Magnesium Oxide nanoparticle was prepared from company nanoshild, USA.

Size: 30 nm.

Purity: 99.9%.

Different concentrations of magnesium oxide nanoparticles from PDA media were used for analyzing the antifungal properties. To do so 1%, 2%, and 3% concentrations of magnesium oxide nanoparticles prepared from deionized water. Then mixing each of them individually to the homogenizer for half an hour to ensure mixing well then the expose each one of them to ultrasonic frequency of 22 - 24 Hz for two minutes to preserve the nanoscale particle size distribution and to ensure homogeneous distribution in the mixture, add all of the concentration separately to the flask 100 ml container of 2.4 g (PDA) and 30 ml of distilled water were added to autoclaved growth medium after its temperature reached approximately 40˚C - 50˚C, agar plugs of uniform size (diameter, 8 mm) containing fungi were inoculated simultaneously at the center of each Petri dish containing Magnesium oxides nanoparticles, followed by incubation at 28˚C ± 2˚C for 14 days. Growth inhibition percent was calculated using the radial growth of mycelium according. The acquired results were compared with those of control group to which no nanoparticles have been added. All tests were conducted in three replications.

Stock Nan silver solution at concentration 4000 ppm was obtained from company MTI, USA. Different working concentration of silver nanoparticles (50 ppm, 100 ppm, 150 ppm) was prepared by diluting the original stock solution with distilled water [7] .

In vitro assay was performed on of growth medium (PDA) treated with different concentrations (50, 100, 150) of silver nanoparticles, The mixing each of them individually to the homogenizer for half an hour to ensure the mixing well then the exposure of the each them to ultrasonic frequency of 22 - 24 Hz for two minutes to preserve the nanoscale particle size distribution and to ensure homogeneous distribution in the mixture. Five ml of AgNPs having different concentrations were poured into growth media prior to plating in a Petri dish (90 × 15 mm). Media containing silver nanoparticles were incubated at room temperature. After 48 hr of incubation, agar plugs of uniform size (diameter, 8 mm) containing fungi were inoculated simultaneously at the center of each Petri dish containing silver nanoparticles, followed by incubation at 28˚C ± 2˚C for 14 days. Growth inhibition percent was calculated using the radial growth of mycelium according to the following equation.

Growth inhibition (%) =, where R is the radial growth of the fungal mycelia

on the control plate and r is the radial growth of fungal mycelia on the plate treated with nanoparticles and chemical.

Dutch selko company experts to affect phylex in preventing fungal growth and mycotoxin. It’s a mixture of organic acids and their salts if it consists of ascorbic acid, formic acid, lactic acid, citric ammonia, materials publisher and water. It is non-toxic extract.

Fourth samples from infected plants were taken from root and stem rot naturally diseased cucurbit plant during 2015. These samples were obtained from four provinces (Babylon. Baghdad. Najaf, Diwaniyah). Samples were cut into 0.5 - 1 cm and washed under running tap water for 30 minute, then surface sterilized in 1% sodium hypochlorite for 2 minute and cultured on Potato dextrose agar (PDA) supplemented with 200 mg\l Tetracycline and incubated at 25˚C ± 1˚C for 7 days, single spore technique was made for each isolate. Isolates were identified to the species level according to their cultural and morphological characteristic [8] .

Field experiment was conducted at the Diwaniyah a province of Iraq in summer season 2015. Soil used for the experiment was sandy loam containing sand 73%, clay 12% silt 13%, pH 7. Electrical conductivity (E C) 0.63 (ds∙m⁻¹) Seeds of melon were sown in plots (3 × 3.5 m) which had 2 rows (1.5 m). Each row contained 2 hills, spaced 50 cm apart. Every hill was sown with 4 sterilized seeds and three replicates were used for each treatment.

The effects of Silver nanoparticles (AgNPs), magnesium oxide nanoparticle and phylex were evaluated on melon growth parameters. Pathogen was prepared by growing them on millet grains in 100 ml flask as follow, the grain were soaked overnight in distilled water, autoclaved twice (121 c for 45 min. and then inoculated with agar discs of 5 days-old fungal culture. flask were incubated at 28 c in dark for 21 days, the fungal inoculate were then mixed with soil.

Surface sterilization was accomplished by immersing the seeds in 5% sodium hypochlorite solution for 5 min and followed by washing by sterile distilled water, all treatment (silver nanoparticles, magnesium oxide nanoparticles, phylex) was accomplished by soaking in with different concentrations for 2 h. There were thirteen groups of treatments with three replications for each treatment. Treatments included: 1―con- troll; 2―silver without; 3―silver with pathogen; 4―magnesium oxide nanoparticles without; 5―magnesium oxide nanoparticles with pathogen; 6―Phylex without; 7― Phylex with pathogen; 8―nanosilver + magnesium oxide with pathogen; 9―nanosilver + magnesium oxide without pathogen; 10―nanosilver + Phylex with; 11―nanosilver + Phylex without; 12―nanosilver + magnesium oxide + Phylex with pathogen; 13― nanosilver + magnesium oxide + Phylex without. The experimental design was complete randomized block with three replicate.

The height of the plant was measured from the base of the hypocotyls to the seedling’s apex. Ruler is used to measure the plant height of each seedling with unit of centimeter (cm).

Scale is used to measure fresh and dry weight of the plant: shoot (SFW) and root (RFW) fresh weight plus shoot (SDW) and root (RDW) dry weight. The samples needed to put in oven at 70˚C ± 1˚C for 72 hours in order to take the dry weight of seedlings.

The growth measures (plant height, fresh and dry weight of plants and roots) were assessed by using the same methods described in the previous section. The plant and root parameters were recorded separately after 45 days of the experiment, where the plants were uprooted and washed under running tap water.

Symptom severity of the areal parts of the plants was assessed (8, 10, 12, 14 days after pathogen inoculation) using the following index 0, no symptoms; 1, yellowing of the cotyledons or the first leaf; 2, yellowing of two leaves; 3, yellowing of three or more leaves; 4, died plant. The disease severity index was calculated using the formula [9] .

Disease severity index (%) = Σ = 4N where ni is the number of plants af-

fected by each degree of severity, si the degree of severity of the attack (0 - 4) and N the total number of plants used for each energy level applied.

Grind the tissue in liquid nitrogen with a mortar and pestle [10] . Transfer the tissue into micro centrifuge tube (FAVORGEN. company protocol). Add 1 ml of FA buffer and resuspend the cell by pipette. Descend the cells by centrifuging at 5.000 × g for min and discard the supernatant completely. Resuspend the cell in 550 μl of FB buffer and add 50 μl of of lyticase solution, mixed well by vortexing incubate the sample at 37 c for 30 min. Add 8 μl of 50 mg /ml RNase A and incubate for 2 min at room temperature. Descend the cell by centrifuging of 5000 g for 10 min. remove the supernatant completely. Add 35o μl TG1 Buffer and mix well by pipetting. Transfer the sample mixture to a bead tube Mix well by plus-vortexing for 5 minute. Add 20 μl of proteinase k (10 mg/ml) and mix well by vortexing. Incubate of 55 c for 15 min, vortex 30 seconds for every 5 minutes incubation.

Descend the cell by centrifuging of 5000 × g for 1 min and transfer 20 μl of supernatant to a new 1.5 ml micro centrifuge tube. Add 200 μl of TG2 Buffer and mix well by pipetting. Add 200 μl of ethanol (96% - 100%) and mix well by pulse-vortexing for 10 seconds. Place a TG mini column in collection Tube Transfer the sample mixture (including only precipitate carefully to TG mini column. Centrifuge at 11000 × g for 30 second and discard the flow through place the TG mini column back to the collection Tube. Add 750 of μl wash Buffer to the TG min column centrifuge of 11000 × g for 30 seconds and discard the flow through place the TG column back to the collection tube Centrifuge at full speed (18000 × g) for an additional 3 min to dry the column. Place the TG mini column to a Elution tube Add 50 - 100 μl of Elution buffer add H₂O to the mem- brane center of the TG mini column. Stand TG mini column for 3 min. Centrifuge of full speed (−18000 × g for 1 min to elute total DNA. Store total DNA at 4˚C or −20˚C.

Primer which has been designed based on 1-α(TEF) gene [11] [12] (Table 1).

RAPD analysis : Amplification reaction were performed in 5 μl master mix (pramago company) 1 μl primer forward and 1 μl primer reverse, 2 μl Genomic DNA and 1 μl distilled water in a final volume of 10 μl (Table 2).

Reaction conditions were 94 c for 10 min followed by 35 cycles of 94 c for 1 min, 56 c for 1 min and 72 c for 1 min. The final extension was at 72 c for 10 min. PCR Products were loaded on a 1.5% agarose gel with 0.1% ethidium bromide in Tri-acetate EDTA buffer, separated for 30 min at 100 v, and visualized under ultraviolet light. Cotoxigeni- city was determined with the polymerase chain reaction (PCR) using master mix 5 μl forward primer 1 μl and 1 μl Reverse DNA 2 μl and 1distilled water to final volume 10 μl.

The reaction was performed which involved 30 cycles PCR 94 c 2 min and 94 c 1 min 72 c 2 min and 72 5 min (Table 3, Table 4).

The molecular size of each fragment was estimated using a standard curve of migration versus the log of the molecular size of 100 bp ladder. Each fragment was scored on

the basis of the presence (1) or absence (0) of particular fragments. A date matrix was constructed based on the presence or absence of the fragments and converted to a similarity matrix.

The results showed that Fusarium solani was predominant in all simple with 100% frequency. Fusarium solani forms white to cream color mycelium on the PDA. Microscopic examination was showed three types of spores, macroconidia were spindle to cylindrical shape containing between three to five septa with distinctly or barely notched basal cells, microconidia formed on false head on long monophialides, growth laterilly on the aerial mycelium. Chlamydospores terminal and intercalars single or in chains Figure 1(a) and Figure 1(b).

All isolates of the Fusarium solani were tested for their pathogenicity on apparently healthy and uniform 7 days old seedling melon in the greenhouse. Roots and stems of the melon seedling were washed in running tap water before inoculation. Conidial suspension of each individual isolate was prepared by pouring sterile distilled water and gently scraping the conidia of 7 days old cultures on PDA plates grown under the standard grown condition. The concentration of the pooled suspension was adjusted to 2 × 10⁶ conidia/ml by using ahaemocytometer.

The roots of seedling were soaking in 20 ml conidial suspension for 20 min for root inoculation technique for stem inoculation technique, 20 ml of the conidial suspension of each Fusarium solani was sprayed on the stems. The control plants were inoculated by booth techniques with ml of sterile distilled water. Three replicates were performed for each isolate and the experiment was repeated twice. Results of the pathogenicity test showed that all isolates of Fusarium solani were highly pathogenic to melon plants. The first wilting occurred 7 days after inoculation in melon plants. 100 percentage of melon plants inoculated with the forty isolates died, inoculated plants exhibited a cortical rot at the base of the stem and upper part of the root system caused discoloration and necrosis then brown rot of the stems. While all uninoculated plants remained asymptomatic, the pathogen was recovered from symptomatic plants, fulfilling Koch s postulates.

Results presented in this study confirm that nanoparticles have significant inhibitory effects and antifungal activity on colony formation from mycelia of Fusarium solani in vitro. The growth rate of strain in the presence of the tested nanoparticles and other compound chemical are summarized in Table 5.

There were significant differences between different concentrations of nanoparticles on growth inhibition of Fusarium solani, while there was no growth inhibition in the negative control. The higher inhibition of fungal growth was recorded at a concentration of 150% ppm from silver nanoparticle was observed on PDA medium. Concentration of 100%, 50% ppm inhibition of the fungal growth is by 98% and 88.9% respectively.

The different concentration of MgO nanoparticles caused inhibition the fungal growth, higher inhibition at 3%, 2% by 100% meanwhile at concentration of 1% by 92.1% (Figure 2).

The higher inhibition of fungal growth was recorded at a concentration of 0.3 from Phylex by 100% and, 91.66%, 81.1% with a concentration 0.2, 0.1 (Figure 3).

The results in the current study are confirmed by the finding of [3] , which reported that the synthesized nanoparticles can significantly inhibit (87.1%, 86.5% and 83.5%) to growth of phytopathogens Colletotricum coccodes (Figure 4).

[13] reported that the controlling effect increases in the administered dosage of nanoparticle and there exists a direct correlation between the administered dosage and controlling effect, the concentration of 2% MgoNps had the greatest effect in both liquid and solid media, [14] reported that the when roots were drenched with MgoNps suspension prior to inoculation with pathogen, the incidence of disease was significantly reduced. Also, these results agree with selko Dutch company research regarding

item phylex inhibitory immune substance for the growth of many fungi. The results are close conformity with finding of [5] . It reported that the inhibitory effected of nanoparticle may be due to the directly attach to and penetrate the cell membrane of the spore and killed them. Also [7] reported that antimicrobial activity of AgNps on microorganism was depended on the concentration of AgNps and was closely associated with the formation of pits in the cell wall of the microorganism than Ag accumulated in the membrane caused the permeability, resulting in the cell death. Inhibition increased at the concentration of AgNps increased. This could be due to high density at which the solution was able to saturate and cohere to fungal hypha and deactivate plant pathogenic fungi.