Abstract

INTRODUCTION

The term 'endophytes' (Greek: endon-within; phyton- plant) was first coined by de Bary (1886). Endophytes relationship with plant varies from symbiotic to pathogenic. Endophytes benefit host plant by preventing pathogenic organism from colonizing. It has been categorized into two main groups (Clavicipitaceous and non-clavicipitaceous) based on differences in evolution, taxonomy, plants host and ecological functions. Clavicipitaceous are able to infect only some species of grasses and non-clavicipitaceous are found in the asymptomatic tissues of other higher plants (Rodriguez et al. 2008Posada F, Vega FE. Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). Mycoscience. 2006; 47: 284-289.). It stimulates plants growth, increase disease resistance, improve the plants ability to withstand environmental stresses and recycle nutrients (Sturz et al. 2000Strobel G, Yang X, Sears J, Kramer R, Sidhu RS, Hess WM. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiol. 1996; 142: 435-440.). Endophytes are rich source of secondary metabolites with multifold importance (Strobel and Daisy 2003Soleimani M, Hajabbasi MA, Afyuni M, Mirlohi A, Borggaard OK, Holm PE. Effect of endophytic fungi on cadmium tolerance and bioaccumulation by Festuca arundinacea and Festuca pratensis. Int J Phytoremediation. 2010; 12: 6, 535 - 549. ). Compare to other endophytic microorganisms, fungal endophytes produce large number of secondary activities (Zhang et al. 2006Webber J. A natural control of Dutch elm disease. Nature. 1981; 292: 449-451.). Endophytes isolated from medicinal plants possess strong fungicidal, bactericidal and cytotoxic metabolites (Wang et al. 2007Vega FE, Posada F, Aime MC, Pava-Ripoll M, Infante F, Rehner SA. Entomopathogenic fungal endophytes. Biological Control. 2008; 46: 72-82.). It produce enzymes which are used for various application like degradation and biotransformation of organic compound (Firáková et al. 2007; Pimentel et al. 2011Patil MP, Patil RH, Patil SG, Maheshwari VL. Endophytic Mycoflora of Indian medicinal plant, Terminalia arjuna and their biological activities. Int J Biotechnol Wellness Ind. 2014; 3: 53-61. ). The endophytes derivatives are used in biotechnological applications (Tomita 2003Tayung K, Barik BP, Jha DK, Deka DC. Identification and characterization of antimicrobial metabolite from an endophytic fungus, Fusarium solani isolated from bark of Himalayan yew. Mycosphere. 2011; 2: 203-213.). It has a wide significance in pharmaceutical science because of its antimicrobial, anticancer and antiviral activities. (Selim et al. 2012Schardl CL. Epichloe festucae and related mutualistic symbionts of grasses. Fungal Genet Biol. 2001; 33: 69-82.).

Anticancer activity

Taxol is a highly functionalized diterpenoid which is widely used as anticancer drug (Wani et al. 1971Wang FW, Jiao RH, Cheng AB, Tan SH, Song YC. Antimicrobial potentials of endophytic fungi residing in Quercus variabilis and brefeldin A obtained from Cladosporium sp. World J Microbiol Biotechnol. 2007; 23: 79- 83. ). It was first isolated from the bark of western yew, Taxus brevifolia. During the cell division, taxol prevent the depolymerization of tubulin. Production of taxol from different genus of endophytes by fermentation is a cheaper method (Page et al. 1999Oses R, Valenzuela S, Freer J, Sanfuentes E, Rodriguez J. Fungal endophytes in xylem of healthy chilean trees and their possible role in early wood decay. Fungal Diversity. 2008; 33: 77-86.). The anticancer drug, taxol has been found in many genera of endophytic fungi (Alternaria, Fusarium, Monochaetia, Pestalotia, Pestalotiopsis, Pithomyces and Taxomyces) (Strobel et al. 1996). More than 15 fungi genera were produced paclitaxel and its analogues (Zhao et al.2010Zhang HW, Song YC, Tan RX. Biology and chemistry of endophytes. Nat Prod Rep. 2006; 23: 753-771.).

Pandi et al. (2011)Page M, Landry N, Boissinot M, Halie MC, Harvey H, Gagne M. Bacterial mass production of taxanes and paclitaxel. US Patent No. WO 99/ 32651. 1999. reported that taxol isolated from Lasiodiplodia theobromae showed activity against breast cancer cell line. Alternariol 9-methyl ether is a major mycotoxin produced by fungi of the genus Alternaria. It can induce mitochondrial apoptosis in human colon carcinoma cells, and induce DNA strand breaks, micronuclei and gene mutations in various cultured mammalian cells (Meng et al. 2012Malinowski DP, Brauer DK, Belesky DP. Neotyphodium coenophialum endophyte affects root morphology of tall fescue grown under phosphorus deficiency. J Agronomy Crop Science. 1999; 183: 53-60.). Giridharan et al. (2012)Giridharan P, Verekar SA, Khanna A, Mishra PD, Deshmukh SK. Anticancer activity of sclerotiorin, isolated from an endophytic fungus Cephalotheca faveolata Yaguchi, Nishim. & Udagawa. Ind J Exp Biol. 2012; 50: 464 - 468. reported that sclerotiorin isolated from Cephalotheca faveolata showed anti-proliferative activity against cancer cells and also it induces apoptosis in colon cancer cells. Extracts from endophytes, Fusarium sp., and Aspergillus fumigatus exhibited inhibitory activity against HeLa cervix cancer cell lines (Ruma et al. 2013Rodriguez RJ, White Jr JF, Arnold A E, Redman RS. Fungal endophytes: diversity and functional roles. New Phytolog. 2009; 182: 314-330.). The endophytic fungi, A. terreus showed cytotoxic effect against HepG2 cancer cell line (Suja et al. 2014Sturz AV, Nowak J. Endophytic communities of rhizobacteria and the strategies required to create yield enhancing associations with crops. Appl Soil Ecol. 2000; 15: 183-190.). Polyketide compounds produced by Phoma sp., had high inhibitory activity against murine leukemia cells (Santiago et al. 2014Sandhiya GS, Sugitha TC, Balachandar D, Kumar K. Endophytic colonization and in planta nitrogen fixation by a diazotrophic Serratia sp. in rice. Indian J Exp Biol. 2005; 43: 802-807.). The anticancer activity was noticed in ethyl acetate extract of Alternaria alternata against human breast cancer cell lines and it also showed good cytotoxicity (Arivudainambi et al. 2014Arivudainambi USE, Kanugula AK, Kotamraju S, Karunakaran C, Rajendran A. Antibacterial effect of an extract of the endophytic fungus Alternaria alternata and its cytotoxic activity on MCF-7 and MDA MB-231 tumour cell lines. Biological Lett. 2014; 51: 7-17. ). Chen et al (2015)Chen ZM, Chen HP, Li Y, Feng T, Liu JK. Cytochalasins from cultures of endophytic fungus Phoma multirostrata EA-12. The J Antibiot. 2015; 68: 23-26. reported that cytochalasins from endophytic fungi, Phoma mutirostrata exhibited moderate anticancer activity against SMMC-7721 (hepatocellular carcinoma cell line), SK-BR-3 (breast cancer cell line), PANC-1 (pancreatic cancer cell line), HL-60 (human myeloid leukemia cell line) and A-549 (lung cancer cell line).

Antimicrobial activity

World health problems caused by drug resistant bacteria and fungi are increasing. Many pathogenic microorganisms have developed resistance due to the misusage or long-term usage of the same class of antibiotics. Intensive search for newer and more effective antibiotics to deal with these problems is now underway. The isolation of novel secondary metabolites from the endophytes is a progressive research field (Huang et al. 2008). Fungal endophytes such as Phaeosphaeria avenaria, Leptosphaeria sp., Fusarium sp., P. chrysanthemicola, Cladosporium sp., Cylindrocarpon sp., Saussurea involucrata, Fusarium solani, Cordyceps memorabilis, P. longicolla and Dothideomycete sp., are found to have antimicrobial activity against human pathogenic bacteria and fungi such as Micrococcus luteus, Enterococus shirae, Escherichia coli, Klebsiella pneumonia, Bacillus subtilis, Staphylococcus aureus, Shigella flexneri, S. epidermidis, Candida tropicalis, C. albicans, Cryptococcus neoformans, A. fumigatus and Salmonella typhi (Lv et al. 2010Lu H, Xou WX, Meng JC, Hu J, Tan RX. New bioactive metabolites produced by Colletotrichum sp., an endophytic fungus in Artemisia annua. Plant Sci. 2000;151: 67-73.;Tayung et al. 2011Suja M, Vasuki S, Sajitha N. Anticancer activity of compounds isolated from marine endophytic fungus Aspergillus terreus. World J Pharm Pharm Sci. 2014; 3: 661-672. ; Rhoden et al. 2012). The Phomopsis sp., exhibited antimicrobial activity against E. coli, K. pneumoniae, B. subtilis, M. luteus and C. albicans (Jayanthi et al. 2011Huang WY, Cai YZ, Hyde KD, Croke H, Sun M. Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Diversity. 2008; 33: 61-75.). Silva et al. (2011) reported that secondary metabolites of A. niger, Curvularia pallescens, Guignardia bidwelii, Paecilomyces variotii and Mycelia sterilia showed antibacterial activity against S. aureus, B. subtilis, Enterococcus faecalis, M. luteus, E. coli and Pseudomonas aeruginosa. Gibberella sp., exhibited the antimicrobial activity against S. aureus and C. neoformans (Liang et al. 2012Li X, Guo Z, Deng Z, Yang J, Zou K. A New α-Pyrone Derivative from Endophytic Fungus Pestalotiopsis microspora. Rec Nat Prod. 2015; 9: 503-508. ). The extracts from Colletotrichum gloeosporioides and Chaetomium globosum exhibited antimicrobial activity against Mycobacterium tuberculosis, Gordonia terrae, S. aureus and E. coli (Bungihan et al. 2013Bungihan ME , Tan MA, Takayama H, dela Cruz TEE, Nonato MG. A new macrolide isolated from the endophytic fungus Colletotrichum sp., Philippine Sci Lett. 2013; 6: http://www.philsciletters.org/pdf/2013n1.8p7.pdf
http://www.philsciletters.org/pdf/2013n1... ). The extracts isolated from C. gloeosporioides showed antimicrobial activity against Streptococcus pyogenes and Enteroccocus faecalis (Nath et al. 2014Meng X, Mao Z, Lou J, Xu L, Zhong L, Peng Y, Zhou L, Wang M. Benzopyranones from the endophytic fungus Hyalodendriella sp. Ponipodef 12 and their bioactivities. Molecules. 2012; 17: 11303-11314.). The extract from Diaporthe arengae showed antimicrobial activity against S. aureus and B. subtilis (Patil et al. 2014Pandi M, Kumaran RS, Choi YK, Kim HJ, Muthumary J. Isolation and detection of taxol, an anticancer drug produced from Lasiodiplodia theobromae, an endophytic fungus of the medicinal plant Morinda citrifolia. African J Biotechnol. 2011; 10:1428-1435. ). The earlier research clearly shows that Endophytic fungi derived compounds are being used in the development of antimicrobial drug.

Insecticidal activity

Endophyti fungi can protect their host plants from pathogens and pests (Arnold et al. 2003Arnold AE, Mejia LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, Herre EA. Fungal endophytes limit pathogen damage in a tropical tree. Proc Natl Acad Sci. 2013; 100: 15649-15654.). The foliar endophytes can reduce herbivory by producing alkaloids which are toxic to insects and vertebrates (Schardl 2001Santiago C, Sun L, Munro MHG, Santhanam J. Polyketide and benzopyran compounds of an endophytic fungus isolated from Cinnamomum mollissimum: biological activity and structure. Asian Pac J Trop Biomed, 2014; 4: 627-632. ). Webber (1981)Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc. 1971; 93: 2325-2327. who was the first researcher to endophytic fungi, P. oblonga to protect elm trees against the beetle Physocnemum brevilineum. The endophtic fungi (Acremonium coenophialum) exhibited insecticidal activity against aphids (Rhopalosiphum padi, Schizaphis graminum) and milkweed bug (Oncopeltus fasciatus) (Johnson et al.1985Jayanthi G, Kamalraj S, Karthikeyan K, Muthumary J. Antimicrobial and antioxidant activity of the endophytic fungus Phomopsis sp. GJJM07 isolated from Mesua ferrea. Int J Curr Sci. 2011; 1: 85-90. ). Cladosporium herbarum, A. alternata, Rhodotorula rubra, Epicoccum nigrum, Cryptococcus sp., Penicillium sp., Fusarium graminearum were act as protectants to plants from herbivores (Larran et al. 2002Kumaresan V, Suryanarayanan TS. Endophytic assemblages in young, mature and senescent leaves of Rhizophora apiculata: evidence for the role of endophytes in mangrove litter degradation. Fungal Diversity. 2002; 9: 81-91. ; dos santos et al. 2003dos Santos RMG, Rodrigues-Fo E, Rocha WC, Teixeira MFS. Endophytic fungi from Melia azedarach. World J Microbiol Biotechnol. 2003; 19: 767-770.). The different genera of entomopathogenic fungus of Acremonium, Beauveria, Cladosporium, Clonostachys and Paecilomyces; were isolated from the coffee plants, among them B. bassiana and Clonostachys rosea were pathogenic to coffee berry borer (Vega et al. 2008Tomita F. Endophytes in Southeast Asia and Japan: their taxonomic diversity and potential applications. Fungal Diversity. 2003; 14: 187-204.). B. bassiana was found to control the borer insects in coffee seedlings (Posada and Vega 2006Pimentel MR, Molina G, Dionísio AP, Junior MRM, Pastore GM. The Use of Endophytes to obtain bioactive compounds and their application in biotransformation process. Biotechnol Res Int. 2011: 1-11. http://dx.doi.org/10.4061/2011/576286
http://dx.doi.org/10.4061/2011/576286... ). Baskar et al. (2012)Baskar K, Raj GA, Mohan PM, Lingathurai S, Ambrose T, Muthu C. Larvicidal and growth inhibitory activities of entomopathogenic fungus, Beauveria bassiana against Asian army worm, Spodoptera litura Fab. (Lepidoptera: Noctuidae). J Entomol. 2012; 9: 155-162. stated that B. bassiana isolated from Puleny exhibited larvicidal and growth inhibitory activities against Spodoptera litura. The endophytic fungi Claviceps purpurea possess a significant insecticidal activity against A. gossypii Glover (Hemiptera:Aphididae) (Shi et al. 2013Senthilkumar N, Murugesan S, Babu DS, Rajeshkannan C. GC-MS analysis of the extract of endophytic fungus, Phomopsis sp. isolated from tropical tree species of India, Tectona grandis L.,. Int J Innov Res Sci Eng Tech. 2014; 3:10176-10179. ). Senthilkumar et al (2014)Selim KA, El-Beih AA, AbdEl-Rahman TM, El-Diwany AI. Biology of eEndophytic fFungi. Curr Res Environm Appl Mycol. 2012; Doi 10.5943/cream/2/1/3. 31-82.
https://doi.org/10.5943/cream/2/1/3... reported that phytochemicals such as Dodecanoic acid, ethylester, Phthalic acid, octyl 2- pentyl ester isolated from Phomopsis sp. are used as insecticide. The Cladosporium oxysporum showed insecticidal activity against A. fabae (Bensaci et al. 2015Bensaci OA, Daoud H, Lombarkia N, Rouabah K. Formulation of the endophytic fungus Cladosporium oxysporum Berk. & M.A. Curtis, isolated from Euphorbia bupleuroides subsp. luteola, as a new biocontrol tool against the black bean aphid (Aphis fabae Scop.). J Plant Protect Res. 2015; 55: 80-87. ). The extracts from Emericella nidulans, A. oryzae, A. tamarii and A. versicolor were applied topically on S. litura larvae and found that all the fungus showed insecticidal activity; notably A. versicolor showed maximum insecticidal activity (Abraham et al. 2015Abraham S, Basukriadi A, Pawiroharsono S, Sjamsuridzal W. Insecticidal activity of ethyl acetate extracts from culture filtrates of mangrove fungal endophytes. Mycobiol. 2015; 43: 137-149. ).

Plant growth stimulants

Endophytes can actively or passively promote the plant growth through a variety of mechanisms, as endophytic metabolites provide a variety of fitness to host plants enhanced by increasing plant resistance to biotic and abiotic stresses, as well as enhance plant growth. Many endophytes are capable of solubilization of phosphate, enhance uptake of phosphorus (P), nitrogen fixation, production of siderophores, and plant hormones such as auxin, abscisins, ethylene, gibberellins, and indole acetic acid (IAA), which are important for plant growth regulations (Goodman et al. 1986Goodman RN, Kiraly Z, Wood RKS. The Biochemistry and Physiology of Plant Disease. University of Missouri Press, Columbia USA. 1986;; Barraquio et al. 1997Barraquio WL, Revilla L, Ladha JK. Isolation of endophytic diazotrophic bacteria from wetland rice. Plant Soil. 1997; 194: 15-24.; Malinowski et al. 1999Ma Y, Prasad MNV, Rajkumar M, Freitas H. Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils. Biotechnol Adv. 2011; 29: 248-258.; Zou et al. 1999; Malinowski and Belesky 2000; Boddey et al. 2003Boddey RM, Urquiaga S, Alves BJR, Reis V. Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant Soil. 2003; 252: 139-149.; Loiret et al. 2004Liang H, Xing Y, Chen J, Zhang D, Guo S, Wang C. Antimicrobial activities of endophytic fungi isolated from Ophiopogon japonicus (Liliaceae). BMC Com Alt Med. 2012; 12: 238. http://www.biomedcentral.com/1472-6882/12/238
http://www.biomedcentral.com/1472-6882/1... , Sandhiya et al. 2005Ruma K, Sunil K, Prakash HS. Antioxidant, anti-inflammatory, antimicrobial and cytotoxic properties of fungal endophytes from Garcinia species. Int J Pharm Pharm Sci. 2013; 5: 889-897. , Firáková et al. 2007). Gibberellic acid (GA) is a potent phytohormone, that regulates plant growth. Fungal endophyte, Cladosporium sphaerospermum from the plant, Glycine max (L) Merr. produce GA3, GA4 and GA7. It induced plant growth in rice and soybean (Hamayun et al. 2009Hamayun M, Khan SA, Ahmad N,Tang D, Kang S, Na C, Sohn E, Hwang Y, Shin D, Lee B, Kim J, Lee I. Cladosporium sphaerospermum as a new plant growth- promoting endophyte from the roots of Glycine max (L.) Merr. World J Microbiol Biotechnol. 2009; 25: 627-632.). A pestalotin analogue isolated from the Pestalotiopsis microspora exhibited significant gibberellin activity against Distylium chinense seeds and increase germination rate (85.56%) (Li et al. 2015Larran S, Perelló A, Simo MR, Moreno V. Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves. World J Microbiol Biotechnol. 2002; 18: 683-686.). The Fusarium tricinctum and A. alternata derivatives of indole acetic acid enhanced the plant growth (Khan et al. 2015Johnson MC, Dahlman DL, Siegel MR, Bush LP, Latch GCM, Potter DA, Varney DR. Insect feeding deterrents in endophyte-infected tall fescue. Appl Environ Microbiol. 1985; 49: 568-571.).

Crop protection

Endophytic fungi are also capable of inducing resistance to diseases, and many mechanisms have been proposed for this resistance. The mechanisms of endophyte induced resistance are related to the nutritional status of the host, and to increase the fitness of plants by enhancing their tolerance to abiotic stress (Aguilar and Barea 1996Aguilar AC, Barea JM. Arbuscular mycorrhizas and biological control of soil-borne plant pathogens - an overview of the mechanisms involved. Mycorrhiza. 1996; 6: 457-464.). Endophytic fungi Cryptosporiopsis cf. quercina and Colletotrichum sp., are found effective against phytopathogens such as Rhizoctonia cerealis, Phytophthora capsici, Pyricularia oryzae, Gaeumannomyces graminis (Li et al. 2000; Lu et al. 2000Loiret FG, Ortega D, Kleiner P, Ortega R, Rodes R, Dong Z. A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane. J Appl Microbiol. 2004; 97: 504-511.).

Phytoremediation

It found to play an important role in the ecological community, with the aim of decreasing the spoilage of land and water caused by excessive toxic organic insecticide, environmental degradation, industrial sewage, poisonous gases and loss of biodiversity. Biological control using endophytes as a new efficient method is being widely used for environmental remediation, and killing insects or pathogens (Guo et al. 2008Guo B, Wang Y, Sun X, Tang K. Bioactive Natural Products from Endophytes: A review. Appl Biochem Microbiol. 2008; 44: 136-142.).

A novel application of endophytes in the area of phytoremediation (plant assisted removal of xenobiotics and heavy metals from the soil) (Ma et al. 2011Lv Y, Zhang Y, Chen Y, Cui J, Xing Y, Li X, Guo S. Diversity and Antimicrobial Activity of Endophytic Fungi Associated with the Alpine Plant Saussurea involucrate. Biol Pharm Bull. 2010; 33: 1300-1306.). However, the success of phytoremediation depends upon microbes and plant ability to tolerate and accumulate high concentrations of pollutant, while yielding a large biomass. Due to the potential of microorganisms for bioaccumulation of heavy metals and other pollutants from environment or its enhancing plant growth and pollutant uptake from soil by plant through mobilization/immobilization of pollutant (Ma et al. 2011). Plants, Festuca arundinacea schreb. and F. pratensis Huds. are infected with endophytic fungi Neotyphodium coenophialum and N. uncinatum and uninfected plants are grown on petroleum contaminated soil. The endophyte infected plants had more shoot and root biomass when compare to the uninfected plants (Soleimani et al. 2010Shi YW, Zhang X, Lou K. Isolation, characterization, and insecticidal activity of an endophyte of drunken horse grass, Achnatherum inebrians. J Insect Sci. 2013; 13:151. doi: 10.1673/031.013.15101.
https://doi.org/10.1673/031.013.15101... a). It proves that the endophytes play a major role in phytoremediation.

Endophytes may play indirect or direct role in phytoremediation process and degradation of environmental toxins, indirectly through enhancing plant growth having ability of phytoremediation and this accelerate phytoremediation process, or directly through degradation and/or accumulating pollutants by itself. Endophytes have a cryptic existence and one of their main role in the ecosystem are decomposers, as they are among the primary colonizers of dead plant tissues (Kumaresan and Suryanarayanan 2002Khan AR, Ullah I, Waqas M, Shahzad R, Hong SJ, Park GS, Jung BK, Lee IJ, Shin JH. Plant growth-promoting potential of endophytic fungi isolated from Solanum nigrum leaves. World J Microbiol Biotechnol. 2015; 31: 1461-1466. ; Oses et al. 2008Nath A, Pathak J, Joshi SR. Bioactivity assessment of endophytic fungi associated with Centella asiatica and Murraya koengi. J Appl Biol Biotechnol. 2014; 2: 6-11. ). The Neotyphodium endophytes enhance the Cadmium tolerance activity in two plant species of Festuca arundinacea and F. pratensis when compared to non endophyte infected plants (Soleimani et al. 2010b). The fungal endophyte Penicillium funiculosum acts against the copper stress and enhances the plant growth; it can be used in bioremediation of pollution in cultivated area by stress mediating endophytes (Khan and Lee 2013).

CONCLUSION

Endophytic fungi have wide application in different fields. It has the potential to produce many bioactive compounds. The secondary metabolites produced by the endophytic fungi have the ability to act as biocontrol agent. Endophytic fungi isolated from the medicinal plants would be a promising source for many pharmaceutical ingredients. Plants are the major preliminary source for pharmaceutical drug products. Isolating a compound from the plants and large scale production of a product is expensive and time consuming. But endophytic fungi originated from medicinal plants have the capability to produce valuable compounds and can be easily cultured and large scale production is possible through fermentation process. In future the products from the endophytic fungi will be a cheap source for medical, agriculture and other industries. It is sure that the research on endophytic fungi will lead to isolate more novel compounds.

ACKNOWLEDGEMENT

The Project was full financially supported by king Saud University, through Vice Deanship of Research Chairs.

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