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This research aimed to use nanotechnology and its applications in agriculture, which gives broad prospects in the future to reduce the excessive use of fertilizers and pesticides and their negative effects on the environment. Therefore, chitosan nanoparticles loaded with nitrogen, phosphorous, and potassium were used as fertilizer and antifungal on potato plant to increase the yield and protect them from microbial infection, which increases the rates of their export from Egypt.
The study was conducted in three parts, namely:
1- Preparation and characterization of chitosan nanoparticles loaded with nitrogen, phosphorous and potassium elements (Nano CS-NPK).
2- Using the foliar application of chitosan nanoparticles (Nano CS-NPK) as fertilizer for the purpose of studying the effect on growth, yield and physiological changes of the potato plants.
3- Study and evaluation the use of foliar spray with chitosan nanoparticles (Nano CS-NPK) as antifungal on the resistance of potato plants infected with Alternaria solani.
The results can be summarized in the following:
1- The chitosan nanoparticles obtained by modified ionotropic gelation method with radical polymerization showed spherical shapes and uniform sizes approximately 33.6 nm in DLS and 25.7nm in the TEM micrographs. The stability of the CS-MAA colloidal suspension was +40 mV. While the diameter size of chitosan nanoparticles incorporation with the N, P and K elements was 51.89 nm in DLS and 40 nm in TEM and stability was +45 mV.
2- The XRD patterns of nano-chitosan incorporated with N, P and K elements indicates disturbance in crystalline form of chitosan due to interaction between chitosan and N, P and K elements. The CS-NPK nanoparticle exhibited three main diffraction peaks at 2θ= 28.46°, 32.02, ° and 40.5°. These peaks are characteristic of N, P and K elements respectively.
3- The FTIR results indicated the existence of electrostatic interactions between –COO– and –NH3 + of the chitosan nanoparticles and the elements N, P and K present in the urea, calcium phosphate, potassium chloride, respectively.
4- In this part of the study, the nano-chitosan loaded with nitrogen, phosphorous and potassium elements (Nano CS-NPK) was used in different concentrations of 10%, 50% and 100% as a nanofertilizer by foliar spraying of potato plants.
5- The absorption and transport of CS-NPK nanoparticle within leaf tissues was detected by TEM and their presence was shown as nanoparticles within the sieve tubes of phloem tissues where they accumulated with increasing concentration.
6- The results showed that the concentration of 10% and 50% Nano CS-NPK fertilizer caused a significant increase in the growth parameters (length, fresh weight and dry matter %) for potato plants at the age of 70 days from the vegetative growth. While a significant decrease in these parameters were recorded in potato plants treated with 100% CS-NPK nanofertilizer.
7- Results showed that foliar application with either conventional fertilizer NPK or Nano CS-NPK at different concentrations significantly increased number of tubers, tuber yield, average tuber weight, dry weight of tubers and tuber dry matter % as compared with control. The highest increments in the yield was recorded in 10% Nano CS-NPK treated plants. It was increased number of tubers, tuber yield, average tuber weight, dry weight of tubers and tuber dry matter % over than untreated control by 256.5% , 200.0% , 158.3 %, 230.2% and 131.5 % respectively.
8- Treatment with concentrations of 10% and 50% Nano-CSK resulted in a higher content of photosynthetic pigments (chlorophyll a and b, carotenoids and total pigments), carbohydrates (sugars, insoluble sugars and total carbohydrates) and mineral elements (Ca, Mg, N, P, K) and 10% wes the most effective.
9- The results showed the positive effect of the Nano CS-NPK fertilizer on the nitrogen metabolism in the leaves and tubers of potatoes as their contents of nitrates, nitrites, ammonium, amino acids and soluble total proteins increased. This was accompanied by an increase in the activity of nitrate reductase (NR) and glutamate dehydrogenase (GDH) compared to the control treatment. Among all treatments, the concentration of 10% of the nanofertilize showed the highest impact.
10- Scanning analysis of SDS-PAGE gel showed the presence of 12 protein bands having molecular weights ranging from 63.38- 12.38 KDa in proteins extracted from leaves of potato plants at the age of 70 days. A set of five protein bands (57.03, 18.63, 17.55, 15.46 and 13.79KDa) appeared in leaves of potato plants as the results of treatment with either conventional NPK or different concentrations of Nano CS-NPK fertilizer and were disappeared in control. In addition, it was found one special protein band having molecular weight 63.38 was induced by 10% and 50 % of Nano CS-NPK and not detected other treatments.
11- The results showed that stimulating growth, yield and metabolic activity in potatoes because of spraying with nano fertilizer CS-NPK is directly related to the high level of endogenous hormones (IAA, GA3, trans-Zeatin and ABA) in the leaves compared to the untreated plant. It was found that the highest hormone content was found in leaves of plants treated with a 10% of the Nano fertilizer.
12- In the third part of the study, Nano CS-NPK was used as antifungal to combat early blight disease in potato plants infected with A. solani and increased their resistance.
13- The efficiency of Nano CS-NPK at concentrations of 10, 50 and 100% was tested as a fungicide for inhibiting the growth of the A. solani. The highest growth inhibition rate (96%) of A. solani was recorded at a concentration of 10% Nano CS-NPK. Therefore, it was found that the concentration of 10% Nano CS-NPK has a strong anti-fungal effect.
14- Based on the results of testing the efficiency of Nano CS-NPK as antifungal in the previous experiment, a 10% concentration of Nano CS-NPK was used by spraying the leaves of potato plants either before or after infection with A. solani to study its effect on increasing potato resistance to early blight disease and stimulating its defense systems and immunity.
15- It was found that treatment of CS-NPK pre-infection was more effective in controlling early blight disease in potato plants than the treatment of CS-NPK post-infection. So, the disease severity index (DSI) has decreased dramatically to 6% compared to the post-infested plants, where the disease severity decreased to 30% at the same age.
16- TEM analysis confirmed the viability of tissue and there were no noticeable changes in the leaves of plants treated with Nano CS-NPK, especially before infection, compared to infected plants.
17- The incidence of Arteraria solani fungus caused a significant decrease in the leaf content of photosynthetic pigments, total soluble sugars and total soluble proteins compared to uninfected control. These contents increased in potato leaves sprayed before or after infection, compared to infected control. The treatment of Nano CS-NPK before infection was better compared to
18- The analysis of SDS-PAGE electrophoretic pattern showed that infection by A. solani induced de novo synthesis of four distinct protein bands (34.91, 27.38, 22.35 and 12.74 KDa) in potato leaves treated with 10% Nano CS-NPK, which not found in the control treatment. While three protein bands having molecular weights 57.03, 52.20 and 18.63 KDa were disappeared in potato leaves as the results of A. solani infection. On the other hand, foliar spraying by 10% Nano CS-NPK pre or post infection led to de novo synthesis of special protein band of molecular weight 123.74 and caused the disappearance of protein band of molecular weight 12.13 KDa.
19- The results showed a noticeable decrease in the level of hydrogen peroxide (H2O2) and malondialdehyde (MDA) and an increase in the activity of lipoxygenase (LOX) in the leaves of potato plants that were sprayed with Nano CS-NPK antifungal, whether before or after infection compared to the infected control. This was accompanied by a noticeable increase in the activity of antioxidant enzymes (superoxide dismutase SOD, catalase CAT and ascorbate peroxidase APX) and the content of non-enzymatic antioxidants (ascorbic acid and flavonoids), especially in the leaves of potato plants treated with the anti-fungus Nano CS-NPK before infection.
20- The response of potato plants to infection with A. solani and and their resistance to the prevalence of the early blight disease resulting in a significant increase in the activity of lignin enzymes (peroxidase POX, poly phenol oxidase PPO and phenylalanine amylase PAL) and the content of total phenolic compounds in the leaves compared to non-infected plants.
21- Compared to the uninfected control, results showed that activity chitinase enzyme increased by 255.5% and 311.1% in leaves of potato plants treated with the antifungal CS-NPK before and after the infection, respectively . While the activity of 1,3-glucanase increased by 275.3% and 328.4% for the same treatments, respectively.
22- The phytoalexin compounds which induced in potato leaves treated with the fungicide Nano CS-NPK were estimated before or after the fungal infection using a Gas chromatography Mass Spectrometry (GC/MS). The results showed that there are eight basic phytoalexins belonging to terpenoids and fatty acids in all treatments. Its percentage increased in potato leaves treated with antifungal, either before or after infection.
23- Also, GC-MS analysis showed the presence of four new compounds of the phytoalexin in potato leaves that were sprayed by Nano CS-NPK either before or after infection and were not found in the extract of leaves in A. solani infected plants which are:
6-Hydroxyhexanoic acid, (4-Nitrophenyl)-1-thio-beta-D-glucopyranoside, N-(4-Methylphenyl) hexopyranosylamine , Oleanolic acid (Caryophyllin).