Assessment of the Inhibitory Effect of Nanoparticles and Salicylic Acid on

Received 01 Apr, 2024

Accepted 23 May, 2024

Published 24 May, 2024

Background and Objective: Alternaria solani is responsible for causing early blight, leaf spot, collar rot, fruit rot and stem canker diseases in solanaceous crops and is very difficult to control. Several management strategies are employed but chemical fungicides are most effective. However, due to their established negative consequences, much greener and eco-friendly options are needed. Nanotechnology could offer new approaches for solving this problem and the present study was carried out to explore the fungicidal activity of twelve nanoparticles against the mycelial inhibition of Alternaria solani. Materials and Methods: The comparative inhibitory effect of twelve Nanoparticles (NPs) viz., Cobalt ferrite (CoFe₂O₄), Elemental copper (Cu^-), Ferric oxide (Fe₂O₃), Nickel ferrite (NiFe₂O₄), Nickel peroxide (NiO₂), Polyamelene (PANI), Polyanelene+ferrite (P600), Tin oxide (SnO₂), Titanium oxide (TiO₂), Zinc ferrite (ZnFe₂O₄), Zinc oxide (ZnO), Zinc peroxide (ZnO₂) and Salicylic acid (SA) were evaluated in vitro against Alternaria solani. All the data were processed by using Analysis of Variance (ANOVA) and R-software at (p<0.05). Results: In vitro, screening of NPs and SA at 5, 10, 15, 20 and 25 μg/mL revealed substantial variability to inhibit the mycelial growth of A. solani. In general, the NPs which caused significant inhibition in fungal growth, their effectiveness increased with increase in the concentrations. Among the NPs, zinc peroxide (20 μg/mL), salicylic acid (20 μg/mL), zinc oxide (20 μg/mL), elemental copper (20 μg/mL), nickel peroxide (25 μg/mL) and ferric oxide (25 μg/mL) caused the 100% inhibition in the mycelia growth of A. solani. Conclusion: This is the first study on the fungicidal effects of nano-zinc peroxide on A. solani. However, the mechanism involved in the fungicidal activity and toxicological studies are required to be confirmed.

INTRODUCTION

Alternaria solani is regarded as an important menace and causes huge damage to solanaceous crops¹. The fungus is responsible for causing diseases like early blight, leaf spot, collar rot, fruit rot, stem canker, etc. and is very difficult to control². Several management strategies are employed against A. solani in various crops³ and chemical fungicides are one of the most effective methods to manage this fungus. However, due to their established negative consequences, much greener and more effective fungicides are needed⁴.

Nanotechnology could offer new approaches for solving this problem as the performance of conventional pesticide/fungicide formulae is often improved by nanotechnology construction^5,6. A multitude of chemical, biological and physical techniques are still being developed which lead to the production of noble nanoparticles, some of which are used against fungal pathogens^7,8. Recent research has demonstrated that the foliar application of nano-CuO, MnO and ZnO significantly declined fungal pathogens without physiological toxicity in a greenhouse⁹. In other studies, antifungal activity of copper-based^10,11, Ag-based¹² against Alternaria alternata and Zn-based nanoparticles against Alternaria mali was shown by Ahmad et al.⁸.

Phenolic compounds including salicylic acid (SA) are involved in plant defence against plant pathogens¹³. Several studies revealed the ability of SA to induce systemic defense in various crops against fungal pathogens⁴. Further, exogenous application of SA induced a strong systemic defense response in rice crops against Rhizoctonia solani^14,15, tobacco against Pythium aphanidermatum¹⁶ and cowpea for root-knot pathogen¹⁷. Jayakumar et al.¹⁸ reported that SA application at 50, 100 and 200 ppm improved plant height, root length, shoot and root weight and significantly decreased root-knot disease in tomato plants.

With the above background, the present in vitro study was carried out to explore the fungicidal activity of twelve nano particles viz., cobalt ferrite, elemental copper, ferric oxide, nickel ferrite, nickel peroxide, polyamelene, polyanelene+ferrite, tin oxide, titanium oxide, zinc ferrite, zinc oxide and zinc peroxide and salicylic acid against the mycelial inhibition of Alternaria solani.

MATERIALS AND METHODS

Study area: The present study was carried out during September-December, 2023 at the Department of Plant Protection, Faculty of Agricultural Science, Aligarh Muslim University, Aligarh, India.

Nanoparticles and their doses: Twelve nanoparticles viz., Cobalt ferrite (CoFe₂O₄), Elemental copper (Cu^-), Ferric oxide (Fe₂O₃), Nickel ferrite (NiFe₂O₄), Nickel peroxide (NiO₂), Polyaniline (PANI), Polyanelene+ferrite (P600), Tin oxide (SnO₂), Titanium oxide (TiO₂), Zinc ferrite (ZnFe₂O₄), Zinc oxide (ZnO) and Zinc peroxide (ZnO₂) were obtained from Department of Chemistry, Aligarh Muslim University, India. Pure and Lab grade salicylic (Qualigen, India) was procured from a chemical dealer in Aligarh, India. To investigate their nematicidal action in terms of mortality and hatching inhibition of M. javanica J2s in vitro, five concentrations viz., 5, 10, 15, 20 and 25 μg/mL of each chemical were used.

Inoculum of Alternaria solani: The isolation, purification and morphological identification of a pathogenic isolate of Alternaria solani AMUAS-1 was previously done and used in the present study¹. Further molecular identification using 18s rDNA was carried out at Macrogen Co., Ltd. (Seol, South Korea) and phylogenetic tree was constructed using the neighbor-joining method in MEGA 11.0¹⁹, with bootstrap values based on 1000 replications. The partial gene sequence was submitted to the NCBI gene bank with accession number KY062572 (Fig. 1).

Determination of minimum inhibitory concentration of nanoparticles and salicylic acid against mycelial growth of Alternaria solani in vitro: The sensitivity of Alternaria solani to twelve nanoparticles (NPs), as mentioned in the previous section and salicylic acid (SA) was tested by poisoned food technique at five different concentrations (5, 10, 15, 20 and 25 μg/mL) as described by Dhingra and Sinclair²⁰. Sterilized potato dextrose agar medium was poured in Petri plates having desired NPs and SA concentrations separately. Thereafter, the medium was allowed to solidify.

Fig. 1: Dendrogram showing the homology similarity search for Alternaria solani AMUAS-1

A mycelial disc (9 mm dia.) of the pure culture of A. solani AMUAS-1 was placed in the middle of the Petri plates and appropriate control was maintained without adding NPs and SA. Every treatment was maintained in five copies, one of which was the untreated control. The plates were incubated at room temperature (25±2°C) and the diameter of colonies was recorded on 7th day and expressed in millimetres (mm). The formula utilized to compute the percent inhibition (PI) was:

Where:

	C	=	Growth of test pathogen (mm) in control
	T	=	Growth of test pathogen (mm) in the amended medium

Statistical analysis: The in vitro study was repeated for more accuracy. Analysis of Variance (ANOVA) was used to process all of the data using R-software for Windows 11. The data were pooled (10 replicates per treatment) because the differences between the two repeated experiments were non-significant at p<0.05. The data were analysed using a two-factor ANOVA, with chemicals (nanoparticle and salicylic acid) as factor one and chemical doses (concentrations) as the second factor and F-values were also examined to identify important treatments at p<0.05.

RESULTS

In-vitro determination of minimum inhibitory concentration of different nanoparticles and salicylic acid against Alternaria solani: In vitro screening of NPs and SA at 5, 10, 15, 20 and 25 μg/mL exhibited substantial variability to inhibit the mycelial growth of A. solani. In general, the NPs which caused significant inhibition, their effectiveness increased with higher concentrations (Table 1). Among the NPs, zinc peroxide, salicylic acid and elemental copper at 20 μg/mL caused the 100% inhibition in the mycelia growth of A. solani (Table 1). While zinc oxide (25 μg/mL), nickel peroxide (25 μg/mL) and ferric oxide (25 μg/mL) exhibited over 90% inhibition in the mycelia growth of test fungus (Fig. 2). The ferrite (cobalt ferrite, nickel ferrite, zinc ferrite and polyanelene+ferrite) were recorded least effective NPs (Table 1). Overall the inhibitory effectiveness of NPs and SA against A. solani at 25 μg/mL were in order of Zinc peroxide>Salicylic acid>Elemental copper>Zinc oxide>Nickel peroxide>Ferric oxide>Tin oxide>Titanium oxide>Polyamelene>Polyanelene+ferrite>Zinc ferrite>Nickel ferrite>Cobalt ferrite (Table 1).

Fig. 2: Effect of nanoparticles and salicylic acid at different concentrations on the mycelial growth of Alternaria solani T1 = Zinc peroxidase, T2 = Salicylic acid, T3 = Elemental copper, T4 = Zinc oxide, T5 = Nickel peroxide and T6 = Ferric oxide

DISCUSSION

The present study evaluated the effectiveness of twelve nanoparticles (NPs) and salicylic acid (SA) against Alternaria solani at five concentration levels in vitro conditions. Screening of NPs has shown significant heterogeneity to cause mycelial growth inhibition of A. solani. Among the twelve NPs evaluated, zinc peroxide, elemental copper, zinc oxide, nickel oxide, ferric oxide and tin oxide were demonstrated to be most effective against the test pathogen and cause 90-100% inhibition of mycelia growth of A. solani. Antifungal activity of copper-based by Ouda¹⁰, Kanhed et al.¹¹ and Krishnaraj et al.¹² also examined the effect of nanoparticles on Alternaria spp., as observed in this study.

Table 1:

Effect of the different concentrations of nanoparticles and salicylic acid on the percentage inhibition of mycelial growth of Alternaria solani in vitro

	Mycelial inhibition (%)
Treatments	5 μg/mL	10 μg/mL	15 μg/mL	20 μg/mL	25 μg/mL	LSD (p<0.05)
Control	1.1	1.3	1.2	1	1.5	0.11
Cobalt ferrite	5	12	15	18	20	1.75
Elemental copper	80	85	93	100	100	8.9
Ferric oxide	56	64	78	85	93	8.7
Nickel ferrite	8	13	19	24	28	2.03
Nickel peroxide	36	47	63	80	94	7.23
Polyamelene	13	15	20	25	39	3.72
Polyanelene+ferrite	20	24	30	33	37	2.77
Salicylic acid	68	80	92	100	100	9.03
Tin oxide	42	61	70	85	90	8.67
Titanium oxide	51	59	63	65	70	6.6
Zinc ferrite	15	19	22	24	29	2.4
Zinc oxide	76	84	93	95	100	9.83
Zinc peroxide	65	78	97	100	100	10.0
LSD p<0.05	2.34	3.46	5.76	6.12	6.27	-
F-values (p<0.05)
NPs (df = 12)	11.3	13.4	15.2	15.6	15.3	-
Dose (df = 4)	9.6	8.9	11.7	9.2	5.9	-
NPs×dose (df = 12)	5.4	4.8	5.1	4.6	NS	-
Data are means of ten replicates, F-values are significant otherwise not significant (NS) at p<0.05 and LSD: Least significant difference

In the current study, zinc peroxide was relatively more effective than salicylic acid and elemental copper. Zinc peroxide is a surgical antiseptic chemical and its antimicrobial activity is well documented historically²¹. However, the anti-fungal properties of zinc peroxide are seldom reported and a majority of the studies are focused on zinc oxide^22-24. In this study also zinc oxide and titanium oxide were found inhibitory to A. solani growth. Khan et al.²⁵ also reported the induction of oxidative stress, growth inhibition and reduction in biofilm formation of Streptococcus mitis by titanium dioxide and zinc oxide nanoparticles.

Malandrakis et al.²⁶ used copper nanoparticles against foliar and soil-borne plant pathogens. In current study, elemental copper was the second most effective NP against A. solani and caused 100% mycelial inhibition at 20 μg/mL concentration. Similarly, the in vitro fungicidal activity of copper nanoparticles against Alternaria spp. was also evaluated by Ouda¹⁰ and Kanhed et al.¹¹, corroborating current study.

Several studies have reported the application of nanoparticles in the management of various plant-pathogenic fungi. Seku et al.²⁷ reported that Candida albicans, Candida parapsilosis, Aspergillus niger and Aspergillus oryzae are subject to antifungal activity by Cu-NP. Rubina et al.²⁸ have found that Cu-chitosan nanocomposites worsen Rhizoctonia solani and S. rolfsii fungal mycelium. Gold nanoparticles are altered and ruptured by the fungal cell membrane of Puccinia graminis tritici, Aspergillus flavus, A. niger and Candida albicans²⁹. Elmer and White⁹ have demonstrated that Fusarium wilt and Verticillium diseases have declined without physiological toxicity in the foliar application of nano-CuO, MnO and ZnO in a greenhouse.

Application of salicylic acid (SA) was also found very effective in suppressing the mycelial growth of A. solani in the present study. The SA has been identified as one of the essential elements in the signal transduction pathway that results in plant resistance to a number of diseases, including Alternaria blight⁷. Several studies revealed that the exogenous application of SA was inhibitory to the fungal pathogens as observed in the present study^14-16.

In the present study, zinc peroxide, elemental copper, zinc oxide and nickel oxide were found most effective against Alternaria solani. However, the mechanism involved in the fungicidal activity and toxicological studies are yet to be confirmed. Further, pot and field trials are required to confirm their performance before commercial use. In addition to this, high processing costs, problems with scalability, industrial production, public perception, environment, health and safety concerns related to the application of nanoparticles are need to addressed properly.

CONCLUSION

Among the NPs screened, zinc peroxide was found most effective in inhibiting the mycelia growth of Alternaria solani. To the best of our knowledge, this is the first report of fungicidal properties of zinc peroxide against early blight fungus, A. solani. Hence, zinc peroxide may be explored as another substitute for chemical fungicide in the management of Alternaria blight, early blight, leaf spot, collar rot, fruit rot and stem canker caused by Alternaria spp. However, the mechanism involved in the fungicidal activity and toxicological studies are required to be confirmed before its commercial use in field conditions.

SIGNIFICANCE STATEMENT

Alternaria solani is regarded as an important fungal pathogen of solanaceous crops and causes severe yield loss to the crop. To explore much greener management options, twelve nanoparticles (NPs) and salicylic acid were evaluated against the mycelial inhibition of Alternaria solani in vitro conditions. Among the NPs screened, zinc peroxide was found most effective and inhibited 100% mycelial growth of Alternaria solani. According to previous studies, this is the first report of fungicidal properties of zinc peroxide against early blight fungus, A. solani. Hence, zinc peroxide may be explored as another substitute for chemical fungicide in the management of Alternaria blight, early blight, leaf spot, collar rot, fruit rot and stem canker caused by Alternaria spp.

ACKNOWLEDGMENT

We wish to acknowledge Dr. Sajid Ali Ansari, King Faisal University Al Hofuf, Saudi Arabia for his valuable input in preparing the manuscript.

REFERENCES

1. Ziaul Haque, S. Zamir, K. Pandey, N. Gupta and R.N. Rajana, 2024. Bio-management of early blight of tomato (Alternaria solani) with solid-state fermentation formulation of Trichoderma isolates. Indian Phytopathol., 77: 323-333
2. Panno, S., S. Davino, A.G. Caruso, S. Bertacca and A. Crnogorac et al., 2021. A review of the most common and economically important diseases that undermine the cultivation of tomato crop in the Mediterranean basin. Agronomy, Vol. 11. 10.3390/agronomy11112188
3. Mamgain, A., R. Roychowdhury and J. Tah, 2013. Alternaria pathogenicity and its strategic controls. Res. J. Biol., 1: 1-9.
4. Haque, Z. and M.R. Khan, 2022. Host resistance and bio-management of tobacco root-rot caused by Pythium aphanidermatum. Indian Phytopathol., 75: 703-712.
5. Khot, L.R., S. Sankaran, J.M. Maja, R. Ehsani and E.W. Schuster, 2012. Applications of nanomaterials in agricultural production and crop protection: A review. Crop Prot., 35: 64-70.
6. de Oliveira, J.L., E.V.R. Campos, M. Bakshi, P.C. Abhilash and L.F. Fraceto, 2014. Application of nanotechnology for the encapsulation of botanical insecticides for sustainable agriculture: Prospects and promises. Biotechnol. Adv., 32: 1550-1561.
7. Singh, D.P., H.B. Singh and R. Prabha, 2016. Microbial Inoculants in Sustainable Agricultural Productivity: Vol. 2: Functional Applications. 1st Edn., Springer, New Delhi, India, ISBN: 978-81-322-2644-4, Pages: 308.
8. Ahmad, H., K. Venugopal, K. Rajagopal, S. de Britto and B. Nandini et al., 2020. Green synthesis and characterization of zinc oxide nanoparticles using eucalyptus globules and their fungicidal ability against pathogenic fungi of apple orchards. Biomolecules, Vol. 10. 10.3390/biom10030425
9. Elmer, W.H. and J.C. White, 2016. The use of metallic oxide nanoparticles to enhance growth of tomatoes and eggplants in disease infested soil or soilless medium. Environ. Sci.: Nano, 3: 1072-1079.
10. Ouda, S.M., 2014. Antifungal activity of silver and copper nanoparticles on two plant pathogens, Alternaria alternata and Botrytis cinerea. Res. J. Microbiol., 9: 34-42.
11. Kanhed, P., S. Birla, S. Gaikwad, A. Gade and A.B. Seabra et al., 2014. In vitro antifungal efficacy of copper nanoparticles against selected crop pathogenic fungi. Mater. Lett., 115: 13-17.
12. Krishnaraj, C., R. Ramachandran, K. Mohan and P.T. Kalaichelvan, 2012. Optimization for rapid synthesis of silver nanoparticles and its effect on phytopathogenic fungi. Spectrochim. Acta Part A: Mol. Biomol. Spectrosc., 93: 95-99.
13. Khan, M.R. and Z. Haque, 2013. Morphological and biochemical responses of five tobacco cultivars to simultaneous infection with Pythium aphanidermatum and Meloidogyne incognita. Phytopathol. Mediterr., 52: 98-109.
14. Saikia, R., R. Kumar, D.K. Arora, D.K. Gogoi and P. Azad, 2006. Pseudomonas aeruginosa inducing rice resistance against Rhizoctonia solani: Production of salicylic acid and peroxidases. Folia Microbiol., 51: 375-380.
15. Anitha, A. and M.A. Das, 2011. Activation of rice plant growth against Rhizoctonia solani using Pseudomonas fluorescens, Trichoderma and salicylic acid. Res. Biotechnol., 2: 7-12.
16. Khan, M.R. and Z. Haque, 2011. Soil application of Pseudomonas fluorescens and Trichoderma harzianum reduces root-knot nematode, Meloidogyne incognita, on tobacco. Phytopathol. Mediterranea, 50: 257-266.
17. Nandi, B., N. Banerjee, N.C. Sukual, P. Das, S. Sengupta and S.P.S. Babu, 2002. Salicylic acid enhances resistance in cowpea against Meloidogyne incognita. Phytopathol. Medit., 41: 39-44.
18. Jayakumar, J., G. Rajendran and S. Ramakrishnan, 2006. Evaluation of salicylic acid as a systemic resistance inducer against Meloidogyne incognita on tomato cv. Co3. Indian J. Nematol., 36: 77-80.
19. Tamura, K., G. Stecher and S. Kumar, 2021. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol., 38: 3022-3027.
20. Dhingra, O.D. and J.B. Sinclair, 1995. Basic Plant Pathology Methods. 2nd Edn., CRC Press, Boca Raton, Florida, ISBN: 9780873716383, Pages: 448.
21. Freeman, B.S., 1940. The use of zinc peroxide in malignant lesions. JAMA, 115: 181-186.
22. Sabir, S., M. Arshad and S.K. Chaudhari, 2014. Zinc oxide nanoparticles for revolutionizing agriculture: Synthesis and applications. Sci. World J., Vol. 2014, 10.1155/2014/925494
23. Ravi, A., V.V.T. Nandayipurath, S. Rajan, S.A. Salim, N.K. Khalid, C.T. Aravindakumar and R.E. Krishnankutty, 2021. Effect of zinc oxide nanoparticle supplementation on the enhanced production of surfactin and iturin lipopeptides of endophytic Bacillus sp. Fcl1 and its ameliorated antifungal activity. Pest Manage. Sci., 77: 1035-1041.
24. Sun, Q., J. Li and T. Le, 2018. Zinc oxide nanoparticle as a novel class of antifungal agents: Current advances and future perspectives. J. Agric. Food Chem., 66: 11209-11220.
25. Khan, S.T., J. Ahmad, M. Ahamed, J. Musarrat and A.A. Al-Khedhairy, 2016. Zinc oxide and titanium dioxide nanoparticles induce oxidative stress, inhibit growth, and attenuate biofilm formation activity of Streptococcus mitis. J. Biol. Inorg. Chem., 21: 295-303.
26. Malandrakis, A.A., N. Kavroulakis and C.V. Chrysikopoulos, 2019. Use of copper, silver and zinc nanoparticles against foliar and soil-borne plant pathogens. Sci. Total Environ., 670: 292-299.
27. Seku, K., B.R. Ganapuram, B. Pejjai, G.M. Kotu and N. Golla, 2018. Hydrothermal synthesis of copper nanoparticles, characterization and their biological applications. Int. J. Nano Dimens., 9: 7-14.
28. Rubina, M.S., A.Y. Vasil’kov, A.V. Naumkin, E.V. Shtykova, S.S. Abramchuk, M.A. Alghuthaymi and K.A. Abd-Elsalam, 2017. Synthesis and characterization of chitosan-copper nanocomposites and their fungicidal activity against two sclerotia-forming plant pathogenic fungi. J. Nanostruct. Chem., 7: 249-258.
29. Jayaseelan, C., R. Ramkumar, A. Abdul Rahuman and P. Perumal, 2013. Green synthesis of gold nanoparticles using seed aqueous extract of Abelmoschus esculentus and its antifungal activity. Ind. Crops Prod., 45: 423-429.