Effect of Difenoconazole and Azoxystrobin on Wheat and Radish Seeds Germination and Tomato Seedling Growth

and also seedling growth. Therefore, the present study was carried out to evaluate the effect of Difenoconazole and Azoxystrobin as fungicides on the germination of monocotyledonous seed (wheat; Triticum aestivum L . ), and dicotyledonous seeds (radish; Raphanus sativus ), then effect on Tomato ( Solanum lycopersicum L.) seedling growth. Seeds of wheat and radish and seedling of tomato were treated with different concentrations of the tested fungicides; recommended, half and the double recommended dose compared with the untreated control. The results showed that the two tested fungicides significantly decreased seed germination%, root and shoot length of both wheat and radish seeds at the different applied doses compared to the control. The highly significant reduction in relative root and shoot length% and the relative germination% of wheat and radish seeds were observed when exposed to Difenoconazole or Azoxystrobin in which double recommended doses being 23.20, 27.88, and 43% or 13.20, 26.02, and 30.10% in wheat seeds, however, the reduction were 22.30, 37.69, and 63.0% or 17.67, 51.67, and 40.70% in radish seeds, respectively. In the case of tomato seedlings; carotene, chlorophyll a, chlorophyll b and total chlorophyll were significantly decreased after three weeks from both fungicides application by the double recommended doses, which recorded 0.04, 2.25, 0.15, 2.40 mg/g fresh weight for difenoconazole or 0.06, 1.66, 0.39, 2.05 mg/g fresh weight for azoxystrobin, respectively. Because Difenoconazole is less hazardous than Azoxystrobin, it might be advised that wheat and radish seeds be treated with it before sowing. Three weeks after being exposed to the fungicides, tomato plants treated with Azoxystrobin displayed a decrease in the a/b ratio at the indicated dose.

systemic fungicides.Azoxystrobin (25%EC) possesses a novel biochemical mode of action and its fungicidal activity results from the inhibition of mitochondrial respiration in fungi.Their biochemical mode of action is the inhibition of electron transport according to Hewitt (1998).Difenoconazole acts as a seed treatment, foliar spray and systemic fungicide.It is taken up through the surface of the infected plant and is translocated to all parts of the plant.It has a curative effect and a preventative effect.Difenoconazole can be applied to winter wheat, oilseed rape, brussels sprouts, cabbage, broccoli/calabrese and cauliflower.It controls various fungi including Septoria tritici, Brown Rust, Light Leaf Spot, Leaf Spot, Pod Spot, Ring Spot and Stem Canker.The mode of action of difenoconazole is a sterol demethylation inhibitor that prevents the development of the fungus by inhibiting cell membrane ergosterol biosynthesis according to Reuveni and Sheglov (2002).Combined with commercial azoxystrobin-, fludioxonil-, difenoconazole-, and tebuconazole-based fungicides commonly used to control early blight (Hatem et al., 2022).Phytotoxicity is the ability of pesticides to cause temporary or permanent damage to vegetative or generative organs, which reduce or totally inhibit germination, and to cause other physiological and morphological changes in sensitive plant species, and/or to certain varieties or genetic lines.Damage occurs in various ways, mostly as chlorosis, i.e. partial or complete destruction of chloroplasts when leaves become chlorotic.Necrosis (burn) is another manifestation of phytotoxicity, which can lead to complete leaf drying or defoliation according to Vuković et al. (2013).Seed germination, shoot and root elongation measurements are quite rapid for use on acute phytotoxicity tests with several advantages: sensitivity, simplicity, low cost, and suitability for reactive chemicals and contaminated soil samples according to Munzuroglu and Geckil (2002).Seed germination and seedling growth are being widely used to test the phytotoxicity of many chemical species such as fungicides which may be released into the environment because it is a crucial stage in plant growth and it is also an important phenomenon in agriculture because it is regarded as a thread of life and plants that ensure its survival according to Iqbal et al. (2016).Therefore, this study aimed to clarify the influence of selected fungicides with different doses on seed germination of radish and wheat as well as seedling growth of tomatoes.

1.Fungicide Solutions Preparation:
Azoxystrobin and difenoconazole were suspended individually in distilled water to make different doses; recommended dose (the Egyptian Ministry of Agriculture official: 50cm/100-liter water for each), half (½D), and double the recommended doses.

Tested Plants:
Two seed types; radish (Raphanus sativus) and wheat (Triticum aestivum L.) were used for the seed germination test.A Tomato seedling (Solanum lycopersicum L.), of super strain B cultivar (45 d., old) was used for the seedling growth test.

3.Seed Germination Assay:
Germination assay was carried out according to the ISTA test (1996).Seeds were immersed in sodium hypochlorite (10%) solution for 10 min to ensure surface sterility then rinsed three times with distilled water.One piece of filter paper was put into a Petri dish (10 mm) and 10 seeds of wheat or radish were put onto the filter paper.Five ml of distilled water (control), or the fungicide suspensions were added/dish.Each exposed dose/plant was replicated three times.The Petri dishes were covered and placed in an incubator for 7 days in the dark at 26±2ºC.After the incubation period, root and shoot length, germination percentage, relative elongation %, relative germination rate and germination index were recorded.The results were compared to the untreated control for each seed type.The relative elongation %, relative germination rate and germination index were calculated according to AOSA (1991)  The experiment was conducted in the greenhouse to evaluate the phytotoxicity effects of the two fungicides on tomato seedlings' pigments and dry weight.Physical and chemical properties and the soil texture of the used soil are summarized in Table (1).Tomato seedlings (45 d. age), were transplanted in the prepared pots and fertilized by adding 1.50g/pot (NPK 20/20/20), weekly.The fungicide suspensions were sprayed on the plants after 2 weeks from transplanting, with six replicates for each dose meanwhile the untreated check was sprayed with water only.Tomato leaves were collected after 1, 2, and 3 weeks from application.After collection, samples were transferred directly to the laboratory for pigments determination.

Dry Weights:
After 4 weeks from treatment, tomato plants were cut off and dried by an electric oven of Fisher Isotemp.(Senior model) at 60 ᵒ C for 2 days.Then dry matter content per tomato plant was recorded by the gram.

Statistical Analysis:
The statistical analysis was done by using a one-way ANOVA by SPSS statistical software according to Landau and Everitt (2004).The results were presented as mean ± SD (standard deviation).Each of the experimental values was compared to the corresponding control.Statistical significance was accepted when the probability of the result assuming the null hypothesis (p) is less than 0.05.

1.Effects of Difenoconazole and Azoxystrobin Suspensions on Wheat Seeds Germination:
Data in Table (2) indicated that difenoconazole and azoxystrobin suspension showed a significant decrease in root and shoot lengths at all treated doses compared with control with a clear significant dose-dependent.The highest significant decrease effect on root and shoot length was obtained by difenoconazole and azoxystrobin when applied at the double recommended dose which recorded the lowest relative root/shoot length % compared to control being 23.20 and 27.88% or 13.20 and 26.02%, respectively.Also, relative germination % and germination index (Fig. 1) Correlated negatively with increasing the applied dose being 43.00% and 9.97%, or 30.10% and 3.97 at the double dose treatment of difenoconazole and azoxystrobin, respectively.The results indicated that wheat seeds were more affected by azoxystrobin treatment and showed the lowest germination measurements than difenoconazole.This is in agreement with Tvaruzek et al. (1995) who indicated that some fungicides such as prochloraz have a severe phytotoxic effect on winter wheat.Also, it could be due to that azoxystrobin affected the membrane stability of plant tissue where the extent of damage was concentration-dependent. Conversely, difenoconazole showed no adverse effect on the integrity of the membrane.However, the loss of electrolytes from the wheat leaves stimulated at high concentrations according to Tripathi et al. (1982).

2.Effects of Difenoconazole and Azoxystrobin Suspensions on Radish Seeds Germination:
Data presented in Table (3) illustrated that difenoconazole and azoxystrobin showed a significant inhibition effect on the root and shoot length of radish seeds at all applied doses compared with the untreated control.Application of difenoconazole and azoxystrobin showed a significant inhibition effect on all tested parameters and the root was more affected than the shoot.The double recommended dose showed the highest significant inhibition effect and recorded the lowest relative root/shoot length% being 22.30 and 37.69% or 17.67 and 51.67%, respectively.Data of relative germination % and Germination index indicated that radish seeds were more sensitive to azoxystrobin than difenoconazole which showed the highest inhibition effect when treated with the double recommended dose, being 40.70% and 9.13 or 63.00% and 14.05, respectively.This is in agreement with Ratsch (1983) who concluded that inhibition of root elongation was a valid and sensitive indicator of environmental toxicity.However, the shoot elongation progressively decreased with increasing concentrations of tested fungicides according to Nithyameenaskshi et al. (2006).

Effects of Difenoconazole and Azoxystrobin Suspensions On Tomato Seedling Pigments:
Data presented in Tables 4-6, indicated that Difenoconazole showed a significant decrease effect when applied at the three application doses on Carotene and Chl.a content compared with the untreated control after 7 days from application, while it did not reveal any significant differences after 14 and 21 d., except that of double recommended dose treatment which showed a highly significant decrease in Chl.a after 21 days and recorded 2.25 mg/g fresh weight.All difenoconazole treatments showed a significant decrease in effect on Chl.b after 14 and 21 days of application.The highest effect was recorded by the double recommended dose compared to the other treatment, recording 0.5 and 0.15 mg/g fresh weight after 14 and 21 days, respectively.Chlorophyll total (Chl.a+b)and Chl.a/b ratio was affected significantly by all applied fungicide doses compared with the control and showed the highest significant value at the double recommended dose after 21 days from application, being 2.4 mg/g fresh weight and 15, respectively.
Azoxystrobin treatment in Tables (4-6), decreased all tested pigments i.e., Carotene, Chl.a, Chl.b, Chl.a+b and Chl.a/b ratio with significant values compared to control at the different tested times.The high significant values were recorded when the double dose was treated after 21 days from the application being 0.06, 1.66, 0.39 and 2.05 mg/g fresh weight, and 4.26, respectively.Values are means of three replicates of each parameter ± standard deviation.Means within each column followed by the same letter are not significant at p > 0.05.* R. Dose: recommended dose by the Egyptian Ministry of Agriculture Table 6.Effect of Difenoconazole and Azoxystrobin suspensions on tomato seedling pigments after three weeks from application (mg/g fresh weight).
Values are means of three replicates of each parameter ± standard deviation.Means within each column followed by the same letter are not significant at p > 0.05 * R. Dose: recommended dose by Egyptian Ministry of Agriculture.

Dry weight:
Data presented in Table ( 7) indicated that difenoconazole and azoxystrobin showed no marked differences between their effects on tomato seedling dry weight.Meanwhile, the seedling's dry weight was significantly decreased by all exposed doses compared with the control.The double recommended dose showed the highest effect and recorded the lowest weight value being 0.76 and 0.76 g/plant for difenoconazole and azoxystrobin, respectively.Moreover, there was no significant difference between the other two treated doses.This is in agreement with the reports showing decreases in biomass production in benomyl fungicides-exposed plants which reduced the growth of Gossypium hirsutum, Helianthus decreannuus, Cucumis sativus, Lactuca sativa and Pinus taeda (Garcia et al., 2003).Also, many fungicides showed phytotoxicity to different field crops under various conditions of their application according to Fungicides (difenoconazole and azoxystrobin) applied at one, two and three times the recommended rates, decreased the plant-growth-promoting attributes of P. putida in the strain PS9 and affected the PGP activities in a concentration-dependent manner.Fungicides at the recommended dose had a minor reducing effect while the doses higher than the recommended dose significantly reduced the PGP activities (phosphate solubilization, salicylic acid, 2,3-dihydroxy benzoic acid, and indole-3-acetic acid production except for exo-polysaccharides, hydrogen cyanate and ammonia production) according to Ahemad and Khan (2012).Vuković et al. (2014) showed the effects of the fungicides: azoxystrobin (Quadris, 0.75 l/ha), mancozeb (Dithane M-70, 2.5 kg/ha) and thiamethoxam (Actara 25-WG, 0.07 kg/ha) by measuring the diameter of chlorosis and/or necrosis around puncture sites and were expressed in mm 2 and found significant differences between treatments.
Difenoconazole (0.3845 mg/L) and Azoxystrobin (2.6019 mg/L) were effective when tested using in vitro assays to control wheat Fusarium crown rot and grain yields were increased (Zhang, et al., 2022).

Conclusion
When wheat and radish seeds are to be treated before planting difenoconazole must be used at the recommended dose because it is less toxic than azoxystrobin.After planting no significant differences between the two fungicides within two weeks from the application were evidenced, whereas after three weeks of the fungicides exposed tomato plants treated with azoxystrobin showed a decrease in a/b ratio at the recommended dose to control.
Singh et al. (2003).Table7.Effect of Difenoconazole and Azoxystrobin suspensions on tomato seedling dry weights (g/plant).Values are means of three replicates of each parameter ± standard deviation.Means within each column followed by the same letter are not significant at p > 0.05.* R. Dose: recommended dose by Egyptian Ministry of Agriculture.

Seedling Growth Assay:
as follows:

Table 1 :
Physical and chemical analyses of the tested soil.

Table 2 .
Effect of Difenoconazole and Azoxystrobin suspensions on wheat seeds germination.

Table 3 .
Effect of Difenoconazole and Azoxystrobin suspensions on radish seeds germination.Values are means of three replicates of each parameter ± standard deviation.Means within each column followed by the same letter are not significant at p > 0.05.
* Relative germination rate compared with control **Germination Index = (Relative germination rate × Relative root length)/100 *** R. Dose: recommended dose by Egyptian Ministry of Agriculture.

Table 4 .
Effect of Difenoconazole and Azoxystrobin suspensions on tomato seedling pigments after one week from application.
** Not significantly different at 0.05

Table 5 :
Effect of Difenoconazole and Azoxystrobin suspensions on tomato seedling pigments after two weeks from the application (mg/g fresh weight).