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DISCUSSION
Wheat (Triticum aestivum L. em Thell.) is the first important and strategic cereal crop for the majority of world’s populations (Laghari et al., 2010 and Curtis and Halford, 2014) and Gazette, 2013). Disease infection is one of the most important biotic constraints limiting wheat production (Fried et al., 1979). Wheat powdery mildew caused by Blumeria graminis f. sp. tritici has recently assumed significance affecting for wheat production under Egyptian conditions, and led to yield reduction about 10 to18% (El-Shamy et al. 2012).
In this study, a survey of powdery mildew disease of wheat, grown under natural conditions, was established during season 2013-2014 in 10 diverse locations in Egypt. The results indicated the presence of the powdery mildew disease in all wheat-growing areas in these ten locations of Egypt. The highest disease severity was recorded in Beheira and Giza governorates on Gemiza 10 and Misr 1 cultivars. However, disease incidence and severity depend on many factors, including cultural practices, weather conditions and the degree of cultivar susceptibility (Fried et al., 1979; El-Shamy et al. 2012). El-Shamy et al. (2016) clarified that the Egyptian wheat cultivars may lack resistance genes and can be modified by inserting genes resistant to powdery mildew through a special breeding program. Development of resistant varieties is the most effective, economically and environmentally friendly approach for disease control (Alam et al., 2013). However, identifying powdery mildew genes that already exist in such cultivars may precede the work in the breeding program. Already, this step was carried out by Emara et al. (2016), who identified three resistant Pm genes, Pm24, Pm35, and Pm37,
in 13 local wheat cultivars, using simple sequence repeat markers. They found that none of the three genes were present in the studied wheat cultivars. Therefore, efforts should be continuing to achieve effective and safe control measures of this disease in Egypt.
Powdery mildew has been managed mainly by synthetic fungicides for a decades (Shalaby and El-Mageed, 2010, Shalaby et al., 2018). However, environmental considerations have necessitated increasing restrictions on the use of pesticides, and therefore environmentally friendly production methods for plant disease suppression need to be developed. The results of this study suggested it may be possible to replace conventional chemical fungicides with safe natural compounds. The hazardous consequences of pesticides have been a major bottleneck in achieving sustainable agriculture, food safety and prolonged human health. Therefore, there is an urgent need for biological control agents that would help in getting rid of pathogens without any adverse effects on the environment (Bramhanwade et al., 2016). Biocontrol refers to any method, product or organism using natural mechanisms in the context of integrated crop protection against bio aggressors (Herth, 2011).
In the present study, different antagonistic microorganisms i.e. Trichoderma harzianum (ThFT1), T. viride (TvGK2), T. hamatum (TmSA2), Pseudomonas fluorescens (PfBN1), Pseudomonas putida (PpFt1), Bacillus subtilis (BsBN3), Bacillus brevis (BbSK1), Paenibacillus polymyxa (PbBB2) and Streptomyces griseus (SgBN2) significantly reduced powdery mildew disease of wheat plants. Area under disease progress curve (AUDPC) was found to be correlated with powdery mildew severity during two growing seasons. The highest reduction of disease severity was obtained with Pseudomonas putida (PpFt1), Bacillus subtilis (BsBN3), T. viride (TvGK2), T. hamatum (TmSA2) and T. harzianum (ThFT1). The strategy for biological control of plant diseases involves the use of antagonistic microorganisms before or after the infection takes place (Cook and Baker, 1983). Commercial biological control agents are available for the seed treatments and soil amendments to protect the plants against soil borne pathogens. Currently, isolates of Pseudomonas spp. and Trichoderma spp. are mostly used for biological control strategies. The importance of environment friendly plant protection methods is greatly emphasized in the sustainable agriculture (Rahman et al., 2012 and Ziaei-Nejad et al., 2015). So the development of suitable and environment friendly control measures against powdery mildew causing fungi may minimize the loss and improve the quality of wheat. The ability of a biocontrol agents (BCAs) to induce resistance to disease has also been suggested as a mechanism of controlling plant pathogens (Harman et al., 2004; Haas and Défago, 2005; Lima and De Cicco, 2006; Nigro et al., 2006; De Curtis et al., 2010a). The use of BCAs as elicitors improved tolerance of wheat against disease (Cook and Baker, 1983; Campbell, 1989; Vidhyasekaran et al., 1997). Application of certain bacterial strains has been reported to promote growth and elicit induced resistance to plants against pathogens.
In the present study, for improving effects of bioagents against powdery mildew disease, we used bacterial and fungal isolates, which isolated from wheat rhizosphere and phyllosphere, to synthesize nano particles of silicon and titanium, using standardized techniques. The word nano technology is generally used when referring to materials with the size of 0.1 to 100 nanometers (Morones et al., 2005). Transmission electron microscopy (TEM) were used to visualize size and shapes of biosynthesized silicon and titanium nanoparticles which have predominantly shown spherical shape structures. Large variations in particle size were observed and average diameter ranged 1.52– 51.67nm. TEM with average diameter ranging 1.52-51.67 nm. Our results indicated Streptomyces griseus (SgBN2), produced the small size of both nano silicon and nano titanium forward by SNPs and TNPs synthesized by P. putida (PpFt1), B. subtilis (BsBN3) and T. harzianum (ThFT1). This result parallel with the result by other researchers (Priyadarshinia et al., 2013 and Zawadzkal et al., 2016), where TEM micrograph of silver nanoparticles obtained after 24 h of incubation showed nanoparticles with variable shape, the size of the particle ranged from 5 to 25 nm. Saifuddin et al. (2009) referred that, the size is within the size range reported earlier for AgNPs from bacteria which varied from 5 to 50 nm in B. subtilis. However, Tian et al. (2014) investigated the antibacterial activity of TiO2 coatings modified by iron NPs and indicated that sensitivity of tested pathogens to nanoparticles depends on the crystalline form of carrier, particle size and surface area metals.
The obtained results point to for characterization of NPs and their pathways of the antibacterial and antifungal activity of tested treatments. Our data also are consistent with other many researchers that many microorganisms could produce nanomaterials in nature (Raliya et al., 2013 and Pantidos and Horsfall, 2014). Nano-based antifungal compounds from zinc oxide (Patra et al. 2012), copper (Kanhed et al., 2014 and Bramhanwade et al., 2016), was used in the effective management of plant pathogenic fungi responsible for crop diseases. Johnston et al. (2013) illustrated the production of pure gold nanoparticles by the bacterium Delftia acidovorans. Reporting the production of a small non-ribosomal peptide, delftibactin, to be responsible for generating the gold nanoparticles (Johnston et al., 2013).
In the preset study, nano silicon and nano titanium, biologically synthesized by these antagonistic microorganisms were applied as seed treatment and foliar spray against powdery mildew disease, under greenhouse conditions. Significant reductions in powdery mildew severity were obtained with all NPs treatments. Disease severity was significantly reduced using nano silicon followed by nano titanium, either under greenhouse compared with bioagents alone.
Under green house and field conditions, methyl jasmonate and arginine significantly reduced powdery mildew disease of wheat plants. The highest reduction was obtained with methyl jasmonate at concentration of 20 mM which reduced disease severity by 85.9%. It has Methyl jasmonate plays an important role in defense mechanisms to protect against both biotic and abiotic stresses (Cheong and De Choi, 2003, Sabbagh et al., 2018 and Li, et al., 2018). In fact, jasmonate induced protein include antifungal protein as phenylalanine ammonia lyase, and thionin, hydroxyproline- and proline – rich cell wall proteins. Another line of evidence for the role of jasmonates in disease resistance comes from their stimulatory effect on secondary metabolite production including alkaloids, terpenes, phenolics and polyamines (Dempsey et al., 1999 and Martin et al., 2002)
Under field conditions in two diverse locations, Gelbana (Sinai) and Nobaria (Beheira), all tested concentrations of nano silicon and nano titanium significantly reduced powdery mildew severity and area under disease progress curve (AUDPC) on wheat plants. The highest disease reduction was obtained with SNPs and TNPs synthesized by P. putida (PpFT1) and B. subtilis (BsBN3) which reduced the area under disease progress curve (AUDPC), followed by SNPs and TNPs synthesized by T. harzianum (ThFT1), T. hamatum (TmSA2) and P. fluorescens (PfBN1). Nanotechnology has been reported as an additional technology which could help in meeting the global demands for sustainable agriculture and prevention of crop losses. Several scientists have concentrated their efforts on the development of non-target, biodegradable and ecofriendly nano-formulations showing strong biological activities against plant pathogens (Singh and Singh 2011 and Oluwaseun and Sarin, 2017).
Silicon is regarded as an essential element for many plant species and Si application has been demonstrated to inhibit various plant pathogens, e.g. Blumeria graminis f. sp. tritici infecting wheat (Bélanger et al., 2003; Rémus-Borel et al., 2005) and Podosphaera fuliginea infecting cucumber (Menzies et al., 1991).
There are several hypotheses concerning the role of Si in inhibiting fungal infection. Si has been thought to protect through mechanical strengthening of the plant (Fauteux et al., 2005). In accordance with this, the enhanced resistance of Si-treated host plants to pathogenic fungi has been suggested to result from more efficient resistance to pathogen penetration of host tissue, e.g. due to the specific deposition of Si compounds in cell walls as suggested for rice blast (Kim et al., 2002) and may play a more active role in plant–pathogen interactions by stimulating other plant defense responses such as deposition of phenolic compounds/phytoalexins with antifungal properties and enhanced production of defense-related enzymes (Rémus-Borel et al., 2005). However, regarding the effect of the disease on grain quality, the highest increase in protein content was observed on plant treated with bio-nano silicon showed the highest increase in protein content.
Titanium oxide (TiO2) photo catalyst technique has great potential in various agricultural applications, including plant protection since it does not form toxic and dangerous compounds and possesses great pathogen disinfection efficiency. Scientists have been trying to improve the phytopathogenic disinfection efficiency of TiO2 thin films by dye doping and other suitable methods (Yao et al., 2007). Although Ti is not toxic for animals and humans, its effects on plants show remarkable concentration dependence. Whereas for plants, it shows beneficial effects on various physiological parameters at low doses (Jaberzadeh et al., 2013).
The results reported herein indicate that nano silica or nano titanum – treated plants show a higher expression of phenolic compounds and oxidative enzymes in compared with bioagents and control plants. Nanoparticles help to produce new pesticides and insect repellants (Owolade et al., 2008).
All tested treatments significantly increased the growth parameters i.e. fresh weight of plant (g) and Spike weight (g) of wheat plants. Furthermore, the highest increase in grain yield was obtained by SNPs and TNPs synthesized by P. putida (PpFT1), B. subtilis (BsBN3) and TNPs synthesized by P. polymyxa (PbBB2) which increased the grain yield more than 211.8 %. Various studies were carried out to understand the effect of nanoparticles on the growth of plants. For example, Lu et al., (2002) studied the effect of mixtures of nano SiO2 and nano-TiO2 on soybean seed. They found that the mixture of nanoparticles increases nitrate reductase in soybean, increasing its germination and growth. Hong et al. (2005) and Yang et al. (2006) reported that a proper concentration of nano-TiO2 was found to improve the growth of spinach by promoting photosynthesis and nitrogen metabolism. Canas et al. (2008) found that nano functionalized carbon nanotubes enhanced root elongation in onion and cucumber (Dhoke et al., 2013). Use of plants and microbes in synthesis of (Ag NPs) is quite novel method as it coast effective and environmental friendly and easily scaled up for large scale synthesis (Sahayaraj and Rajesh, 2011 and Duhan, et al. 2017).
Increases in peroxidase, polyphenol oxidase and chitinase activities was recorded in wheat plants treated by SNPs and TNPs, synthesized by P. putida (PpFT1), Bacillus subtilis (BsBN3) and T. harzianum (ThFT1) in addition to methyl jasmonate. As for total protein and phenols, the most effective treatments were SNPs and TNPs synthesized by P. putida (PpFT1), Bacillus subtilis (BsBN3) which increased the total protein and phenol more than 100.0 % as compared with untreated plants. Several authors also revealed significant increases in activities of plant defense related enzymes, in treated plants compared (Haggag, 2005, Gutiérrez et al., 2013 and Al-Ani, 2018). Microbial elicitors derived from some biocontrol agents promote biomass and induce metabolites biosynthesis and production in plant suspension cells (Wang et al., 2006).

The use of fungi in producing nanoparticles has received significant interest as they offer certain advantages (Bhainsa and D’Souza, 2006). Although extracellular formation has its advantages, such as lower cost, simpler downstream processing (Das et al., 2014), intracellular formation can also be of great importance. In the case of bioremediation, heavy metals such as Cu and Pt need to be removed from contaminated environments. Trichoderma asperellum and Trichoderma reesei are both fungi that produce AgNPs when exposed to silver salts (Mukherjee et al., 2008) and have been proven to be nonpathogenic which makes them ideal for use commercially (Nevalainen et al., 1994). In fact, T. reesei has already been used widely in sectors such as food, animal feed, pharmaceuticals, paper and textile industries (Nevalainen et al., 1994).

The development of eco-friendly technologies in material synthesis is of considerable importance to expand their biological applications. An alternative way of synthesizing nanoparticles is by using living organisms such as bacteria and fungi. To survive in environments containing high levels of metals, organisms have adapted by evolving mechanisms to cope with them. These mechanisms may involve altering the chemical nature of the toxic metal so that it no longer causes toxicity, resulting in the formation of nanoparticles of the metal concerned. This “green” method of biological nanoparticle production is a promising approach that allows synthesis in aqueous conditions, with low energy requirements and low-costs. There are important links between the way nanoparticles are synthesized and their potential uses. The most important feature of nanoparticles is their surface area to volume aspect ratio, allowing them to interact with other particles easier (Narayanan and Sakthivel, 2010 and Thakkar et al., 2010)
Generally, the results of this study demonstrate that biologically synthesized nanoparticles of silicon and titanium, could be used as an alternative potent agent against powdery mildew and other diseases of wheat and to increase plant growth and yield.

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