Introduction

Sunflower (Helianthus annuus L) is the most important globally oil crop which is grown over

22 million hectares, with a production of 26 million tonnes throughout the worldwide

(Shirshikar, 2005; Skoric et al., 2007). It belongs to a family Asteraceae and belongs to a

genus of Helianthus. One race of sunflower is Heliabthus annuus which is cultivated for

edible seed (Khan et al., 2007). In Pakistan, sunflower introduced during 1960 with the object

of bridging the gap between production and consumption of edible oil in the country (Burney

et al., 1990). Sunflower soyabean found to have a potential to raise the productivity of edible

oil (Nasir, 2003). In Pakistan sunflower also became an important crop for farmers and

consumers and it is also considered the important cash crop in all over the country (shah et

al., 2005).

Available varieties of sunflower contain oil in the seed from 39 to 49%. Sunflower is

considered as premium oil because of its high level of unsaturated fatty acid, light color and

lack of linolenic acid. Several reasons such as occasional adverse, climate conditions and

prevalence of diseases are the causes of low yield of sunflower (Mirza and Beg, 1983).

Sunflower is suffered from a charcoal rot disease which is caused by fungus Macrophomina

phaseolina (Jalaluddin, 2008). Charcoal rot disease is considered most prevalent than the

other diseases on sunflower. M. phaseolina is a soil born fungus and infects 500 species or

more of 7 families of worldwide which includes legumes, fiber crops, vegetables, and fruits

(Kumar et al., 1986; Sinclair and Backman, 1986; Salik, 2007). M. phaseolina is responsible

to cause losses on more than 500 cultivated and wild plant species, and has a wide host range

(Indera et al., 1986). This fungus also affects the fibrovascular system of the roots, blocking

the transport of water and nutrients to plant upper part. 12% annual loss in the yield is

estimated in 12 million hectares of the world due to diseases of sunflower (Kolte, 1985).
M. phaseolina survives in the soil as free sclerotia and embedded in host crop or in dry soil

for a long period (Sinclair, 1982). Sclerotium is a cushion shaped and 1-8 mm in diameter.

When field is flooded for irrigation sclerotia floats freely (Keim and Webster, 1974; Webster

et al., 1976). Due to charcoal rot disease sudden wilting of plants occur after pollination. On

the stem brown to dark brown spots appears, due to which the disease gets the name

“charcoal rot”. Black microsclerotia found in the pith if the stem split due to infection. The

plants also become dry, weak and show ashy or light gray black discoloration of stems.

Vascular fibers shredded and pith disintegrates and covered with this fungus (Sinclair, 1982;

Yang and Owen, 1982; Hoes, 1985; Kolte, 1985).

Various methods are used to combat pathogenic fungi. These methods are chemical,

biological, cultural and physical methods (Sharma, 1996; Kata, 2000). Chemical method is

considered as a good method to control the plant diseases than the others. Chemical

pesticides provide quick, effective and economic management of plant diseases. But

fungicides do not protect the crops for long period (Jeyarajan et al., 1991). Today there are

strict regulations on chemical pesticides use, and there is political pressure to remove the

most hazardous chemicals from the market. Additionally, the spread of plant diseases in

natural ecosystems may preclude successful application of chemicals, because of the scale to

which such applications might have to be applied. Consequently, some pest management

researchers have focused their efforts on developing alternative inputs to synthetic chemicals

for controlling pests and diseases (Pal et al., 2006). Because of increase in cost of chemical

pesticides emphasis is given on biological control agent against plant pathogen (Agrios,

2004).

The terms “biological control” and its abbreviated synonym “biocontrol” have been used in

different fields of biology, most notably entomology and plant pathology. In entomology, it

has been used to describe the use of live predatory insects, entomopathogenic nematodes, or
microbial pathogens to suppress populations of different pest insects. In plant pathology, the

term applies to the use of microbial antagonists to suppress diseases as well as the use of

host-specific pathogens to control weed populations. In both fields, the organism that

suppresses the pest or pathogen is referred to as the biological control agent (Pal et al.,

2006).

Genus Trichoderma was first described by Persoon in 1794. Persoon’s classification was

found to be doubtful so, Rifai made first attempt in 1969. Trichoderma is a septate fungus

and produces conidiophores which have a pyramidal and conical outline. Trichoderma

species form many colonies of different color like yellow, green and white (Rifai, 1969).

Trichoderma species are free living and can be found in large number in decaying wood and

in agricultural soil (Irina and Christian, 2004).

Trichoderma species are the successful antagonists and having the ability of biocontrol

against soil-borne pathogens and plant parasites (Kushwaha and Verma, 2014; Shahid et al.,

2014). These species attacks on plant pathogens and reduces the infection of M. phaseolina

and promotes the growth of plants and roots (Harman, 2006). The most important species of

trichoderma are in the field are T. harzianum, T. virens and T. asperellum (Benitezet et al.,

2004). Many isolates of T. viride and T, harzianum was obtained from the rhizosphere soil of

the plantation crops and forest soil (B. N. Chakraborty et al., 2010). Trichoderma species act

as a guard for their hosts, and also fulfill the needs of its host and help the host to increase

their growth and protection from pathogens (Seidl et al., 2009). Recent studies showed that

Trichoderma species induces localized and systemic resistance in plants against attack of

variety of plant pathogens or damage caused by insects or treatment with different chemical

inducers (Kuc, 2001; Oostendorp et al., 2001). Strains of Trichoderma are well known in

their ability to colonize roots but conidia of Trichoderma when applied to fruit, flowers and

foliage can control plant diseases (Harman, 2000; Dubos, 1987; Elad, 1994). The present
study was attempted to use of Trichoderma species as a seed coating for the control of

charcoal rot fungus on sunflower.

Nanotechnology is considered to be the advanced technique that has become an important

part of our lives, playing a major role in the agricultural production (Biswas and Wu, 2005).

The Greek noun “nano” which means dwarf or short from which the word nano is derived. In

medicine, the particle size of nanoparticles (NP) ranges from 5-250nm (Moghini et al., 2005).

At the present time, in all fields of science nanotechnology has extensive horizon. They are

usually used in preparation of the industrial products, pharmaceuticals, in the fields of

engineering, medicine and in agriculture (Biswas and Wu, 2005). These nanoparticles are the

aggregation of atom and molecules having dimension range which lie between 1-100 nm

(Wiesner et al., 2006).

They have modified physical and chemical properties because in compare to bulk materials it

has large surface to volume ratio (Nel et al., 2006). At nano scale, the activities of most

substances in which chemical and biological activities are included have increased

(Mazaherinia et al., 2010).

Nanotechnology can also be used in improvement of crop production and growth and in

increasing yield (Reynolds, 2002). The nanoparticles synthesis is thought to be as a dynamic

area in research and the application of these nanoparticles. There are many different methods

of nanoparticle synthesis including these three methods; physical, chemical and biological

methods. The nanoparticles synthesis through biological method is relatively widely used

because it has advantages including low cost and having friendly environment without toxic

effect. The application of nanoparticles through biological method is an easy way for

nanoparticles green synthesis. (Thunugunta et al 2015). The proved nanoparticles application

to plants can be valuable for growth and development because of its ability for higher

absorbance and high reactivity (Liu and Lal, 2015).

Silver nanoparticles are the most widely used nanomaterials (Dakal et al., 2016). Sizes of

AgNPs vary and are between 1 and 100 nm in three dimentions with positive and negative

charge depending on their coating material (Graf et al., 2003). Sunflower exhibit a strong

potential to form Ag+ from AgNPs as compared to Basella alba, Saccharum officinarum and

Oryza sativa (Leela and Vivekanandan, 2008). Silver nanoparticles at concentrations of 20,

40, 60, 80 and 100 mg kg-1 impacted on plant growth parameters such as leaf surface area,

carbohydrate and chlorophyll, all of which affected growth of the common bean and corn and

promoting plant growth at low doses and inhibition at high concentrations (Salama, 2012).

Trichoderma viride produces many hydrolytic enzymes and is used against the pathogenic

fungi. This fungus also produces many enzymes like chitinases, proteases and glucanase.

T.viride is used to senthetize biogenic AgNPs because it is a nonpathogenic fungi and

environmentally friendly fungus (Grondona et al., 1997; Lunge AG and Anita SP, 2012).

T.harzianum is also used as a stabilizer in the biogenic synthesis of AgNPs. Silver

nanoparticles were characterize to confirm the synthesis and determine parameters including

size distribution and morphology (Singh and Raja, 2011; Ahluwalia et al., 2014).
Agrios, G.N, 2004. Plant Pathology. 5th Edition. Elesvier, Academic Press. pp. 922.

Bhattacharya, D.T.K., Dhar, K.A.I. Siddiqui and E. Ali. 1994. Inhibition of Seed Germination

by Macrophomina phaseolina is related to phaseolinone production. Journal of Applied

Bacteriology, 77(2): 129-133.

Ahluwalia, V., Kumar, J., Sisfodia, R., Shakil, N. A. & Walia, S. Green Synthesis of Silver

Nanoparticles by Trichoderma harzianum and Their Bio-efficacy Evaluation Against

Staphylococcus aureus and Klebsiella pneumonia. Ind. Crops Prod. 55, 202–206 (2014).

Benitez T, Rincon AM, Limon MC, Codon AC., 2004. Biocontrol Mechanisms of

Trichoderma strains.Int.l J. Micro. 7: 249–260.

Biswas, P., & Wu, C. Y. (2005). Nanoparticles and the Environment. Journal of the air &

waste management association, 55(6), 708-746.

Burney, K., Ahmad, I. and Aslam, M. 1990. Charcoal Rot and Important Disease of

Sunflower and Its Control. Progressive Farming 10: 34–6.

Dubos, B. (1987). In: Innovative Approaches to Plant Disease Control (ed. Chet, I.) 107–135

(John Wiley and Sons, New York).

Elad, Y. (1994). Control of Grape Grey Mould by Trichoderma harzianum. Crop Protect. 13,

35–38.

Grondona I, Hermosa MR, Tejada M, et al., Physiological and Biochemical Characterization

of Trichoderma harzianum, a Biocontrol Agent Against Soil Borne Fungal Plant Pathogens.

Appl Environ Microbiol. 1997;63:31893198.

Harman, G. E. (2000). Myths and Dogmas of Biocontrol. Changes in Perceptions derived

from research on Trichoderma harzianum T22. Plant Dis., 84: 377–393.

Harman, G.E., 2006. Overview of Mechanisms and Uses of Trichoderma spp.

Phytopathology 96, 190-194.

Hoes JA, 1985. Macrophomina phaseolina Causal Agent of Charcoal Rot of Sunflower and

Other Crops. Agriculture Research Station, Modren Manitoba, Canada.

Indera K, Singh T, Machado CC, Sinclair, JB, 1986. Histopathology of Soybean Seed

Infection by Macrophomina phaseolina. Phytopathology 76: 532-535.

Irina, D. and P.K. Christian. 2004. Species and Biodiversity in Trichoderma and Hypocera:

from Aggregate Species to Species Clusters. J. of Zhejiang Uni. Sci., 6: 100-112.

Keim R, Webster RK, 1974. Effect of Soil Moisture and Temperature on Viability of

Sclerotia of Sclerotium Oryzae. Phytopathology 64: 1499-1502.

Khan, S. N., Ayub, N., Ahmad, I., Shehzad, I. and Asad. 2007. Effect of Climatic Conditions

on life cycle of Charcoal Rot infected Sunflower plant. Pakistan Journal of Botany 39:2657-

2662.

Kolte, S.J. (1985). Sunflower diseases of annual oilseed crops, Vol. III, CRC Press, Inc. Boca

Raton, Florida, pp. 33-44.

Kolte, S.J. (1985). Sunflower diseases of annual oilseed crops, Vol. III, CRC Press, Inc. Boca

Raton, Florida, pp. 33-44.

Kuc, J. (2001). Concepts and Direction of induced Systemic resistance in plants and its

application. Eur. J. Plant Pathol., 107: 7–12.

Kunwar, I. R., T. Sinyh, C.C. Machado and J.B. Sinclair (1986). Histopathology of Soybean

seed and seedling infection by Macrophomina phaseolina. Phytopathology, 76: 532 − 535.
Kushwaha, M., Verma, A.K., 2014. Antagonistic activity of Trichoderma spp, (a bio-control

agent) against isolated and identified plant pathogens. Internationl Journal of Chemical and

Biological Sciences 1, 1-6.

Liu, R., & Lal, R. (2015). Potentials of engineered Nanoparticles as fertilizers for increasing

agronomic productions. Science of the total environment, 514, 131-139.

Lunge AG, Anita SP. Characterization of efficient chitinolytic enzyme producing

Trichoderma species: a tool for better antagonistic approach. Int J Sci Environ Technol.

2012;1:37385.

Mazaherinia, S., Astaraei, A. R., Fotovat, A., & Monshi, A. (2010). Nano iron oxide particles

efficiency on Fe, Mn, Zn and Cu concentrations in Wheat plant. World Applied Sciences

Journal, 7.

Mirza, M. S. and Beg, A. 1983. Diseases of Sunflower in Pakistan in 1982. Helia. 6: 55- 56.

Moghimi, S. M., Hunter, A. C., & Murray, J. C. (2005). Nanomedicine: current status and

future prospects. The FASEB journal, 19(3), 311-330.

Nasir, N. 2003. Detecting seed borne fungi of Soybean by different incubation method.

Pakistan Journal of Plant Pathology 3: 114- 118.

Nel, A., Xia, T., Mädler, L., & Li, N. (2006). Toxic potential of materials at the

nanolevel. science, 311(5761), 622-627.

Oostendorp, M., W. Kunz, B. Dietrich and T. Staub (2001). Induced disease resistance in

plants by chemicals. Eur. J. Plant Pathol., 107: 19–28.

Pal, K. K. and B. McSpadden Gardener, 2006. Biological Control of Plant Pathogens The

Plant Health Instructor. DOI: 10.1094/PHI-A-2006-1117-02.

Persoon CH.1794. Dispositamethodicafungorum. Römer'sNeues Mag Bot. 1794;1:81–128

Reynolds, G. H. (2002). Forward to the Future: Nanotechnology and regulatory policy.

Pacific Research Institute.

Rifai MA. 1969. A revision of the genus Trichoderma.Mycological Papers.116: 1-56.

Salik, N. K. (2007). Macrophomina phaseolina as causal agent for Charcoal rot of Sunflower.

Mycopath., 2, 111 − 118.

Seidl V, Song L, Lindquist E, Gruber S, Koptchinskiy A, Zeilinger S, Schmoll M, Martinez

P, Sun J, Grigoriev I, Herrera-Estrella A, Baker SE, Kubicek CP. 2009b. Transcriptomic

response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal

prey.BMC Genomics. 10: 567-572.

Shah, N.A.,Shah, H. and Akmal, N. 2005. Sunflower area and production variability in

Pakistan: opportunities and constraints. Helia. 28: 165-178.

Shahid, M., Srivastava, M., Singh, A., Kumar, V., Rastogi, S., Pathak, N., Srivastava, A.,

2014. Comparative study of biological agents, Trichoderma harzianum (ThAzad) and

Trichoderma viride (01PP) for controlling wilt disease in pigeon pea. Journal of Microbial

and Biochemical Technology 6, 110-115.

Shirshikar, S.P. 2005. Mildew disease in India. Helia. 28:153-158.

Sinclair JB, 1982. Compendium of Soybean disease. 2nd Ed. by American Phytopathology

Society, St. Paul, Minnesota, USA.

Sinclair, J. B. and P. A. Backman (1986). Compendium of Soybean diseases. Third edition

The American Phytopathological Society, 30 − 33.

Sinclair. 1982. Compendium of Soyabean diseases. 2nd ed. Publ. by American

Phytopathological Society 3340 Piolt Knob road, St. Paul Minnesota, USA.

Singh, P. & Raja, R. B. Biological synthesis and characterization of silver nanoparticles using

the fungus Trichoderma harzianum. Asian J. Exp. Biol. Sci. 2, 600–605 (2011).

Skoric, D., Jocic, S., Lecic, N. and Sakac, Z. 2007. Development of Sunflower hybrids with

different oil quality. Helia. 30:205-212.

Webster RK, Bolstad J, Wick CM, Hall DH, 1976. Vertical distribution and survival of

Sclerotium oryzae under various tillage conditions. Phytopathology 66: 97-101.

Wiesner, M. R., Lowry, G. V., Alvarez, P., Dionysiou, D., & Biswas, P. (2006). Assessing

the risks of manufactured Nanomaterials.

Yang SM, Owen DF, 1982. Symptomology and detection of Macrophomina phaseolina in

Sunflower plants parasitized by Cylendrocopturus adspersus larvae. Phytopathology 72: 819-

821.