot plate magnetic stirrer and then precipitated with NaOH solution.
 
Addition of R. solani and F. incarnatum inoculum to the pots
 
Five g of the pathogenic fungi inoculum carried on millet seeds was added to the soil distributed in plastic pots with a capacity of 9 kg of soil/pot. After three days, the eggplant seedlings were planted at 4-5 weeks old with three replicates for each treatment, as three seedlings were planted in each pot and then thinned to two seedlings. Three replicates were also left for the control treatment (the eggplant seedlings were planted without adding the fungal inoculum).
 
Field experiment
 
A factorial experiment was designed in a randomized complete block design (RCBD) and included two factors; the first factor involved extracts of R. graveolens and S. aromaticum at three concentrations (0%, 2% and 4%)  along with zinc oxide nanoparticles loaded and unloaded onto the plant extracts against R. solani and F. incarnatum and the second factor included the presence and the absence of the pathogenic fungi. Plants were sprayed with the extracts concentrations when infection appeared after 10 days until saturated, followed by a second spray after 20 days.
 
Infection incidence of eggplant seedlings (%)
 
The infection incidence was calculated using the following equation:
 
  
 
Percentage of infection severity (%)
 
The percentage of infection severity was estimated 30 days after planting according to the 5-point disease index described by Otunola (2022).
0 = Healthy plants, no disease symptoms.
1 = 1-10% show symptoms of wilting of the lower leaves.
2 = More than 11-25% show symptoms with slight stunting.
3 = More than 26-50% show brown discoloration with moderate stunting of the vegetative parts.
4 = More than 50% show dark discoloration with severe stunting of the vegetative parts.
 
  
       
The findings of infection incidence, infection severity, wet and dry vegetative weight and chlorophyll were taken 30 days after planting.
 
Statistical analysis
 
The statistical data were analyzed using a randomized complete block design (RCBD) with a factorial experiment using the statistical program SPSS to analyze according to Duncan’s multinomial test at a probability level of 0.05.

RESULTS AND DISCUSSION

Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on infection incidence of R. solani and F. incarnatum in the eggplant
 
The results in Table 2 indicate that there were significant differences among treatments in infection incidence as compared to the control, as treatment of R. graveolens extract 4% + nano zinc oxide achieved the lowest infection incidence, reaching 7.41%, followed by other treatments, while the control treatment recorded the highest infection incidence at 66.67%. In spite of no significant differences being observed in infection incidence between the pathogenic fungi R. solani and F. incarnatum, but it outperformed the treatment without fungi. Regarding the interaction between the treatments and the presence or absence of the pathogenic fungi. R. graveolens extract 4% + nano zinc oxide achieved the lowest infection incidence at 11.11% against R. solani and F. incarnatum, besides the treatment of S. aromaticum extract 4% + nano zinc oxide at 11.11% against F. incarnatum as compared with the control at 100% to each fungus.

 

Table 2: Effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO on infection incidence of Rhizoctonia solani and Fusarium incarnatum in the eggplant.


 
Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on infection severity of R. solani and F. incarnatum in the eggplant
 
The results in Table 3 showed that there were significant differences among treatments in infection severity as compared to the control, as treatment of R. graveolens extract 4% + nano zinc oxide recorded the lowest infection severity, reaching 8.33 %, followed by R. graveolens extract 2% + nano zinc oxide and S. aromaticum extract 2% + nano zinc oxide, which amounted to 10.18%, while the control treatment recorded the highest infection severity at 54.63%. The infection severity achieved the lowest value in F. incarnatum at 27.77% with significant differences from R. solani at  29.70% and the absence of fungi at 0%. The treatment of R. graveolens extract 4% + nano zinc oxide recorded the lowest infection severity with F. incarnatum at 11.11%, whereas the control with R. solani  recorded the highest infection severity at 83.33%, when the interaction between the treatments and the presence or absence of the pathogenic fungi.

Table 3: Effect of Ruta graveolens and Syzygium aromaticum extracts and their nanoparticles of ZnO on infection severity of Rhizoctonia solani and Fusarium incarnatum in the eggplant.


 
Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on chlorophyll content of eggplants that are infected with R. solani and F. incarnatum
 
The result in Table 4 revealed that the chlorophyll content in the leaves is significantly different among treatments as compared to the control. The treatments of S. aromaticum extract 4% + nano zinc oxide, R. graveolens extract 4% + nano zinc oxide and nano zinc oxide from S. aromaticum extract 4% have the highest chlorophyll content, reaching 60.48, 59.46 and 59.01 SPAD, respectively, compared to the control, which recorded 38.19 SPAD. The plants with the absence of the pathogenic fungi achieved the highest content of chlorophyll at 60.55 SPAD, followed by plants with F. incarnatum at 53.83 SPAD and with R. solani at 51.10 SPAD. The interaction between the treatments and the presence or absence of the pathogenic fungi showed that the treatments of R. graveolens extract 4% + nano zinc oxide and the control with the absence of the pathogenic fungi have the highest content of chlorophyll, reaching 62.30 SPAD, while the plants with R. solani and F. incarnatum recorded the lowest content of chlorophyll at 25.30 and 26.97 SPAD respectively.

Table 4: Effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO on chlorophyll content of eggplants that are infected with Rhizoctonia solani and Fusarium incarnatum.


 
Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on wet vegetative weight of eggplants that are infected with R. solani and F. incarnatum
 
The results in Table 5 indicate that  the wet vegetative weight is significantly different among treatments as compared to the control. The treatment of S. aromaticum extract 4% + nano zinc oxide recorded the highest wet vegetative weight, reaching 333.8 g, while the control recorded the lowest wet vegetative weight, 173.0 g. The plants with the absence of the pathogenic fungi achieved the highest wet vegetative weight at 357.8 g, followed by plants with F. incarnatum at 269.8 g and with R. solani at 240.7 g. The interaction between the treatments and the presence or absence of the pathogenic fungi showed that the control with the absence of the pathogenic fungi has the highest wet vegetative weight, amounting to 370.0 g, followed by the treatments of S. aromaticum extract 4% + nano zinc oxide with the absence of the pathogenic fungi and with R. solani, reaching 363.8 g, while the plants with R. solani and F. incarnatum recorded the lowest wet vegetative weight at 68.7 g and 80.4 g, respectively.

Table 5: Effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO on wet vegetative weight of eggplants that are infected with Rhizoctonia solani and Fusarium incarnatum.


 
Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on dry vegetative weight of eggplants that are infected with R. solani and F. incarnatum
 
The results in Table 6 showed that the dry vegetative weight is significantly different among treatments as compared to the control. The treatments of S. aromaticum extract 4% + nano zinc oxide and R. graveolens extract 4% + nano zinc oxide recorded the highest dry vegetative weight, reaching 30.77 g and 30.54 g, respectively, while the control recorded the lowest dry vegetative weight, 21.0 g. The plants with the absence of the pathogenic fungi achieved the highest dry vegetative weight at 31.16 g, followed by plants with F. incarnatum at 26.43 g and with R. solani at 24.99 g. The interaction between the treatments and the presence or absence of the pathogenic fungi showed that the treatment of S. aromaticum extract 4% + nano zinc oxide in the absence of the pathogenic fungi has the highest dry vegetative weight, amounting to 34.10 g, while the plants with R. solani and F. incarnatum recorded the lowest dry vegetative weight at 14.13 g and 14.87 g, respectively.

Table 6: Effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO on dry vegetative weight of eggplants that are infected with Rhizoctonia solani and Fusarium incarnatum.


       
Ethyl acetate extract from R. graveolens leaves shows strong antifungal activity against Botrytis cinerea, Fusarium oxysporum and Colletotrichum spp. The extract of R. graveolens exhibits fungicidal properties against key agricultural pathogens including Colletotrichum fragariae, C. gloeosporioides, C. acutatum, Botrytis cinerea and Fusarium oxysporum (Otunola, 2022; Yadav and Ghasolia, 2022).
       
In addition to preventing the mycelial growth of pathogenic fungus, zinc nanoparticles can either kill or stop germs from germinating (Pandey et al., 2011). The survival of cotton seedlings was developed when ZnONPs was used against R. solani. In the pots study, foliar ZnONP spray outperformed seed covering in increasing plant dry weight and managing the complex disease in beetroot roots, Beta Vulgaris L., which is caused by Pectobacterium betavasculorum, Meloidogyne incognita and R. solani (Salim et al., 2016; Bashyal et al., 2022). One primary mechanism of ZnONP toxicity against R. solani is mechanical encapsulation, which is believed to be how ZnONPs interact with pathogens) Aye and Matsumoto, 2011). This effect was evidenced by electron microscopy, which revealed clear deformations in the fungal hyphae of F. oxysporum treated with ZnO nanoparticles (Sivasankarapillai et al., 2023). In their study of the antifungal activity of zinc nanoparticles (ZnONPs) against F. oxysporum on tomato plants, (Suresh et al., 2015; Hong et al., 2022) found that ZnONPs at concentrations of 1500-3000 g/ml produced the lowest disease severity and the disease incidence rate.     
       
The use of zinc nanoparticles (ZnONPs) has greatly improved the survival rate of cotton plants affected by seedling damping off disease, leading to increased height and dry weight. They may create an antifungal layer around seeds, providing protection against fungi (Yehia and Ahmed, 2013). The application of zinc oxide nanoparticles (ZnO-NPs) as foliar sprays on tomato plants showed effective control of Botrytis cinerea infection, leading to reduced disease severity and increased plant biomass, indicating ZnO-NPs’ growth-promoting capabilities. Treated plants exhibited elevated catalase and peroxidase activities, suggesting that ZnO-NPs may enhance resistance against B. cinerea, thereby providing an alternative to traditional fungicides for managing gray mold disease in tomatoes (Zaki et al., 2021).

CONCLUSION

The study’s results demonstrated that the treatment of R. graveolens extract 4% + nano zinc oxide was superior in reducing the incidence and  disease severity and increasing chlorophyll, whereas the treatment of S. aromaticum extract 4% + nano zinc oxide was outperformed in increasing wet and dry vegetative weight. It can be concluded that Ruta graveolens and Syzygium aromaticum extracts, along with ZnO nanoparticles, could protect eggplant seedlings from damping-off disease and improve their growth characteristics.

CONFLICT OF INTEREST

The authors declare that no conflict of interest.

REFERENCES


  1. Abdelaziz, A.M., Salem, S.S., Khalil, A.M.A., El-Wakil, D.A., Fouda, H.M. and Hashem, A.H. (2022). Potential of biosynthesized zinc oxide nanoparticles to control Fusarium wilt disease in eggplant (Solanum melongena) and promote plant growth. BioMetals. 35: 601-616. https://doi.org/10.100 7/s10534-022-00391-8.

  2. Ahmed, S., Annu, Chaudhry, S.A. and Ikram, S. (2017). A review on biogenic synthesis of ZnO nanoparticles using plant extracts and microbes: A prospect towards green chemistry. Journal of Photochemistry and Photobiology B: Biology. 166: 272-284. https://doi.org/10.1016/j.jphotobiol.2016. 12.011.

  3. Al-Dhabaan, F.A., Shoala, T., Ali, A.A.M., Alaa, M. and Abd-Elsalam, K. (2017). Chemically-produced copper, zinc nanoparticles and chitosan-bimetallic nanocomposites and their antifungal activity against three phytopathogenic fungi. International Journal of Agricultural Technology. 13(5): 753-769.

  4. Alharbi, F.N., Abaker, Z.M. and Makawi, S.Z.A. (2023). Phytochemical substances-mediated synthesis of zinc oxide nanoparticles (ZnO NPS). Inorganics. 11(8): 328. https://doi.org/10. 3390/inorganics11080328.

  5. Aye, S.S. and Matsumoto, M. (2011).  Effect of some plant extracts on Rhizoctonia spp. and Sclerotium hydrophilum. Journal of Medicinal Plants Research. 5(16): 3751-3757.

  6. Bashyal, B.M., Kharayat, B.S., Parmar, P., Gupta, A.K., Dubey S.C. and Aggarwal, R. (2022). Influence of temperature on the pathogenesis and gene expression of rhizoctonia solani causing web blight/wet root rot disease in mungbean. Legume Research. 45(1): 104-109. doi: 10.18805/LR- 4342.

  7. Hassan, W.A. and Yousif, A.A. (2013). Soil recolonization by saprophytic fungi after solarization and soil amendments. Science Journal of University of Zakho. 1(2): 427-436.

  8. He, L., Liu, Y., Mustapha, A. and Lin, M. (2011). Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum. Microbiological Research. 166(3): 207-215. https://doi.org/10.1016/j.micres.2010.03.003.

  9. Hong T.T., Nhi N.T.H., Day P.V. (2022). Effect of Solanum procumbens L. extract on survival, growth performances, immune responses and against vibrio parahaemolyticus causing acute hepatopancreas necrosis disease in white leg shrimp (Litopenaeus vannamei). Indian Journal of Animal Research. 56(12): 1513-1518. doi: 10.18805/IJAR.BF-1566.

  10. Milesi, S.B.M., Massot, B., Gontier, E., Bourgaud, F. and Guckert, A. (2001). Ruta graveolens L. a promising species for the production of furanocoumarins. Plant Science. 161(1): 189-199. https://doi.org/10.1016/S0168-9452(01)00413-7.

  11. Otunola, G.A. (2022). Culinary spices in food and medicine: An overview of Syzygium aromaticum (L.) Merr. and LM Perry [Myrtaceae]. Frontiers in Pharmacology. 12: 793200. https://doi.org/10.3389/fphar.2021.793200.

  12. Pandey, P., Mehta, A. and Hajra, S. (2011). Evaluation of antimicrobial activity of Ruta graveolens stem extracts by disc diffusion method. Journal of Phytology. 3(3): 92-95. https://update publishing.com/journal/index.php/jp/article/view/2233.

  13. Salim, H.A., Salman, I.S. and Jasim, B.N. (2016). IPM approach for the management of wilt disease caused by Fusarium oxysporum f. sp. lycopersici on tomato (Lycopersicon esculentum). Journal of Experimental Biology and Agricultural Sciences. 4: 742-747. http://dx.doi.org/10. 18006/2016.4(VIS).742.747.

  14. Sivasankarapillai, V.S., Krishnamoorthy, N., Eldesoky, G.E., Wabaidur, S.M., Islam, M.A., Dhanusuraman, R. and Ponnusamy, V.K. (2023). One-pot green synthesis of ZnO nanoparticles using Scoparia dulcis plant extract for antimicrobial and antioxidant activities. Applied Nanoscience. 13: 6093- 6103. https://doi.org/10.1007/s13204-022-02610-7.

  15. Suresh, D., Nethravathi, P.C., Udayabhanu, Rajanaika, H., Nagabhushana, H. and Sharma, S.C. (2015). Green synthesis of multifunctional zinc oxide (ZnO) nanoparticles using Cassia fistula plant extract and their photodegradative, antioxidant and anti bacterial activities. Materials Science in Semiconductor Processing. 31: 446-454. https://doi.org/10.1016/j.mssp. 2014.12.023.

  16. Yadav, L.S. and Ghasolia R.P. (2022). Management of root rot of fenugreek caused by rhizoctonia solani using plant extracts and bio-agents. Legume Research. 45(3): 391-395. doi: 10.18805/LR-4438.

  17. Yehia, R.S. and Ahmed O.F. (2013). In vitro study of the antifungal efficacy of zinc oxide nanoparticles against Fusarium oxysporum and Penicilium expansum. African Journal of Microbiology Research. 7(19):1917-1923. https://doi. org/10.5897/AJMR2013.5668.

  18. Zaki, S.A., Ouf, S.A., Albarakaty, F.M., Habeb, M.M., Aly, A.A. and Abd-Elsalam, K.A. (2021). Trichoderma harzianum- mediated ZnO nanoparticles: A green tool for controlling soil-borne pathogens in cotton. Journal of Fungi. 7: 952. https://doi.org/10.3390/jof7110952. 
Published In
Indian Journal of Agricultural Research
Article Metrics
Metrics Icon
11
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Effectiveness of Ruta graveolens and Syzygium aromaticum Extracts and the Nanoparticles of ZnO against Rhizoctonia solani and Fusarium incarnatum Causing the Damping-off Disease on Eggplants

    N
    Noor Tariq Abid1,*
    R
    Rabab Majead Abed1
    N
    Najm Abdullah Alzubaidy1
    1Faculty of Education for Pure Sciences, University of Diyala, Iraq.

    ABSTRACT

    Background: Rhizoctonia and Fusarium are the most prevalent soil-borne fungi that lead to decline in a number of plants. Rhizoctonia solani is a pathogenic parasite that causes dangerous diseases on various vegetative crops. To evaluate the impact of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO against damping-off disease caused by Rhizoctonia solani and Fusarium incarnatum.

    Methods: A pot experiment was conducted in the nursery of the Diyala Agriculture Directorate, Diyala Province, Iraq, in the season 2024-2025 to evaluate the effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO against the disease of damping off caused by Rhizoctonia solani and Fusarium incarnatum.

    Result: The treatment of R. graveolens extract 4% + nano zinc oxide recorded the lowest infection incidence and disease severity with F. incarnatum, which reached 11.11%. The same treatment with the absence of pathogenic fungi achieved the highest chlorophyll at 62.30 SPAD followed by the treatment of S. aromaticum extract 4% + nano zinc oxide with Fusarium incarnatum  at 61.37 SPAD. The treatment of S. aromaticum extract 4% + nano zinc oxide with the absence of pathogenic fungi recorded the highest wet and dry vegetative weight, which were 363.8 g and 34.10 g, respectively.

    INTRODUCTION

    Eggplant is one of the important crops in Iraq, both nutritionally and economically. It is grown in many regions of Iraq. Rhizoctonia and Fusarium are the most prevalent soil-borne fungi that lead to decline in a number of plants. Rhizoctonia solani is a pathogenic parasite that causes dangerous diseases on various vegetative crops. It exists spontaneously as vegetative hyphae and sclerotia due to the absence of conidia (Abdelaziz et al., 2022). One of the most potent and destructive soil-borne diseases is Fusarium wilt, which is caused by the Fusarium fungus. By infecting host roots and colonizing xylem vessels, it causes plant wilting and substantial economic losses (Ahmed et al., 2017). Plant extracts are the best alternatives to the hazardous chemicals, where decreasing their use is crucial to reducing the pollution. Therefore, it is necessary to choose friendly manners to promote plant growth (Al-Dhabaan et al., 2017). Ruta graveolens, a plant belonging to the Rutaceae family, has the ability to produce furanocoumarins, which are phytoalexins play a role in the activity of antifungal and antimicrobial (Alharbi et al., 2023). Clove (Syzygium aromaticum L.) is an important aromatic and medicinal plant belonging to the Myrtaceae family. It is rich in phenolic compounds, primarily eugenol, as well as terpene compounds and volatile esters such as eugenyl acetate, which give clove its unique aromatic and medicinal properties (He et al., 2011).
           
    ZnO-NPs nanoparticles possess a high capacity to penetrate fungal cell walls and interact with their internal vital components such as proteins, nucleic acids and enzymes, leading to disruption of their basic vital processes, damage to the cellular structure, breakdown of plasma membrane functions and ultimately fungal cell death. Recent studies have confirmed that ZnO-NPs cause cell wall lysis, induce anatomical changes in the internal structure of fungal cells and reduce reproduction and germination rates, making them an effective agent in halting the spread of pathogenic fungi (Hassan and Yousif, 2013).

    One of the most important aspects of using ZnO-NPs in modern agriculture is the biogenic synthesis of these nanoparticles using plant extracts as natural reducing agents and stabilizers. This method is an environmentally safe alternative to traditional chemical methods that may leave toxic residues or contribute to soil and water pollution. Studies have shown that using plant extracts such as Ruta graveolens and Syzygium aromaticum in ZnO-NP synthesis produces stable and effective particles characterized by low toxicity to beneficial organisms and greater sustainability when applied in agricultural environments (He et al., 2011). The aim of this study was to evaluate the impact of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO against damping-off disease caused by Rhizoctonia solani and Fusarium incarnatum.

    MATERIALS AND METHODS

    A pot experiment was conducted in the nursery of the Diyala Agriculture Directorate, Diyala Province, Iraq, in the season 2024-2025. The study fungi  were acquired from the Directorate of Diyala Agriculture’s Plant Pathology Lab. They were molecularly identified using the PCR technique, with accession numbers OQ357844-OQ357845 for R. solani and OQ357846-OQ357847 for F. incarnatum.
     
    Preparation and sterilization of soil
     
    Soil from one of the fields in Diyala Governorate was used. It was cleaned and sifted to remove plant and weed residues. The soil was moistened and sterilized with formalin at a concentration of 3% of the commercial solution (37%). It was covered with nylon for 7 days and then ventilated for 7 days to get rid of the formalin vapors (He et al., 2011).
     
    Soil analysis
     
    The soil was analyzed to identify some of its physical and chemical properties, as shown in Table (1), at the Soil and Water Laboratory-Diyala Agriculture Directorate and the Agricultural Research Department laboratories.

    Table 1: Physical and chemical properties of soil.


     
    Preparing the fungal inoculum
     
    An inoculum of R. solani and F. incarnatum was prepared, each individually, by growing on local millet seeds (Panicum miliaceum L.) after sterilizing the millet seeds inside 250 ml glass flasks using an autoclave (at a temperature of 121°C for 20 minutes) twice consecutively. Then, they were inoculated with 5 discs with a diameter of 0.5 cm, taken from the edges of fungal cultures grown on PDA medium at 7 days old. Then, the flasks were incubated at a temperature of 25±1°C for 10 days, taking care to shake continuously from time to time to ensure aeration and uniform distribution of the inoculum on all seeds (Milesi et al., 2001).
     
    Preparation of R. graveolens and S. aromaticum extracts
     
    150 millilitres of deionised water (DW) and 350 millilitres of ethanol were mixed with ten grams of powdered R. graveolens and S. aromaticum. After that, the mixture was heated to 70°C for three hours while being stirred magnetically. Aluminium foil needed to be used to seal the glass flask. The extract was then stored in the refrigerator after the precipitation was separated using filter sheets (Whitman No. 1, pore size 25 μm).
     
    Biosynthesis of zinc oxide nanoparticles from R. graveolens and S. aromaticum extracts
     
    According to Otunola (2022), ZnO NPs were prepared by dissolving 10 g of Zn(NO3)2. 6H2O in 150 ml of D.W. while stirring for 30 minutes. Next, add 45 ml of either R. graveolens or S. aromaticum and stir for 60 minutes. After that, 20 ml of D.W. was mixed with 2.5 g of sodium hydroxide (NaOH) drop by drop. The mixture was then agitated for ten minutes until a milky precipitate formed. To get rid of any contaminants, the precipitate was cleaned four times using D.W. and ethanol before being dried for an hour at 150°C in an oven. Lastly, the white powder was calcined for three hours at 550°C.
     
    Preparation of zinc oxide nanoparticles loaded on extracts of R. graveolens and S. aromaticum
     
    Ten g of the previously prepared powder for each extract was weighed and dissolved in 100 ml of deionized distilled water; then 100 ml of each extract was added with continuous stirring on a hot plate magnetic stirrer and then precipitated with NaOH solution.
     
    Addition of R. solani and F. incarnatum inoculum to the pots
     
    Five g of the pathogenic fungi inoculum carried on millet seeds was added to the soil distributed in plastic pots with a capacity of 9 kg of soil/pot. After three days, the eggplant seedlings were planted at 4-5 weeks old with three replicates for each treatment, as three seedlings were planted in each pot and then thinned to two seedlings. Three replicates were also left for the control treatment (the eggplant seedlings were planted without adding the fungal inoculum).
     
    Field experiment
     
    A factorial experiment was designed in a randomized complete block design (RCBD) and included two factors; the first factor involved extracts of R. graveolens and S. aromaticum at three concentrations (0%, 2% and 4%)  along with zinc oxide nanoparticles loaded and unloaded onto the plant extracts against R. solani and F. incarnatum and the second factor included the presence and the absence of the pathogenic fungi. Plants were sprayed with the extracts concentrations when infection appeared after 10 days until saturated, followed by a second spray after 20 days.
     
    Infection incidence of eggplant seedlings (%)
     
    The infection incidence was calculated using the following equation:
     
      
     
    Percentage of infection severity (%)
     
    The percentage of infection severity was estimated 30 days after planting according to the 5-point disease index described by Otunola (2022).
    0 = Healthy plants, no disease symptoms.
    1 = 1-10% show symptoms of wilting of the lower leaves.
    2 = More than 11-25% show symptoms with slight stunting.
    3 = More than 26-50% show brown discoloration with moderate stunting of the vegetative parts.
    4 = More than 50% show dark discoloration with severe stunting of the vegetative parts.
     
      
           
    The findings of infection incidence, infection severity, wet and dry vegetative weight and chlorophyll were taken 30 days after planting.
     
    Statistical analysis
     
    The statistical data were analyzed using a randomized complete block design (RCBD) with a factorial experiment using the statistical program SPSS to analyze according to Duncan’s multinomial test at a probability level of 0.05.

    RESULTS AND DISCUSSION

    Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on infection incidence of R. solani and F. incarnatum in the eggplant
     
    The results in Table 2 indicate that there were significant differences among treatments in infection incidence as compared to the control, as treatment of R. graveolens extract 4% + nano zinc oxide achieved the lowest infection incidence, reaching 7.41%, followed by other treatments, while the control treatment recorded the highest infection incidence at 66.67%. In spite of no significant differences being observed in infection incidence between the pathogenic fungi R. solani and F. incarnatum, but it outperformed the treatment without fungi. Regarding the interaction between the treatments and the presence or absence of the pathogenic fungi. R. graveolens extract 4% + nano zinc oxide achieved the lowest infection incidence at 11.11% against R. solani and F. incarnatum, besides the treatment of S. aromaticum extract 4% + nano zinc oxide at 11.11% against F. incarnatum as compared with the control at 100% to each fungus.

     

    Table 2: Effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO on infection incidence of Rhizoctonia solani and Fusarium incarnatum in the eggplant.


     
    Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on infection severity of R. solani and F. incarnatum in the eggplant
     
    The results in Table 3 showed that there were significant differences among treatments in infection severity as compared to the control, as treatment of R. graveolens extract 4% + nano zinc oxide recorded the lowest infection severity, reaching 8.33 %, followed by R. graveolens extract 2% + nano zinc oxide and S. aromaticum extract 2% + nano zinc oxide, which amounted to 10.18%, while the control treatment recorded the highest infection severity at 54.63%. The infection severity achieved the lowest value in F. incarnatum at 27.77% with significant differences from R. solani at  29.70% and the absence of fungi at 0%. The treatment of R. graveolens extract 4% + nano zinc oxide recorded the lowest infection severity with F. incarnatum at 11.11%, whereas the control with R. solani  recorded the highest infection severity at 83.33%, when the interaction between the treatments and the presence or absence of the pathogenic fungi.

    Table 3: Effect of Ruta graveolens and Syzygium aromaticum extracts and their nanoparticles of ZnO on infection severity of Rhizoctonia solani and Fusarium incarnatum in the eggplant.


     
    Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on chlorophyll content of eggplants that are infected with R. solani and F. incarnatum
     
    The result in Table 4 revealed that the chlorophyll content in the leaves is significantly different among treatments as compared to the control. The treatments of S. aromaticum extract 4% + nano zinc oxide, R. graveolens extract 4% + nano zinc oxide and nano zinc oxide from S. aromaticum extract 4% have the highest chlorophyll content, reaching 60.48, 59.46 and 59.01 SPAD, respectively, compared to the control, which recorded 38.19 SPAD. The plants with the absence of the pathogenic fungi achieved the highest content of chlorophyll at 60.55 SPAD, followed by plants with F. incarnatum at 53.83 SPAD and with R. solani at 51.10 SPAD. The interaction between the treatments and the presence or absence of the pathogenic fungi showed that the treatments of R. graveolens extract 4% + nano zinc oxide and the control with the absence of the pathogenic fungi have the highest content of chlorophyll, reaching 62.30 SPAD, while the plants with R. solani and F. incarnatum recorded the lowest content of chlorophyll at 25.30 and 26.97 SPAD respectively.

    Table 4: Effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO on chlorophyll content of eggplants that are infected with Rhizoctonia solani and Fusarium incarnatum.


     
    Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on wet vegetative weight of eggplants that are infected with R. solani and F. incarnatum
     
    The results in Table 5 indicate that  the wet vegetative weight is significantly different among treatments as compared to the control. The treatment of S. aromaticum extract 4% + nano zinc oxide recorded the highest wet vegetative weight, reaching 333.8 g, while the control recorded the lowest wet vegetative weight, 173.0 g. The plants with the absence of the pathogenic fungi achieved the highest wet vegetative weight at 357.8 g, followed by plants with F. incarnatum at 269.8 g and with R. solani at 240.7 g. The interaction between the treatments and the presence or absence of the pathogenic fungi showed that the control with the absence of the pathogenic fungi has the highest wet vegetative weight, amounting to 370.0 g, followed by the treatments of S. aromaticum extract 4% + nano zinc oxide with the absence of the pathogenic fungi and with R. solani, reaching 363.8 g, while the plants with R. solani and F. incarnatum recorded the lowest wet vegetative weight at 68.7 g and 80.4 g, respectively.

    Table 5: Effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO on wet vegetative weight of eggplants that are infected with Rhizoctonia solani and Fusarium incarnatum.


     
    Effect of R. graveolens and S. aromaticum extracts and the nanoparticles of ZnO on dry vegetative weight of eggplants that are infected with R. solani and F. incarnatum
     
    The results in Table 6 showed that the dry vegetative weight is significantly different among treatments as compared to the control. The treatments of S. aromaticum extract 4% + nano zinc oxide and R. graveolens extract 4% + nano zinc oxide recorded the highest dry vegetative weight, reaching 30.77 g and 30.54 g, respectively, while the control recorded the lowest dry vegetative weight, 21.0 g. The plants with the absence of the pathogenic fungi achieved the highest dry vegetative weight at 31.16 g, followed by plants with F. incarnatum at 26.43 g and with R. solani at 24.99 g. The interaction between the treatments and the presence or absence of the pathogenic fungi showed that the treatment of S. aromaticum extract 4% + nano zinc oxide in the absence of the pathogenic fungi has the highest dry vegetative weight, amounting to 34.10 g, while the plants with R. solani and F. incarnatum recorded the lowest dry vegetative weight at 14.13 g and 14.87 g, respectively.

    Table 6: Effect of Ruta graveolens and Syzygium aromaticum extracts and the nanoparticles of ZnO on dry vegetative weight of eggplants that are infected with Rhizoctonia solani and Fusarium incarnatum.


           
    Ethyl acetate extract from R. graveolens leaves shows strong antifungal activity against Botrytis cinerea, Fusarium oxysporum and Colletotrichum spp. The extract of R. graveolens exhibits fungicidal properties against key agricultural pathogens including Colletotrichum fragariae, C. gloeosporioides, C. acutatum, Botrytis cinerea and Fusarium oxysporum (Otunola, 2022; Yadav and Ghasolia, 2022).
           
    In addition to preventing the mycelial growth of pathogenic fungus, zinc nanoparticles can either kill or stop germs from germinating (Pandey et al., 2011). The survival of cotton seedlings was developed when ZnONPs was used against R. solani. In the pots study, foliar ZnONP spray outperformed seed covering in increasing plant dry weight and managing the complex disease in beetroot roots, Beta Vulgaris L., which is caused by Pectobacterium betavasculorum, Meloidogyne incognita and R. solani (Salim et al., 2016; Bashyal et al., 2022). One primary mechanism of ZnONP toxicity against R. solani is mechanical encapsulation, which is believed to be how ZnONPs interact with pathogens) Aye and Matsumoto, 2011). This effect was evidenced by electron microscopy, which revealed clear deformations in the fungal hyphae of F. oxysporum treated with ZnO nanoparticles (Sivasankarapillai et al., 2023). In their study of the antifungal activity of zinc nanoparticles (ZnONPs) against F. oxysporum on tomato plants, (Suresh et al., 2015; Hong et al., 2022) found that ZnONPs at concentrations of 1500-3000 g/ml produced the lowest disease severity and the disease incidence rate.     
           
    The use of zinc nanoparticles (ZnONPs) has greatly improved the survival rate of cotton plants affected by seedling damping off disease, leading to increased height and dry weight. They may create an antifungal layer around seeds, providing protection against fungi (Yehia and Ahmed, 2013). The application of zinc oxide nanoparticles (ZnO-NPs) as foliar sprays on tomato plants showed effective control of Botrytis cinerea infection, leading to reduced disease severity and increased plant biomass, indicating ZnO-NPs’ growth-promoting capabilities. Treated plants exhibited elevated catalase and peroxidase activities, suggesting that ZnO-NPs may enhance resistance against B. cinerea, thereby providing an alternative to traditional fungicides for managing gray mold disease in tomatoes (Zaki et al., 2021).

    CONCLUSION

    The study’s results demonstrated that the treatment of R. graveolens extract 4% + nano zinc oxide was superior in reducing the incidence and  disease severity and increasing chlorophyll, whereas the treatment of S. aromaticum extract 4% + nano zinc oxide was outperformed in increasing wet and dry vegetative weight. It can be concluded that Ruta graveolens and Syzygium aromaticum extracts, along with ZnO nanoparticles, could protect eggplant seedlings from damping-off disease and improve their growth characteristics.

    CONFLICT OF INTEREST

    The authors declare that no conflict of interest.

    REFERENCES


    1. Abdelaziz, A.M., Salem, S.S., Khalil, A.M.A., El-Wakil, D.A., Fouda, H.M. and Hashem, A.H. (2022). Potential of biosynthesized zinc oxide nanoparticles to control Fusarium wilt disease in eggplant (Solanum melongena) and promote plant growth. BioMetals. 35: 601-616. https://doi.org/10.100 7/s10534-022-00391-8.

    2. Ahmed, S., Annu, Chaudhry, S.A. and Ikram, S. (2017). A review on biogenic synthesis of ZnO nanoparticles using plant extracts and microbes: A prospect towards green chemistry. Journal of Photochemistry and Photobiology B: Biology. 166: 272-284. https://doi.org/10.1016/j.jphotobiol.2016. 12.011.

    3. Al-Dhabaan, F.A., Shoala, T., Ali, A.A.M., Alaa, M. and Abd-Elsalam, K. (2017). Chemically-produced copper, zinc nanoparticles and chitosan-bimetallic nanocomposites and their antifungal activity against three phytopathogenic fungi. International Journal of Agricultural Technology. 13(5): 753-769.

    4. Alharbi, F.N., Abaker, Z.M. and Makawi, S.Z.A. (2023). Phytochemical substances-mediated synthesis of zinc oxide nanoparticles (ZnO NPS). Inorganics. 11(8): 328. https://doi.org/10. 3390/inorganics11080328.

    5. Aye, S.S. and Matsumoto, M. (2011).  Effect of some plant extracts on Rhizoctonia spp. and Sclerotium hydrophilum. Journal of Medicinal Plants Research. 5(16): 3751-3757.

    6. Bashyal, B.M., Kharayat, B.S., Parmar, P., Gupta, A.K., Dubey S.C. and Aggarwal, R. (2022). Influence of temperature on the pathogenesis and gene expression of rhizoctonia solani causing web blight/wet root rot disease in mungbean. Legume Research. 45(1): 104-109. doi: 10.18805/LR- 4342.

    7. Hassan, W.A. and Yousif, A.A. (2013). Soil recolonization by saprophytic fungi after solarization and soil amendments. Science Journal of University of Zakho. 1(2): 427-436.

    8. He, L., Liu, Y., Mustapha, A. and Lin, M. (2011). Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum. Microbiological Research. 166(3): 207-215. https://doi.org/10.1016/j.micres.2010.03.003.

    9. Hong T.T., Nhi N.T.H., Day P.V. (2022). Effect of Solanum procumbens L. extract on survival, growth performances, immune responses and against vibrio parahaemolyticus causing acute hepatopancreas necrosis disease in white leg shrimp (Litopenaeus vannamei). Indian Journal of Animal Research. 56(12): 1513-1518. doi: 10.18805/IJAR.BF-1566.

    10. Milesi, S.B.M., Massot, B., Gontier, E., Bourgaud, F. and Guckert, A. (2001). Ruta graveolens L. a promising species for the production of furanocoumarins. Plant Science. 161(1): 189-199. https://doi.org/10.1016/S0168-9452(01)00413-7.

    11. Otunola, G.A. (2022). Culinary spices in food and medicine: An overview of Syzygium aromaticum (L.) Merr. and LM Perry [Myrtaceae]. Frontiers in Pharmacology. 12: 793200. https://doi.org/10.3389/fphar.2021.793200.

    12. Pandey, P., Mehta, A. and Hajra, S. (2011). Evaluation of antimicrobial activity of Ruta graveolens stem extracts by disc diffusion method. Journal of Phytology. 3(3): 92-95. https://update publishing.com/journal/index.php/jp/article/view/2233.

    13. Salim, H.A., Salman, I.S. and Jasim, B.N. (2016). IPM approach for the management of wilt disease caused by Fusarium oxysporum f. sp. lycopersici on tomato (Lycopersicon esculentum). Journal of Experimental Biology and Agricultural Sciences. 4: 742-747. http://dx.doi.org/10. 18006/2016.4(VIS).742.747.

    14. Sivasankarapillai, V.S., Krishnamoorthy, N., Eldesoky, G.E., Wabaidur, S.M., Islam, M.A., Dhanusuraman, R. and Ponnusamy, V.K. (2023). One-pot green synthesis of ZnO nanoparticles using Scoparia dulcis plant extract for antimicrobial and antioxidant activities. Applied Nanoscience. 13: 6093- 6103. https://doi.org/10.1007/s13204-022-02610-7.

    15. Suresh, D., Nethravathi, P.C., Udayabhanu, Rajanaika, H., Nagabhushana, H. and Sharma, S.C. (2015). Green synthesis of multifunctional zinc oxide (ZnO) nanoparticles using Cassia fistula plant extract and their photodegradative, antioxidant and anti bacterial activities. Materials Science in Semiconductor Processing. 31: 446-454. https://doi.org/10.1016/j.mssp. 2014.12.023.

    16. Yadav, L.S. and Ghasolia R.P. (2022). Management of root rot of fenugreek caused by rhizoctonia solani using plant extracts and bio-agents. Legume Research. 45(3): 391-395. doi: 10.18805/LR-4438.

    17. Yehia, R.S. and Ahmed O.F. (2013). In vitro study of the antifungal efficacy of zinc oxide nanoparticles against Fusarium oxysporum and Penicilium expansum. African Journal of Microbiology Research. 7(19):1917-1923. https://doi. org/10.5897/AJMR2013.5668.

    18. Zaki, S.A., Ouf, S.A., Albarakaty, F.M., Habeb, M.M., Aly, A.A. and Abd-Elsalam, K.A. (2021). Trichoderma harzianum- mediated ZnO nanoparticles: A green tool for controlling soil-borne pathogens in cotton. Journal of Fungi. 7: 952. https://doi.org/10.3390/jof7110952. 
    In this Article
      Contact us
      Follow us
      Published In
      Indian Journal of Agricultural Research

      Editorial Board

      View all (0)