UNIVERSITY OF AGRICULTURE, FAISALABAD.

Department of Plant Pathology

Synopsis for M.Sc. (Hons.) Agriculture Degree

Title: Chemotherapic Management of Macrophomina phaseolina causing Charcoal Root Rot in

Sesame (Sesamum indicum L.)
Name of student : Asad Mehmood

Regd. No : 2013-ag-3826

ABSTRACT

In Pakistan Sesame ( Sesamum indicum L.) is an vital and antique oilseed crop. The origin of
sesame crop is East Africa and South Asia, originated about 5000 years ago. Charcoal root rot,
Blight of seedlings and Charcoal stem rot are caused by fungus Macrophomina phaseolina (Tassi)
Goidanich. It can also infect of more than 500 crop and non-crop species. During 2015-16
Worldwide, sesame cultivation area was 8.26 million hectares and the Production of 3.31 million
tons. In Pakistan total cultivated area was 80 thousand hectares with production of 32.4 thousand
tons annually. The pathogen attacks a broad spectrum of economically important crops, in the
pathogenic stage the fungus is a nonspecific pathogen. Worldwide it infects more than 500 plant
species of about 100 families including major oil crops (sesame, soybean, sunflower) pulse crops
(green gram, common bean) food crops (sorghum, maize) fiber crops (cotton, jute). Sesame
production facing a lot of limitations such as fungal diseases. Among these limitations, M.
phaseolina causes Root rot of sesame is an important destructive thermophilic, polyphagus and
soil born fungal pathogen. It causes charcoal rot Root rot of roots and lower stem. This pathogen
cause massive losses of about 146 million USD in Nigeria and 40-57% disease incidence. In
Pakistan it cause 50-75% disease incidences. Under favorable soil and environmental conditions
pathogen growth and development rate increased which result in failure of crop. Isolation,
identification, purification and management of pathogen by using various fungicides will be done
in this experiment. Research area of Department of Plant Pathology will be used to conduct field
trial for this purpose at University of Agriculture, Faisalabad following Randomized Complete
Block Design (RCBD). After this, noted data will be examined.

UNIVERSITY OF AGRICULTURE, FAISALABAD

 Department of Plant Pathology
Synopsis for the degree of M.Sc. (Hons.) Plant Pathology

Title: Chemotherapic Management of Macrophomina phaseolina causing Charcoal Root Rot in

Sesame (Sesamum indicum L.)

a) Date of Admission : 28-09-2017

b) Date of Initiation : 20-06-2018
c) Probable Duration : 4 semesters

Personnel:
a) Name of the student : Asad Mehmood
: 2013-ag-3826
b) Supervisor : Dr. Nasir Ahmed

Supervisory Committee:
a) Dr. Nasir Ahmed : (Chairman)
c) Dr. Khali Naveed : (Member)
d) Dr. Rana Muhammad Atif : (Member)

Need for Project

Sesame (Sesamum indicum L.) also known as Til, is among the important edible oilseeds crop.
Sesame seeds are rich source of oil (around 50%) and proteins (18-20%) (Hwang, et al 2005).
In addition to the protein and oil content, Sesame plant seeds contains carbohydrates, mineral
substances and fatty acids, too ( Nzikou et al., 2009; Alyemeni et al., 2011). The nutritional
value of 100gm seeds of sesame is describe as proteins 17gms, lipid contents 48gms,
carbohydrates 25.7gms, fiber 14gms and ash 6gms approximately. Sesame seeds possess
minerals in large amounts such as Phosphorous, Calcium, Potassium and Magnesium. Sesame
seeds are also rich source vitamins such as vitamin B-6, Thiamin, Niacin, and Riboflavin
(USDA Nutrient Database, 2015). In irrigated and dry areas, sesame is successfully cultivated.
Due to short period of vegetation, low production cost and the possibility of its being
cultivated as second crop the importance of this cultivated plant is gradually increasing
.(Arıoğlu et al., 2010). Sesame is belonging to the Pedaliaceae family (Falusi & Salako et al
2001). The origin of sesame species is Africa and South Asia, sesame species was originated
about 5000 years ago (Alghuthaymi MA, et al. 2016). In ancient era sesame is cultivated in
Africa, after that it spread in India at a very early date (Alegbejo et al., 2003; TundeAkintunde
et al. 2012). Tropical and subtropical regions of Asia, Africa and South America are known
to cultivated regions of sesame crop (Anilakumar et al 2010). In production, sesame ranks
third among the oilseed crops. These are top ten countries China, India, Ethiopia Myanmar,
Nigeria, Uganda, Somania , Niger, United Republic of Tanzania, and Burkina Faso of sesame
growing by production of Sesame seeds. (FAOSTAT, 2011). In 2010 Average global yield of
sesame crop was 3.84 million metric tons, cultivated on an area of 7.8 million hectares. In
2013 Burma was the leading producer in 2013 of sesame seeds. World’s leading exporter was
India and Japan was the leading importer of sesame seeds (FAOSTAT, 2013). Among major
sesame producing countries Pakistan ranks fourteen (FAO et al. 2014). In Pakistan 88
thousand hectares was cultivated with production of 19.3 thousand tons approximately (Shah
et al., 2014). 9,398,770 ha Worlds total cultivation area under sesame, yield 4.76 million tons
of sesame seeds (FAO, 2013). In 2007 China, Sudan, India Ethiopia, Uganda, Burma and
Nigeria, were major countries in sesame seeds production. In 2001 the leading producers of
sesame seeds were India and China followed by Sudan (3 million tons) and Burma (Myanmar)
(4.2 million tons). Africa and Asia produced 26% and 70% of world sesame, respectively
(Hansen, et al. 2011). The sesame crop cultivated for its edible seeds (Weiss et al 2000) and
oil which has high behenic acid content. A sesame seed possesses nutritive, preventive and
curative properties. Seeds of sesame are used for cooking and also in medicinal products.
Sesame seeds contains phyto-nutrients with health promoting properties includes flavonoid
phenolic anti-oxidants, omega-6 fatty acids, dietary fiber with potent anti-cancer and
Vitamins. There are number of fungal diseases which effect the sesame plant among these the
most destructive disease caused by Macrophomina phaseolina is charcoal root rot .It is a soil
borne fungus causes charcoal Root rot of roots and lower stem. Important factors which
reduced Production and minimizing the sesame competition in crop rotation is the fungal
pathogen M.phaseolina which causes charcoal root rot a most destructive disease of sesame
plant(Abdou et al., 2001). If management is not taken to control ,heavy yield losses may cause
 by pathogen in sesame ranging from 50-100%. ( El Bramaway et al 2006; El Shakess and
Khalifa et al 2007).
M.phaseolina with wide host range is the most destructive fungal pathogen of the crops (
Dinakaran & Mohammed 2001; Gaige.et al, 2010). The pathogen attacks a broad spectrum of
economically important crops, in the pathogenic stage the fungus is a nonspecific pathogen
Worldwide it infects more than 500 plant species of about 100 families including major oil
crops (sesame, soybean, and sunflower) pulse crops (green gram, common bean) food crops
(sorghum, maize) fiber crops (cotton, jute) ( Khan, 2007; Dinakaran and Mohammad, 2001;
Aly et al, 2007).

Objective

The experiment has following objective;

 We will conduct experiment for Isolation and identification of the causal agent from root
of sesame infected plant.
 Experiment conduct for Pathogenicity tests of fungal pathogen on sesame plant.
 In lab study of different ways of spreading of the disease causative agent.
 In lab study the effect of various fungicides on growth and development of disease causing
pathogen and disease development.

In order to accomplish the objectives following line of work was adopted.

 Survey of sesame growing areas of Chak # 37 Gardana Faisalabad and collection of

samples.
 Isolation, identification, purification and preservation of pathogen linked with sesame plant
roots.
 Against isolated pathogen evaluation of various chemicals in vitro.

Review of literature

The phylum of fungus Macrophomina phaseolina (Tassi) Goid is ascomycetes, class

Dothideomycetes, order Botryosphaeriales, belongs family Botryosphaeriaceae, genus
Macrophomina and species phaseolina. It has septate hyphae filliform, hyaline at the first and later
becoming grey to black producing jet black oval to round microsclerotia its diameter ranges from
80-90 μm .(Akhtar et al, 2011; Mahmoud and Budak, et al 2011). M. phaseolina a soil borne
fungal pathogen causing Charcoal root rot of sesame.It is a most important disease on sesame crop
cause serious losses of seedlings and mature plants (Abdou et al, 2001). M. phaseolina is a
pathogen of many crops causing a serious diseases, the well known disease is charcoal root rot.
Host range of M. phaseolina is more than 500 plant families. With heterogeneous host specificity
and non-uniform distribution in the soil it can infect monocots and dicots. (Mayek-Perez et al.,
2001; Su et al., 2001). Both in agricultural and natural ecosystems M. phaseolina is the very
drstructive soil and seed borne pathogen. M. phaseolina has heterogeneous geographical and host
ranges. Pathogenicity variations of isolates acquire from both a single plant and a single host
species has confirmed its variability (Babu et al., 2010; Jana et al., 2005; Saleh et al., 2010). M.
phaseolina were not divided into sub-species owing to large variations in pathogenicity and
morphology (Das et al., 2008; Khan, 2007). Pre- and postemergence plant mortality is caused by
Macrophomina infection. Disease symptoms are the development of lesions which are spindle
shaped, microsclerotia are present which has small sized pinhead with dark border and light gray
centre and sometimes pycnidia. For a long periods of time fungus have ability to stay alive in the
soil due to presence of sclerotia (Baird et al., 2003). M. phaseolina is a plant as well as human
pathogen. Many medical reports have been established that M. phaseolina causative agent of skin
and many fungal infections in humans.(Tan et al., 2008; Srinivasan et al., 2009).This pathogen
infect seedlings and lower roots and their color changes from brown to black depend on disease
intensity (Manjunatha et al., 2013). For better cultivation and high yield of Sesame crop, there are
several limitations (Langham and Wiemers, et al 2002). Charcoal root rot caused by this pathogen
is very crucial disease of sesame among all the limitations (Dinakaran & Mohammed, et al 2001).
Under field conditions there were 57% losses, and the disease incidence was 40% whereas 5-100%
yield losses has also observed under favorable climatic conditions (EL-Bramawy and Abdul
Wahid, et al 2006). In nature M.phaseolina is necrotrophic and thermophillic phytopathogen
(Salik 2007). Within the genus Macrophomina a single specie phaseolina is recognized but among
various isolates from different hosts a immense variability in morphology and pathogenicity has
seen.(Thippeswamy et al., 2003). Through microsclerotia or pycnidia pathogen mainly can
reproduces. Through soil as well as seed the fungus spread immediately, as temperature rise from
28-35°C, severity of disease enhances as in soil and air (Khan, 2007). The characteristic disease
symptoms are development of lesions, central part of leaves is light grey with dark border,
secondary roots are black, occurance of sudden wilting and death of growing plants (Khalili et al.,
2016).

There are lot of managements to control disease include cultural practices, tolerant varieties,
biological control method, crop rotation soil solarization, minimum supply of soil moisture and
systemic induce resistance to reduce disease incidence of pathogen M. phaseolina
which cause charcoal root rot, these measures require long time and highly skilful accuracy
(Infantino et al., 2006). A suitable and simple technique is used in application of systemic
fungicides, which are quick in action against this disease. In this practice, soil drenching method
is used for applying mixed fungicides solution with irrigation water. (El-Fiki et al., 2004).For
getting high economic return and high yield from crop farmers sensibly apply fungicides for better
production and protection of crop (Arriel et al., 2007). The fungicide application on crops to
obstruct disease losses, possesses relatively low cost and more effectiveness (Azeez and
Morakinyo , et al 2010). Application of systemic fungicides helps in Plant protective measures`
for soil born diseases. To reduce the disease occurance and for managing and reducing the attack
of thermophillic plant pathogen that are soil borne are main part of study of this research. In
existing era the demand of environmentally friendly systemic fungicide is more. Therefore, there
is need of a suitable concentration of systemic fungicides for charcoal root rot of sesame. Various
concentrations of fungicides like Nativo, Topsin-M Score, Mancozib, Topass and Antracol were
used to control sesame infection caused by M.phasiolina known as Charcoal root rot. Fungicide
named Nativo was used that disturbed the metabolism of fungus and by hindering development
and growth reduce the fungal radial colony growth, for this purpose 150 ppm concentration of
fungicide is used that reduced 1.26 cm radial colony of pathogen. These fungicides attach with
sclerotia by forming covalent bond and disturb the ionic concentration of pathogen. The other
fungicides like Trifloxystrobin 25% + Tebuconazole 50% with various concentrations were used
for fungus M. phasiolina but these fungicides did not showed better results in reducing radial
colony growth of pathogen. Nativo fungicide showed considerable results in reducing radial
colony growth of pathogen. Likewise, by using food poison technique researchers applied six
different fungicides named mancozeb, carbendazim, propiconazole, tebuconazole, hexaconazole
and cheshunt against fungus M. phasiolina using five different concentrations of 50, 100, 250, 500
and 1000 ppm, the results were that Tebuconazole, carbendazim and propiconazole hindered
growth of mycelium at concentration of 50 ppm as compared to all other concentrations. Thus, in
this research efficient concentrations of systemic fungicides were used to reduced disease losses
of sesame crop (Muhammad R. Bashir et al , 2017).

Material and method

Collection of Diseased Sample

Roots of sesame plants shows characteristics diseased symptoms of charcoal root rot. Diseased
sample will be collected from the sesame field. To avoid from dehydration these diseased samples
store in polythene bags and will be bring to the Lab of Mycolog, University Of Agriculture
Faisalabad.

Results
Isolation, identification, purification and management of pathogen by using various fungicides will
be done in this experiment. Research area of Department of Plant Pathology will be used to conduct
field trial for this purpose at University of Agriculture, Faisalabad following Randomized
Complete Block Design (RCBD). After this, noted data will be examine.

Isolation of Pathogen
Isolation of pathogen will be done by firstly we will washed the diseased roots sample with tap
water to get rid of the contaminats like sand, dust particles and weeds etc. Then the diseased roots
sample will be cutted into small pieces by using scissor. We will used a beaker having distilled
water to washed the sample again. We will put these small pieces for surface sterilization into
another beaker having bleach mixed in distilled water for 60 second. Again wash the small roots
pieces with distilled water and dry the sample by putting these pieces on sterilized filter paper. At
least three pieces of diseased roots will be placed in a petri plate having Potato Dextrose Agar
(PDA) in sterilized Laminar flow. After that we will wrap the petri plates and labeled the petri
plate by writing the name of sample, date and name of student. Then we will place the plates into
incubator for three to four days at 30oc temperature.

Identification of Pathogen
Identification of pathogen will be done by we will prepare various slides of pathogen using petri
plates having pathogen growth. After this we use compound microscope to examined slides having
pathogen.

Purification of Pathogen
Petri plates will be checked after three to four days of incubation for fungus growth. The pathogen
growth occur in petri plates.To get pure culture of the pathogen we will transfer the pathogen in
an other sterilized petri plates having Potato Dextrose Agar (PDA). For this purpose we will
prepared PDA and pour it into petri plates and leave these plates to solidify PDA for five to ten
minutes in Laminar flow chamber. Then we will take a minute part of pathogen growth by using
anoculating needle and transfer it to another petri plates having PDA. After that we will write name
of pathogen, Date, name of student and wrap the petri plates with cling film and transfer these petri
plates into incubator at optimum temperature 4oc. After some days we will checked petri plates to
getting pure culture of pathogen.

Inoculation of Pathogen to Healthy Sesame Plants

In field healthy plants of sesame will be grow for inoculation of pathogen and then these plants
will be transfer into pots on which inoculation process will be done. By using proper technique we
will make a suspension of pathogen and apply this pathogen suspension in soil around the
rhizosphere after getting pure culture of pathogen.After some days the sesame plants show
characteristics symptoms when the infection occur, note the infection data on regular basis.

Re-isolation of Pathogen from the Diseased Sesame Plants After Inoculation

When the process of inoculation occur after few days the sesame healthy plants show
characteristics symptoms of disease and infection will be occurred. The infected root sample will
be collected again and these sample for re-isolation will brought to the Lab. Re-isolation of
pathogen is done in order to confirm the re-isolated pathogen is same as the pathogen we incolated
earliar and to confirmed the fourth point of Koch's Postulates that was identification of causative
agent in particular disease.

Use of Fungicides to Control Of Root Rot of Sesame Disease

After inoculation of pathogen in healthy sesame plants disease will be developed. Then we will
use different fungicides with various concentrations to manage charcoal root rot disease of the
sesame plant.

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